Practical Guide to Chemical Safety (Rapra Practical Guides)

Practical Guide to Chemical Safety Testing Regulatory Consequences - Chemicals, Food Packaging and Medical Devices

Edited by Derek J. Knight and Mike B. Thomas

Rapra Technology Limited Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 http://www.rapra.net

First Published in 2003 by

Rapra Technology Limited Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

©2003, Rapra Technology Limited

All rights reserved. Except as permitted under current legislation no part of this publication may be photocopied, reproduced or distributed in any form or by any means or stored in a database or retrieval system, without the prior permission from the copyright holder. A catalogue record for this book is available from the British Library.

ISBN: 1-85957-372-X

Typeset and printed by Rapra Technology Limited Cover printed by The Printing House

Preface and Acknowledgements

This ‘Practical Guide to Chemical Safety Testing Regulatory Consequences – Chemicals, Food Packaging and Medical Devices’ covers the basis of toxicology in relation to the safety assessment and regulatory requirements for chemicals, plastics and rubber. It is aimed at manufacturers, distributors and users and hence covers industrial and household chemicals, food packaging and medical devices. The emphasis is on providing a basis of understanding of toxicology, ecotoxicology and physico-chemical properties for hazard assessment and interpretation within risk assessment and regulatory frameworks. We want to acknowledge the contributions of many people who have helped us with this work. Dr Sally Humphreys, Technical Editor at Rapra, encouraged us to make the proposal for this project and was patient with us while the work was in progress. The Safepharm Laboratories Board of Directors permitted us and our various colleagues the time and resources to make this book possible. Jacqueline Billing administered the project for us extremely thoroughly and efficiently, after taking over from early co-ordination work by Dr Carlo Poncipe. Jane Burrows typed several of the chapters from scratch and reformatted the rest and made our various editorial changes. Matthew Pearce indexed about half the chapters and prepared the annex of acronyms and his sister, Hayley Pearce, thoroughly checked the page proofs. We are most grateful for all this help. Finally, we want to thank the authors of the chapters, who as experts in the various specialist fields, have made this book possible. We would like to dedicate this book to the memory of Derek Knight’s parents, the late Dorothy and Keith Knight. Derek Knight and Mike Thomas January 2003

i

Contributors

Derek J. Knight is the Director of Regulatory Affairs at Safepharm Laboratories Ltd., a leading UK contract research organisation, specialising in safety assessments of chemicals, biocides, and agrochemical pesticides. He heads a team of regulatory affairs professionals who deal with a wide range of registration projects covering many product types for regulatory compliance in all the key markets globally. As such he has gained an overall perspective into commercial issues associated with the regulation of the chemical industry. His doctoral studies at the University of Oxford were in organosulfur chemistry. Mike B. Thomas is the Marketing Director for Safepharm Laboratories. A graduate in Zoology and Chemistry from London University, he began his career in toxicology at Consultox Laboratories Ltd. in London, conducting short-term toxicity studies. Eventually he became Head of Toxicology at this company, with responsibility for a wide range of mammalian and genetic toxicity studies. Prior to joining Safepharm in 1982, he was Director of Biological Services at International Consulting and Laboratory Services Ltd., of London. Malcolm P. Blackwell BSc PhD MIBiol CBiol FIAT has 20 years experience working in toxicology safety testing, including 18 years in the contract research industry. His specialist fields are inhalation toxicology and repeated dose toxicology. He has held various senior positions at Safepharm Laboratories and is currently Director of Mammalian Toxicology. Having started his career as a technician he has considerable practical experience as well as academic knowledge gained from graduate training, PhD research and interpretation of study data. Eric Wood has worked in toxicology for 25 years, in the pharmaceutical, agrochemical and contract research industries. For the past 20 years he has specialised in reproductive toxicology, and has been Head of the Reproductive Toxicology Department at Safepharm Laboratories for 11 years. He is a member of the UK Reproductive Toxicology Discussion Group and the European Teratology Society. Peter C. Jenkinson began his career in Genetic Toxicology at the ICI Central Toxicology Laboratories in 1978. After graduating from university, he worked in two contract research organisations, in the UK and in Spain, before joining the British Industrial Biological Research Association. During his 5 years there he continued to work in the field of genetic toxicology and also completed a collaborative PhD with Professor Dennis Parke

iii

Practical Guide to Chemical Safety Testing at the University of Surrey. He joined Safepharm Laboratories in 1987 to establish the new Department of Genetic Toxicology. He is an active member of the UK Environmental Mutagen Society and Industrial Genotoxicology Group, and is the current Secretary of the former and past Chairperson of the latter. John W. Handley is the Head of Ecotoxicology at Safepharm Laboratories. He has some 20 years experience of conducting ecotoxicology studies. Following the completion of his Masters research into acid rain and the toxicity of aluminium to rainbow trout, he worked at the University of Cardiff on contract to the EU, developing methods for chemical registration. From there he moved to Huntingdon Research Centre where he was Deputy Head of Aquatic Toxicology for 4 years. He moved to Safepharm Laboratories in 1989 to design and set up the ecotoxicology facility. He is a member of the Society of Environmental Toxicology and Chemistry, and participates on UK shadow OECD discussion panels for ecotoxicology and biodegradation. He is a EUROTOX registered toxicologist. Darren M. Mullee is the Head of the Analytical and Physico-Chemical Properties Department at Safepharm Laboratories, with 13 years experience of performing Good Laboratory Practice (GLP) studies in support of new chemical notifications and agrochemical registrations. He has previously gained over 7 years experience in analytical chemistry working for a major pharmaceutical company. He is currently an active member of the Physico-Chemical Regulatory Characterisation group, and participates in collaborative ring tests and analytical method data generation for various regulatory bodies. He has a degree in chemistry and holds the designatory CChem MRSC qualification. Karmel P. Biring graduated from the University of Southampton in 1995 with a degree in Chemistry. He is currently in his seventh year working for Safepharm Laboratories. Building on his roots within quality assurance, he quickly progressed to become a Registration Executive where his day to day activities include advice both in-house and to clients on global strategies for notification of new substances, with an emphasis on the key area of pharmaceutical intermediates. Damien Breheny graduated from the National University of Ireland in 1998 with an Honours Degree in Microbiology and in 2001 qualified with First Class Honours for an MSc in Toxicology from Athlore Institute of Technology, Ireland. In August 2000 he worked as an In Vitro Toxicologist at Safepharm Laboratories, and is currently working as a genetic toxicologist at British American Tobacco in Southampton. Dr Paul Illing is Principal of Paul Illing Consultancy Services, a consultancy specialising in toxicology for occupational health, product safety and the environment which he set up in 1999. He is also an honorary lecturer in the Centre for Occupational and iv

Contributors Environmental Health of the University of Manchester and Chairman of the Royal Society of Chemistry Environment Health and Safety Committee Shadow Group for the UK Government’s Chemicals Stakeholders Forum. From 1982 to 1998 he was employed by the UK Health and Safety Executive, where he held posts that included Principal Toxicologist, Head of Toxicology, Head of Biocides and Biological Agents and (on secondment) Secretary to the Government/Research Councils Initiative on Risk Assessment and Toxicology. Prior to this he carried out toxicokinetic studies on new chemical entities in the pharmaceuticals industry. Robert Diderich has been involved in environmental risk assessment of chemical substances since 1992, when he joined the German Federal Environmental Agency. He has been working in France since 1995, first at the Ministry of the Environment and then at the National Institute for Industrial Environment and Risks, where he is currently studying the environmental risks of industrial chemicals and biocides. He is involved in the continuous development of the EU technical guidance documents for the environmental risk assessment of chemicals. John M. Hislop gained a degree in Applied Chemistry from Nottingham Trent University, graduating in 1994. He has since worked in the Department of Registration Services at Safepharm Laboratories, and currently holds the position of Senior Registration Executive. He specialises in the worldwide notification of industrial chemicals. He is a Member of the Royal Society of Chemistry and the British Institute of Regulatory Affairs. Dr Carlo Poncipe obtained his PhD in chemistry from the University of Surrey in 1985. After 3 years working in the development of novel polyurethane elastomers, he spent the next 13 years working in the medical device industry, both in the UK and the US. Having joined Safepharm Laboratories as a Registration Officer nearly two years ago, he now specializes in high production volume (HPV) chemical registrations under both the US and the International Council of Chemical Associations (ICCA) programmes. John Moore joined ICI in 1968 and worked in research for 15 years before becoming involved in health and environmental safety in 1983. As a result of demergers, he became part of Zeneca Specialties and then Avecia, without actually moving location. He has held the post of Regulatory Manager for the past 8 years. Jeremy Tinkler is Principal Expert in Biosciences and Implants at the UK Department of Health’s Medical Devices Agency, which is becoming part of the Medicines and Healthcare Products Regulatory Agency in April 2003. Before joining the Department of Health in 1987, as its first toxicologist to specialise in medical devices, he worked for ten years as a toxicologist in contract research organisations and at the Health and Safety Executive.

v

Practical Guide to Chemical Safety Testing Dr Sandra Costigan has an MSc in Life Sciences from Wageningen Agricultural University in the Netherlands and a PhD with post-doctoral research in electrophysiology from Limbergs University in Belgium. She has worked as a toxicologist at Procter & Gamble’s European Technical Centre. She then carried out further post-doctoral research in biochemistry at Imperial College, London and Bristol University, before joining the UK Medical Devices Agency in January 2001. Lesley A. Creighton has worked within SafePharm Laboratories for 11 years providing regulatory support to the chemical industry for the notification of new chemical substances, food contact materials and cosmetic products. Before working in regulatory affairs, she was based in the laboratory, mainly working on the determination of physicochemical properties. She has a combined science degree in chemistry and mathematics. Dr A. Mel Cooke is the founder of Alchemy Compliance, an independent consultancy offering advice on a wide range of regulatory issues, especially relating to the supply of chemicals and biocides, notification of new chemicals, classification and labelling, and existing chemicals programmes. He gained over six years of regulatory experience at Safepharm Laboratories Ltd., reaching the position of Deputy Head of Registration Services. He is co-editor of a book, ‘The Biocide Business’ published in 2002 by WileyVCH. He has previously gained industrial experience in the Ciba-Geigy laboratories in Basel. He researched the synthetic organic chemistry and pharmacology of secondary messengers to gain his PhD from the University of Leicester.

vi

Contents

Preface and Acknowledgements .............................................................

i

Contributors .............................................................................................

iii

1.

Introduction ..................................................................................................

1

1.1

Purpose of the Book ........................................................................

1

1.2

Purpose of Safety Evaluation ..........................................................

1

1.3

Safety Studies ..................................................................................

2

1.4

Risk Assessment and Safety Data ..................................................

5

1.5

Regulatory Schemes .......................................................................

6

1.6

Summary .........................................................................................

8

Part I. Safety Testing and Assessment 2.

Mammalian Toxicology ................................................................................

9

2.1

Introduction ......................................................................................

9

2.2

Acute Toxicity Studies ..................................................................... 2.2.1 Nature and Relevance of Tests ........................................ 2.2.2 Methodology ..................................................................... 2.2.3 Acute Oral Toxicity Studies ............................................... 2.2.4 Dermal Toxicity Studies .................................................... 2.2.5 Inhalation Toxicity Studies ................................................ 2.2.6 Alternative Acute Oral Toxicity Methods ........................... 2.2.7 Local Tolerance Tests ....................................................... 2.2.8 Contact Sensitisation ........................................................

9 9 11 12 13 14 15 17 18

2.3

Repeated Dose Toxicity Studies ...................................................... 2.3.1 Nature and Relevance of Tests ........................................ 2.3.2 Importance of Repeated Dose Toxicity ............................. 2.3.3 Methodology .....................................................................

20 20 22 23

2.4

Reproduction Toxicology ................................................................. 2.4.1 Nature and Relevance of Tests ........................................ 2.4.2 Methodology ..................................................................... 2.4.3 Alternative Approaches .....................................................

25 25 26 29

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vii

viii

3.

4.

Contents 2.5

Carcinogenicity ................................................................................ 2.5.1 Nature and Relevance of Tests ........................................ 2.5.2 Methodology ..................................................................... 2.5.3 Dose Levels ...................................................................... 2.5.4 Conduct of Study .............................................................. 2.5.5 Data Evaluation ................................................................. 2.5.6 Risk Assessment .............................................................. 2.5.7 Alternative Approaches .....................................................

30 30 31 32 32 33 33 34

2.6

Medical Device Testing .................................................................... 2.6.1 Exposure Routes .............................................................. 2.6.2 Dose Preparation .............................................................. 2.6.3 Cytotoxicity Testing of Medical Devices ............................

34 35 35 35

Genetic Toxicology ......................................................................................

43

3.1

Introduction ......................................................................................

43

3.2

Mechanisms of Mutation – Genes and Chromosomes ....................

44

3.3

Standard Genetic Toxicology Assays ..............................................

47

3.4

Bacterial Mutagenicity Assays .........................................................

47

3.5

Chromosome Aberration Tests in Vitro ............................................

50

3.6

Mammalian Cell Gene Mutation Assays in Vitro ..............................

53

3.7

The in Vivo Micronucleus Test .........................................................

55

3.8

The Unscheduled DNA Synthesis Assay .........................................

57

3.9

Conclusions .....................................................................................

59

Ecotoxicology ..............................................................................................

63

4.1

Introduction ......................................................................................

63

4.2

Bacterial Toxicity Testing .................................................................

65

4.3

Biodegradation Tests ....................................................................... 4.3.1 Ready Biodegradation Tests ............................................. 4.3.2 Inherent Biodegradation Tests .......................................... 4.3.3 Simulation Tests ............................................................... 4.3.4 Anaerobic Biodegradation Tests .......................................

65 66 70 71 71

4.4

Aquatic Toxicity Testing ................................................................... 4.4.1 Acute Tests ....................................................................... 4.4.2 Analytical Measurements .................................................. 4.4.3 Difficult Substances .......................................................... 4.4.4 Chronic Tests ....................................................................

72 73 77 78 79

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5.

Contents

ix

4.5

Fish Bioaccumulation Test ...............................................................

81

4.6

Sediment Toxicity Tests ...................................................................

81

4.7

Terrestrial Toxicity Tests .................................................................. 4.7.1 Earthworms ....................................................................... 4.7.2 Bees and Beneficials ........................................................ 4.7.3 Plant Growth Tests ...........................................................

82 82 82 83

4.8

Microcosm and Mesocosm Studies .................................................

83

4.9

Conclusion .......................................................................................

83

Physico-Chemical Properties ......................................................................

87

5.1

Introduction ......................................................................................

87

5.2

Performance of the General Physico-Chemical Tests ..................... 5.2.1 Melting Temperature/Melting Range (OECD Test Guideline 102) ................................................................... 5.2.2 Boiling Point (OECD Test Guideline 103) ......................... 5.2.3 Vapour Pressure (OECD Test Guideline 104) .................. 5.2.4 Water Solubility (OECD Test Guideline 105) .................... 5.2.5 Partition Coefficient (OECD Test Guidelines 107 and 117) ................................................................................... 5.2.6 Adsorption Coefficient (OECD Test Guidelines 106 and 121) ............................................................................ 5.2.7 Density/Relative Density (OECD Test Guideline 109) ...... 5.2.8 Particle Size Distribution (OECD Test Guideline 110) ...... 5.2.9 Hydrolysis as a Function of pH (OECD Test Guideline 111) ................................................................................... 5.2.10 Dissociation Constant (OECD Test Guideline 112) .......... 5.2.11 Surface Tension (OECD Test Guideline 115) ................... 5.2.12 Fat Solubility (OECD Test Guideline 116) ........................

89 89 90 91 93 95 97 99 100 101 103 104 106

5.3

Performance of the Polymer Specific Physico-Chemical Tests ....... 106 5.3.1 Number-Average Molecular Weight and Molecular Weight Distribution of Polymers (OECD Test Guideline 118) ................................................................... 107 5.3.2 Solution/Extraction Behaviour of Polymers in Water (OECD Test Guideline 120) .............................................. 108

5.4

Performance of the Hazardous Physico-Chemical Tests ................ 108 5.4.1 Flash Point (EC Method A9) ............................................. 109 5.4.2 Flammable Solids (EC Method A10) ................................. 109 This page has been reformatted by Knovel to provide easier navigation.

x

Contents 5.4.3

5.4.4 5.4.5

5.4.6

6.

7.

Flammable Gases (EC Method A11), Flammable Substances on Contact with Water (EC Method A12) and Substances Liable to Spontaneous Combustion (EC Method A13) .............................................................. Explosive Properties (EC Method A14) ............................ Auto-Ignition Temperature, Liquids and Gases (EC Method A15) and Relative Self–Ignition Temperature, Solids (EC Method A16) ................................................... Oxidizing Properties (EC Method A17) .............................

110 111

112 113

5.5

Order in Which Physico-Chemical Tests are Performed ................. 114

5.6

Conclusion ....................................................................................... 115

Alternatives to Animal Testing for Safety Evaluation ................................... 119 6.1

Introduction ...................................................................................... 119

6.2

Validation of Alternative Methods .................................................... 120

6.3

Aspects of Human Toxicity Targeted by in Vitro Assays ................. 122 6.3.1 Systemic Toxicological Properties .................................... 122 6.3.2 Validated Tests Currently in Use in the EU ....................... 125

6.4

Structure-Activity Relationships and Prediction of Properties .......... 129

6.5

Strategies to Minimise Use of Animals ............................................ 131

6.6

Future Developments and Conclusions ........................................... 132

Toxicological Assessment within a Risk Assessment Framework .............. 137 7.1

Introduction ...................................................................................... 137

7.2

Definitions and Concepts ................................................................. 137 7.2.1 Risk ................................................................................... 138 7.2.2 Toxicology ......................................................................... 144

7.3

Exposure Scenarios ......................................................................... 148 7.3.1 Routes of Administration ................................................... 148 7.3.2 Exposure Prediction .......................................................... 151

7.4

Judgements ..................................................................................... 7.4.1 The ‘Precautionary Principle’ ............................................ 7.4.2 What Test and When? ...................................................... 7.4.3 The Interpretation of Toxicity Test Results for Classification and Labeling Purposes ............................... 7.4.4 Risk Assessment and Risk Evaluation – Interpretation of General Toxicity ............................................................

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152 153 154 154 155

Contents 7.4.5

8.

xi

Mutagenicity, Carcinogenicity and Reproductive Toxicity .............................................................................. 157

7.5

Risk Management ............................................................................ 159

7.6

Final Word ....................................................................................... 160

Environmental Risk Assessment ................................................................. 163 8.1

Introduction ...................................................................................... 163

8.2

Exposure Assessment ..................................................................... 8.2.1 Identification of the Target Compartments ........................ 8.2.2 Estimation of Emissions or Releases ................................ 8.2.3 Distribution and Degradation in the Environment (Environmental Fate) ........................................................ 8.2.4 Predicted Environmental Concentrations ..........................

164 165 167 168 175

8.3

Effects Assessment ......................................................................... 178 8.3.1 Estimating PNECs by Applying Uncertainty Factors ......... 179 8.3.2 The Statistical Extrapolation Method ................................ 182

8.4

Risk Characterisation ....................................................................... 184

8.5

Conclusion ....................................................................................... 184

Part II. Regulatory Framework 9.

EU Chemical Legislation ............................................................................. 191 9.1

EU Legislation within the European Economic Area and Europe ............................................................................................. 191

9.2

Notification of New Substances ....................................................... 9.2.1 History of the Notification Process .................................... 9.2.2 Data Sharing ..................................................................... 9.2.3 Base Set Studies for Full Notification ................................ 9.2.4 Reduced Notification Studies ............................................ 9.2.5 Level 1 and Level 2 Notification Studies ........................... 9.2.6 The Notification Summary Form ....................................... 9.2.7 The Sole-Representative Facility ...................................... 9.2.8 Polymers ........................................................................... 9.2.9 Derogations/Exemptions from Notification ........................ 9.2.10 Confidentiality ...................................................................

9.3

Risk Assessment ............................................................................. 202 9.3.1 Human Health Risk Assessment ...................................... 203 9.3.2 Environment Risk Assessment ......................................... 204 This page has been reformatted by Knovel to provide easier navigation.

192 193 193 193 194 194 199 200 200 200 201

xii

Contents 9.4

Existing Chemicals Regulation ........................................................ 9.4.1 Data Collection .................................................................. 9.4.2 Priority Setting ................................................................... 9.4.3 Risk Assessment ..............................................................

207 208 211 211

9.5

Chemical Hazard Communication ................................................... 9.5.1 Classification and Labeling of Dangerous Substances ....................................................................... 9.5.2 Classification and Labeling of Dangerous Preparations ...................................................................... 9.5.3 Safety Data Sheets ...........................................................

212

9.6

Transport Regulations ..................................................................... 9.6.1 Introduction ....................................................................... 9.6.2 The United Nations Transportation Classification Scheme ............................................................................. 9.6.3 Transport of Marine Pollutants ..........................................

212 214 215 215 215 217 218

9.7

National Chemical Control Measures .............................................. 219 9.7.1 National Product Registers ............................................... 220 9.7.2 German Water Hazard Classification Scheme ................. 220

9.8

Other EU Legislation for Specific Product Types ............................. 9.8.1 Control of Cosmetics in the EU ......................................... 9.8.2 Detergents ........................................................................ 9.8.3 Offshore Chemical Notification Scheme: Oslo and Paris Convention for the Protection of the North East Atlantic ..............................................................................

9.9

222 223 224

225

Summary and Future Developments ............................................... 226

10. Chemical Control in Japan .......................................................................... 235 10.1

Introduction to the Japanese Regulatory Culture ............................ 235

10.2

The Ministry of Economy, Trade and Industry and Ministry of Health, Labour and Welfare Chemical Substances Control Law 10.2.1 Introduction ....................................................................... 10.2.2 The Inventory of Existing Substances ............................... 10.2.3 Exemptions from Notification ............................................ 10.2.4 Standard Notification ......................................................... 10.2.5 Polymer Notification .......................................................... 10.2.6 Class I and II Specified and Designated Substances .......

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236 236 238 239 241 244 245

Contents

xiii

10.3

The Ministry of Health, Labour and Welfare Industrial Safety and Health Law ....................................................................................... 246

10.4

Hazard Communication and Product Liability .................................. 247

10.5

Other Chemical Legislation .............................................................. 249

10.6

Summary ......................................................................................... 252

11. Chemical Control in the US and the Rest of the World ............................... 255 11.1

Introduction ...................................................................................... 255

11.2

US Chemical Legislation: the Toxic Substances Control Act (TSCA) ............................................................................................. 11.2.1 Key Objectives of TSCA ................................................... 11.2.2 The TSCA Inventory ......................................................... 11.2.3 Testing of Existing Substances ......................................... 11.2.4 Manufacturing and Processing Notices ............................ 11.2.5 PMN Requirements ........................................................... 11.2.6 Significant New Use Rules (SNURs) ................................ 11.2.7 Exemptions from PMN ......................................................

256 256 256 261 261 261 262 262

11.3

US Occupational Safety and Health Act (OSHA) ............................ 265

11.4

The US Chemical Right-to-Know Initiative for High Production Volume Chemicals ........................................................................... 11.4.1 Voluntary Challenge Programme ...................................... 11.4.2 Persistent Bioaccumulative Toxic (PBT) Chemicals ......... 11.4.3 US Voluntary Children’s Chemical Evaluation Program ............................................................................

11.5

11.6

Chemical Control Legislation in Canada .......................................... 11.5.1 The Canadian Environmental Protection Act .................... 11.5.2 Inventories ........................................................................ 11.5.3 Environmental Assessment Regulations .......................... 11.5.4 Data Requirements for Notification ................................... 11.5.5 Significant New Activity Notice .......................................... 11.5.6 Administration ................................................................... 11.5.7 Inspection, Enforcement and Penalties ............................ 11.5.8 Future Changes ................................................................ 11.5.9 The Workplace Hazardous Materials Information System ..............................................................................

266 266 268 269 270 270 271 272 273 273 273 275 275 276

Chemical Control Legislation in Switzerland .................................... 276 11.6.1 The Federal Law on Trade in Toxic Substances .............. 276 This page has been reformatted by Knovel to provide easier navigation.

xiv

Contents 11.6.2 11.7

11.8

11.9

The Federal Law on Environmental Protection ................. 277

Notification of New Chemical Substances in Australia .................... 11.7.1 National Industrial Chemicals (Notification and Assessment) Scheme ....................................................... 11.7.2 Inventory ........................................................................... 11.7.3 Data Requirements for Notification ................................... 11.7.4 Existing Substances .......................................................... 11.7.5 Hazard Communication ....................................................

280

Chemical Control in Korea ............................................................... 11.8.1 The Toxic Chemicals Control Law and Ministry of Environment Notification ................................................... 11.8.2 The Industrial Safety and Health Law and Ministry of Labour Toxicity Examination ............................................. 11.8.3 Hazard Communication ....................................................

283

280 281 281 282 282

283 285 286

Chemical Control in the Philippines ................................................. 11.9.1 The Toxic Substances and Hazardous and Nuclear Wastes Control Act ........................................................... 11.9.2 Inventory ........................................................................... 11.9.3 Data Requirements for Notification ................................... 11.9.4 Administration ................................................................... 11.9.5 Priority Chemicals List (PCL) ............................................

286

11.10 Chemical Control in the People’s Republic of China ....................... 11.10.1 Latest Developments ........................................................ 11.10.2 First Import and Toxic Chemicals Regulations ................. 11.10.3 Inventory ........................................................................... 11.10.4 Hazard Communication ....................................................

290 290 291 291 292

11.11 Chemical Control in New Zealand ................................................... 11.11.1 Toxic Substances Act ....................................................... 11.11.2 Resource Management Act .............................................. 11.11.3 Hazardous Substances and New Organisms Act ............. 11.11.4 Data Requirements for Notification ................................... 11.11.5 Hazard Communication ....................................................

292 292 292 293 294 295

286 287 287 288 290

11.12 Mexico ............................................................................................. 295 11.12.1 Legislation ......................................................................... 295 11.12.2 Safety Data Sheets ........................................................... 296

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11.13 Singapore ........................................................................................ 296 11.14 Malaysia ........................................................................................... 297 11.15 Thailand ........................................................................................... 297 11.16 Indonesia ......................................................................................... 297 11.17 Taiwan ............................................................................................. 297 11.18 HPV Programmes ............................................................................ 298 11.18.1 OECD ................................................................................ 298 11.18.2 International Council of Chemical Associations Global Initiative ............................................................................. 299 11.19 Useful Web Sites ............................................................................. 300 12. Notification of Polymers Worldwide ............................................................. 307 12.1

Introduction ...................................................................................... 307

12.2

North America .................................................................................. 308 12.2.1 USA ................................................................................... 308 12.2.2 Canada ............................................................................. 311

12.3

Asia Pacific ...................................................................................... 12.3.1 Japan ................................................................................ 12.3.2 Australia ............................................................................ 12.3.3 New Zealand ..................................................................... 12.3.4 Korea ................................................................................ 12.3.5 Philippines ......................................................................... 12.3.6 China .................................................................................

12.4

Europe ............................................................................................. 331 12.4.1 EU ..................................................................................... 331 12.4.2 Switzerland ....................................................................... 340

12.5

Overall Comparison of the Nine Polymer Notification Schemes ...... 341

312 312 320 324 325 328 331

13. Medical Device Regulation .......................................................................... 345 13.1

Introduction ...................................................................................... 345

13.2

European Economic Area ................................................................ 13.2.1 Background ....................................................................... 13.2.2 Before Marketing ............................................................... 13.2.3 After Marketing ..................................................................

13.3

United States of America ................................................................. 359 13.3.1 Background ....................................................................... 359 13.3.2 Before Marketing ............................................................... 359 This page has been reformatted by Knovel to provide easier navigation.

345 345 351 357

xvi

Contents 13.3.3

After Marketing .................................................................. 362

13.4

Japan ............................................................................................... 363 13.4.1 Background ....................................................................... 363 13.4.2 Before Marketing ............................................................... 364 13.4.3 After Marketing .................................................................. 365

13.5

Conclusion ....................................................................................... 366

14. Regulation of Food Packaging in the EU and US ........................................ 367 14.1

Introduction ...................................................................................... 367

14.2

Control of Food Packaging in the EU .............................................. 14.2.1 EU Framework Directive ................................................... 14.2.2 Food Contact Plastics in the EU ....................................... 14.2.3 Future Developments for Food Plastics in the EU ............ 14.2.4 Other EU Food Packaging Measures ............................... 14.2.5 Strategy for Food Contact Plastic Approval in the EU ......

368 368 369 376 377 378

14.3

National Controls on Food Packaging in EU Countries ................... 14.3.1 Germany ........................................................................... 14.3.2 France ............................................................................... 14.3.3 The Netherlands ............................................................... 14.3.4 Belgium ............................................................................. 14.3.5 Italy ...................................................................................

379 379 381 381 381 382

14.4

Council of Europe Work on Food Packaging ................................... 14.4.1 Introduction ....................................................................... 14.4.2 Completed Council of Europe Resolutions ....................... 14.4.3 Council of Europe Ongoing Work .....................................

382 382 383 385

14.5

Food Packaging in the USA ............................................................. 14.5.1 Introduction ....................................................................... 14.5.2 History and Development of US Food Packaging Legislation ......................................................................... 14.5.3 The FDA Petition ............................................................... 14.5.4 Threshold of Regulation Process ...................................... 14.5.5 The Pre-Marketing Notification Scheme ...........................

388 388 389 391 394 395

15. Regulation of Biocides ................................................................................. 401 15.1

Introduction ...................................................................................... 401

15.2

Control of Biocides in the EU ........................................................... 402 15.2.1 Introduction ....................................................................... 402 This page has been reformatted by Knovel to provide easier navigation.

Contents 15.2.2 15.2.3 15.2.4

xvii

Main Features of the Directive .......................................... System of Approval ........................................................... Assessment for the Inclusion of Active Substances in Annex I of the Biocidal Products Directive ........................ Authorisation of Biocidal Products .................................... Hazard Communication .................................................... The Review Programme for Existing Active Substances ....................................................................... Technical Guidance ..........................................................

403 403

15.3

Control of Biocides in the USA ........................................................ 15.3.1 Introduction ....................................................................... 15.3.2 Data Requirements for Registration .................................. 15.3.3 Registration Applications .................................................. 15.3.4 Data Compensation .......................................................... 15.3.5 Re-Registration of Existing Pesticides .............................. 15.3.6 Petition for a Pesticide Tolerance ..................................... 15.3.7 Regulation of Food Contact Biocides ................................

409 409 410 411 413 413 415 415

15.4

Regulation of Biocides in Other Countries ....................................... 417

15.2.5 15.2.6 15.2.7 15.2.8

404 405 406 406 407

Abbreviations and Acronyms ............................................................... 421 Index ....................................................................................................... 435

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Introduction

1

Introduction Mike B. Thomas and Derek J. Knight

1.1 Purpose of the Book Chemicals play a vital role in modern industrial society, and with an estimated 100,000 synthetic chemicals in commerce today, the monitoring and regulation of these materials with respect to their safety has become a significant task involving many scientific disciplines. This ‘Practical Guide to Chemical Safety Testing, Regulatory Consequences – Chemicals, Food Packaging and Medical Devices’ covers the basis of toxicology in relation to the safety assessment and regulatory requirements for chemicals, plastics and rubber. It is aimed at manufacturers, distributors and users and hence covers industrial and household chemicals, food packaging and medical devices. The emphasis is on providing a basis of understanding of toxicology, ecotoxicology and physico-chemical properties for hazard assessment and interpretation within risk assessment and regulatory frameworks. Many acronyms are used in this field, and these are collated as a complete list at the end of the book for ease of reference. There is a complex path from development to eventual successful marketing of new chemical products involving many functions within the innovator company. The process is costly in terms of money, time and human resources, and there are many potential pitfalls. An important element is regulatory planning, which requires a regulatory strategy to meet the commercial aspirations for the products. Such a strategy will include a testing programme covering the various national notification or other regulatory schemes and minimising the use of experimental animals. Usually a commercial decision has to be taken on what to prioritise: minimising the cost of testing, reducing the time to marketing or achieving certain regulatory compliance.

1.2 Purpose of Safety Evaluation The toxicological properties of products ranging from pharmaceuticals to agrochemicals, biocides and industrial household chemicals including toiletries and cosmetics, have to

1

Practical Guide to Chemical Safety Testing be determined to make sure they may be used safely. Traditionally the hazardous properties are determined by testing using standardised procedures, normally with the studies conducted in compliance with Good Laboratory Practice (GLP). These studies are, in effect, a model for the impact that the chemical may have on the systems of concern, namely humans and the environment. Knowledge of the toxicological properties affecting human health and the ecotoxicology and environmental fate properties, which can affect the environment, are required. To provide safe products is undoubtedly of the utmost importance, but this aim has been brought into conflict with strong public opinion, especially in Europe, against animal testing. Hence industry, academia and regulators have been working in partnership to find other ways of evaluating the hazardous properties of chemicals. Test data are used to identify hazardous properties, which are described by various classification and labelling schemes using stylised interpretation criteria. The magnitude of hazardous properties from many of the laboratory tests can then be used to predict the adverse effects on humans or the environment. Hence, when combined with the predicted exposure, a risk assessment can be performed, in which the chance of undesirable effects from the chemical in the real world is assessed. The outcome of risk assessment may be risk management, if necessary, to prevent harm to humans or the environment. The risk assessment is one of the main factors in deciding whether the product can be approved in schemes that require marketing authorisation, such as biocides. It is very important to appreciate that risk assessment and regulatory approval is usually an iterative process, i.e., a cycle of successive improvements, where the assessment is revised several times. If the initial outcome is unfavourable, the risk assessment can be made more realistic and less precautionary either by improving the exposure assessment, for example by revising use conditions to lower exposure, or by generating more safety data to improve the knowledge of the hazardous properties.

1.3 Safety Studies The chemical structure and the physico-chemical properties of the substance to be evaluated for safety are of key importance. These features can help decide what safety studies to conduct, and they will certainly need to be considered in interpreting many of the studies and extrapolating the results of the standard laboratory tests to predict effects on man or the environment. The chemical properties and structure may be such that it is possible to determine the toxicological or ecotoxicological properties of the chemical with enough confidence to avoid testing, but more often these predictions are used in making professional judgements for general safety assessment or in range-finding work for the definitive studies and in assessing the reliability of existing studies of uncertain quality.

2

Introduction Chapter 5, by Darren Mullee and Karmel Biring, covers testing of physico-chemical properties, both general properties and those which determine if the chemical is hazardous. The results of the general properties tests do not lead directly to classification and labelling of a substance, but they often affect the choice of further physico-chemical, toxicological and ecotoxicological studies. Furthermore, many of the endpoints measured are pivotal for environmental fate modelling and occupational exposure assessment. However, the hazardous properties tests do determine the classification of the substance. Estimation methods for physico-chemical endpoints are sometimes employed, and these usually make use of thermodynamic and empirical relationships. There are also quantitative structureactivity relationships (QSARs) to estimate physico-chemical endpoints instead of testing. Such predictions may be used in deciding which of the experimental methods for a test is appropriate where there is more than one method available, or for providing an estimate or limit value in cases where the experimental method cannot be applied for technical reasons. Alternatively, a calculation can help identify cases where omitting experimental measurement is justified and so is a key to obtaining a data waiver for that particular endpoint. The toxicological investigation of the hazardous properties of chemicals is largely conducted using experimental animals. The aim is to obtain sufficient data on their toxic properties which, taken with a knowledge of the mechanisms involved, allow prediction of the likely adverse effects in humans. Chapter 2, by Malcolm Blackwell and Eric Wood, provides an overview of the mammalian toxicology studies that are used to identify hazards associated with chemicals or other substances. Mammalian toxicity studies have been devised to assess most of the important, recognised human health hazards associated with exposure to chemicals. These studies are generally conducted in the form of a tiered testing strategy, progressing dependent on the nature and use of a chemical and, in many cases, the volume of manufacture and risk assessments associated with the substance. Thus, most chemical substances will be subjected to short-term (acute) toxicity and local tolerance tests. The longer and more expensive studies designed to determine hazards, such as carcinogenicity or reproductive toxicity, are reserved for chemicals with a high potential for human exposure, or where an indication of hazard has been identified in earlier tests. Genetic toxicology, which is covered in Chapter 3 by Peter Jenkinson, is the study of the potential of chemicals to induce damage to deoxyribonucleic acid (DNA). Damage to DNA may result in error-free repair, mutation or cell death. Cell death is of concern because of the effect that it may have on ageing and the degenerative diseases associated with old age and exposure to chemicals. However, the primary concern that has driven genetic toxicology over the last three decades is the link between mutation and cancer. The other major concern related to exposure to genotoxic chemicals is the induction of mutations in germ cells, either in sperm after exposure of the male or oocytes after exposure of females.

3

Practical Guide to Chemical Safety Testing Any mutations that occur in germ cells may be inherited by future generations, which may have adverse effects on their morbidity, fertility or life span. Hence the purpose of genetic toxicology tests is to identify chemicals that may have the potential to cause cancer or other mutagen-related disease. Various regulatory authorities have developed genetic toxicology testing strategies in order to identify such chemicals. It is of the utmost importance to ensure that the wide range of chemicals in commerce today can be used safely without injuring the human population or the environment. However, in recent times this objective has been brought into conflict with public opinion, which has shown a growing concern over the use of animal experiments to investigate toxicity, particularly of chemicals which are perceived to be relatively safe, e.g., cosmetics, toiletries, and household products. Hence, industry, academia and regulators have been working in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals and the severity of tests using them. There is a long way to go before all the potential hazardous properties can be evaluated without any animal studies, but considerable progress has been made using a combination of in vitro (i.e., non-animal) tests and prediction of properties based on chemical structure. Chapter 6, by Derek Knight and Damien Breheny, describes these important and worthwhile developments in various areas of toxicology testing, with a focus on the European regulatory framework for general industrial and household chemicals. Alternative tests are used in different contexts, from screening tests to eliminate substances from further development on the basis of their potential toxicity, to regulatory safety evaluation. There are great incentives to develop in vitro alternatives to supplement and even replace the existing animal toxicology methods, with the key consideration being animal welfare. Effects of chemicals on the environment are predicted using standard ecotoxicity studies, as described in Chapter 4 by John Handley. Ecotoxicology is the study of the adverse effects of chemicals in the environment and on ecological systems. It is mostly carried out in standardised laboratory tests designed to be robust yet reproducible, sensitive and reliable. It is clearly impractical to study the effect of the chemical on all the organisms in the various relevant ecosystems, so regulatory ecotoxicity studies therefore focus on indicator species, to allow hazard assessments to be made which in turn can be used to define the risk of a chemical to the environment. The aquatic environment is normally the primary route of exposure, either from direct run-off or by discharge from production or sewage treatment facilities. Hence ecotoxicity testing for regulatory compliance concentrates mostly on the aquatic environment. Firstly, the likely toxic effect of chemicals on the microorganisms responsible for the degradation of the chemical is assessed, followed by the investigation of biodegradation itself. Next assessments are made of the toxic effects of chemicals to the different trophic levels in the environment.

4

Introduction

1.4 Risk Assessment and Safety Data The information used to classify a chemical substance as ‘dangerous’, either to health or the environment, can be used for hazard assessment, which can be combined with chemical exposure predictions to produce a risk assessment. Further information on toxicity or exposure may be needed to refine the risk assessment before any necessary risk management action is taken to ban or restrict the use of the chemical. Defined hazard and risk assessment procedures may be used by regulators, or informal assessments based on practical experience can be undertaken by chemical users, either voluntarily or to fulfil statutory obligations. The procedures for risk assessment of chemicals in the EU are illustrative of how risk assessments are undertaken by other regulators. In the EU, risk assessment of both new and existing substances follows these steps for human health (toxicology and physicochemical properties) and the environment: a) Assessments of effects, comprising: • identification of the intrinsic hazardous properties of the substance. • elucidation of the dose (or concentration)–response (or effects) characteristics, quantitatively or qualitatively. b) Exposure assessment for the human populations (i.e., workers, consumers and man exposed indirectly via the environment), and for the different environment compartments (water, soil and air) likely to be exposed to the substance. c) Comparison of information on hazardous properties with exposure levels in order to characterise the degree of risk posed by the substance to human health or to the environment. In conducting an exposure assessment, the assessor takes into account those human populations or environmental spheres for which exposure to the substance is known or reasonably foreseeable in the light of available information on the substance, with particular regard to manufacture, transport, storage, formulation into a preparation or other processing, use and disposal, or recovery. Also, for certain particular effects such as ozone-depleting potential for which the above steps are impracticable, risk assessment is conducted on a case-by-case basis. The risk assessment identifies the significant hazardous effects, and also the significant routes of exposure. From this information it is possible to identify which specific stages in the manufacture, distribution, use or disposal of the chemical give rise to particular risks, thus enabling an effective risk reduction strategy to be devised.

5

Practical Guide to Chemical Safety Testing Chapter 7 on human health risk assessment, by Paul Illing, and Chapter 8 on environmental risk assessment by Robert Diderich, are in essence a bridge between the earlier chapters on safety testing and the second part of the book on the specific regulatory frameworks within which human health and environmental risk assessments are conducted as part of the decision-making process. Although safety studies are necessary for the assessment and evaluation of risk, they are not sufficient alone. Exposure information is also required in order to conduct risk assessments. Thus a key element of these chapters is an examination of exposure scenarios used in regulatory risk assessment.

1.5 Regulatory Schemes The safety studies discussed in the first part of the book are normally conducted within a regulatory context to meet various legal obligations. Chemical control legislation, including the degree of its practical enforcement, varies between countries, as does the associated official advice and voluntary industry codes of practice. These systems for chemical control are being continuously developed and improved, sometimes with a view to international harmonisation. In practice, the supply, use and disposal of chemicals throughout their full life cycle is regulated in most of the developed world. However, there are still many differences in chemical control measures between countries, and careful monitoring of developments is essential. There are also other separate legislative controls and voluntary schemes for specific product types. Demands from customers for product information and reassurance of compliance with such voluntary schemes further exacerbates costs. The second part of the book describes the regulations and controls affecting the chemical and related industries, with the EU, Japan and the USA covered in detail, together with important countries throughout the world. There is a separate chapter for polymers (Chapter 12 by John Moore), because although the various chemical notification schemes make a clear distinction between polymer and non-polymeric substances, the strategies employed by the different regions and their data requirements vary significantly. These differences range from what constitutes new and existing polymers, to the actual data required for their notification. Chemical control measures in the EU are described in Chapter 9 by John Hislop. EU chemical legislation is of key importance, not only in view of the importance of the EU market, but because it influences developing legislation in other countries. As the EU expands, other countries in Central and Eastern Europe will follow these schemes directly. Other countries, notably those of the European Economic Area and in due course Switzerland, follow EU chemical legislation although they are not in the EU. Other countries adopt their own chemical control legislation based on that of the EU, for example

6

Introduction hazard communication in Australia. The direct controls on chemicals, and the other measures affecting the chemical industry, are especially complex in Europe, because controls in the EU are based on a network of legislation for hazard communication and safety assessment. This EU legislation is brought into force in individual Member States by national laws, regulations and administrative procedures and hence, although chemical control is fundamentally harmonised, there can be minor differences between countries. Chapter 10 by Derek Knight covers chemical control in Japan. The regulatory systems governing the manufacture and supply of chemicals in Japan are relatively complicated, being covered by various laws which have developed over time and which are administered by different authorities. Moreover for successful notification of new chemicals in Japan it is necessary to grasp the social, business and legal culture in which these systems operate. Hence, for the non-Japanese supplier seeking to notify a new chemical in Japan, it is advisable to use the services of an experienced local representative. Chemical control in the USA is featured in Chapter 11, by John Hislop, Derek Knight and Carlo Poncipe. This chapter also provides a summary of the remaining important chemical control schemes in place in the rest of the world, i.e., in the USA, Canada, Switzerland, Australia, Korea, the Philippines, China and New Zealand, followed by a brief summary of developing schemes in Mexico, Singapore, Malaysia, Thailand, Indonesia and Taiwan. There is a discussion of the various programmes developed to evaluate high production volume (HPV) chemicals, which have become increasingly important since the mid 1990s. Many of these substances have been marketed for many years, but without the same degree of safety testing as required for new notifiable substances. A key element in deciding whether to market new chemicals or support existing HPV chemicals is the cost of the safety testing. The studies are generally based on the Organisation for Economic Co-operation and Development (OECD) minimum pre-marketing data set (MPD), but vary between schemes. For notification of new substances, testing is generally supply driven, with less needed for supply typically below 1 tonne per annum (or 10 tonne per annum in some schemes). Additional testing may be required at higher supply levels, as in the EU. The standard testing programmes for the various schemes are given in this chapter as a resource for general reference. Medical device regulation is described in Chapter 13 by Sandra Costigan and Jeremy Tinkler. The wide variety of products to be controlled means that regulatory systems have to be flexible and allow for product improvement and development. Many regions adopt the use of international standards and all incorporate risk evaluations in the regulatory approval process, and thus allow for less stringent regulation for lower risk devices. In the EU the risk evaluation is the responsibility of the manufacturer.

7

Practical Guide to Chemical Safety Testing Regulation of food packaging in Europe and the USA is described in Chapter 14 by Lesley Creighton and Derek Knight. Food packaging and other articles and materials, which come into contact with food during storage, preparation, cooking and serving, are a potential source of contamination. Chemicals could leach from packaging into food, and these might cause health effects from long-term exposure. Regulation of food packaging is one aspect of this general scheme to ensure food safety. The approaches vary, depending partly on the type of packaging and food involved, but even more so on the particular country or region of supply. Control measures for food packaging seem to have developed gradually, perhaps as safety issues have arisen, based on, or at least within the framework of, previous national food legislation. These measures differ drastically between Europe and the US, and indeed controls on many types of packaging differ between countries within the EU, although there is a strong harmonising influence from the Council of Europe as well as the European Commission. Food packaging regulations are continually under revision, as the work planned to deal with existing products progresses, but also as the need arises to deal with new types of packaging. The final chapter of the book, Chapter 15 by Derek Knight and Mel Cooke, covers the regulation of biocides in the key markets of the EU and the USA, with a brief description of other countries. Biocides are intended to control harmful or unwanted organisms, and as such they are biologically active and therefore potentially pose a risk to human health or the environment. There are an extremely wide variety of uses, with different potential exposure patterns.

1.6 Summary National chemical control systems vary considerably, in spite of attempts at harmonisation, the situation is made more complex by frequent regulatory changes. Furthermore, chemicals are generally regulated throughout their entire life cycle, with controls at each separate stage. Consequently, it can be difficult to ensure that all the regulatory requirements are met for worldwide supply of a chemical. The cost of not satisfactorily accomplishing this complex task could be a considerable delay in marketing the product, or even legal penalties for non-compliance with the requirements, with the associated bad publicity. Planning to meet all the multiple regulatory obligations is essential early in the development of a new chemical product. Input from the regulatory authorities or an experienced regulatory affairs professional will be needed to make sure that the proposed testing programme is not only adequate to meet the regulatory requirements but does not include unnecessary studies, which are clearly undesirable in terms of cost and animal welfare.

8

PART 1:

SAFETY TESTING AND ASSESSMENT

Mammalian Toxicology

2

Mammalian Toxicology Malcolm P. Blackwell and Eric Wood

2.1 Introduction This chapter is intended to provide an overview of the mammalian toxicology studies that are used to identify hazards associated with chemicals or other substances. The information obtained from such studies is used to make an assessment of product safety and ultimately used in formal regulatory risk assessment processes. Further information on the use of data obtained from mammalian toxicity studies is given in Chapter 7. Mammalian toxicity studies have been devised to assess most of the important, recognised human health hazards associated with exposure to chemicals. These studies are generally conducted in the form of a tiered testing strategy, progressing dependent on the nature and use of a chemical and, in many cases, the volume of manufacture and risk assessments associated with the substance. Thus, most chemical substances will be subjected to shortterm (acute) toxicity and local tolerance tests. The longer and more expensive studies designed to determine hazards such as carcinogenicity or reproductive toxicity are reserved for chemicals with a high potential for human exposure, or where an indication of hazard has been identified in earlier tests.

2.2 Acute Toxicity Studies

2.2.1 Nature and Relevance of Tests Acute toxicity studies are designed to determine the adverse effects of a chemical following a single exposure. They are of particular value in establishing effects that may occur in man following accidental exposure, perhaps in the workplace, or deliberate intoxication, for example, in suicide attempts. Relatively high doses are investigated in this type of study to ensure that results are relevant to high level human exposures.

9

Practical Guide to Chemical Safety Testing In their crudest form, acute toxicity studies have historically involved determination of the LD50 value, which is the dose level determined experimentally to cause 50% mortality in the species tested (for inhalation studies the equivalent nomenclature is LC50 representing the exposure concentration causing 50% mortality). In reality, these studies have long been used to obtain other information about the nature of systemic toxic effects induced, target organs affected and, in some cases, mechanisms of toxicity. In recent years concern has mounted about the severity of these studies and the number of animals used to obtain a precise LD50 value with statistical confidence limits. A number of alternative methods have now been developed which use fewer animals. One of these is designed to classify chemicals based on acute toxicity without using death as an endpoint. These methods are discussed in more detail later in this chapter. There are a great many national and international systems for classification and labelling of chemicals based on their acute toxicity. Generally, chemicals are assigned to categories according to their LD50 (in dose per kilogram of body weight) and these categories are used as the basis for appropriate precautionary labelling. A good example of such a system is that operated within the European Union and defined in Annex VI of Council Directive 67/548/EEC (the Dangerous Substances Directive) [1]. Under this scheme, chemicals are categorised as ‘Harmful’, ‘Toxic’, or ‘Very Toxic’ and assigned a standard risk phrase for labelling purposes, as shown in Table 2.1. Provision is also made under this scheme for classification using new alternative methods for determining acute oral toxicity. Other countries have similar schemes but often use different cut-off values for assignment of toxic categories. Under the auspices of the OECD [2], a globally harmonised system for chemical classification has recently been proposed which will allow a single classification to be valid throughout the world. Several countries have already adopted this system but global acceptance is probably some years away. Most acute toxicity studies are conducted in the rat, which is the preferred species under all appropriate regulatory guidelines for chemical safety testing. There are no longer any regulatory requirements for acute toxicity testing of chemicals in a second species. Most regulatory guidelines allow the use of another species (usually another rodent species) if this can be scientifically justified. In the author’s own experience, use of a species other than the rat is extremely uncommon. Due to their smaller body size, occasionally mice may be used if a test substance is in short supply.

10

EU risk categories Oral toxicity Dermal toxicity Inhalation toxicity Mammalian Toxicology

Table 2.1 EU risk categories for chemicals HARMFUL Route

LD50/LC50

Risk Phrase

Oral

200 < LD50 ≤ 2000 mg/kg

R22 Harmful if swallowed

Dermal

400 < LD50 ≤ 2000 mg/kg

R21 Harmful in contact with skin

Inhalation (aerosols or particulates)

1 < LC50 ≤ 5 mg/litre/4h

R20 Harmful by inhalation

Inhalation (gases or vapours)

2 < LC50 ≤ 20 mg/litre/4h

R20 Harmful by inhalation

R out e

LD50/LC50

Risk Phrase

Oral

25 < LD50 ≤ 200 mg/kg

R25 Toxic if swallowed

Dermal

50 < LD50 ≤ 400 mg/kg

R24 Toxic in contact with skin

Inhalation (aerosols or particulates)

0.25 < LC50 ≤ 1 mg/litre/4h R23 Toxic by inhalation

Inhalation (gases or vapours)

0.5 < LC50 ≤ 2 mg/litre/4h

R23 Toxic by inhalation

Route

LD50/LC50

Risk Phrase

Oral

LD50 ≤ 25 mg/kg

R28 Very toxic if swallowed

Dermal

LD50 ≤ 50 mg/kg

R27 Very toxic in contact with skin

Inhalation (aerosols or particulates)

LC50 ≤ 0.25 mg/litre/4h

R26 Very toxic by inhalation

Inhalation (gases or vapours)

LC50 ≤ 0.5 mg/litre/4h

R26 Very toxic by inhalation

TOXIC

VERY TOXIC

2.2.2 Methodology Acute toxicity studies follow a generally similar design regardless of route of exposure, although significant differences exist for acute oral studies since this is the only exposure route for which alternative methods have been developed and adopted by regulatory authorities.

11

Practical Guide to Chemical Safety Testing Animals are exposed to the test substance on a single occasion. On the day of treatment clinical observations are carried out at regular intervals followed by at least daily observations for a subsequent recovery/observation period, normally for 14 days. Clinical observations must be conducted by trained operators and good training is essential to ensure consistency. Many regulatory guidelines provide lists of particular signs of toxicity that should be considered such as: changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. In practice, any changes in behaviour or physical condition compared with untreated animals of the same strain and age should be recorded. There is generally no requirement for control groups in acute toxicity studies so it is essential that observers are familiar with the appearance of normal animals. Animals are weighed at intervals during the observation period (at least once weekly) in order to calculate bodyweight gain. At the end of the study a gross necropsy examination is conducted with detailed macroscopic examination of major organs. Histopathology (microscopic examination of tissues by a pathologist) may be performed on selected tissues, particularly those showing macroscopic abnormality, but most acute studies do not include a histopathology element.

2.2.3 Acute Oral Toxicity Studies

2.2.3.1 Administration Techniques For oral toxicity studies, dosing is by gavage with the test substance administered by a rigid or flexible catheter attached to a syringe and passed down the oesophagus into the stomach. Recommended maximum dose volumes are often given in regulatory guidelines and guidance on good practice for dose volumes in toxicity studies, by various routes, has been published [3]. For oral studies, dose volumes greater than 20 ml/kg bodyweight (for aqueous solutions) or 10 ml/kg bodyweight (non-aqueous) should not be used.

2.2.3.2 Preparation Liquid substances can be administered undiluted or formulated in a suitable vehicle, whereas solids must be formulated to produce a solution or suspension suitable for dosing. Commonly used vehicles include vegetable oils, water (with or without a suitable thickening agent such as methyl cellulose to aid suspension) and polyethylene glycols. The toxicological properties of the vehicle must be well established and use of an unusual vehicle may necessitate inclusion of a vehicle control group in the study.

12

Mammalian Toxicology

2.2.3.3 Special Considerations Some considerable skill is required to avoid injury or death of the animal caused by improper dosing. However, these skills can be readily obtained and, in the hands of experienced operators, such problems are uncommon. Limitations on the dose volumes that can be administered sometimes mean that doses need to be given in smaller fractions divided over a period not exceeding 24 hours. This is only necessary for extremely insoluble and difficult-to-formulate substances.

2.2.4 Dermal Toxicity Studies

2.2.4.1 Administration Techniques Dermal studies are performed by spreading the test substance over the shaved dorsal surface skin (normally approximating to 10% of the total body surface area). The skin site is then covered by a suitable dressing held in place for a period of 24 hours. The most common form of dressing is semi-occlusive, consisting of a gauze patch covered by a self-adherent bandage. Occlusive dressings can also be used, maximising potential for absorption.

2.2.4.2 Preparation As for oral administration, liquids may be administered undiluted or formulated in a suitable vehicle. Solids must be formulated in order to ensure good contact with the skin. The use of water as a vehicle in dermal studies is less effective because retention at the site of administration can be difficult.

2.2.4.3 Special Considerations Animals must be housed individually during the exposure period to avoid cage-mates interfering with the dressing and potentially ingesting the test substance. Skin reactions at the site of administration should be recorded using a graded scale such as that described by Draize [4]. Substances that have previously been shown to be corrosive should not be tested by this route.

13

Practical Guide to Chemical Safety Testing

2.2.5 Inhalation Toxicity Studies

2.2.5.1 Administration Techniques Inhalation studies involve exposure of test animals to controlled atmosphere concentrations of the test substance. Various exposure systems may be used including whole-body exposure, nose-only or head-only exposure. Nose-only or head-only systems are preferred since they avoid concurrent exposure by other routes, specifically oral and dermal. In these systems, animals are held in restraining cones inserted into the wall of an exposure chamber. An atmosphere is generated inside the chamber under dynamic (continuous flow) conditions. The normal exposure period for acute studies is 4 hours but shorter exposures are sometimes used to mimic specific human exposure scenarios.

A B

C

D

D

E

A - Metered air supply B - Generation system C - Observation port

D - Animal restraint tube E - Metered vacuum exhaust

Figure 2.1 Typical nose-only inhalation exposure chamber

14

Mammalian Toxicology

2.2.5.2 Preparation Wherever possible, test substances should be administered as supplied, but formulation in a suitable vehicle may be necessary to improve aerosolisation or particle size distribution. Solid materials may need to be ground to achieve a particle size distribution in the respirable range for the species used.

2.2.5.3 Special Considerations The conduct of inhalation toxicity studies is technically challenging requiring specialist expertise and equipment. Atmospheres need to be produced in the form of aerosols (liquids or dusts), vapours and gases. The chamber concentration needs to remain reasonably stable during the exposure period, preferably within ± 25% of the mean, and a suitable method for analysis of chamber concentrations must be developed. For materials of very low volatility (most dusts and some liquids) gravimetric analysis can be used, but where this is inappropriate a suitable method of chemical analysis is required. In aerosol studies, the particle size distribution of the test atmospheres needs to be determined using a technique that provides a size distribution based on aerodynamic diameter. Respirability and deposition within the respiratory tract is dependent on aerodynamic diameter and to achieve comparable deposition in rats to that expected in humans, particle sizes may need to be reduced in rat studies. For example, it is widely recognised that particles with aerodynamic diameter less than 10 µm can achieve alveolar deposition in man [5]; whereas particles greater than 4 µm are unlikely to be deposited anywhere other than the external nasal cavity in rats [6]. The US Environmental Protection Agency (EPA) is currently the only regulatory body that has a definitive requirement for particle size distribution in inhalation studies. For acute studies, the mean mass aerodynamic diameter (MMAD) should be between 1 and 4 µm. This is based on the recommendations of the American Society of Toxicology, Inhalation Speciality Section [7] and, in principle, should be followed for all inhalation studies. This said, it must be recognised that a conflict exists between achieving these particle size characteristics and reaching the high chamber concentrations required in acute inhalation studies on materials of low toxicity.

2.2.6 Alternative Acute Oral Toxicity Methods Three alternative methods for determination of acute oral toxicity have been developed and adopted as OECD guidelines. These are designed to replace the traditional acute oral toxicity study (LD50 determination) described in OECD Guideline 401 [8]. The

15

Practical Guide to Chemical Safety Testing methods are refined to enable the necessary information to be obtained using less severe methodology and/or smaller numbers of animals. OECD Guideline 401 was deleted in November 2002.

2.2.6.1 The Fixed Dose Procedure The fixed dose procedure is detailed in OECD Guideline 420 [9]. This method was originally developed by the British Toxicology Society and represents a significant refinement of the traditional method because it does not focus upon determination of a lethal dose. Instead, animals are treated at one of a range of fixed doses to ascertain the dose at which ‘evident toxicity’ is observed. The fixed doses were designed to allow classification under the EU classification and labelling system and have recently been amended to encompass the Globally Harmonised System (GHS) [2]. In terms of animal welfare, the fixed dose procedure has significant advantages over the other methods. Its main drawback is that it does not provide a lethal dose value and so the results cannot readily be used for classification schemes other than those for which it is devised.

2.2.6.2 The Acute Toxic Class Method The acute toxic class method (OECD 423) [10] is a stepwise procedure in which groups of three animals of a single sex are treated at each step, and based on the mortality seen, further groups are treated as necessary. On average, 2 to 4 groups need to be treated to enable adequate assessment of acute toxicity for classification purposes. This represents a significant reduction in animal numbers compared with the traditional study design. The method is not intended to allow calculation of a precise LD50 but can determine dose levels at which lethality is expected. It can therefore be applied to different classification systems more easily than the fixed dose procedure although it was originally designed to coincide with the EU system and has now been updated in line with the GHS. The focus of this method on lethality as an endpoint means that it is substantially more severe than the fixed dose procedure. It is, however, a popular method because it enables comparisons to be made with older data from traditional LD50 studies whilst using reduced numbers of animals.

2.2.6.3 The Up-and-Down Procedure The up-and-down procedure (OECD 425) [11] is the only one of the alternative methods that can provide a point estimate of the LD50 and the slope of the dose-response curve. A single animal is treated at a dose level a step below the best estimate of the LD50. Further 16

Mammalian Toxicology single animals are then treated at dose levels either a step up or a step down depending on the results in the initial animal. To judge when sufficient animals have been dosed, the guideline provides stopping criteria based on reversal in response. In most cases, testing is completed with only 4 animals after initial reversal in outcome and the test is designed to use no more than 15 animals. A supplemental test can be conducted using further multiple testing sequences at doses below the LD50 established in the primary test, to provide an estimate of the slope of the dose-response curve and 95% confidence limits for the LD50.

2.2.7 Local Tolerance Tests

2.2.7.1 Skin and Eye Irritation Assessment of the potential of chemicals to cause irritation is clearly essential and often represents the first stage of hazard determination. Chemicals may induce reversible or irreversible damage on contact with skin or eyes, so irritation/corrosion tests are conducted to identify this hazard. The traditional methods for determining irritation and corrosion involve application of the substance to skin or eyes of rabbits, with determination of effects by a graded system of assessment of reactions. In recent years the testing procedures have been significantly refined. Chemicals that can be predicted to be corrosive due to extreme pH do not need to be tested in animals and a number of in vitro and ex vivo tests have been validated that can provide adequate assessment of corrosivity without the use of live animals. The same cannot be said for irritation reactions which involve a more complex inflammatory response that cannot, as yet, be adequately reproduced in non-animal systems. A considerable research effort is being directed at developing alternative irritation tests and several of these were recently subjected to pre-validation studies under the auspices of the European Centre for Validation of Alternative Methods (ECVAM). None were considered suitable to proceed to full validation [12] (further information on alternatives is given in Chapter 6). Nevertheless, the procedures used have been refined to incorporate a tiered testing strategy. Initial considerations are made using available human and animal data on the substance or related substances and, where possible, structure-activity relationships are investigated. Physico-chemical properties are also taken into account before proceeding with skin testing. Eye irritation tests do not need to be conducted on substances which produce severe irritation to the skin. Most regulatory classification systems include some form of categorisation based on irritation potential and this is invariably based on the results obtained from studies in rabbits. Classification may range from ‘non-irritant’ to ‘severely irritant’ or ‘corrosive’.

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Practical Guide to Chemical Safety Testing

2.2.7.2 Skin Irritation Tests In general, skin irritation tests involve direct application of the test chemical to an area of shaved skin. The test sites are then covered by a dressing to prevent loss of the chemical from the site or ingestion by the animal during grooming. At the end of the exposure period (normally 4 hours), dressings are removed and the skin sites are gently decontaminated. Detailed observations of dermal reactions are then made over a period of a few days or until reactions have subsided. The skin responses are semi-quantified by a graded visual assessment of the severity of oedema and erythema. For most regulatory requirements a test in three rabbits is sufficient and, in practice, this should be carried out in a step-wise fashion. A single rabbit is treated first, with subsequent animals only exposed to the test chemical if severe reactions are not seen in the first animal.

2.2.7.3 Eye Irritation Tests For assessment of eye irritation, test chemicals are applied directly into the rabbit eye. One eye only is treated with the other acting as a concurrent control. The treated eye is carefully observed for an appropriate period after administration and reactions are recorded using graded scales for corneal and conjunctival changes. Corneal effects can be further characterised by ophthalmoscopic or slit-lamp biomicroscope examinations. As with skin irritation studies, a three rabbit test is normally sufficient and, again, a single rabbit is treated first in case of severe reactions. If severe irritation is seen in the first animal it is normally possible to classify the test chemical without treatment of further animals. In addition to this, the first animal should be observed for any initial pain response to treatment. Some substances can induce significant pain on introduction to the eye even though they are not severely irritant. In such cases, local anaesthetic can be applied to the eye prior to the exposure of subsequent animals.

2.2.8 Contact Sensitisation Certain substances, particularly high molecular weight chemicals and reactive chemicals that are capable of binding to proteins, can induce specific adverse reactions in the skin that only occur following repeated exposure. These are allergic responses, which occur as a result of an immunologically mediated hypersensitivity reaction. Such reactions are well known and typified by the common reaction to nickel in belt buckles and fasteners used on clothing. The erythematous response is unmistakable and occurs only after initial contact with the substance induces an immune response and subsequent exposure of sensitised individuals produces the skin reaction. Repeated exposure to sensitising chemicals can result in severe effects in exposed individuals, so sensitisation testing is an important component of hazard identification. 18

Mammalian Toxicology

2.2.8.1 Guinea Pig Sensitisation Tests Contact sensitisation is a Type IV delayed hypersensitivity response mediated by cells in the epidermis acting via local lymph nodes to induce an inflammatory response. Most sensitisation studies are conducted in guinea pigs and involve initial administration (induction) at one skin site followed by dermal challenge at another site. There are several different study designs with the main variations being the technique used for induction. The most commonly employed test is the maximisation test [13], which aims to maximise the potential for a sensitisation response by the use of an exaggerated induction exposure. The test chemical is administered by intradermal injection in combination with Freund’s complete adjuvant (a water in oil emulsion containing killed mycobacterium tuberculosum, a potent immune enhancer which is extremely inflammatory). The adjuvant is thought to increase the potential for immune response, presumably by stimulating a general inflammatory response. A subsequent, topical induction exposure is then carried out one week later. Two weeks after the topical induction, animals are challenged by topical application at a remote skin site. It is important that non-irritant concentrations of the test chemical are used for the challenge and this dose level must be determined by preliminary ‘sighting’ tests. There are a number of problems with the maximisation test, not least of which are animal welfare considerations. It is by nature a severe test because the induction procedures are designed to maximise the inflammatory response. Severe reactions, including ulceration, at the induction sites are not uncommon. Furthermore, the results of these studies can be difficult to interpret with irritant responses at challenge sites producing apparent false positive results. The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) has issued a guidance document on skin sensitisation testing [14], which addresses this issue at some length. In general terms, it is the persistence of reactions (characteristic of the delayed hypersensitivity response) that is important. Skin reactions that are apparent 24 hours after exposure but not at the subsequent 48-hour post exposure observation may not be indicative of sensitisation. Following this guidance will go some way to reducing false positive results, but this remains an important drawback with the maximisation test. Nevertheless, some regulatory authorities have a strong preference for this test, in view of the precautionary principle (see Section 7.4.1), because it is unlikely to fail to detect skin sensitisers, although some chemicals which are not human skin sensitisers may test positive in this model.

2.2.8.2 Local Lymph Node Assay In the late 1980s Kimber and co-workers [15, 16, 17] described an alternative method for identification of skin sensitising chemicals, the local lymph node assay (LLNA). This

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Practical Guide to Chemical Safety Testing test is conducted in mice and utilises the antigen specific immune response in lymph nodes draining the site of exposure, which is characteristic of the induction phase of contact sensitisation. The clonal expansion of T lymphocytes in these lymph nodes correlates closely with the degree of sensitisation potential of the administered substance. This cell proliferation in lymph nodes can be readily measured by a radiochemical assay that quantifies uptake of injected 3H-thymidine in lymph node cells. The main advantage of this test is that it directly measures induction of a specific immunological reaction and it does not require the use of adjuvant or subsequent challenge exposures. Thus, the procedures used are substantially less severe than those in the maximisation test and, although animals are still required, the numbers used are usually reduced. An OECD Guideline (Guideline 429) has recently been adopted and a number of regulatory authorities are already accepting this test in lieu of guinea pig studies. It seems likely that this will become the preferred test for determination of skin sensitisation potential in most circumstances.

2.3 Repeated Dose Toxicity Studies

2.3.1 Nature and Relevance of Tests Repeated dose toxicity studies are designed to determine the adverse effects of a chemical following repeated exposures of either short or long duration. This information is relevant to human exposure to chemicals in a wide range of situations such as: workplace exposure, chemicals used in the home, and exposure via food and drugs for repeated administration. The purpose of the studies is to identify target organ toxicity or other adverse effects that may occur following repeated exposure at dose levels lower than those which cause toxicity in acute studies and to identify dose levels at which such toxic effects are absent – the ‘no observed effect level’ (NOEL) or ‘no observed adverse effect level’ (NOAEL). The effects of repeated exposure to chemicals cannot be predicted from single dose, acute studies. Repeated dose toxicity studies are a crucial component in many regulatory hazard identification schemes and provide vital evidence for use in risk assessment. Most repeated dose studies are conducted in rats although mouse studies are sometimes needed for preliminary information prior to a carcinogenicity study in this species. For some regulatory purposes, repeated dose toxicity studies are required in a second, non-rodent species. The duration of the studies depends on the nature of the chemical and the regulatory purpose but the most common study types are 28-day and 90-day studies. Under the

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Inhalation Mammalian Toxicology European notification of new substances regulations the duration of the tests depends on supply levels of the chemical with a 28-day study required at 1 tonne per annum (or 5 tonnes cumulative) and a 90-day study at 10 or 100 tonnes per annum (50 or 500 tonnes cumulative) – depending on risk assessment. For chemical notification in Japan, a 28-day study is the only toxicity study required and, for this reason, the regulatory authorities interpret the results strictly. For food additives or food contact materials a 90-day study is more appropriate and for other purposes, such as medical device testing, the length of study may be related to the anticipated duration of human exposure. The results of repeated dose toxicity studies are used to make a variety of regulatory decisions such as banning a chemical for a specific use or imposing conditions relating to production and use. In addition to this, repeated dose studies may be needed for appropriate classification and labelling. Under the EU system, severe adverse effects seen in repeated dose toxicity studies may trigger the ‘R48’ risk phrase, ‘Danger of serious damage to health by prolonged exposure’ in conjunction with ‘Toxic’ or ‘Harmful’ classification depending on the dose level at which the severe effects are seen (see Table 2.2). The oral route of exposure is applicable in most cases using either gavage or dietary administration. Usually, the oral route is most relevant to potential human exposure. Dermal studies may be required if extensive, prolonged dermal exposure is anticipated but, in the author’s experience, dermal studies are rarely required except for pesticides

Table 2.2 EU classification and labelling scheme (Annex VI of Council Directive 67/548/EEC) R48 risk phrase Trigger dose levels for ‘harmful’ + R48 Route

90-Day study

28-Day study

Oral

≤ 50 mg/kg/day

≤ 150 mg/kg/day

Dermal

≤ 100 mg/kg/day

≤ 300 mg/kg/day

≤ 0.25 mg/l, 6h/day

≤ 0.75 mg/l, 6h/day

Inhalation

Trigger dose levels for ‘toxic’ + R48 Route

90-Day study

28-Day study

Oral

≤ 5 mg/kg/day

≤ 15 mg/kg/day

Dermal

≤ 10 mg/kg/day

≤ 30 mg/kg/day

≤ 0.025 mg/l, 6h/day

≤ 0.075 mg/l, 6h/day

Inhalation

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Practical Guide to Chemical Safety Testing and dermally administered pharmaceuticals. Inhalation studies may be appropriate if this is considered the most likely route of human exposure, e.g., gases, volatile liquids and powders of respirable particle size.

2.3.2 Importance of Repeated Dose Toxicity The toxicity of many substances is profoundly different following repeated exposure than would be predicted from acute (single dose) toxicity studies alone. The main reasons for these differences are as follows:

2.3.2.1 Bioaccumulation Some substances, particularly those that show high lipid solubility, may accumulate in body tissues or fat following repeated exposure. This results in progressively higher concentrations at target tissue sites, which may overwhelm endogenous protective mechanisms or simply result in greater exposure of receptor sites to the foreign (xenobiotic) substance.

2.3.2.2 Biotransformation Humans and animals have well-developed protective mechanisms to defend them against the many foreign chemicals to which they are exposed. In general, these protective mechanisms, usually mediated by enzymes, are capable of transforming lipophilic chemicals into more hydrophilic metabolites that can be readily excreted. The purpose of these mechanisms is detoxification and the enzymes responsible may be subject to induction following repeated exposure to a particular chemical. In other words, the body responds to the repeated insult by increasing production of the appropriate metabolising enzyme(s). In some circumstances, the metabolites formed during biotransformation may themselves show significant toxicity and in these cases enzyme induction can result in exaggerated toxicity.

2.3.2.3 Prolonged Exposure The most obvious effect of repeated doses is to prolong the exposure of target tissues to the xenobiotic substance. Effects that may be mild or reversible after a single exposure could be severe and/or irreversible following repeated insult.

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Mammalian Toxicology

2.3.3 Methodology Protocols for repeated dose toxicity studies tend to be broadly similar regardless of regulatory purpose or national requirements. There are some minor differences in study design that are required by certain regulatory authorities. For example, the US Food and Drug Administration (FDA) has specific, stringent requirements for studies on food additives and food contact materials, whilst the Japanese authorities have a particular 28-day study design for new chemical notifications. With these exceptions, the design of repeated dose toxicity studies generally follows the OECD Guidelines for Testing of Chemicals; primarily OECD 407 (28-day study) [18] and OECD 408 (90-day study) [19]. Both protocols have been revised recently to include a specific neurotoxicity assessment and the requirement for histopathology on the 28-day study has been significantly increased compared to the previous guideline.

2.3.3.1 Administration Animals are dosed (usually) once per day with the test substance for the duration of the study. Some regulatory guidelines allow for dosing to be done 5 days per week, based on purely practical considerations. This practice should not be encouraged, however, because a 2-day break in the dosing regime can allow for significant recovery to occur. Most laboratories now conduct repeated dose studies with 7 days per week exposure.

2.3.3.2 Observations Clinical observations are carried out on the animals at least daily with a detailed record made of changes in behaviour and general appearance. These observations have now been supplemented with specific observations designed to detect neurotoxicity: the socalled ‘Functional Observation Battery’. This comprises a defined set of detailed behavioural observations made on individual animals in an open-field arena, away from the home cage. Quantitative or semi-quantitative measurements are also made of a number of functional performance parameters: motor activity, grip strength and sensory reactivity to stimuli. Bodyweight change and food (and possibly water) consumption are monitored, usually once weekly.

2.3.3.3 Haematology and Clinical Chemistry For all repeated dose toxicity studies, blood samples are taken for haematology and clinical chemistry. Many substances can exert effects on haematological parameters either

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Practical Guide to Chemical Safety Testing through direct interaction with blood cells or through effects on target tissues such as bone marrow or kidneys. Clinical chemistry parameters can be sensitive indicators of organ damage and a range of parameters are measured to indicate changes in organ function or cellular damage resulting in intracellular substances being released into plasma. Regulatory guidelines are similar but not identical in the list of parameters that need to be evaluated. A harmonised approach to haematology and clinical chemistry determination in toxicity studies has been proposed by the Joint Scientific Committee for International Harmonization of Clinical Pathology Testing [20].

2.3.3.4 Opthalmoscopy Certain study types, primarily studies of 90-days or longer duration, need to include ophthalmoscopic examination of animals to observe any ocular changes occurring as a consequence of treatment. Such examinations need to be conducted by an ophthalmoscopist experienced in examination of the species and strain of animal used. Examinations are usually carried out prior to the start of treatment, to establish any existing ocular conditions, and towards the end of the treatment period.

2.3.3.5 Pathology Pathology determinations carried out at necropsy include a detailed macroscopic examination of all animals with records made of all observed abnormalities. Organ weights are measured for a series of organs variously defined in regulatory guidelines. It is reasonable to regard the lists given in OECD Guidelines 407 and 408 as best practice since these have recently been revised to include representative reproductive and immune system organs. Organ weights are generally expressed relative to bodyweight and changes in these relative weights can be sensitive indicators of target organ toxicity. All repeated dose toxicity studies include a requirement for histopathology and the histopathology results are often regarded as the most definitive measure of the toxic effects of a substance. Most regulatory guidelines provide a prescriptive list of tissues for histopathological examination and the examinations are normally conducted in a stepwise fashion. The full list of tissues from high dose and control animals is examined first with subsequent examination of any affected tissues (target organs) in the other dose groups. These histopathology determinations are time consuming and constitute a significant portion of the time taken to conduct a repeated dose toxicity study, but they are essential in identifying and characterising adverse effects of treatment. Such adverse effects may include changes indicative of effects on reproductive performance or preneoplastic changes that may influence the need for subsequent studies.

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Mammalian Toxicology

2.4 Reproduction Toxicology

2.4.1 Nature and Relevance of Tests As part of a safety testing program, the effects of chemicals upon the reproductive system are evaluated using specific protocol designs. The studies are required to either evaluate one specific aspect of reproduction or the entire reproductive cycle. Figure 2.2 outlines those aspects of the reproductive cycle that are evaluated in these studies. Although there are standard guidelines for the assessment of the effects of test materials upon reproduction, it is not uncommon to design unique studies to evaluate one particular aspect of reproduction. The primary purpose of the reproductive toxicology studies is to identify potential hazards to reproduction. For classification and labelling purposes these are divided into effects upon fertility and reproductive performance or effects upon offspring development. It is necessary to qualify these potential hazards with other toxic effects that may, or may not, be associated with reproductive hazard. Under normal circumstances the study design requires the inclusion of a dose level that can be expected to induce a minimum level of toxicity to the adult. The purpose of this is to establish if the effects upon reproduction are a consequence of a non-specific toxicity to the adult. This is also the case with

Gametogenesis

Post weaning

Lactation Pre weaning

Parturition

Mating

Pre implantation

Post implantation

Figure 2.2 Reproductive cycle

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Practical Guide to Chemical Safety Testing developmental toxicity. It is necessary to establish whether any observed effect on offspring development is a unique event that is not a consequence of general toxicity to the adult. The induction of supernumerary ribs is an examples of a skeletal defect that has been associated with toxicity to the adult [21]. The results of other toxicity studies may give an indication of the type of findings to be expected in reproduction assays. For example a strong mutagen/clastogen (causing mutations or structural chromosome damage), may affect embryo development in utero. It may also be expected to affect spermatogenesis. This profile of effects may therefore be used as a model for risk assessment.

2.4.2 Methodology For the testing of chemicals, most regulatory authorities require two forms of study; the one or two generation (fertility) study and the developmental toxicity study.

2.4.2.1 Generation (Fertility) Studies These may be either the one generation study [22] or the two generation (multigeneration) study [23, 24, 25]. The former is exclusive to the notification of chemicals in Europe. The basic study design is outlined in Figures 2.3 and 2.4. Most commonly the species of choice is the rat because of the practicality and the amount of background data available. In the one generation study, three treated and one control group of 20 or more adult animals are exposed to the test material prior to mating. For males this period is at least 70 days, which is considered to cover the entire spermatogenic cycle of the rat. For females the period of 14 days allows completion of at least 2 oestrous cycles. Dosing then continues throughout subsequent mating, gestation and lactation phases. Mating performance is usually assessed on the ability to produce a single litter of offspring. The viability, growth and development of the offspring are assessed during the lactation period up to weaning. Based on litter size at birth and offspring bodyweights on Day 1 and 4 post partum, it is possible to determine if there has been an effect on offspring survival and growth in utero. Post mortem studies such as histopathology and organ weight analysis can determine target organ effects on the reproductive system. At this stage the study is normally complete. If effects upon any aspect of reproduction are observed, it may be possible to continue the study by either re-mating the adults from the first generation or deriving a second generation by selecting offspring to form a second generation. The methods used reflect the normal procedures undertaken if a two generation study was originally designed.

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Mammalian Toxicology

Figure 2.3 One generation study

Dosing period

Figure 2.4 Two generation study

In the two generation study, usually 3 dose groups plus one negative control group of adult males and females are exposed to the test material for at least 10 weeks prior to mating and then subsequently throughout mating, pregnancy and lactation for one mating phase. At weaning individual offspring are selected from the litters produced from the parental generation. The selected offspring are designated the F1 generation. The F1 generation are then exposed to the test material for 10 weeks prior to mating and then through mating, gestation and lactation for one complete mating phase. Both study designs outlined in Figures 2.3 and 2.4 are designed to cover all aspects of the reproductive process. The two generation study obviously allows more investigations to

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Practical Guide to Chemical Safety Testing be performed and does allow the study of effects of cumulative exposure to a test material through more than one generation. This is important if there is concern for prolonged exposure to the general population. Recent modifications to the study design of the two generation study have added to the complexity of the design. Particular emphasis has been placed on the evaluation of male and female gametogenesis. This includes computer assisted sperm analysis [26] and histopathological oocyte evaluation [27]. Offspring evaluations have also been extended to include sexual maturation [28]. These additions to the testing guideline reflect concern about the effects of endocrine disruption on the reproductive process.

2.4.2.2 Developmental Toxicity Studies Developmental toxicity study designs are generally a development of the traditional studies referred to as teratology studies. This is not just changing the name for the sake of correctness, but reflects the need to evaluate all aspects of embryo/foetal development and not just look for malformations. Figure 2.5 represents the standard approach to this type of study. The basic study design involves the dosing of groups of twenty or more pregnant animals from the time of embryo implantation into the wall of the uterus to one day prior to the caesarean delivery of offspring. The dosing period may be extended to include the post mating period up to embryo implantation if the test material is not expected to severely affect embryo development and implantation. The most commonly used species is the rat but, if there are valid scientific reasons, the mouse may be used as a rodent species. The most commonly used second species is the rabbit. Rats and rabbits are primarily

Figure 2.5 Developmental toxicity study in the rat

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Mammalian Toxicology chosen for practical reasons and the vast amount of background data available. Many other species have been used for developmental toxicity studies but the frequency of their use suggests that for the testing of chemicals, there is no particular demand or reason for alternative species. The development of the embryo and the foetus in utero is the primary subject of investigation in this study. Offspring viability is assessed by the number of embryo/foetal deaths. Malformations of embryo/foetal development are the primary concern for evaluation of offspring but, in addition, the degree of foetal development is of equal importance. Development is assessed by the evaluation of foetal weight and by postfixation evaluation of the foetal skeleton. Following processing and staining of the foetus [29] it is possible to determine the amount of development that has occurred. Delays in skeletal development are commonly associated with foetal toxicity. Foetuses are also evaluated for effects upon the viscera using standard dissection techniques [30]. As with effects upon the skeleton, delays in development of some organs can be attributed to foetal toxicity. As a completely separate investigation, the assessment of developmental neurotoxicity can be performed [31]. The purpose of the study is to assess the effects of a test material on the prenatal and postnatal neural development of offspring. The study involves a series of functional tests to assess aspects such as motor activity and learning/memory in the offspring of females exposed to the test material together with detailed neuropathology.

2.4.3 Alternative Approaches The size and complexity of study designs for reproductive toxicology has led to the consideration of possible alternatives to conventional studies. Certain in vitro tests which look at single aspects of reproduction, particularly screening for effects upon embryo development, have been evaluated, but to screen the reproductive process in one study has required the use of an in vivo study [32]. This study design has been developed with the aim of screening a large number of existing chemicals that have no reproduction toxicity data, particularly high production volume chemicals. The design is an abbreviated version of the single generation study. Groups of 10 adult males and females are dosed for 2 weeks prior to mating with dosing continuing through the mating, gestation and early lactation phases. The approximate study length is 56 days, which is half the time for a normal single generation study. Fertility is assessed on the ability to produce a litter of offspring. Offspring viability and growth are assessed during a four-day period after birth. As with the single generation study, it is possible to assess in utero offspring viability and growth. The histopathology of reproductive organs is assessed post mortem.

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Practical Guide to Chemical Safety Testing This study design can be further developed to evaluate those elements of a standard repeat dose toxicity study by inclusion of evaluations for neurotoxicity, haematology and blood chemistry, as well as extending the histopathology investigations to other organs [33]. This type of study is therefore designed with the intention of deriving the maximum amount of information from as few animals as possible, and fulfils one criteria in the ‘Replacement, Reduction and Refinement’ of animal studies (see Section 6.1). Other variations on study designs that have been adopted on a more ad hoc basis include combining a ninety-day repeat dose study with a single generation reproduction study. The protocol does require some minor revisions and does make assumptions that target organ responses are not affected by pregnancy. A number of short-term tests are now being proposed to evaluate the effects of chemicals with suspected endocrine disruption potential. Many of these are in vitro assays but there are also a number of in vivo methods available [34]. On example is the uterotrophic assay [35]. This test is performed on either the juvenile female or an ovarectomised adult. The assay is designed to examine the effects of test material on uterine weight. A significant increase in uterine weight is associated with oestrogenic activity.

2.5 Carcinogenicity

2.5.1 Nature and Relevance of Tests The purpose of the conventional in vivo carcinogenicity study is to determine if a test material induces one or more of the following: (i) An increase in the overall incidence of neoplasms (tumours). (ii) An increase in the time to appearance of tumours in a population of animals. (iii)A reduction in the time to appearance of tumours in a population of animals. All these evaluations require comparison to both concurrent control and historical control data. Although the designs of these studies are outlined in the various regulatory guidelines [36], their conduct requires considerable attention to detail in the study design phase.

30

Mammalian Toxicology

2.5.2 Methodology The conventional study design requires the administration of test material to animals throughout the majority of the life span for that particular animal model. A series of issues needs to be addressed before undertaking such a time consuming and expensive procedure. 1. Animal model: Studies require administration of test material for the majority of the animal’s life span and, for practical reasons, this limits the choice of species for most chemicals. Rats, mice or hamsters are regarded as the species of choice with rat and mouse being the most commonly used. The selection of an appropriate strain has led to much debate over the use of inbred, outbred or hybrid strains. There are seen to be drawbacks to each strain [37]. The only requirement is that the selected strain should be susceptible to carcinogenic effects but that the spontaneous tumour rate is not so high that meaningful assessments cannot be performed. Also, as stated above, the amount of background data, particularly with regard to tumour incidence, is an important consideration. The guideline requirement is for 50 animals per sex for each dose group. 2. Available toxicity/chemical data: Prior to the start of the study the results of shorterterm toxicity studies will need to be assessed in order to establish appropriate dose levels and also to identify any potential target organs. It may be possible to identify preneoplastic change in tissues from subchronic toxicity studies. The results of the in vitro and in vivo genotoxicity assays may also indicate potential carcinogenic activity. Other aspects such as the physico-chemical characteristics of the test material may also be of relevance in determining both dose levels and the expected route of exposure, especially when considering the most likely route of human exposure. 4. Route of exposure: The practicalities of test material administration do influence the methods used. For chemicals, the primary requirement would be to use a route that would most likely result in systemic exposure. Wherever possible the route would be the same as that used for subchronic toxicity studies. The oral route is most commonly used with test material administered in the diet or, less commonly, in drinking water. Oral gavage is also to be considered as a potential method of administration. A second route of exposure would be via inhalation, which may be particularly relevant

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Practical Guide to Chemical Safety Testing to worker-safety evaluation for industrial chemicals. The options for method of administration are usually either using a ‘whole body’ exposure chamber or a ‘noseonly’ chamber. Dermal exposure may also be considered but there is a question over the degree of systemic exposure to test material. The route of administration does require justification when designing this type of study. A number of other methods have been used. The implantation of medical devices or the instillation of products into the rectum, vagina, trachea or nasal passages are rare studies, which are designed for a specific product under investigation. 5. Duration of exposure: The test material should be administered to the test model for the majority of its life span. Usually the quoted periods in regulatory guidelines are 104 weeks for rats and 78 weeks for mice. It should be noted that the numbers of surviving animals within each dose group (including negative controls) should not be below 25% of the original group size at the end of the required dosing period. In practice, this has meant that, particularly for certain strains of rat, the study has had to be terminated before the end of the expected dosing period. The argument in defence of these studies is that the strain of rat has a shorter life span and therefore the study is still valid even if the duration of exposure is shorter than for other strains. Conversely there are strains of rat that have an expected life span in excess of 104 weeks. The argument is that the study should be therefore extended for these particular strains although opinion may be divided on this issue.

2.5.3 Dose Levels As with most regulatory toxicity studies, guidance on dose level selection is provided in the appropriate test guidelines. In practice the highest dose level is expected to show some evidence of toxicity to the animals under test. The level of toxicity should not be so high that it induces mortality. A benchmark figure is a 10% reduction in bodyweight gain over the life span of the animals. The lowest dose level should provide an adequate safety margin for human exposure.

2.5.4 Conduct of Study The animals are randomly allocated to dose groups and are acclimatised to the laboratory conditions prior to the start of the study. Test material administration begins at approximately 4 to 6 weeks of age. Alternatively, exposure may begin during pregnancy. During the course of the study, regular assessments of bodyweight, food consumption and water consumption are performed. All animals are regularly monitored for clinical signs of ill health or reaction to the test material. Blood samples may be taken at intervals,

32

Mammalian Toxicology primarily for haematological investigations but also, if required, for blood chemistry investigations or immunotoxicological screening. During the in-life phase all animals are palpated regularly in order to detect any tumour development. The site, size and progression of each tumour is monitored regularly. At termination a detailed macroscopic post mortem examination of all animals is performed, including organ weight analysis, followed by full histopathological examination.

2.5.5 Data Evaluation The data evaluation from carcinogenicity studies is a time consuming and detailed process. All results need to be assessed to determine whether confounding issues may have influenced the result [38]. These confounding issues will include the influence of environmental conditions on results. Issues such as the effects of diet [39] have to be taken into account. The interpretation of histopathological findings requires some standardisation and guidelines for this process have been published [40]. Some of the aspects of tumour analysis that require evaluation are: (i) Classification of benign or malignant neoplasms. (ii) The number of tumours that develop in one or multiple sites. (iii) The time of onset of tumour development. (iv) The influence of tumour development on the mortality rate. The classification of tumours and the procedures for data presentation are the subject of much effort on behalf of many institutions [40] as is the statistical analysis of tumour data. The statistical analysis is of particular importance because under some conditions, an increase in the incidence of a rare tumour may only occur amongst small numbers of animals. The relevance to man of the types of tumours seen must also be taken into account. There are certain classes of rodent carcinogens such as the hepatic peroxisome proliferators where it is likely that these findings may not be seen amongst humans.

2.5.6 Risk Assessment The extrapolation of findings from the rodent carcinogenicity bioassay is a separate area of expertise. Various mathematical models have been prepared for the purpose of low

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Practical Guide to Chemical Safety Testing dose extrapolation of findings [41], in order to establish suitable safety margins for human exposure or to determine what is an acceptable risk. In the case of many industrial chemicals the necessity is simply to determine the hazard presented by a test material for the purposes of classification and hazard labelling.

2.5.7 Alternative Approaches The traditional battery of in vitro and in vivo genetic toxicity assays that are performed as part of a safety testing programme for most chemical entities is expected to identify any suspected genotoxic carcinogens. This obviously fails to identify those materials that can be classed as non-genotoxic carcinogens, co-carcinogens or tumour promoters. In vitro assays are under development that may detect more types of potential carcinogen. There are computer based structure-activity relationship programmes that are able to determine if the molecule contains any ‘structural alerts’ for mutagenicity and carcinogenicity. One of the practical difficulties with carcinogenicity bioassays is the time required to perform the study. One option to decrease the time of the assay is to use transgenic animals such as the mouse. Three tumour models have been seen as potentially of value: (i) The TG.AC skin model (ii) The p53 ‘knockout’ model (iii)The ras H.2 model. The time of development of tumours in these models is significantly reduced, by months. As with all new testing methods, much validation work is required prior to the use of such approaches.

2.6 Medical Device Testing The basic principles for mammalian toxicology testing of medical devices are identical to those described for chemical substances, but special considerations are necessary regarding route of exposure and dose preparation. The regulation of medical devices is discussed in Chapter 13.

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Mammalian Toxicology

2.6.1 Exposure Routes The route of exposure used for testing medical devices is designed to be representative of human exposure. For studies of systemic toxicity, administration may be by parenteral injection: intravenous, subcutaneous, intramuscular or even intra-articular, or by implantation of a sample of the medical device. For local tolerance studies, direct application, intracutaneous injection or implantation may be used.

2.6.2 Dose Preparation Many toxicity studies on medical devices are conducted using extracts prepared from the test device. This is done by incubating the device in nonpolar and polar solvents for a period of time and at a temperature relevant to the nature of the device. The resulting extracts are considered to contain any substances that can be readily leached from the materials used to manufacture the device. These extracts can then be injected or applied to the animals in the toxicity tests. For some studies, the most appropriate method of application is by implantation. A representative sample of the medical device is surgically implanted or inserted using a wide gauge hypodermic needle and trocar. The most common of these studies is the muscle implantation test, in which implants are made into the paravertebral muscles of rabbits to assess local tolerance, but subcutaneous implantation or even implantation into bone may be required. Further information on this subject can be found in the International Organization for Standardization (ISO) guidelines [42].

2.6.3 Cytotoxicity Testing of Medical Devices In addition to the more traditional toxicity studies, medical devices are often subjected to in vitro cytotoxicity testing. The most commonly used cell line for these tests is the L929 mouse fibroblast line, but other cell lines may be used such as CCL 81 or MRC 5 cells. The term ‘medical devices’ includes a diverse range of products from simple catheters and wound dressings to complex blood product recovery sets, and yet, whatever the product type it is almost certain that a cytotoxicity test will be required as part of the regulatory package necessary for registration. Indeed, the matrix of evaluation tests suggested for consideration in Part 1 of the International Standards Organization (ISO) 10993 guidelines includes cytotoxicity tests for all category types and contact durations. Cytotoxicity tests detect the in vitro toxic effects of the extractable components of medical

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Practical Guide to Chemical Safety Testing devices. This is true for whichever method is used except perhaps for the direct contact method in which immobilised toxic components could also exert toxic effects on the cells that they touch. The ISO 10993 standards give quite detailed guidance on the technical procedures associated with cytotoxicity tests and describe three main methods: the extract test, the direct-contact test and the indirect-contact test. The choice of test depends on the usepattern of the device, and it should be remembered that, where possible, the device should be tested in its final form. Thus for a device that comes into indirect contact with the patient, such as a fluid line or container, the appropriate method would be the extract test. This is because the device itself does not come into contact with the patient and it is the fluid contained in, or transported by, the device that may carry extractable materials from the device to the patient. For devices that are used on intact or unbreached skin, then the indirect-contact method is appropriate and for devices that are used on mucosal membranes, breached skin or for internal applications, the direct-contact method should be used. In some cases the method of choice is not immediately obvious, or is not technically possible. For example, stainless steel prostheses come into contact with the living cells of the tissues of the hip joint and the direct-contact method should be selected. However, the size of the device would make such a test extremely difficult to perform and may give misleading results because of the physical or mechanical effects that may damage the cells. In this example the extract method would be selected and justified by the obvious technical considerations.

2.6.3.1 Extract (Elution) Method The extract method is used primarily for devices that do not come into direct contact with the patient, although it may also be used for direct contact devices as described above. In addition, it is the method with perhaps the oldest pedigree and therefore has a long history of use and many users continue with this method because of their historical data, even though it may not be the obvious choice. The method involves preparing an extract of the device, or a sample of the device, and exposing cultures of cells to the extract for a period of time, usually 24 or 48 hours. The extractant may be culture media, as used for growing the cells, saline or water. In the case of water or saline the final extract cannot be used in its neat form because the cells would die and it is therefore diluted with culture medium. The extract is also prepared for a certain time period and at a set temperature, as follows: (a) not less than 24 hours at 37 ºC (b) 72 hours at 50 ºC

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Mammalian Toxicology (c) 24 hours at 70 ºC (d) 1 hour at 121 ºC The extract conditions are selected as those that best simulate or exaggerate the conditions of clinical use without causing significant changes in the test material. For most medical devices this means that an extract temperature of 37 ºC for 24-72 hours is selected, using serum-supplemented culture medium. The test material (or a sample thereof) is prepared by determining the surface area and calculating the volume of extractant. For most devices the ratio of the surface area to volume is 3 or 6 cm2/ml depending on its thickness. For some materials, such as non-woven fabrics, it is impossible to calculate the surface area, so weight can be used, i.e., 0.1 or 0.2 g/ml. Furthermore, some materials are absorbent and in these cases it is necessary to determine the pre-hydration value and adjust the volume of extractant accordingly. Once the extract is prepared it may need to be filter sterilised, depending upon the sterility status of the device, and then it may be tested either in its neat form only or as a series of dilutions. Cell cultures prepared 24 hours earlier are exposed to the extract and then evaluated for cytotoxic effects at the end of the exposure period. Concurrent negative, positive and extractant controls are included for comparative purposes. The cells are examined using an inverted microscope and graded according to the degree of cytotoxicity and the proportion of cells affected. The evaluation is essentially subjective but may be classified into a numerical scheme whereby a non-cytotoxic response is grade 0 and a severe response affecting the majority or all of the cells is given a grade of 4. Intermediate effects are graded between 1 and 3. The evaluation of the cells may be done on the living cells or they may be fixed and stained.

2.6.3.2 Indirect Contact Method The indirect method is used for devices that come into contact with the unbroken skin of the patient, e.g., an electrocardiogram sensor. In this procedure the cells are grown in tissue culture dishes and the culture medium discarded and replaced with a mixture of fresh culture media and tissue culture grade agar. The agar sets to a gel that is sufficiently firm to support a sample of the device and is designed to mimic, in a simplistic way, the protective effect of the epidermis. In most cases the device has to be cut into 1 cm2 pieces which are tested in duplicate or triplicate. After a suitable exposure period (normally 24 hours) the samples are removed and the cells examined for toxic effects. The grading of cytotoxicity takes into account both the toxicity and the size of the zone of the effect. Thus if the cells immediately beneath the sample are dead but the zone does not extend

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Practical Guide to Chemical Safety Testing beyond the perimeter of the sample, then a cytotoxicity grade of 2 may be applied. However, a modest toxic effect that extends across the whole diameter of the tissue culture dish would warrant a score of grade 4. This method can be technically the most difficult because the cells do not grow so well under agar and it can be difficult to distinguish weak cytotoxic effects from reduced growth caused by the agar. Another factor that can often affect the results of the inexperienced is the temperature at which the agar/medium mixture is applied. It is necessary to hold the agar at approximately 45 ºC before adding the culture medium and then the mixture has to be applied swiftly to the cells before it sets firm. If the temperature is too high then heat stress will affect the morphology of the cells, and if the temperature is too low then the agar may set unevenly and make it difficult to visually assess the cells.

2.6.3.3 Direct Contact Method This is the simplest version of the cytotoxicity test and is normally used for devices that contact breached skin, mucosal membranes or are implanted into living tissue. Cells are grown on the surface of a tissue culture dish and then all but a small volume of the culture media is removed so that the cells do not dry out, but samples of the device may be placed in contact with the cells. The samples are usually square or circular pieces of the device approximately 1 cm2 in area and have a low weight or density so as not to affect the cells through mechanical means. After 24 hours the samples are removed and the cells examined and scored using a similar grading scale as used for the indirect method. As before, vital stains may be used to aid evaluation, or the cells may be fixed and stained to prepare permanent specimens.

2.6.3.4 Conclusion The descriptions of cytotoxicity methods given above are highly simplified and it is recommended that only experienced tissue culture technicians perform these tests. There are many pitfalls that can trap the unwary and give rise to false positive or negative results. It is important to prehydrate samples that are hygroscopic for example; otherwise the cells may be dehydrated or suffer from osmotic effects. The orientation of the sample should be borne in mind so that it properly represents the use pattern of the device. If the device contains any additives that may affect the pH or osmolarity of the culture medium then these should be taken into account. The in vitro cytotoxicity test is a highly sensitive method and the significance of the results need to be put into the context of the use pattern of the device and of any other

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Mammalian Toxicology data that are available. However, it can also be used to track down production problems or to identify the most promising candidate of a series of pre-production devices. When a series of tests, both in vitro and in vivo are required then it is always prudent to do the cytotoxicity test first so that if any adverse findings are detected then the problem can be resolved before proceeding with the animal tests.

Acknowledgement The authors would like to thank Dr Peter Jenkinson for his contribution on cytotoxicity testing of medical devices.

References 1.

Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, Official Journal of the European Communities, 21:8:01, L225, 263.

2.

Harmonised Integrated Hazard Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures, OECD Series on Testing and Assessment, Number 33, OECD, Paris, France, 2001.

3.

K-H. Diel et al., Journal of Applied Toxicology, 2001, 21, 15.

4.

J. H. Draize et al., Journal of Pharmacology and Experimental Therapeutics, 1944, 82, 377.

5.

EPA-540/09-88-101, Hazard Evaluation Division Standard Evaluation Procedure: Inhalation Toxicity Testing, 1988.

6.

O.G Raab et al., Annals of Occupational Hygiene, 1988, 32, 53.

7.

Technical Committee of the Inhalation Speciality Section, Society of Toxicology, Fundamental and Applied Toxicology, 1992, 18, 321.

8.

OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 401, 24 February 1987.

9.

OECD Guideline for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 420, 20 December 2001.

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Practical Guide to Chemical Safety Testing 10. OECD Guideline for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 423, 20 December 2001. 11. OECD Guideline for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 425, 20 December 2001. 12. J.H. Fentem et al., Toxicology in Vitro, 2001, 15, 57. 13. B. Magnusson and A.M. Kligman, Journal of Investigative Dermatology, 1969, 52, 268. 14. ECETOC Technical Report No. 78, Skin Sensitisation Testing: Methodological Considerations, 1999. 15. I. Kimber et al., Food and Chemical Toxicology, 1986, 24, 585. 16. I. Kimber et al., Contact Dermatitis, 1989, 21, 215. 17. I. Kimber and D.A. Basketter, Food and Chemical Toxicology, 1992, 30, 165. 18. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 407, 27 July 1995. 19. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 408, 21 September 1998. 20. K. Weingand et al., Fundamental and Applied Toxicology, 1996, 29, 198. 21. R.J. Kavlock, N. Chernoff and E.H. Rogers, Teratogenesis, Carcinogenesis and Mutagenesis, 1985, 5, 3. 22. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Test Guideline 415, 26 May 1983. 23. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects Test Guideline 416, 22 January 2001. 24. United States Environmental Protection Agency, Health Effects Test Guidelines OPPTS 870.3800, August 1998. 25. Japan Ministry of Agriculture Forestry and Fisheries, Agrochemical Test Guidelines, Multigeneration Toxicity (2-1-17), 2001. 26. V.L. Slott, J.D. Suarez and S.D. Perreault, Reproduction Toxicology, 1991, 5, 449.

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Mammalian Toxicology 27. T. Pedersen and H. Peters, Journal of Reproduction and Fertility, 1968, 17, 555. 28. C.C. Korenbrot, I.T. Huhtaniemi and R.I. Weiner, Biology of Reproduction, 1977, 17, 298. 29. A.B. Dawson, Stain Technology, 1926, 1, 123. 30. J.G. Wilson in Teratology: Principles and Techniques, Eds., J.G. Wilson and J. Warkany, University of Chicago Press, Chicago, 1965, 262. 31. United States Environmental Protection Agency, Health Effects Test Guidelines, OPPTS 870.6300, August 1998. 32. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects Test Guideline 421, 27 July 1995. 33. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects Test Guideline 422, 22 March 1996. 34. G. Ankley et al., Environmental Toxicology and Chemistry, 1998, 17, 1, 68. 35. J. Odum et al., Regulatory Toxicology and Pharmacology, 1997, 25, 176. 36. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects Test Guideline 451, 12 May 1981. 37. F.J.C. Roe, Laboratory Animals, 1994, 28, 148. 38. UK Department of Health, Guidelines for the Evaluation of Chemicals for Carcinogenicity, HMSO, London, 1991. 39. M.J. Tucker, Toxicology Letters, 1985, 25, 131. 40. R. Peto et al. in Long term and short term screening assays for carcinogens: a critical appraisal, IARC Monographs, Supplement 2, International Agency for Research on Cancer, Lyon, 1980, 311. 41. F.R. Johnannsen, Critical Reviews in Toxicology, 1990, 20, 5, 341. 42. ISO 10993, Biological Evaluation of Medical Devices, 1997.

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Genetic Toxicology

3

Genetic Toxicology Peter C. Jenkinson

3.1 Introduction Genetic toxicology is the study of the potential of chemicals to induce damage to deoxyribonucleic acid (DNA). Damage to DNA may result in error-free repair, mutation or cell death. The first endpoint is of little concern because there is no net change to the genotype (the complete genetic code of an individual) or phenotype (the physical appearance of an individual that results from the expression of the genes in the genotype); although the possibility does exist that the cell-cycle delay resulting from the time taken to repair the damage may have some adverse consequence. This may be of particular concern to the developing embryo, in which the timing of cell replication may be crucial to the correct flow of embryogenesis. Cell death is of concern because of the effect that it may have on ageing and the degenerative diseases associated with old age and exposure to chemicals. This may be exemplified by the appearance of the faces of heavy smokers who often seem to be older than their years. The contribution of exposure to mutagens to the development of degenerative diseases has perhaps been underestimated in previous years and may become the focus of more research in the future. The primary concern that has driven genetic toxicology over the last three decades is the link between mutation and cancer. Mutations in somatic cells may change the phenotype of the cell such that it loses its ‘identity’ and the regulatory control of the body over that cell is diminished. The consequences are that the cell is either, identified by the body as a cancer cell and eliminated, or that the change goes unnoticed and the cell develops into a tumour that may result in morbidity or the death of the individual. The International Agency for Research on Cancer (IARC) has officially recognised 87 chemicals as human carcinogens and the US National Toxicology Program (NTP) lists 198 chemicals as known or anticipated human carcinogens. However, the US Occupational Safety & Health Administration (OSHA) has listed 4-500 chemicals as ‘select carcinogens’ and many more chemicals have been shown to be genotoxic carcinogens in animal studies. Human exposure to these chemicals is minimised by exposure limits in the workplace and by exclusion/containment to the wider environment and the general public.

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Practical Guide to Chemical Safety Testing The other major concern related to exposure to genotoxic chemicals is the induction of mutations in germ cells, either in sperm after exposure of the male or oocytes after exposure of females. Any mutations that occur in germ cells may be inherited by future generations and all of the individual’s cells will be affected by the mutation. This may have adverse effects on morbidity, fertility or the life span of the individual. So far there has been no clear epidemiological evidence for the existence of a human germ cell mutagen, although such chemicals have been identified in animal tests and are presumed to have similar effects in humans. The purpose of genetic toxicology tests is to identify chemicals that may have the potential to cause cancer or other mutagen-related disease. Various regulatory authorities have developed genetic toxicology testing strategies in order to identify such chemicals, and thereby to regulate their use and distribution in the environment. Genetic toxicology is fortunate to have been in the vanguard of the development of in vitro assays, and consequently a vast selection of assays have been developed for the detection of mutagens. However, of the hundreds of assays that have been proposed, only a small group have been sufficiently well validated to be accepted by the regulatory and testing community. These assays have proven themselves, over many years of use in hundreds of laboratories throughout the world, as being reliable indicators of mutagenic potential. Needless to say these assays can be abused and misused and inexperience, carelessness or design flaws can readily produce false negative or false positive results.

3.2 Mechanisms of Mutation – Genes and Chromosomes In order to understand how genetic toxicology assays work it is necessary to have a basic knowledge of the structure of DNA and chromosomes. DNA is a linear molecule consisting of a sequence of nucleotide bases linked to a sugar-phosphate backbone (see Figure 3.1). The DNA molecule is double-stranded and the paired strands are complementary in terms of their base sequence. There are four bases in DNA; two of which are the purines, adenine (A) and guanine (G), and the other two are the pyrimidines, thymine (T) and cytosine (C). Hydrogen bonds, that preferentially form between A and T or G and C, link the paired strands. The sequence of bases forms three-base ‘codons’ and each of the twenty amino acids of proteins have one or more specific codons used as a template for transcription. It should be remembered that the primary purpose of DNA is to act as a template, or blueprint, for proteins. Proteins that are transcribed from DNA (via the translation apparatus of ribonucleic acid (RNA)) do all the actual work of a cell in terms of enzyme activity, structural functions, etc.

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Genetic Toxicology

Figure 3.1 The structure of DNA Reproduced from US Department of Energy Human Genome Program, www.ornl.gov/hgmis

Although the DNA molecule is linear it has a spiral three-dimensional form commonly known as a double helix. The double helix is further coiled and super coiled to compress the molecule into an extremely small space. When the DNA is replicated or is being transcribed into RNA, the degree of coiling is reduced and the DNA molecule is more ‘open’ to attack by electrophilic chemicals. Thus growing or dividing cells are more sensitive to mutagens than non-dividing cells. This difference in sensitivity forms the basis of the therapeutic mode of action of many cytotoxic drugs used in cancer treatment. In cancer, the tumour cells are often growing at a much faster rate than non-tumour or normal cells and therefore they are more likely to be killed by DNA damaging chemicals. This characteristic is also reflected in the requirement for the use of an actively growing population of cells in in vitro genetic toxicology assays in order to maximise the sensitivity of the assay. It is also important to recognise the difference between the organisational structure of bacterial DNA (the prokaryotic genome) and the genome of eukaryotes such as mammalian cells. Bacterial DNA is a single large closed circular molecule, which is tightly packed into a structure called a nucleoid and has no nuclear membrane. The eukaryotic genome is composed of chromosomes, the number of which depends upon the species.

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Practical Guide to Chemical Safety Testing Humans have 46, or 23 pairs of chromosomes, twenty-two of the pairs of chromosomes are matched in both sexes and carry the same genes as each other. Thus for each gene there are two copies in each cell, although the gene copies are not necessarily identical. The sex chromosomes make up the 23rd pair and in human females they are both X chromosomes, whereas in the male they are the unmatched X and Y chromosomes. The number of chromosomes in a cell is known by either the number of pairs of chromosomes, the haploid number (23 in humans), or the number of actual chromosomes, the diploid number (46 in humans). The number of chromosomes is important because the number of gene copies in a cell influences gene expression. Each chromosome is a single DNA molecule that has a beaded structure formed of lengths of DNA twice-wrapped around histone proteins, which are together known as nucleosomes. The nucleosomes are joined together by stretches of ‘linker’ DNA and this structure is packed by super coiling into a membrane bound nucleus. This difference in DNA organisation is one reason why bacterial assays are insufficient as a screen for genetic toxicology. Bacteria simply do not have the degree of complexity in their DNA to act as an adequate surrogate for mammalian cells. Thus bacterial genes are specific sequences of nucleotide bases carried upon the DNA molecule and the sequence of genes is ‘read’ by the transcription apparatus of the cell to produce proteins. Any change in the sequence of DNA bases, or the ‘message’, will result in a change in the transcription and is called a mutation. This could mean that the incorrect amino acid is incorporated into the protein sequence and the consequence may be that the protein works less efficiently or not at all. The change in the message may be a base-pair deletion or insertion (a ‘frameshift’ mutation) in which the reading frame of the gene is changed by the addition or loss of a specific nucleotide and it’s complementary base on the opposite strand. Alternatively, a base sequence may be altered by substitution with a different base: a purine-to-purine change is termed a transition mutation and a purine to pyrimidine change (or vice versa) is termed a transversion mutation. Frameshift and substitution mutations are collectively known as point mutations and they may also occur in eukaryotic cells. The genes of eukaryotic cells are also specific sequences of nucleotide bases but these are carried on the DNA molecule organised as chromosomes within the nucleus. Consequently they are subject not only to change by point mutations, but also by chromosome mutations and genomic mutations. Chromosome mutations are structural changes to the chromosome that result in an interruption of the base sequence of a gene or the repositioning of a gene that affects the expression of the gene. Gene expression is the transcription of the gene into a functional protein and up-regulation, suppression or inappropriate timing of expression may alter the normal expression of a gene. Structural chromosome mutations may often be visualised by viewing chromosome preparations under a light microscope.

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Genetic Toxicology Genomic mutations are changes to the number of chromosomes in a cell or an organism. The change may be limited to the addition or loss of a single chromosome, known as aneuploidy, or the addition of a multiple of the normal number of chromosomes, known as polyploidy. Aneuploidy in germ cells can result in the birth of abnormal offspring, an example being Down’s syndrome, which is caused by the presence of three sets of chromosome 21, i.e., trisomy 21. In somatic cells, changes in chromosome number are associated with tumourigenicity. The adverse consequences of polyploidy are less clear. Polyploid human embryos are not viable and in somatic cells polyploidy is a common normal phenomenon in many different tissues such as the liver. The observation of increases in the number of polyploidy cells in in vitro chromosome aberration tests is considered by some to be a marker for possible aneuploidy-inducing potential.

3.3 Standard Genetic Toxicology Assays Genetic toxicology testing strategies consist of a battery of tests arranged in a more or less sequential series. The series generally begins with one, two or three in vitro assays followed by one or more in vivo assays, which are usually only required if positive activity had been detected in vitro. If the in vitro testing programme shows a complete absence of activity then, in most cases, in vivo testing is not required. Many chemicals are not directly mutagenic, however, they may be metabolised by enzymes and the metabolites may be DNA reactive. Bacteria and most cells used in in vitro mammalian cell assays have limited metabolic activity. Consequently, metabolic activation systems are incorporated into in vitro assays to mimic mammalian metabolism. These activation systems are generally prepared from the livers of rats in which the enzyme levels have been increased by prior exposure to inducing chemicals such as Aroclor 1254 or a mixture of phenobarbitone and ß-naphthoflavone. The liver preparation is mixed with enzyme co-factors (salts and a nicotinamide adenine dinucleotide phosphate (NADPH) generating system) prior to use, and these provide an energy supply for the enzymes. Almost uniquely in toxicology, positive control chemicals are used routinely in in vitro genotoxicology assays, both to confirm the performance of the test and the activity of the metabolic activation system.

3.4 Bacterial Mutagenicity Assays The most widely known genetic toxicology assay is a bacterial mutation test commonly known as the ‘Ames Test’ after Professor Bruce Ames of the University of California, who developed the method in the early 1970s. At that time, the link between mutation and cancer had been recognised and the test was seen as a potential screen for carcinogens

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Practical Guide to Chemical Safety Testing [1]. Ames and his co-workers developed a series of strains of Salmonella typhimurium that were dependent on the addition of histidine to their culture medium in order to grow. Ironically, the strains were his- mutants and were back mutated, or reverted, to wild-type following exposure to mutagens. A typical regulatory bacterial mutation assay, conforming with the OECD 471 test method guideline, would use five different strains of Salmonella or four strains of Salmonella and one strain of Escherichia coli. The choice of strains, from the many variants that have been developed, is based on scientific principles, the cumulative experience of many scientists and the results of collaborative trials. The different strains are sensitive to various classes of mutagen and some chemicals are strain specific whereas others may be detected in several strains. The five strains that are commonly used (known as TA100, TA1535, TA102, TA98 and TA1537) will detect the majority of mutagens that can be detected in bacterial assays and knowledge of the chemical class or structure can indicate when an alternative strain should be considered. The practical aspects of the assay include a preliminary toxicity test in 1 or 2 strains to determine an appropriate dose range for the first mutation experiment. Typically, the maximum dose level will be 5,000 µg/plate, the lowest precipitating dose level (if nontoxic) or the lowest dose level that gives clear evidence of toxicity. In addition, it is usual to include four dose levels that are non-toxic, using a 2-3 fold dilution series. The first mutation experiment will use all five strains of bacteria, exposed to the test chemical both in the presence and absence of a metabolic activation system (commonly known as S9-mix). The name S9 is derived from the fact that it is prepared from the supernatant of homogenised rat liver centrifuged at 9000g. The ‘mix’ is the mixture of salts and cofactors added to the S9 to enable the enzymes to function. Each strain is exposed to several concentrations of the test chemical, the vehicle control and a positive control. Each concentration is tested in triplicate, i.e., three agar plates per dose level. The concentrations may be different for the exposure in the presence of S9-mix and the exposure without S9-mix, because the toxicity of the test chemical may be quite different in the two exposure groups. The bacteria may be exposed to the test chemical in either of two common procedures, the plate incorporation method or the pre-incubation method. The former is the most common although the pre-incubation method is recommended for some chemical classes such as azo dyes, aliphatic N-nitroso compounds and alkaloids. Azo dyes may also require a modification to the S9-mix known as the ‘Prival-Mitchell modification’ [2]. It has been suggested that the pre-incubation method should be used in preference to the plate incorporation method and the Japanese Regulatory agencies are said to have a preference for the former procedure. In our laboratories we use plate incorporation as the default method, but if an equivocal result is obtained then the pre-incubation method is used to

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Genetic Toxicology clarify the result. However, our experience is, that for most equivocal chemicals, the plate method gives the stronger response and only a small minority show a quantitative difference in favour of the pre-incubation procedure. Either procedure involves the addition of the test chemical solution, the S9-mix (or buffer), the bacterial culture and a small volume of top agar. These are mixed and spread onto the top of a minimal agar plate that contains salts and a carbon source that is sufficient to support the growth of his+ bacteria (tryptophan+ in the case of E. Coli) but not of the non-revertant strains. In the plate incorporation method the bacteria, test material solution, S9-mix or buffer and the top agar are mixed at the same time before immediate application to the base-agar plate. In the pre-incubation method the bacteria, test solution and S9mix or buffer are mixed and incubated at 37 °C for 20 to 60 minutes before addition of the top agar and addition to the base-agar plate. The top agar contains a trace amount of amino acid that is sufficient to promote the limited growth of the non-revertant bacteria. This is important for several reasons, firstly to allow a ‘background lawn’ of bacterial growth that enables an evaluation of toxicity to be made. Secondly, cell division is required to increase the sensitivity of the cells to the mutagen and to enable any induced mutations (back mutations) to be expressed. The agar plates are incubated at 37 °C for 48-72 hours and then the number of revertant colonies is counted, usually with the aid of an electronic colony counter. Each strain of bacteria has a characteristic frequency of spontaneous mutations that may be used as a quality control feature and as a comparison for the evaluation of the significance of any increased frequencies seen after exposure to the test chemical. Originally, Ames proposed a 2-fold rule for the determination of a positive response, thus if any of the test material concentrations induced more than twice the number of revertant colonies seen in the concurrent control then a positive response was indicated. However, since the introduction of cheap and powerful computers it is now possible to use statistical packages to analyse the data more scientifically. The current recommendation is to examine the data and to look for dose-response relationships and reproducibility as the prime indicators of a positive response [3]. Statistics are only used as an aid to the interpretation of the data. If a clear positive response is obtained in the first experiment then the need for a second, or confirmatory experiment, may be obviated. If a negative response or equivocal response occurs then it is necessary to perform a second experiment, or even additional experiments for some difficult test chemicals. The result of a bacterial mutation assay, whether positive or negative, is insufficient to describe the mutagenic potential of a test chemical because it does not indicate whether or not activity will be detected in mammalian cells. However, it is useful as a first screening test because it is economical, rapid and robust in that it presents few false positive results.

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Practical Guide to Chemical Safety Testing This is because there are very few mechanisms by which increases in the numbers of revertant colonies can be induced without the involvement of mutagenicity [4].

3.5 Chromosome Aberration Tests In Vitro Structural and numerical changes to the chromosome complement of a cell are implicated in malignancy and cause heritable effects when they occur in germ cells. Consequently, the evaluation of clastogenicity (structural chromosome damage) is an important feature of genetic toxicology screening programmes. It may also be important to know whether a chemical can induce numerical changes in the genome, particularly in terms of aneuploidy. The analysis of potential aneuploidy is not so readily addressed as clastogenicity and has largely been ignored until recently [5]. Methods for the analysis of chromosome aberrations have been available for many years and it is perhaps the most widely validated of all genetic toxicology methods after the Ames test. The OECD 473 test method requires the use of a growing culture of cells that has a consistent chromosome number and karyotype (number and appearance of the chromosomes). For historical reasons, the different geographical areas of the world have developed their own preferences for particular cell types. Thus, in Japan the Chinese hamster lung (CHL) cell line is dominant because much of the validation and development of the chromosome aberration test in Japan was done by Professor Ishidate and coworkers using this cell line [6]. In the USA Chinese hamster ovary (CHO) cells are very popular and in Europe primary cultures of human lymphocytes are in common use. The latter choice has the added benefit of being derived from the species with which we are most concerned. However, it may also be argued that lymphocytes are less sensitive than CHL or CHO cells and that for screening purposes the most sensitive available system should be used. In practice, it probably makes little difference which cell line is used, so long as the protocol design is robust. Lymphocytes are a little unusual in that they do not normally divide in culture unless they are stimulated by the addition of a mitotic stimulant such as phytohaemagglutinin. One of the most important factors in protocol design and data interpretation for these studies is that of the cell cycle. Mammalian cells proceed through a clearly defined cycle that has a crucial impact on the time points used in a chromosome aberration study. The duration of the cell cycle depends on the cell line, culture conditions and culture density, and also on the toxicity of the test chemical. The cell cycle may be represented as shown in Figure 3.2. Cells that are not actively dividing are said to be in the G0 phase of the cell cycle and when they are recruited into the dividing cell population they enter the G1 phase, or first

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Genetic Toxicology

M G1 S G2 G0 n 2n

= mitosis, separation of paired chromosomes culminating in cell division = gap phase; general cell metabolic activities and DNA transcription = DNA synthesis phase = gap phase, preparation for division = withdrawal from cell cycle by differentiated cells = haploid = diploid

Figure 3.2 The cell cycle

gap phase. This phase is highly variable in duration and may be affected by toxicity and adverse culture conditions. However, once the cell enters the S phase, or DNA synthesis phase, it is committed to cell division at a fixed rate. During S phase, the DNA of the cell is replicated and the majority of mutagenic chemicals require the completion of an S phase during or after exposure before aberrations develop. The chromosomes can only be clearly visualised during the metaphase subdivision of the mitosis phase (M phase). The complete cell cycle has a duration of approximately 12-18 hours depending on the cell type and mitosis may last for only 30 minutes. Therefore, in order to obtain sufficient cells for analysis, a chemical that arrests the cells in metaphase is used. It is difficult to over emphasise the importance of the cell cycle in this test. Toxicity-induced cell cycle delay is a feature observed with most test chemicals and it has a marked influence on the dose-response curve and the time point where the largest response is obtained. Bell shaped or inverse dose-response curves are often seen with chemicals that are toxic, and toxicity is practically inevitable with clastogenic chemicals, although the reverse is not true.

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Practical Guide to Chemical Safety Testing In a typical chromosome aberration test there is a need to use both short-term and longterm exposures. This is because chemicals have been identified that are only detected as clastogens using either a short or long exposure. The short-term exposure normally lasts for 2-6 hours depending on the protocol specification. The exposure is performed both with and without the addition of S9-mix and the cells are washed following exposure and subsequently cultured in the absence of the chemical for a total period of approximately 1.5 cell cycles (approximately 24 hours in most cases). About 2 hours prior to the end of the culture period the mitotic arresting agent is added and finally the cells are harvested by fixation in a methanol/acetic acid mixture. At this point the cells are stable and stained slide preparations can be made when convenient. The slides are evaluated for quality and then coded so that the slide scorer is not aware of the identity of the exposure. This is necessary because the technique for slide scoring has an element of subjectivity and knowledge of the exposure group may result in ‘scorer bias’. Toxicity may be estimated using cell counts or mitotic index (MI). Reductions in cell counts may indicate a slow-down in cell growth or loss of cells due to cell death. The MI is a measure of the proportion of cells in the metaphase stage of the cell cycle and reductions in MI indicate a slowing of the cell cycle as a result of toxicity. The aim is to achieve a range of three or more dose levels that go from low toxicity to approximately 50% cell growth inhibition. The test chemical vehicle is used as the negative control and positive control chemicals are used in both exposure groups. The level of response achieved by the positive control should be clear but not so obvious as to render the slide code redundant. The cells are evaluated for structural chromosome aberrations including gaps, breaks and exchanges (see Figure 3.3). The different aberration types are totalled to give a global frequency of cells with aberrations that is used as the measure of the clastogenic effect. If a clear clastogenic response is obtained in either exposure group then it is not necessary to do further testing. However, if a negative or equivocal response is observed then a second experiment is normally required. In the second experiment the long-term exposure group is used, often in tandem with a repeat of the with-S9 exposure group, perhaps with a modification to the S9-mix. The long-term exposure is for approximately 1.5 cell cycles, which typically equates to 24 hours. It is not possible to do a long-term exposure in the presence of S9 because the S9 itself is toxic and inhibits cell growth. Chromosome aberration tests in vitro are susceptible to the generation of false positive and false negative results if certain precautions are not taken. For example, extremes of pH and osmolarity may cause an increase in the frequency of cells with aberrations and it is important to control any test chemical-induced changes in pH or osmolarity within defined limitations [7]. It has also been suggested that extreme levels of toxicity may induce chromosome aberrations. However, the situation is complicated by the fact that many chemicals do not induce aberrations at extreme toxicity and it may be that the mechanism of toxicity is more important than the actual level of toxicity. In any case,

52

Genetic Toxicology

Figure 3.3 Example of a metaphase cell with a chromatid exchange aberration

most laboratories limit the upper level of toxicity to approximately 50% cell growth inhibition to avoid any possibility of an artefactual response.

3.6 Mammalian Cell Gene Mutation Assays In Vitro These assays detect mutagenic potential by monitoring the activity of a particular gene. Typically, the gene in question is present, or functional, in a single copy in the cells used in the test. Thus, the gene may be situated on the X chromosome, which is present as one copy in males and as one active copy in females. Alternatively, the target gene may be hemizygous (single copy) or heterozygous (2 copies but only one is active). Mutant cells may be selected by killing non-mutant cells with the use of a toxic analogue of the substrate of the enzyme coded by the target gene. There are several well-validated mammalian cell gene mutation assays that may be used to comply with regulatory requirements for genetic toxicology and the OECD 476 test method guideline gives details of the appropriate experimental design. However, the mouse lymphoma assay (MLA) is now the most widely used and is recommended for

53

Practical Guide to Chemical Safety Testing most purposes. One of the main alternatives to the MLA is the hypoxanthine phosphoribosyl transferase (HPRT) assay in CHO cells. This assay is considered to be particularly insensitive and is not recommended for routine use. For the remainder of this section only the MLA will be discussed. The target gene in the MLA is the thymidine kinase or TK gene, which resides on chromosome number 11 of the mouse lymphoma cell line known as L5178Y. This cell line is heterozygous for thymidine kinase because one copy of the gene is inactive whilst the other gene is active and produces the enzyme thymidine kinase. Thymidine kinase is a salvage enzyme that recovers nucleic acid breakdown products for re-use. It is not an essential enzyme because other enzymes may be used to synthesise these products de novo. If the active TK gene is mutated to an inactive form then the cell is transformed into one that is homozygous for non-production of thymidine kinase, and is termed a TK -/- cell. These cells are resistant to trifluorothymidine (TFT), a toxic analogue of thymidine. The MLA has the advantage that the morphology of the mutant colonies may be an indication of the mutational mechanism involved in its origin. Thus ‘large’ colonies often result from point mutations whereas ‘small’ colonies are caused by chromosome changes and are suggestive of clastogenic activity. A growing culture of cells, of sufficient population size, is exposed to the test chemical for both short- and long-term periods. These typically equate to 3-4 hours and 24 hours and the short-term exposure is performed both with and without the inclusion of S9mix. After exposure the cells are washed and cultured for an expression period, which is normally for 48 hours. The expression period is essential to allow pre-existing TK enzyme to degrade so that the ‘phenotype’ of the cell is the same as the new mutant ‘genotype’. Thymidine kinase has a relatively short half-life whereas HPRT is much more stable and an expression period of 7 days is necessary to deplete the enzyme. During the expression period the cell numbers are counted and diluted daily so that the culture density does not exceed the optimum level. At the end of the expression period the cells are plated in culture plates in two different conditions. In the first set of plates the cell density is very low and normal culture medium is used. At the end of a 10-14 day culture period the number of colonies on these plates gives an estimate of the cloning efficiency of the cells, i.e., what proportion of the cells is actually able to grow and form a colony. In the second set of plates a much higher number of cells are plated and the culture medium contains the selective agent, e.g., TFT. In these plates the colonies that grow are those that were either TK-/- before the exposure to the chemical or those that mutated to the TK-/- genotype during or after the exposure period. A calculation using both the mutation frequency and the cloning efficiency produces a mutant frequency per viable cell and may be used to compare the mutation frequency in the control culture with that of a treated culture. This takes into account any toxicity of the chemical that may reduce the cloning efficiency of the cells. This assay is a little different from the chromosome

54

Genetic Toxicology aberration test in that it is considered necessary to achieve a toxicity level of 80-90% in order to be certain not to have obtained a false negative response. There are two common methods for this assay, the agar method and the microtitre method. In the agar method the post-expression period cells are cultured in small plastic dishes in a medium that is mixed with a soft agar gel. The purpose of the agar is to immobilise the cells so that multiple colonies do not arise from single mutant cells following a few cell replications. The alternative method is the microtitre method. This uses 96-well microtitre plates and the number of cells plated into each well is restricted so that, statistically speaking, only a single cell will form a colony in a single well. In order to calculate the cloning efficiency and mutant frequency it is necessary to determine the number of wells with colonies versus the number of wells without colonies. The agar method pre-dates the microtitre method and has the advantage that the mutant colonies may be counted using electronic colony counters. However, it has the big disadvantage that cells generally do not grow as well in agar as they do in culture medium without agar. This means that the cloning efficiency of the agar method is reduced and that the recovery of small mutant colonies is depressed. Both methods have their advocates but the consensus view is that either method is acceptable.

3.7 The In Vivo Micronucleus Test Micronuclei are small, membrane-bound pieces of DNA that are detached from the nucleus when it divides just prior to cell division. The micronucleus test is essentially a chromosome aberration test because most micronuclei are formed following chromosome breakage. However, micronuclei may also develop from whole chromosomes and consequently may also be used as a marker for aneuploidy. It is not possible to distinguish whether a micronucleus is a whole chromosome or is a fragment using standard staining procedures, although it has been tried to correlate the size of a micronucleus with its mode of formation. Centromeric stains may be used on micronuclei to determine the origin of the micronucleus but are not used in routine assays. The centromere is the part of the chromosome that is used to attach it to the nuclear spindle, the structure that is essential for correct nuclear division. Consequently, acentromeric chromosome fragments are easily lost from the nucleus and become free floating nuclear bodies that are observed as micronuclei. Chromosomes, which are by definition centromeric, may become detached from the nuclear spindle either by chance or by the action of a spindle poison or inhibitor. There are several tissues that have been used as targets for the micronucleus test including the liver and the intestine. However, the mouse or rat bone marrow is predominantly used because it is convenient to sample and technically the most simple; the OECD 474

55

Practical Guide to Chemical Safety Testing Guideline [8] is the standard regulatory test method. In the bone marrow there is a population of dividing stem cells that generate the red and white blood cells needed to supply and replenish the peripheral blood. Early stem cells have the ability to develop into either leucocytes (white cells) or erythrocytes (red cells), however, following a particular point in their development they become committed to develop into one or the other. Micronuclei are most easily detected in immature erythrocytes because in these cells the main nucleus is expelled following the final cell division of the preceding erythroblast. Erythrocytes have no nucleus, and have a relatively short life span of 6-10 weeks, consequently there is a continuous high rate of erythrocyte production in the bone marrow. Immediately after the expulsion of the nucleus the erythrocyte can be stained to give a bluish purple colour due to the residual DNA and RNA nucleotides that were associated with the nucleus prior to enucleation. During the next 20-30 hours the concentration of these molecules falls so that the cell loses it’s bluish staining appearance and develops a pale pink colour. At this point the cell is known as a normochromatic erythrocyte (NCE) as opposed to the blue staining polychromatic erythrocyte (PCE). The relative ratio of these two cell types may be used to estimate the toxicity of the test chemical to the bone marrow. Prior to the micronucleus test it is necessary to determine the maximum tolerated dose level of the test chemical, either through the use of a preliminary toxicity test or by reference to existing data. Existing data, even if from a different species, may often be used to establish a starting dose level lower than would be used if no information were available. This is desirable from an ethical perspective because it may mean that fewer animals are required and the severity of clinical observations is reduced. If there is no significant difference in the toxicity of the test chemical to the two sexes then it is acceptable to use a single sex in the micronucleus test. Historically the male is chosen because it has been shown to be marginally more sensitive than the female in comparative studies [9]. The route of exposure is also selected during the preliminary toxicity test and this may be oral, intraperitoneal, or intravenous. In the main study of the micronucleus test, groups of mice are dosed with the test chemical, the vehicle control or a positive control. The group sizes are a minimum of five per sex, although if a single sex is used group sizes of seven are recommended to increase the statistical power of the study. If a single dose is given then it is usual to include two timepoints in the study; a primary time-point at 24 hours and a second one at 48 hours. Three dose levels are included at the 24-hour time-point, unless the test chemical is nontoxic, when a single maximum dose level of 2000 mg/kg may be used. The three dose levels are typically the maximum tolerated dose level (MTD), MTD/2 and MTD/4. The MTD is defined as a dose level that is tolerated well by the animal, i.e., the clinical signs are moderate rather than severe. A single dose level is used in the 48-hour exposure group and this is normally the same as the MTD for the 24-hour group. Vehicle control

56

Genetic Toxicology groups are included at both time-points and a positive control is included at the 24-hour time-point only. At the end of the exposure time, the animals are humanely killed and the bone marrow tissue is collected. The cells are spread onto microscope slides, stained and subsequently observed for the frequency of micronucleated PCEs and the ratio of PCEs to NCEs. The ratio of the two cell types may give an indication of any toxic effects in the target tissue. An increase in the number of NCEs may occur following a ‘wound response’ and the consequent influx of peripheral blood into the bone marrow. Statistical analysis may be used to determine whether or not there has been a significant increase in the micronucleus frequency or a significant change in the ratio of erythrocytes. The micronucleus test is the primary in vivo mutagenicity test that is currently in use and has been extensively validated in many multi-centre studies. It is included in all regulatory testing strategies for chemicals and pharmaceuticals. However, many chemicals that give positive responses in in vitro tests are shown not to induce micronuclei in the bone marrow of mice or rats. In these cases it is often necessary to confirm the absence of a positive response in a second in vivo test to ensure that the result is not a false negative.

3.8 The Unscheduled DNA Synthesis Assay Scheduled DNA synthesis is that which proceeds prior to cell division and occurs during the S-phase of the normal cell cycle so that the complete DNA content of the nucleus of a cell is replicated. Unscheduled DNA synthesis (UDS) takes place when DNA repair mechanisms are invoked following DNA damage after exposure to a genotoxic chemical. There are a number of key DNA repair mechanisms in mammalian cells that operate in different ways and respond to various types of DNA damage or to lesions on the DNA molecule. Most of the DNA repair systems involve a degree of DNA synthesis of a new length of DNA that is used to replace the damaged section. This process can be monitored by the utilisation of appropriate labelling techniques to identify the presence and amount of DNA repair activity. Inappropriate levels of DNA repair indicate that elevated levels of DNA damage have taken place and this may be linked to exposure to a genotoxic chemical. UDS assays are most frequently performed in the rat liver and this has the virtue of being both a second species and a second tissue when utilised as a follow up to the mouse micronucleus test. Furthermore, the liver is the primary organ for the metabolism of chemicals following exposure and is therefore of great toxicological importance. The rat liver UDS assay is actually an ex vivo procedure because although the exposure to the chemical occurs in the live animal, the detection of UDS activity takes place in cultured

57

Practical Guide to Chemical Safety Testing hepatocytes harvested from the animals using a surgical procedure. The majority of chemicals that induce UDS in the rat liver do so with a peak effect some 12-16 hours after dosing. However, some rapidly absorbed chemicals, such as water-soluble nitrosamines, may have a peak response much earlier, i.e., within 2-4 hours after dosing. Consequently it is necessary to include both time-points in the assay to be certain of detecting all positive chemicals. It is usual to do the later time-point first and if a positive response is obtained then the earlier time-point may be omitted. It is also possible to dose the animals twice and to harvest the hepatocytes 2 hours after the second dose, which is given 14 hours after the first dose. Thus both time-points can be examined in the same animals and a reduction in the number of animals that are used is possible. This latter procedure has not yet been universally accepted and is typically used as a screening procedure. The regulatory requirements for a UDS assay are described in the OECD 486 test method guideline. As in the micronucleus study, it is necessary to determine the MTD before the UDS assay can be initiated and it is good ethical practice to utilise existing data where possible. Two dose levels are generally used in the UDS study, the MTD and the MTD/3, and negative and positive controls are included. The positive controls are selected so as to be preferentially detected at the early or late harvest time-point. Most studies are performed on male rats only, but females may be included if there is a significant sex difference in toxicity. The primary route is oral but the intravenous route may be used if appropriate. The intraperitoneal route is often reported as not being an option in this assay because of the location of the liver in the peritoneal cavity. However, the intraperitoneal route has been successfully used when there is no evidence of absorption via the oral route and the chemical formulation cannot be given via the intravenous route. Following euthanasia, the hepatocytes are isolated from the liver by a 2-stage perfusion procedure that utilises a collagenase enzyme to disassociate the cells from the matrix of the organ to give a single-cell suspension. The cells are then cultured in an incubator following their attachment onto microscope slides. The cells are incubated in a culture medium containing radiolabelled (tritiated) thymidine, which is absorbed by the cell and incorporated into the DNA at sites of repair. A period of culture in the absence of labelled thymidine and an excess concentration of unlabelled thymidine (the so called ‘cold-chase’) is used to reduce the background levels of labelling. After the appropriate period of culture, the cells are fixed and the slides overlaid with a thin film of photographic emulsion. A subsequent storage period of 7-14 days in the dark allows the ß particles emitted from the labelled thymidine to cause the deposition of silver grains in the emulsion layer. The emulsion is then developed and the cells are counter-stained to produce differential staining of the cytoplasm and the nucleus. The slides are then evaluated for the relative incidence of grains that can be detected in the emulsion over the cytoplasm and over the nucleus. The ‘cytoplasmic’ grains result

58

Genetic Toxicology from residual labelled thymidine that was absorbed by the cell during the labelling procedure. The ‘nuclear’ grains are produced by the labelled thymidine absorbed into the nucleus and incorporated into the DNA of the nucleus. The number of grains over the nucleus is counted first, either manually or more typically by the use of an image analysis system. Then an estimate of the number of grains in an equivalent area of the cytoplasm is determined and the net grain count for the cell is calculated by subtracting the cytoplasmic count from the nuclear count. Most negative control cells have a negative value for the net grain count because the cytoplasm normally has more grains than the nucleus. However, when DNA damage has been induced and repaired the nuclear count increases and positive net grain counts are obtained. Statistical analysis of the data is not always necessary, a simple evaluation of the incidence of cells in repair and the mean net grain count is usually sufficient to give a conclusion to the study. A negative result in the UDS assay may be used to confirm the result of a micronucleus test and to conclude that the test material poses little or no hazard to humans even though positive results were obtained in vitro. A positive result in the UDS assay is clear evidence of mutagenic hazard and may indicate the necessity to evaluate the test material for effects in the germ cells. This may be done by the use of further in vivo tests such as the dominant lethal test or a chromosome aberration test in germ cells. In the dominant lethal test, male mice or rats are dosed with the test material and then mated to nonexposed females. Before the end of term, the females are examined for the number of live and dead foetuses. An increase in the number of dead foetuses may indicate that dominant lethal mutations have been induced in the male germ cells that lead to the premature death of a foetus. Such mutations are normally associated with either changes in chromosome number or structure.

3.9 Conclusions The in vitro test battery for genotoxicity has been developed and refined over the last three decades into a reliable, sensitive and ethical strategy for evaluating the genotoxic potential of test materials. Some may say that the in vitro tests are overly sensitive and that false positive results are all too frequent. It is true that the Ames test has a positive hit-rate in the region of 8-15% of new chemicals tested. The mammalian cell assays have a higher hit-rate of up to 25%, depending on the particular assay and the source of the chemicals being tested [10]. Pharmaceutical chemicals tend to have a higher hit-rate than industrial chemicals because they are designed to have biological effects. Conversely, the in vivo tests such as the micronucleus and UDS, have much lower frequencies of positive results, ~3-11% [11]. Pharmaceuticals again score higher than industrial chemicals, but the problem is the discrepancy between the in vitro and in vivo results.

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Practical Guide to Chemical Safety Testing It is difficult to know how to handle such data; do we take comfort from negative in vivo data and assume little hazard, or do we consider that the in vivo tests are insensitive and give false negative results? The problem with the in vivo assays is that we may be looking in the wrong place or at the wrong time for the effects that we expect to see. In particular, the target tissue may be completely different to the one that is examined and perhaps what is required is an assay in which the site of contact may be investigated. A relatively new assay that is currently being validated by many laboratories is the single cell gelelectrophoresis assay, or COMET assay for short. In this procedure, any tissue that can be prepared to provide a single cell suspension may be examined for DNA damage. Perhaps surprisingly most tissues will provide single cells using fairly simple techniques and because only small numbers of cells are required, the yield may be low. The cells are suspended in a gel and mounted on a microscope slide, the cell membrane is removed and the DNA unwound by the use of a very high pH buffer. An electric current is then passed across the gel and this causes DNA fragments to migrate towards the anode. Subsequent staining reveals a nuclear ‘head’ and a DNA fragment ‘tail’, which looks very similar to astronomical comets, and hence the test is called the COMET assay. Analysis of the mean amount of DNA in the tail and/or the length of the tail may be used to estimate the relative amount of DNA damage, in both qualitative and quantitative terms. It is probable that there will now be little further development of in vitro tests, except perhaps for specific applications such as high-throughput screening assays. However, it is perhaps equally probable that new or refined in vivo tests will appear to enable us to better understand the results of the in vitro tests.

References 1.

B.N. Ames, W.E. Durston and E. Yamasaki, Proc. Natl. Acad. Sci. USA, 1973, 70, 2, 281.

2.

M.J. Prival and V.D. Mitchell, Mutation Res., 1982, 97, 103.

3.

OECD Guideline for Testing of Chemicals, 471 Bacterial Reverse Mutation Test, 1997.

4.

E. Gocke and S. Albertini, Mutation Res., 1996, 350, 51.

5.

Department of Health (UK) Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, Draft Guidelines on the Strategy for Testing of Chemicals for Mutagenicity, 2000.

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Genetic Toxicology 6.

M. Ishidate Jr. et al., Food and Chemical Tech., 1984, 22, 623.

7.

D. Scott et al., Mutation Res., 1990, 257, 147.

8.

OECD Guideline for Testing of Chemicals, 474 Mammalian Erythrocyte Micronucleus Test, 1997.

9.

The Collaborative Study Group for the Micronucleus Test, Mutation Res., 1986, 172, 151.

10. R.D. Snyder and J.W. Green, Mutation Res., 2001, 488, 151. 11. D.J. Kirkland and L. Müller, Mutation Res., 2000, 464, 137.

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Ecotoxicology

4

Ecotoxicology John W. Handley

4.1 Introduction Ecotoxicology is the study of the adverse effects of chemicals in the environment and on ecological systems. These effects may include both lethal (mortality) and sublethal (effects on growth and development for example) and can be expressed in quantitative or qualitative terms. The study of ecotoxicology for new and existing chemicals is mostly carried out in standardised laboratory tests designed to be robust yet reproducible, sensitive and reliable. Ecotoxicological studies should by definition be ecologically relevant, however to study the effect of a chemical on all the organisms present in all the different ecosystems of the world would clearly be unrealistic. Regulatory ecotoxicity studies therefore focus on indicator species, to allow hazard assessments to be made which in turn can be used to define the risk of a chemical to the environment. In order that the tests can be reproducible and comparable (to enable ranking of chemicals) much of the environmental relevance is removed as these factors serve more to confuse the investigator than to aid the interpretation of environmental effects. For example all aquatic ecotoxicity studies are conducted on single species in defined media in standard laboratory glassware in the absence of sediments or plants. Regulatory ecotoxicity studies are conducted following defined test guidelines published by the EU, OECD, US EPA, Japanese Ministry of Economy, Trade and Industry (METI) and Ministry of Agriculture, Forestry and Fisheries (MAFF), although reference will be made primarily to OECD methods. This chapter will briefly discuss the most commonly conducted studies and will focus on how tests are conducted on new chemical substances. The testing of effluents, whilst based on similar methodology, has specific elements relating to the testing of mixtures of chemicals that are outside the scope of this publication. Ecotoxicity testing for regulatory compliance concentrates mostly on the aquatic environment (at lower production tonnages of chemicals) and is based on the assessment of effects at different trophic levels. The amount and complexity of testing is dependent upon the regulatory scheme followed and the amount of test compound likely to be produced in a given period. The aquatic environment is the primary route of disposal of

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Practical Guide to Chemical Safety Testing most man-made chemicals either from direct run-off or via end of pipe discharge from production or treatment facilities. Indeed, most regulatory schemes are based on exposure of the freshwater environment, although discharge to the marine environment is equally important and there are specific notification schemes for the use of chemicals in the marine environment. Essentially the same test methods are employed, but with marine species as opposed to freshwater species. Firstly the likely toxic effect of chemicals on the microorganisms responsible for the degradation of the chemical is assessed followed by the investigation of biodegradation itself. Next assessments are made of the toxic effects of chemicals to the different trophic levels in the environment. Initially the effect on primary producers (algae) is assessed, then primary consumers (invertebrates) and then the secondary consumers (fish). Clearly if a chemical is toxic to algae but not to invertebrates and fish it will cause an environmental effect, since the food source of the invertebrates and subsequently the fish will be removed. Further sublethal effects may lead to an accumulation of a chemical in one species that may then be magnified up the food chain. The choice of test species is governed not only by sensitivity to chemicals, but also by ease of culture or availability, ability to withstand laboratory conditions and the ease of determination of the toxicological end points. For regulatory ecotoxicity tests it is important to establish that the test organisms are not exposed directly to the test chemical, as they are with mammalian tests, but the organisms are exposed via their environment. Thus when assessing the quantitative measurement of toxicity, the data will be expressed in terms of the lethal concentration (LC) as opposed to the lethal dose (LD). The complexity and type of studies conducted to assess the ecological impact of a chemical are dependent upon the production tonnages of the chemical and the regulatory body making the assessment of the tests. For example, under the Japanese Chemical Substance Law notification scheme if a chemical is found to be non-biodegradable then the next study to be conducted is a fish bioaccumulation test. However, under the European New Chemical Notification scheme fish bioaccumulation studies are not conducted until what are known as the Level 1 or 2 trigger points based on annual production levels of the chemical. Correct dosing and exposure assessment are of vital importance in the testing of chemicals for ecotoxicological effects. The majority of chemicals tested are of low aqueous solubility or show some form of instability in aqueous media. Thus knowing what the organisms have been exposed to and ensuring that during exposure the test chemical is presented in such a way that it is bioavailable is critical. Detailed advice documents have been published to aid the investigator in the design and conduct of ecotoxicological studies [1, 2, 3].

64

Ecotoxicology The object of these tests is to provide data on exposure that can be used in conjunction with use pattern data, release estimations and environmental fate data to generate or refine an environmental risk assessment (see Chapter 8).

4.2 Bacterial Toxicity Testing The most common form of bacterial toxicity test conducted for regulatory compliance is the Activated Sludge Respiration Inhibition Test, OECD Guideline No 209 [4]. In essence this test consists of an exposure vessel containing activated sewage sludge and the test material, which is then aerated for a period of 3 hours. A synthetic sludge, made of peptone, urea, meat extract and a buffer solution, is added to provide a respiratory substrate for the microorganisms and a positive control (3,5-dichlorophenol) is run in parallel. The rate of respiration is then determined for each control and test vessel at the end of the exposure period. The rates of respiration are then compared and percentage inhibition values calculated. Similar criteria to those used to assess aquatic toxicity testing (see Section 4.4) should be used to determine the toxicity of the chemical to the microorganisms present. Concentrations up to 1000 mg/l may be tested for hazard identification at sewage works. In order to satisfy the requirements of all Competent Authorities worldwide, the study is often conducted using two methods of dispersing the test material in the test system; one using a single test concentration equal to the water solubility, the other with test concentrations up to a maximum of 1000 mg/l prepared by direct dispersion in test water. Using this approach, the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge microorganisms’ exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge. In addition, the use of a single test vessel at the limit of water solubility of the test material can show whether soluble test material has an adverse effect on the activated sewage sludge.

4.3 Biodegradation Tests Ready biodegradation tests are often considered to be screening biodegradation tests making the basic discrimination between readily biodegradable materials and other compounds. They are all basic batch tests using the test material as the only source of organic carbon and a low concentration of a non-adapted inoculum. The amount of carbon in the test from the inoculum is kept to a minimum and allowance is made for the endogenous activity of the microorganisms by running parallel blanks. A reference

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Practical Guide to Chemical Safety Testing compound is run in parallel to confirm the suitability of the test conditions. For the ready biodegradation tests the major difference between the tests is how the degradation is monitored. This may be by means of oxygen uptake, carbon dioxide evolution or removal of dissolved organic carbon (DOC). Compound specific chemical analysis may also be used to assess primary degradation. Ready biodegradation tests normally run for a period of 28 days. The results of ready biodegradation tests do not correlate directly with those from environmental systems as the ready tests usually underestimate the potential for degradation in the environment. Positive results indicate the compound to be degradable under natural conditions without difficulty. However, a negative result does not mean the contrary, just that further studies, i.e., inherent biodegradation tests could be conducted to obtain a fuller assessment of degradation potential. Inherent tests (of a more complex nature than the initial ready screening tests) are used to determine whether biodegradation of the test chemical is possible. Concentrations of both the inoculum and test chemical are higher. The tests are conducted in either batch or semibatch conditions, sometimes in the presence of other readily biodegradable compounds. The final level of testing is that of simulation tests which are used to confirm positive or negative results from previous tests. Much of the work has focused on the development of activated sludge systems as opposed to biological filters.

4.3.1 Ready Biodegradation Tests Table 4.1 briefly provides a summary of the test conditions of ready biodegradation tests. All studies should be conducted in the dark to avoid the possibility of photodegradation. The concept of the 10-day window, peculiar to ready tests, is used to define pass levels along with the specific criteria of each. Compounds must achieve the pass level of the test 10 days after first attaining 10% degradation. As will be discussed for aquatic toxicity testing the delivery of the test compound is critical in determining an accurate measure of degradation. Quite clearly the removal of dissolved organic carbon is not used for water insoluble substances, as no test material will be in the dissolved form. Also volatile substances are not tested in systems that are constantly aerated such as the CO2 evolution test. For insoluble materials it may be possible to disperse the compound evenly throughout the exposure vessel, thereby giving a greater chance of microbial attack, by using an inert carrier. Handley and co-workers [5] describe how this may be performed using finely ground silica gel to aid the dispersion of insoluble viscous liquids and the International Standards Organization (ISO) and ECETOC have produced advice documents [2, 6] on the treatment of poorly water soluble compounds in biodegradation tests. Table 4.2 shows the applicability of each test method to the testing of difficult substances.

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Ecotoxicology

Table 4.1 A summary of the test conditions in OECD ready biodegradation tests (adapted from OECD guidelines) DOC DieAway

Test concentration

CO2 Evolution

10-40 mg/l 5-20 mg C/l

Manometric Respirometry

Modified OECD Screening test

Closed Bottle test

METI test

100 mg/l

10-40 mg C/l

2-5 mg ThOD/l

100 mg/l

≤ 30 mg SS/l

Concentration of inoculum

30 mg SS/l

≤ 100 ml effluent/l

0.5 ml effluent/l

approx 107–108 cells per ml

< 5 ml effluent/l

approx 105 approx 104 cells per ml cells per ml

approx 107–108 cells per ml

22 ± 2

Temperature (ºC)

25 ± 1

SS = suspended solids ThOD = theoretical oxygen demand effluent = sewage effluent

Table 4.2 Suitability of ready biodegradation tests for difficult substances Test method

Analytical measurement

Suitable for compounds which are: Poorly watersoluble

Volatile

Adsorptive to glassware or inoculum

DOC Die-Away DOC

-

-

✓

CO2 Evolution

CO2 production

✓

-

✓

METI

Oxygen consumption

✓

✓ (-)

✓

Closed Bottle test

Dissolved oxygen

-

√

✓

Modified OECD Screening test

DOC

-

-

✓

Manometric Respirometry

Oxygen consumption

✓

✓ (-)

✓

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Practical Guide to Chemical Safety Testing

4.3.1.1 OECD 301A DOC Die-Away Test [7] This test, and the modified OECD Screening test, are very similar. The major difference is the concentration of the inoculum (see Table 4.1) with this test having the higher inoculum level. The test material must be truly soluble at the levels used in the test as DOC removal is used to monitor degradation. The solution is incubated in a shaken system for 28 days at 20-25 ºC in the dark and degradation is followed by the DOC analysis of samples taken periodically throughout the test. Compounds giving > 70% degradation within the 10-day window are considered to be readily biodegradable.

4.3.1.2 OECD 301B CO2 Evolution Test [8] In this test, also known as the Modified Sturm test, a solution or dispersion of the test chemical is incubated in the presence of the inoculum under constant aeration with CO2 free compressed air. The CO2 in the exhaust air is trapped in sodium hydroxide solutions and the amount determined with inorganic analysis and expressed as a percentage of the theoretical CO2 production. This is a good test for poorly water-soluble substances since the test vessel size allows accurate weighing of small amounts of test chemical that can then be dispersed directly to the test vessel. Compounds giving > 60% degradation within the 10-day window are considered as readily biodegradable.

4.3.1.3 OECD 301C METI (I) Test [9] The METI (Ministry of Economy, Trade and Industry, Japan, previously known as MITI, Ministry of International Trade and Industry) test is based on the measurement of biological oxygen demand (BOD), i.e., oxygen consumption, the analysis of residual chemicals as DOC and specific chemical analysis. The 10-day window criterion does not apply to this test. Compounds giving > 60% degradation are considered as readily biodegradable. The results of the specific chemical analysis can often cause serious problems with notification since all degradation products must be identified and quantified, as further evaluation, possibly involving further studies, may be required on stable degradants. An automated respirometer gives a continuous recording of BOD and the test requires the use of a specific inoculum derived from 10 different sampling sites, grown on glucose-peptone medium.

4.3.1.4 OECD 301D Closed Bottle Test [10] This is probably the most stringent of all the ready biodegradation tests, having very low test compound and inoculum concentration requirements. The test uses standard BOD

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Ecotoxicology bottles and follows degradation by the consumption of oxygen using either an oxygen electrode or titrametric analysis. The very low test material concentration would seem to indicate that this would be a good test for poorly water-soluble compounds, but in fact this is not the case. The practicalities of conducting this traditional BOD test are such that a concentrated stock solution must first be produced and then diluted as part of the preparation procedure to obtain the final concentration. The compound must be in solution for this test to be valid. Compounds giving > 60% degradation within the 10day window are considered as readily biodegradable.

4.3.1.5 OECD 301E Modified OECD Screening Test [11] This is a very similar test to the DOC Die-Away test except that it uses a lower inoculum rate and is aerated rather than shaken. The test material must be fully soluble at the levels used in the test as DOC removal is used to monitor degradation. The solution is incubated in a shaken system for 28 days at 20-25 ºC in the dark and degradation is followed by the DOC analysis of samples taken periodically throughout the test. Compounds giving > 70% degradation within the 10-day window are considered to be readily biodegradable.

4.3.1.6 OECD 301F Manometric Respirometry Test [12] This test is essentially the same as the METI test except that use of an automated respirometer and a specific inoculum are not mandatory requirements. Normal activated sludge is typically used. This test is also recommended for poorly water-soluble compounds. Compounds giving > 60% degradation within the 10-day window are considered as readily biodegradable.

4.3.1.7 Seawater Biodegradation Test (OECD 306) [13] This is essentially the same as the Closed Bottle test [9] but adapted for marine use. Compounds being registered for use off-shore under the OSPARCOM Scheme (Oslo Paris Convention, Harmonized Off-shore Chemical Notification) require an additional biodegradation test using a marine inoculum. The inoculum is provided by using natural seawater and so the bacterial loading is quite low. The test method is then the same as for the Closed Bottle test.

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Practical Guide to Chemical Safety Testing

4.3.2 Inherent Biodegradation Tests

4.3.2.1 OECD 302A Semi-Continuous Activated Sludge Test (SCAS) [14] This test is only applicable to water-soluble test compounds. Activated sludge, the test chemical and settled raw sewage are aerated for 23 hours and then allowed to settle for 1 hour. The supernatant liquor is then removed and analysed for DOC. The remaining sludge is then mixed with a further aliquot of test compound and sewage and the cycle repeated. The length of this ‘fill and draw’ procedure should be at least 12 weeks for compounds showing a low degradation. Whilst the OECD Guidelines do not specify a pass level, a compound showing > 20% loss of DOC may be considered to be inherently biodegradable and compounds showing > 70% are considered to have shown evidence of ultimate degradation. This procedure can also be used to good effect to produce an adapted inoculum to be used in other tests. The method provides favourable conditions for both acclimatisation and extensive biodegradation but does not simulate conditions during common sewage treatment.

4.3.2.2 OECD 302B Modified Zahn-Wellens/EMPA Test [15] Activated sludge, the test compound and a mineral medium are aerated in a static batch culture for up to 28 days. Samples are removed periodically and DOC analysis performed on filtered samples. Whilst the OECD Guidelines do not specify a pass level, compounds showing > 20% of DOC may be considered to be inherently biodegradable and compounds showing > 70% are considered to have shown evidence of ultimate degradation.

4.3.2.3 OECD 302C METI (II) Test [16] This is essentially the same as the METI (I) test except that the sludge and test compound concentrations are reversed to give 30 mg/l of test compound and 100 mg/l of inoculum. This is the only inherent biodegradation study suitable for poorly water-soluble compounds. In practice this study is rarely used given the great difference between environmental (sewage treatment facilities) degradation and the test.

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Ecotoxicology

4.3.3 Simulation Tests

4.3.3.1 OECD 303 Coupled Units Test [17] This is recommended as a test for the determination of ultimate biodegradation of compounds under conditions that simulate the treatment in an activated sludge plant. Two continuously operated lab-scale activated sludge units with complete mixing are operated in parallel; the parallelism is enhanced by a transinoculation procedure. The test compound is added in one unit, while the other is fed only synthetic sewage. Some experts say that better results are obtained using raw domestic sewage. The DOC of both effluents is measured. The test compound is tested at a concentration of 20 mg DOC/l with a mean retention time of 3 to 6 hours and a solids content of 2.5 g/l. The working-in period should not exceed 6 weeks and the test period should not be less than 3 weeks. Degradation is expressed as percentage removal of DOC. The difference between the DOC effluent values is due to non-degraded or partially degraded test compound. Further tests are necessary if a distinction between biodegradation (or partial degradation) and adsorption is necessary.

4.3.3.2 Porous Pot Test This is essentially another form of lab-scale simulation test, which measures biological degradation of a chemical by the removal of DOC. The porous pot is constructed from sheets of porous polyethylene, which are made into cylinders with a conical base. The porous pot is contained in an impervious vessel with a supporting ring made of plastic around the top of the inner vessel so that there is an effluent space of 0.5 cm between the inner and outer vessels. The pots are mounted in a thermostatically controlled water bath and have an air supply to the base of the inner vessel with a diffuser. A constant flow of synthetic sewage is pumped into the vessels. Similar run conditions to the coupled units test are employed.

4.3.4 Anaerobic Biodegradation Tests Currently the need for anaerobic biodegradation testing is limited to compounds which have reached the higher tonnage triggers. The rationale for this testing is based on the compound’s ability to adsorb to sewage sludge. If the compound has a high adsorption coefficient (See Chapter 5), then during normal sewage treatment it is likely that a significant amount of compound will remain within the sewage treatment facility. It is becoming more common for the further degradation of sewage solids by anaerobic

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Practical Guide to Chemical Safety Testing digestion. There are a variety of different methods based on methane production from anaerobic degradation. One of the more simple [18] uses 250 ml serum bottles containing an anaerobic sludge and test material, sealed and vented with nitrogen to maintain anaerobic conditions. Degradation is followed by monitoring the increase in headspace pressure (i.e., gas production) using a handheld pressure meter. The study is conducted at 35 ºC for a period of 56 days.

4.4 Aquatic Toxicity Testing Tests for aquatic toxicity are conducted following standard methods recommended by the EU, OECD or US EPA Office of Prevention, Pesticides and Toxic Substances (OPPTS). Most studies are carried out using freshwater species but the tests can be modified easily to accommodate marine species, where the hazard to the marine environment needs to be assessed. As briefly discussed for the biodegradation tests the method of substance delivery to the test system is critical in the determination of aquatic toxicity. Coupled with this is the need for accurate measurement of the exposure concentrations and determination of the bioavailable fraction (dissolved portion) of the preparation. When testing fish, ethical issues must be addressed before testing commences. Testing on vertebrate species should be avoided wherever possible and if considered to be necessary, should be minimised. Testing with invertebrate species should be conducted in preference to fish. The use of quantitative structure-activity relationships to aid dose level selection is encouraged to reduce animal numbers. For acute tests the LC50 or EC50 values are determined (EC50 is an effective concentration that causes a 50% response in the population) as well as a no observed effect concentration (NOEC). Toxicity can be ranked into 4 levels based on the L(E)C50 values. This ranking follows closely the guidelines for the classification and labelling of chemicals as dangerous to the environment (see Table 4.3).

Table 4.3 Classification of chemicals as dangerous to the environment Rating Highly toxic to aquatic life Toxic to aquatic life Moderately toxic to aquatic life Relatively non-toxic

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LC50 < 1 mg/l > 1 < 10 mg/l > 10 < 100 mg/l > 100 mg/l

Ecotoxicology Testing is carried out up to a maximum concentration of 100 mg/l or the limit of water solubility for poorly water-soluble compounds. If in preliminary trials no toxicity is observed at either 100 mg/l or the limit of water solubility then a Limit Test is conducted at the appropriate level to confirm an absence of toxicity.

4.4.1 Acute Tests

4.4.1.1 Algal Growth Tests This test allows for an acute effect measurement, but as cell division (reproduction) occurs this test can also be used as an indicator of chronic toxicity. The test can be conducted using either freshwater or marine species and can last for 72 or 96 hours depending upon the regulatory guideline followed. Typically studies are conducted following the OECD method [19] with one of two freshwater species (Scenedesmus subspicatus or Pseudokirchneriella subcapitata) or a marine species (Skeletonema costatum). To provide additional effect data studies may also be conducted with species from a second taxonomic grouping, such as the diatom Navicula. The effect of the test chemical is determined by the growth (or lack of growth) of the algal cells. The test system is set up using an algal cell culture in log phase growth. The test chemical is mixed with the algal culture using a defined culture media, such that the initial cell density in the test is approximately 1 x 104 cells per ml. At the end of the test, the cell density can rise up to 108 cells per ml depending on the duration of the test. The validation criteria of the test state that the cell density must increase by a factor of 16 after 72 hours in the controls, and that the pH of the control vessels should not vary by more than 1.5 pH units over the test period. Samples are removed on a daily basis and the cell density determined by direct cell counting using either a microscope and counting chamber or an electronic particle size counter. The test vessels are incubated at 24 ºC under constant illumination (approximately 7000 lux) and shaking (approximately 150 rpm). Temperature and pH are monitored at least at the start and termination of the test. The test is conducted under static test conditions and the test substance concentration is determined at the start and end of the exposure period. At the end of the exposure period, the cell density data is used to calculate the effect concentration (EC) for the study. This can be based on biomass (EbC50) or growth rate (ErC50) and the lowest value should be used as the indicator of toxicity. The no observed effect concentration (NOEC) is also determined as the highest test concentration showing no significant difference from the control in terms of total cell density.

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Practical Guide to Chemical Safety Testing In standard laboratory algal growth inhibition tests conducted on dyestuffs [19], it is not possible to differentiate between reduced growth of green algae due to real toxic effects of the dyestuff, or reduced growth caused by the indirect effect of light absorption of the coloured test solution. Thus, the results of these tests, the EC50 values and NOEC, depend on both effects, the toxicity and reduced light intensity. Risk assessment of chemicals to the environment is based on such test results, however if the reduction of algal growth is solely due to light absorption, this is probably not relevant to the estimation of environmental risk. Moreover, in the criteria of environmental classification of substances, the inhibition of algal growth solely by light absorption is specifically excluded as a classification criterion. This means that EC50 values should not be used as a basis for classification where it can be demonstrated that the inhibition is the result of a pure light effect. The modified test system (developed by Memmert and co-workers [20]) enables quantification of the pure light filter effect of a coloured test substance on algal growth, and the total growth inhibition by both light absorption and toxicity, by the use of two experimental methods set up in parallel. The differences in algal growth between the two experimental methods are interpreted as the real toxic effect of the dyestuff on algal cell growth.

4.4.1.2 Acute Invertebrate Tests The standard freshwater invertebrate used in toxicity tests is Daphnia magna. The method [21] uses animals at the most sensitive life stage, i.e., the first instar that are less than 24 hours old. The animals are derived from parthenogenically breeding laboratory cultures that are maintained on reconstituted water and fed a high quality algal feed. Typically the test is conducted using 250 ml containers each containing 10 animals. The test is conducted in a temperature controlled laboratory at 21±1 ºC for a period of 48 hours with a 16 hour light: 8 hour dark light cycle incorporating a 20 minute dawn and dusk transition period. The animals are not fed during the exposure period. Dissolved oxygen, temperature and pH are monitored at least at the start and termination of the test. The test vessels receive no auxiliary aeration during the test and providing the test media is aerated before use and dissolved oxygen remains > 3 mg O2/l at the end of the test to meet the validation criterion. Further, immobilisation in the controls must not exceed 10%, the Daphnia should not be trapped at the water surface and the pH should not vary by more than 1 unit. Typically, for an EC50 determination, 9 test concentrations, plus a control and solvent control if necessary, are used, each having two replicates. For a Limit Test four replicate vessels are set up at the required test concentration. The test is typically conducted under static test conditions, however, for unstable substances the test can be conducted

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Ecotoxicology under semi-static (daily) renewal of the test media or even flow-through conditions. These test organisms are relatively small and a true determination of mortality is not possible. The effect criterion is immobilisation which is defined as ‘those animals which are unable to swim within 15 seconds after gentle agitation of the test container’. Often the animals can still be alive but are unable to swim. In order to confirm absolute death, microscopic examination of the animal is needed to confirm a heart beat and this is considered to be excessive for such an acute test. The number of immobilised Daphnia, found at 24 and 48 hours, is used to calculate the EC50 and 95% confidence limits using an appropriate statistical technique. Computer software packages are available that encompass all the most commonly used methods for this purpose. The NOEC is also determined as the highest test concentration showing no immobilisation or sublethal effects at each time point. The marine equivalent test uses Acartia tonsa as the test species.

4.4.1.3 Acute Fish Tests The toxicity test can be conducted using a variety of test species that are listed in Table 4.4. The requirements for water temperature and size of the fish are different for OECD [22] tests and US OPPTS [23] methods and these are highlighted in Table 4.4. Typically, most acute tests for regulatory compliance are conducted using Rainbow trout. The US EPA often require that tests be conducted on a cold water and a warm water species and here the second species would typically be the Bluegill sunfish or the Fathead minnow. Marine species such as Turbot and Sheepshead minnow (an estuarine species) may also be used. Species such as Guppy and Zebra fish are rarely used, whilst certain regulatory schemes require specific species, for example Killifish are used in various Japanese notification schemes. Another major difference between US OPPTS and OECD/EU methods is the number of fish used in the test. OECD/EU requires 7 to 10 fish per test group whilst US OPPTS requires that 20 fish per group (two replicates of 10 fish each) be used. Thus ethical considerations must be taken into account when planning regulatory testing in order that the minimum numbers of fish are used wherever possible. The test is conducted in a temperature controlled laboratory at the required temperature for the species (see Table 4.4) for a period of 96 hours with a 16 hour light: 8 hour dark light cycle incorporating a 20 minute dawn and dusk transition period. Typically 20 litre test vessels (glass aquaria) are used, but larger vessels may be used to allow for increased volumes for samples for analysis. The test media can be either a reconstituted water, or

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Practical Guide to Chemical Safety Testing

Table 4.4 Summary of requirements for regulatory fish tests Species

Temperature (ºC)

Size (total length of test animal in cm)

OECD/EU

US OPPTS

OECD/EU

US OPPTS

Zebra fish (Brachydanio rerio)

21-25

21-25

2.0+/-1.0

2.0+/-1.0

Fathead minnow (Pimephales promelas)

21-25

21-25

2.0+/-1.0

2.0+/-1.0

Common carp (Cyprinus carpio)

20-24

20-24

3.0+/-1.0

3.0+/-1.0

Japanese Killifish (Oryzias latipes)

21-25

21-25

2.0+/-1.0

2.0+/-1.0

Bluegill sunfish (Lepomis macrochirus)

21-25

20-24

2.0+/-1.0

2.0+/-1.0

Rainbow trout (Onchorhynkus mykiss)

13-17

10-14

5.0+/-1.0

5.0+/-1.0

Guppy (Poecilia reticulata)

21-25

21-25

2.0+/-1.0

2.0+/-1.0

Golden Orfe (Leuciscus idus)

21-25

-

6.0+/-2.0

-

Turbot (Scotpthalmus maximus)

13-15

-

5.0+/-1.0

Sheepshead minnow (Cyrinodon variegatus)

-

20-24

-

2.0+/-1.0

more typically, a domestic tap water which is uncontaminated by harmful concentrations of chlorine, heavy metals, pesticides or other substances. Detailed water quality data should be provided in the test report. Dissolved oxygen, temperature and pH are monitored daily (pre- and post-media change for semi-static tests, daily for flow-through tests). The aquaria are constantly aerated except when testing volatile compounds, when completely filled and sealed test vessels should be used. The fish are acclimatised to the test temperature and water for at least 12 days prior to the test and are fed commercial fish foods which is discontinued 24 hours prior to test initiation. During exposure the animals are not fed. Typically for an LC50 determination, 5 test concentrations, plus a control and solvent control if necessary, are used. For a Limit Test two replicate vessels at the required test concentration are prepared. Usually, the test is conducted under semi-static (daily renewal) test conditions, however, for unstable substances the test can be conducted under flowthrough conditions. Static test conditions can be employed, however the test chemical must be stable in water for a period of 96 hours and the test volume must be sufficient to

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Ecotoxicology dilute any nitrogenous products of the fish excreta, so that these do not have an adverse effect on the test. Mortality and sublethal effects are determined at regular inspections of the exposure vessels. The data is used to calculate the LC50 and 95% confidence limits after 24, 48, 72 and 96 hours exposure. The NOEC at 96 hours is also determined as the highest test concentration showing no mortalities or sublethal effects. Depending on the type of media renewal and chemical stability in water, samples of the test chemical concentration are taken for analysis at regular intervals during the test. Unstable and/or difficult compounds require a greater amount of analytical input. The validation criteria of the test state that mortality in the controls must not exceed 10%, dissolved oxygen concentrations in the controls must be > 60% air saturation value (ASV) throughout the test and that the pH should not vary by more than 1 unit. The OECD has published a test that looks at a more sensitive life stage of fish, in the embryo and fry stage of development. This method, the ‘Fish, short-term toxicity test on embryo and sac-fry stages’ [24], is rarely conducted at low supply volumes of chemicals, but is worthwhile considering at higher tonnages to help refine the assessments of effects.

4.4.2 Analytical Measurements An accurate determination of the test substance concentration in the exposure vessels in the assessment of aquatic toxicology is critical. Many substances are stable in water and provide no problems in the assessment of the effect concentrations however, equally as many are not. Others may hydrolyse, oxidise, photodegrade or biodegrade under the conditions of the test and it is vital that a suitable method of analysis is developed in order to measure the test material concentration and, where possible, the degradation products of the substance. Initial range-finding tests define the concentrations to be used in the main study. At this stage a suitable method of analysis has to be developed. Wherever possible this should be a compound-specific, stability-indicating method. It is essential to establish that there is acceptable linearity of response and recovery from the chosen analytical method. There also has to be stability analysis to confirm that the substance concentration is maintained under test conditions in open and closed vessels over a period equivalent to the period of test media renewal. Once this analytical information is available, a decision can be made on which media renewal system to employ, the frequency and amount of analysis and whether to test the parent substance or its degradation products. Further analysis should be used to determine the exact concentration of test material that is bioavailable and hence causing the toxicity observed. This can often be difficult to determine visually for substances of limited water solubility and requires that the samples are either filtered or centrifuged prior to analysis in order to determine the true concentration of test material that is present in the dissolved form. The test methods

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Practical Guide to Chemical Safety Testing define validation criteria for test compound concentrations in the exposure vessels as being in the range 80% to 120% of the nominal value. In some cases this is not possible, and providing sound scientific reasons can be presented and the results are based on the measured test concentrations in the test, then the data will be acceptable to regulatory bodies.

4.4.3 Difficult Substances Many substances are classed as difficult compounds for a variety of reasons. It may be due to their low aqueous solubility or as a result of hydrolytic instability. Furthermore, complex mixtures and substances that adsorb to organic matter or glassware are also classed as difficult substances. A full review of difficult substances is outside the scope of this chapter and reference should be made to the OECD and EPA Guidance documents [1, 25]. How testing is approached and the amount and complexity of analysis should be dealt with on a case-by-case basis. Poorly water-soluble substances should not be tested above the level of water solubility, but where this is not technically possible to achieve, then stable homogenous dispersions may be tested with analysis of centrifuged or filtered samples to determine the bioavailable test concentrations. It may be necessary to use solvent carriers to aid the dosing or dispersion of poorly water soluble or highly toxic chemicals. In such cases the amount and type of solvents are restricted. Concentrations of solvent up to a maximum of 100 µl/l may be used and the solvent must be of known low toxicity to aquatic life. The use of saturated solutions produced by constant shaking of an excess of test material (removed by filtration or centrifuge) or column generators may be used instead of solvents. Often, for unstable substances, flow-through testing is required. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media may be performed. In cases where the test material is a complex mixture and is poorly-soluble in water and in the permitted auxiliary solvents, an approach endorsed by several important regulatory authorities in the EU and elsewhere [1, 3, 26], is to expose organisms to a water accommodated fraction (WAF) of the test material. Complex mixtures whose individual components are significantly different in physico-chemical characteristics may be tested using a WAF. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work should ensure that sufficient mixing time is given to allow equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by mid-depth siphon and the test organisms exposed to the aqueous phase or WAF. Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period

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Ecotoxicology (loading rate) irrespective of the actual concentration of test material in the WAF. Ideally regulatory advice should be sought before the use of a WAF, as whilst the data generated is suitable for classification and labelling it is not applicable for risk assessment purposes.

4.4.4 Chronic Tests In order to further refine risk assessments and to identify long-term environmental hazard, chronic tests are conducted when chemical production volumes increase. Chronic tests employ lower test concentrations than acute tests and examine more complex end points that can involve reproductive effects. The tests are longer in duration and require that the test organisms are fed. Often flow-through test conditions are essential for animal health and welfare as well as maintaining test substance concentrations. The test concentrations are often based on the results of acute toxicity tests conducted at a previous threshold.

4.4.4.1 Aquatic Plant Tests The standard algal growth bioassay allows some measure of chronic toxicity since by its very nature it measures cell division. Additional end points for aquatic plants can be determined by studying the effect of the test chemical on a higher plant such as Lemna, or duckweed. Here the growth of the test organism, determined by frond (or leaf of a floating plant) increase, is followed in the presence of the test chemical. The test is conducted at 24 ºC under constant illumination (approximately 7000 lux) over a study duration of 7-14 days depending on the protocols followed [27, 28]. Regular media changes and analysis of freshly prepared and old test media ensure that an accurate assessment of the exposure concentration is made.

4.4.4.2 Daphnia Reproduction Tests Following from the results of an acute test, the Daphnia reproduction test investigates the effect on the growth of first instar Daphnia to adult stages and the subsequent effect on the reproduction of these organisms [29]. Five test concentrations plus a control, and solvent control, if necessary, are assigned to the test based on the 48-hour EC50 value from an acute test. Daphnia commence reproduction from approximately 7 days old. Each test concentration has ten replicate test vessels each containing a single daphnid. The test media are replaced every two or three days and the animals are fed with a concentrated algal suspension at a rate of 0.1-0.2 mg carbon/daphnid/day. Exposure conditions are as previously described for the acute test. The numbers of young produced

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Practical Guide to Chemical Safety Testing on a daily basis are removed and counted. An assessment of the size and condition of the parental and young Daphnia is also made to aid in the interpretation of the NOEC. EC50 values for toxicity in the parental generation and also for reproductive effect are determined. Analysis of freshly prepared and old test media ensure that an accurate assessment of the exposure concentration is made.

4.4.4.3 Fish Growth Tests Using the results of an acute toxicity test, the study [30] is designed to follow the growth of rainbow trout over a 28-day period. Test concentrations are selected such that mortality is avoided and that chronic effects are monitored. Fish 1-5 g in weight are used as opposed to the smaller fish in acute tests. The exposure conditions are the same as described for the acute tests except that the fish are held in groups of 16 and are fed a daily ration of commercial fish food at a rate of up to 4% bodyweight per day. Feeding is usually split into two equal portions per day to ensure that all food is eaten. Preferably the test is conducted using flow-through test conditions for fish health reasons as well as maintaining test material concentrations. Uneaten portions of food and fish excreta are removed twice daily so that adsorption of the test material to organic material is reduced to a minimum. The fish are weighed and their length recorded on day 0. On day 14 the fish are removed and anaesthetised prior to weight and length determinations. Fish are then allowed to recover for a short period in clean water before being returned to the exposure period. Food rations are then adjusted to allow for the weight change of the test organisms. The length and weight of the fish is determined again at termination of the test. Regression analysis is used to determine the EC20 (the effective concentration giving a 20% response in the population) and the NOEC is determined by statistical analysis (analysis of variance). Regular analysis of the test media ensure that an accurate assessment of the exposure concentration is made.

4.4.4.4 Fish, Early Life Stage Tests As an alternative to the study of fish growth, and allowing investigation of chronic effects on a more sensitive life stage of fish, OECD has published the ‘Fish, early-life stage toxicity test’ [31]. In this test freshly fertilised eggs are exposed under flow-through test conditions to concentrations of the test material likely not to cause mortality in an acute test. Development (survival and growth) of the eggs and fry are followed up to approximately 35 days post-hatch depending on the species used. Typically, fathead minnows are used in this test as they can easily be reared under laboratory conditions to give a year round supply of freshly fertilised eggs. Once hatched, the fry are fed live food (e.g., the protozoan, Paramecium or brine shrimp) to ensure maximum growth. The test consists of two replicate

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Ecotoxicology vessels for each test concentration, each containing 30 eggs. The remaining exposure conditions are as for the acute tests previously described. At the end of the test, the fry numbers, length and weight are determined and statistical analysis conducted to determine the lowest observed effect concentration (LOEC) and NOEC values. (The NOEC is the highest test concentration at which no effect is seen, whilst the LOEC is the lowest test concentration at which an effect is seen. The NOEC must be less than the LOEC.)

4.5 Fish Bioaccumulation Test For test materials with a high n-octanol:water partition coefficient (i.e., log Pow > 3), if the use pattern data indicate a prolonged aquatic exposure at higher tonnage levels there may be a requirement to investigate the potential of material to accumulate in the food chain at higher tonnage levels. The fish bioaccumulation test [32] should be conducted at concentrations of 1/100th and 1/1000th of the acute LC50 value in the species under investigation. Numerous species may be used but possibly the two most commonly selected are Rainbow trout and Common carp. The study is run for a minimum of 28 days and a maximum of 8 weeks but may be terminated at any point during the exposure period if three consecutive determinations show that the compound has achieved steady state concentrations in the fish tissue. The study is conducted using flow-through conditions. Fish are fed daily at a rate of 2% bodyweight per day. The fish used are larger than used in acute toxicity tests (Rainbow trout = 8 ± 4 cm and Common carp = 5 ± 3 cm) and are maintained at the optimum temperature for growth of that species. Regular analysis of the test media for test concentration is conducted and periodically fish are sacrificed and the test material concentration in either the whole fish or separate portions (head and internal organs and the body muscle or fillet) determined. This data is used to determine the bioconcentration factor (BCF), which is the end point of the test. BCF values of greater than 500 should give cause for concern, whilst a BCF of > 1,000 would indicate that the test material can be considered likely to bioaccumulate in the aquatic environment.

4.6 Sediment Toxicity Tests For substances that exhibit a potential for adsorption to organic matter (a high adsorption coefficient, Koc) the effect of the chemical on sediment dwelling organisms may need to be investigated at higher tonnage levels. Currently OECD has published methods using Chironomus species [33], but other species such as Lumbriculus variegatus [34] may be used. Testing on Lumbriculus is usually requested when the risk assessment has predicted exposure and/or risk to the sediment compartment, and hence information on the effects of the chemical on sediment-dwelling organisms is needed. It is useful to have test results from more than one taxa and the testing of Lumbriculus is usually accompanied by

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Practical Guide to Chemical Safety Testing testing on Chironomus. Lumbriculus will feed primarily from the sediment, whilst Chironomus will feed primarily from the pore-water. Direct exposure through ingestion of the sediment may be important, particularly for chemicals with high log n-octanol:water partition coefficient (Pow) which will tend to partition to the sediment. Exposure of Lumbriculus to the test chemical is therefore different to Chironomus and testing on this species may provide further information on the effect of the test substance on sedimentdwelling organisms. In essence both tests involve the use of artificial sediment spiked with test material and an overlying water column of a pre-defined medium. The test organisms are added and, in the case of the Chironomus test the end point is emergence of the adult flies, whilst for the Lumbriculus test the end point is survival and reproduction (i.e., increase in animal numbers).

4.7 Terrestrial Toxicity Tests As has been indicated for anaerobic degradation and sediment toxicity tests, compounds that have a potential to adsorb to organic matter may have an impact on the terrestrial environment via sewage sludge. Currently sewage sludge solids are disposed of via anaerobic digestion, landfill or application to arable land as a fertiliser. The need to make an assessment of the hazard to terrestrial organisms may therefore be necessary when production tonnages of the chemical meet the higher trigger values.

4.7.1 Earthworms The study of the effect of a test chemical on earthworms is performed in an artificial soil to ensure reproducibility and replication. The test species of choice, Eisenia foetida [35], is not a native soil dwelling organism but is more commonly found in manure piles. This species is chosen in favour of more traditional species due to its relatively short breeding cycle and its ease of breeding under laboratory conditions. The test material is mixed into an artificial soil and the worms added to the surface of the prepared soil. Exposure is maintained for 14 days at 21 ºC under continuous lighting to ensure that the worms remain within the soil. The worms are not fed during the exposure period. The end point of the study is survival and weight change in 40 worms (four replicates of 10 worms) per test concentration.

4.7.2 Bees and Beneficials For industrial chemicals direct exposure to bees and other beneficial organisms (natural predators of insect pests) is unlikely and so discussion of these tests is outside the scope

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Ecotoxicology of this publication. It is of note however that, for pesticides where there is direct exposure to these non-target organisms, these tests are mandatory.

4.7.3 Plant Growth Tests Where sewage sludge is applied to arable land as a fertiliser then the effect on plant growth of compounds adsorbed to the sludge may need to be investigated when tonnage volumes increase. The test as published by OECD [36] uses three species (two monocotyledons and a dicotyledon). The test chemical is mixed with a defined soil and seeds are grown for a known period of time under conditions suitable for each species to germinate and grow shoots. The end point of the test is seedling emergence and shoot weight.

4.8 Microcosm and Mesocosm Studies It is highly unlikely that an industrial chemical would require testing in either a microcosm or mesocosm test. In these tests, environmental conditions are modelled either in a bounded and partially enclosed outdoor experimental unit that closely simulates the natural environment (mesocosm test) or a smaller-sized outdoor pond/tank or small laboratory multispecies test system (microcosm test). Great care is required when designing and conducting these tests which employ multiple species and have a long duration. As with bee and beneficial organism testing, these tests are conducted usually on agrochemicals rather than industrial chemicals.

4.9 Conclusion The study of the ecotoxicological effects of a test chemical can be simple or complex depending on the nature of the test chemical and the test method. Experienced and skilled staff are required to undertake the tests necessary for chemical notification, and the ability to measure the test chemical concentration in the exposure vessels is essential in making a true assessment of the hazard of the chemical. Moreover, the effect of the chemical on the environment should not be judged on the results of a single study, but should be based on the data from different species, taking into account the ability of the chemical to degrade either biologically or chemically and its likely potential to accumulate in the food chain.

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References 1.

OECD, OECD Series on Testing and Assessment Number 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

2.

ISO 10634, Water Quality – Guidance for the Preparation and Treatment of Poorly Water-soluble Organic Compounds for the Subsequent Evaluation of their Biodegradability in an Aqueous Medium, 1996.

3.

ECETOC Monograph No. 26, Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances, 1996.

4.

OECD, Guideline No. 209, Activated Sludge Respiration Inhibition Test, 1984.

5.

J.W. Handley, C. Mead, G.A. Rausina, L.J. Waid, J.C. Gee and S.J. Herron, Chemosphere, 2002, 48, 529.

6.

ECETOC, Technical Report No. 20, Biodegradation Tests for Poorly-soluble Compounds, 1986.

7.

OECD, Guideline No. 301A, Ready Biodegradability – DOC Die-Away Test, 1992.

8.

OECD, Guideline No. 301B, Ready Biodegradability – CO2 Evolution (Modified Sturm Test), 1992.

9.

OECD, Guideline No. 301C, Ready Biodegradability – METI (I) Test (Ministry of Economy, Trade and Industry, Japan, 1992.

10. OECD, Guideline No. 301D, Ready Biodegradability – Closed Bottle Test, 1992. 11. OECD, Guideline No. 301E, Ready Biodegradability – Modified OECD Screening Test, 1992. 12. OECD, Guideline No. 301F, Ready Biodegradability – Manometric Respirometry Test, 1992. 13. OECD, Guideline No. 306, Biodegradability in Sea Water, 1992. 14. OECD, Guideline No. 302A, Inherent Biodegradability Modified SCAS Test, 1981. 15. OECD, Guideline No. 302B, Zahn-Wellens/EMPA Test, 1992.

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Ecotoxicology 16. OECD, Guideline No. 302C, Inherent Biodegradability – Modified MITI Test (II), 1981. 17. OECD, Guideline No. 303, Proposal for Updating Guideline No. 303; Simulation Test – Aerobic Sewage Treatment : 303A Activated Sludge Study – 303B : Biofilms, 2001. 18. ECETOC Technical Report No. 28, Evaluation of Anaerobic Biodegradation, 1988. 19. OECD, Guideline No. 201, Algal, Growth Inhibition Test, 1984. 20. U. Memmert, H. Motschi, J. Inauen and B. Wüthrich, Inhibition of algal growth caused by coloured test substances, RCC Interim Report Project 460302, RCC Ltd., Switzerland, 1994. 21. OECD, Guideline No. 202, Daphnia sp. Acute Immobilisation Test and Reproduction Test, 1984. 22. OECD, Guideline No. 203, Fish, Acute Toxicity Test, 1992. 23. US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances (OPPTS) Ecological Effects Test Guidelines 850.1075, Fish Acute Toxicity Test, Freshwater and Marine, 1996. 24. OECD, Guideline No. 212, Fish, Short-term Toxicity Test on Embryo and SacFry Stages, 1998. 25. US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances (OPPTS), Ecological effects test guidelines 850.1000, Special considerations for conducting aquatic laboratory studies, 1996. 26. M.M. Singer, D. Aurand, G.E. Bragin, J.R. Clark, G.M. Coelho, M.L. Sowby and R.S. Tjeerdema, Marine Pollution Bulletin, 2000, 40, 1007-1016. 27. OECD, Draft Guideline, Lemna Growth Inhibition Test, 2000. 28. US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances (OPPTS), Ecological effects test guidelines 850.4400, Aquatic plant toxicity test using Lemna spp. Tiers I and II, 1996. 29. OECD, Guideline No. 211, Daphnia magna Reproduction Test, 1998. 30. OECD, Guideline No. 215, Fish, Juvenile Growth Test, 2000. 85

Practical Guide to Chemical Safety Testing 31. OECD, Guideline No. 210, Fish, Early-life Stage Toxicity Test, 1992. 32. OECD, Guideline No. 305, Bioconcentration Flow-through Test, 1996. 33. OECD, Draft Guideline No. 219, Sediment-water Chironamid Toxicity Test using Spiked Water, 2001. 34. G.L. Phipps, G.T. Ankley, D.A. Benort and V.R. Mattson, Environmental Toxicology and Chemistry, 1993, 12, 269. 35. OECD, Guideline No. 207, Earthworm, Acute Toxicity Test, 1984. 36. OECD, Guideline No. 208, Terrestrial Plants, Growth Test, 1984.

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5

Physico-Chemical Properties Darren M. Mullee and Karmel P. Biring

5.1 Introduction Physico-chemical testing can be divided into two areas; testing for ‘general physical properties’ and testing for ‘hazardous physical properties’. In this chapter, the regulatory impact and practical aspects of both general and hazardous physico-chemical properties will be considered. It is important to note that the results of the general properties tests do not lead directly to classification and labelling of a substance, but they often affect the choice of further physico-chemical, toxicological and ecotoxicological studies on a substance. This will be discussed individually with each test within this chapter. Furthermore, many of the end points measured are pivotal for environmental fate modelling and occupational exposure assessment, and whilst brief details are discussed here, further discussion can be found in Chapters 7 and 8. In contrast, to the general properties test, the hazardous properties tests do determine the classification of the substance based on the hazardous properties measured. Again, further details will be discussed within this chapter. The OECD Test Guidelines [1] for the determination of general physico-chemical properties are internationally recognised as the standards used for chemical testing purposes. These cover qualifying criteria for acceptability of the results. It is the OECD Minimum Premarketing Data-Set (MPD) [2] which details the core set of general physico-chemical test requirements, as required by regulatory authorities across the world. In particular the OECD Guidelines are encompassed in the OPPTS test methods as used for regulatory purposes in the USA and are closely followed in the EU ‘Annex V’ test methods [3]. Further direct comparisons are found in DIN (German), ASTM (USA), BS (UK) and JIS (Japanese) standards. (Table 11.1 in Chapter 11 gives further details on differences between testing requirements internationally.) For the purposes of this chapter the OECD MPD physicochemical tests will be considered together. Adsorption/desorption, dissociation constant, hydrolysis and particle size distribution will also be considered, as they are usually conducted

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Practical Guide to Chemical Safety Testing in tandem with the other tests described. These studies make up the most commonly conducted studies for regulatory purposes on new chemical substances. There are no OECD guidelines for determination of hazardous properties, and the most widely used methods are the UN Transport Tests [4] which allow a substance to be assigned a particular ‘class’ for transport. It should be noted that in the EU there are methods for determination of hazardous physico-chemical properties but these differ slightly to those of the UN scheme. This often leads to duplicate testing when UN classification and EU registration is required, however, it is usually possible to suggest a UN class from the EU Annex V test results. For the purposes of this chapter the EU Annex V hazardous physico-chemical tests will be considered and comparisons drawn to the UN methods. It is hoped that the present classification systems will, in time, be harmonised as the OECD Global Harmonisation Programme continues. Physico-chemical testing must be conducted according to the principles of Good Laboratory Practice (GLP). It is sometimes the case that chemical companies obtain data in-house and these studies are not conducted to GLP or perhaps do not follow the OECD guidelines. Under most circumstances, regulatory bodies will not accept such non-GLP data, unless they are satisfied that a valid method has been used and that the data are generated via an appropriate quality system. Several jurisdictions around the world now allow use of quantitative structure-activity relationships (QSARs) for estimation of physico-chemical endpoints in place of test data, where appropriate. Examples are the US EPA who have been directly involved in the development of prediction methods, the EU and the OECD existing chemicals programme. Qualitative structure-activity relationship data, often referred to as ‘read-across’ data in the EU, is also now acknowledged as an alternative means of predicting physico-chemical properties. The many different methodologies for prediction of physico-chemical endpoints are not discussed in detail here but there are several papers on the subject that may be of use to the reader [5-8]. Estimation methods for physico-chemical endpoints are sometimes employed and these methods usually make use of thermodynamic and empirical relationships [9]. Such calculations may be used in deciding which of the experimental methods for a test is appropriate where there is more than one method available, or for providing an estimate or limit value in cases where the experimental method cannot be applied for technical reasons. Alternatively, a calculation can help identify cases where omitting experimental measurement is justified and so is a key to obtaining a data waiver for that particular endpoint, especially when used in conjunction with QSAR and read-across. It is especially important where prediction and calculation methods have been used, to ensure that the results shown by the method are consistent with one another and are

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Physico-Chemical Properties reasonable based on chemical structure. The same is true for those endpoints which have been measured directly, as an unexpected result for the particular structure will lead to lack of self-consistency and perhaps negative regulatory impacts based on submission of a potentially deficient study. A final issue to consider is physico-chemical testing for polymers, and methods will be described here. Polymers are generally considered to be a special category of chemical substance, which potentially pose less risk to health and the environment than other substances, due to their inherent inertness and certain physical properties. Nonetheless, under most regulatory schemes some polymer specific physico-chemical data are required in addition to the standard physico-chemical data. Refer to Chapter 12 for more details of the specific requirements for polymer testing worldwide.

5.2 Performance of the General Physico-Chemical Tests The following general physico-chemical tests are listed with the relevant OECD test method and a brief description of the techniques used in their determination. The relevance of the results of each test will highlight how the results are used in regulatory submissions.

5.2.1 Melting Temperature/Melting Range (OECD Test Guideline 102) [10]

5.2.1.1 Definition The melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs. Ideally this temperature also corresponds to the freezing temperature. As the phase transition of many substances takes place over a temperature range, it may be described as the melting range.

5.2.1.2 Methods The method used for determination of melting/freezing temperature depends upon the physical state and composition of a substance at room temperature. The temperature limits are normally in the range of –20 ºC to 360 ºC. In practice the most commonly used techniques, are the differential scanning calorimetry (DSC) method, capillary method with liquid bath, freezing temperature method and pour point method.

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Practical Guide to Chemical Safety Testing The DSC method is a thermal analysis method mainly applied to substances that are solids at room temperature. It measures the energy change during transition from the solid to liquid state. The use of DSC is generally preferred to other techniques as it is quick, very accurate and other useful data can be obtained such as observation of loss of volatile components and assessment of purity. The observation of exothermic reactions may lead to concerns over explosivity and so may act as a useful pre-screen for this property. The capillary method with liquid bath is a traditional technique but the results can be very subjective for some substances. The freezing temperature method generally applies to substances that are liquids at room temperature. The freezing point is taken as the point at which a plateau of constant temperature is obtained following continuous cooling to a temperature of –20 °C. The pour point method was developed for petroleum oils but is relevant for any viscous liquid, especially where other techniques such as freezing temperature are deemed not suitable due to practical reasons. The technique involves heating the sample to well above the point at which it will pour and then cooling. The lowest temperature at which pouring of the substance will occur is the pour point.

5.2.1.3 Relevance of the Results Melting/freezing temperature data is used to define the physical characteristics of a substance. Although a relatively simple test to perform, the result may be pivotal in determining which further physico-chemical tests are required on a substance. If no melting occurs up to 360 ºC then the boiling point and vapour pressure tests are irrelevant on the grounds of non-volatility. Also, the result determines which hazardous physico-chemical tests may be relevant for the material; for example, flammability and autoflammability studies.

5.2.2 Boiling Point (OECD Test Guideline 103) [11]

5.2.2.1 Definition The boiling point is defined as the temperature at which the vapour pressure of a liquid is equal to standard atmospheric pressure (101,325 Pa).

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5.2.2.2 Methods There are many methods available for the determination of the boiling temperature, over the normal range of ambient to 400 °C. The most commonly used techniques are thermal methods such as DSC or differential thermal analysis (DTA), distillation methods and the ‘Siwoloboff’ method. The DSC/DTA method is as previously described for the determination of melting/freezing temperature (Section 5.2.1). Distillation methods are favoured for substances that are complex reaction mixtures containing components with different physical properties. They may also be adapted for substances that undergo decomposition at atmospheric pressure, by performance at reduced pressure. The technique involves distillation using conventional laboratory apparatus and measurement of the vapour re-condensation temperature and the amount of distillate produced. In the Siwoloboff method, the boiling temperature is the temperature at which, on momentary cooling, a string of bubbles stops and liquid material suddenly rises into the capillary of the apparatus.

5.2.2.3 Relevance of the Results Boiling temperature data is used to define the physical characteristics of a substance. The value obtained is taken into account during tests such as vapour pressure, flash point and autoflammability. There should also be a correlation between the boiling point and the vapour pressure result given that the log of vapour pressure is indirectly proportional to the boiling temperature, according to the Clausius-Clapeyron equation.

5.2.3 Vapour Pressure (OECD Test Guideline 104) [12]

5.2.3.1 Definition The vapour pressure of a substance is defined as the saturation pressure above a solid or liquid substance. At the thermodynamic equilibrium, the vapour pressure of a pure substance is a function of temperature only.

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5.2.3.2 Methods The technique used depends upon the physical state of the material and the expected degree of volatility. There is no single measurement procedure applicable to the entire range of possible vapour pressures, however, a variety of methods are available which, when used together, cover the range 105 to < 10-4 Pa. The following three methods can be applied in different vapour pressure ranges and cover the normal range required for regulatory purposes. The Isoteniscope method is used for liquids with relatively high vapour pressures, however, it is not suitable for multicomponent substances. The recommended range covered is 100 to 105 Pa. The technique is simple and involves heating the substance and taking corresponding readings of vapour pressure on a manometer. The vapour pressure at 25 ºC can then be obtained by extrapolation from a calibration curve. The static method is used for solids or liquids and may be suitable for pure and multicomponent substances. The recommended range covered is usually 10 to 105 Pa, but this can be extended to cover the range 1 to 10 Pa. The vapour pressure balance method, which is an effusion method, can be applied to non-volatile liquids or solids. The recommended range covered is 10-3 to 1 Pa, however, in practice lower limits of 10-5 Pa can also be used for completely non-volatile substances. The technique involves heating a sample of accurate mass at various temperatures and determining the mass lost due to volatilisation by use of a highly sensitive microbalance. The required vapour pressure is determined by extrapolation from a calibration curve. Calculation methods may be used where determination of vapour pressure by experiment is impossible, or for determination of the appropriate experimental method to use. Such calculations can make use of the Clausius-Clapeyron equation (see Section 5.2.2.3).

5.2.3.3 Relevance of the Results The vapour pressure gives a direct measure of volatility and thus the choice of test method for other studies can be significantly influenced by the vapour pressure result. The method chosen for the biodegradation test and conduct of ecotoxicology studies may be affected, as steps must be taken to reduce evaporative losses of a material of high volatility. Such losses are also possible during analytical determinations.

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Physico-Chemical Properties The appropriate exposure route for mammalian toxicity testing may be influenced by the vapour pressure. It is reasonable to assume that for volatile substances used in the industrial workplace humans are most likely to be exposed via the inhalation route rather than the oral or dermal route. Thus, the vapour pressure data may be of primary importance in the choice of single dose and repeated dose toxicity studies, which are conducted to investigate the overall local and systemic toxicity of a substance. The vapour pressure result is also used in environmental fate modelling and hence in risk assessment. In particular, Henry’s constant, which is derived from vapour pressure, molecular weight and water solubility, may be used in models for determining the fate of a chemical in the effluent treatment plant.

5.2.4 Water Solubility (OECD Test Guideline 105) [13]

5.2.4.1 Definition The water solubility is defined as the saturation mass concentration of a substance in water at a given temperature and is usually expressed as units of mass per volume of solution (g/l).

5.2.4.2 Methods There are two commonly used techniques, which depend on solubility and physical state. These are the column elution method and the flask method. The choice of method is decided after performing a preliminary estimate of water solubility. The column elution method applies where solubility is less than 10 mg/l and a substance is stable in water for the test duration. Experience has shown that this method is not suitable for liquids, as there are problems with circulation of water through the microcolumn used in the test. Therefore this method is only used for solids. The flask method is applicable to substances with a solubility above 10 mg/l and is suitable for both liquids and solids. In this technique, samples containing excess of a substance are shaken for extended periods to ensure saturation is obtained. For very high solubility substances, neither of the methods may be suitable, as it may be impossible to obtain saturated solutions that can be separated from the excess material. In such cases only a visual estimate of the solubility may be possible.

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Practical Guide to Chemical Safety Testing For either of the above methods for determining solubility it is essential that a suitable analytical method is available, as it will determine the limit of detection and hence the limit of solubility. Full chemical composition data will enable the most appropriate analytical method to be developed and other physico-chemical properties to be assessed. The demands of regulatory bodies have increased over the years and it is advisable that water solubility values should be measured to as low a level as practically possible. Information on vapour pressure, dissociation constant, hydrolysis, partition coefficient, oxidation, photolysis and adsorption properties should be known, where possible, prior to the commencement of the test, since these properties may affect the results. For substances that are not pure, problems can arise from impurities that affect the solubility of the main component. This is especially true for reaction mixtures and polymers. To obtain true water solubility values for each individual component of a mixture is practically impossible and thus a simplistic approach of using total organic carbon (TOC) analysis is usually adopted.

5.2.4.3 Relevance of the Result The water solubility is probably the most important physico-chemical endpoint that is measured and is crucial in deciding on the methodology of other regulatory physicochemical tests, mammalian toxicology tests and ecotoxicology tests. The risk assessment of a substance may also be influenced by the value obtained. With respect to other physico-chemical tests, the water solubility value firstly decides whether or not the surface tension study is performed and, if it is performed, the route of sample preparation. Further, it can be used in predicting a preliminary estimate of partition coefficient. Also, studies such as dissociation constant and abiotic degradation are dependent on the water solubility value as they may not be feasible for substances of low water solubility. In ecotoxicology studies, test concentrations should not exceed the water solubility, as problems with insolubility can give rise to effects due to physical toxicity rather than the true intrinsic toxicity of a substance. For mammalian toxicology the water solubility value can help decide the most suitable vehicle for preparing the formulations used in dosing. High and low water solubility values can present problems when performing environmental risk assessments; in particular, poorly soluble substances will not be released

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Physico-Chemical Properties to the aquatic compartment at the levels expected. Highly soluble substances of indeterminable solubility may require upper limits of solubility to be set such that Henry’s constant can be calculated for use in environmental fate modelling. For environmental classification purposes, poorly soluble substances in general will not exhibit high levels of toxicity to aquatic organisms when compared to soluble substances. Poorly soluble substances may, however, show high Pow values, which will lead to concerns over the potential for bioaccumulation.

5.2.5 Partition Coefficient (OECD Test Guidelines 107 and 117) [14, 15]

5.2.5.1 Definition The partition coefficient is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In OECD tests the solvents used are n-octanol and water. The partition coefficient is usually referred to as Pow or Kow and results of tests are often quoted in the form of a logarithm to base 10 (log Pow).

5.2.5.2 Methods There are two main methods used to obtain Pow values, namely the shake-flask and the high performance liquid chromatography (HPLC) method. The applicability of each method depends upon the chemical composition and properties of the material. In the shake-flask method the partition coefficient is calculated from the ratio of the concentration of the substance found in the two phases used. The method allows the determination of log10 Pow in the approximate range of -2 to 5. It may be possible to extend the range beyond these values, if the detection limits of the analytical procedure are sufficiently sensitive enough. The HPLC method involves use of a series of calibration reference standards of known Pow under pre-defined HPLC parameters. The substance Pow is estimated from a calibration curve of the reference standards where HPLC retention time is relative to hydrophobicity. The method allows the Pow to be estimated in the range log10 Pow from 0 to 6 (see Figure 5.1).

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Figure 5.1 A typical example of a HPLC Pow calibration curve which can be used in the derivation of the Pow of a substance The result of a surface tension study is useful prior to initiation of the test, as this, together with the chemical structure, enables an assessment to be made on the validity of the partition coefficient result. The test methods described in the guidelines are not suitable for surface-active materials. In such cases an estimate by a calculation method is the only valid route to obtain a result. The pH at which the partition coefficient is performed is critical and may have implications for the classification and risk assessment. Ideally, the Pow should be obtained on the nonionised form of a material, however, a Pow value measured in the environmentally significant range (pH 5 to 9) is usually considered appropriate. Salts, zwitterionic and metal complexes should be tested using the shake-flask method. In cases where a substance is an organic salt, ideally both the cation and anion are monitored individually. It is reasonable to assume that the behaviour of the cation and anion will be totally independent following extensive dilution and physical separation in the environment. If a substance contains a mixture of components, the HPLC method should be the preferred choice, as the Pow value can be determined for each component of the mixture. For substances that have low solubility in n-octanol and water the HPLC method is the preferred method. The substance should, however, be soluble in typical reverse phase HPLC solvents at sufficient concentration to allow detection.

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Physico-Chemical Properties For substances prone to hydrolysis, the HPLC method is again preferred as it minimises the amount of contact of the substance with water. Also, the mobile phase consists predominantly of organic solvent, which will suppress the rate of hydrolysis that can occur. In addition, as this method only requires qualitative analysis (peak retention time data of the parent material), any degradation will be irrelevant as test concentrations are not used in determination of the Pow result. Information on the Pow of the hydrolysis products can also be obtained from the same data.

5.2.5.3 Relevance of the Results Where no experimental bioconcentration factor (BCF) has been determined, Pow can be used as an indication of the potential for bioaccumulation in fat in vivo, assuming that biotransformation is low. If the log Pow is greater than a set value, usually 3 or 4, the material is considered to have a potential to accumulate in organisms and the environment and may pose concerns through indirect environmental exposure. This may lead to environmental classification. The partition coefficient is used in prediction of the environmental fate; materials which are lipophilic (of high Pow) will be found in sludge and consequently be present at higher levels within the terrestrial compartment. The adsorption coefficient may be calculated from the Pow where a measured value is not available. Adsorption coefficient is discussed further in Section 5.2.6. The Pow value is of use in estimating dermal absorption of chemicals, in human health risk assessment and is used by the pharmaceutical industry to indicate the best way of introducing a drug to the body. Substances with high Pow values can generally be administered as ointments (partition into the body through the skin). Substances with lower values may be applied orally or intravenously.

5.2.6 Adsorption Coefficient (OECD Test Guidelines 106 and 121) [16, 17]

5.2.6.1 Definition The adsorption coefficient is defined as the ratio between the concentration of the substance in soil/sludge and the concentration of the substance in the aqueous phase at adsorption equilibrium. The adsorption coefficient normalised to the organic carbon content of the soil/sludge is defined as the Koc. The result is often quoted as log Koc.

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5.2.6.2 Methods Methods 106 and 121 differ considerably in study design. Method 121 is an HPLC estimation method only, whereas Method 106 is a batch equilibrium method and provides Koc data on different soil types. Method 121 is procedurally similar to the HPLC method for the determination of partition coefficient (Section 5.2.5.2). The only differences of note are the column type and choice of reference standards. It can provide estimates for substances for which the batch equilibrium method is not possible, such as insoluble, volatile and adsorbing substances, mixtures and substances that are unstable in aqueous media or difficult to quantify at low levels. The method is not recommended for substances that may react with the eluent or the stationary phase, are surface active, inorganic or strong acids and bases. The determination should be performed on a substance in its unionised form and at an environmentally relevant pH. For mixtures, the range and the individual Koc values can be obtained for each component together with an estimate of the proportion of the component present in the mixture. TheHPLC method will allow the Koc to be determined based on the calibration performed (see Figure 5.2). Method 106 involves the determination of Koc values on five different types of soils, each differing with respect to organic carbon content, clay content and pH. It is performed in

Figure 5.2 A typical example of a HPLC Koc calibration curve which can be used in the derivation of the Koc of a substance

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Physico-Chemical Properties three different ‘tiers’ depending upon the regulatory requirements, and information on desorption is also obtained. It is normally the required method for pesticide registration. For this method, a substance specific analytical method is required that is capable of detecting at least two orders of magnitude below that of the original working concentration, which may be problematic for water insoluble materials. Due to the low levels of detection and interference obtained from the soil matrices, mass selective detection techniques are usually required, for example liquid chromatography mass spectrometry (LCMS) or gas chromatography mass spectrometry (GCMS). Whilst this study is usually performed where the HPLC method or a calculation is deemed invalid, other factors, as described in the HPLC method above, make the batch equilibrium method difficult or impossible to perform for some types of substances.

5.2.6.3 Relevance of the Result The value is used in the risk assessment to determine the distribution and ultimate fate of a substance in the environment and may be used in calculation of other solid-water partition coefficients, for example for soil, sediment and suspended matter. Stable, nonvolatile materials with high water solubility and low Koc are likely to remain in the aquatic compartment, whereas poorly soluble materials with a high Koc are likely to be associated with the terrestrial compartment and hence increase concern over toxicity for terrestrial organisms.

5.2.7 Density/Relative Density (OECD Test Guideline 109) [18]

5.2.7.1 Definition The density of a substance is defined as the quotient of its mass and its volume at a specific temperature and is expressed in units of kg/m3. The relative density is defined as the ratio between the mass of a volume of material, determined at 20 ºC, and the mass of the same volume of water, determined at 4 ºC, and has no units.

5.2.7.2 Methods The method used depends upon the physical state of the substance at room temperature.

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Practical Guide to Chemical Safety Testing The two most commonly used techniques are the pycnometer method and the gas comparison pycnometer method. The pycnometer method is generally used for liquids but can be difficult to apply to very viscous liquids. The density is calculated from the difference in mass between the full and empty pycnometer and its known volume. The volume of the pycnometer is determined using water from its known density at the test temperature and the measured mass required to fill the pycnometer. The gas comparison pycnometer method applies to solids in any form. The volume of a known mass of material is measured in an inert gas, within a cylinder of calibrated volume.

5.2.7.3 Relevance of the Results Density data is used to define the physical characteristics of a substance. The data may be of relevance for the choice of fire extinguishers recommended for a substance, as in general water based extinguishers are not recommended for liquids with a density of less than one. The density value is often requested in order to validate ‘read-across’ in the EU.

5.2.8 Particle Size Distribution (OECD Test Guideline 110) [19]

5.2.8.1 Definition The range of the diameters of particles present in a typical sample of a substance is represented by the particle size distribution.

5.2.8.2 Methods No single method is available for determination of particle size. In practice, sieving methods and methods that measure aerodynamic particle size ranges of a solid, glean the most useful results for regulatory purposes. The OECD Guideline focuses mainly on fibres and there is no EU Annex V method for the test, however, guidance for EU notification has been published [20]. The sieving method is a manual technique and whilst the results are variable, it serves as a useful screening test to determine if further particle size testing is justified. Where a significant number of particles pass through a sieve of reasonable pore size (100 µm) then further testing via aerodynamic methods is suggested.

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Physico-Chemical Properties Aerodynamic particle size methods involve dispersal of the substance in air and measurement of a range of particle sizes by weight differences in a series of sieves. The method can be unreliable where particles readily aggregate and the choice of sampling method can affect the result.

5.2.8.3 Relevance of Results The results of the study should determine if a substance is inhalable or respirable in man. A substance is usually considered to be inhalable if significant proportions of the particles are below 100 µm in size and respirable if significant proportions of the particles are below 10 µm in size. This will affect the choice of route of exposure in mammalian toxicity studies as noted for vapour pressure results.

5.2.9 Hydrolysis as a Function of pH (OECD Test Guideline 111) [21]

5.2.9.1 Definition Hydrolysis can be defined as the rate of chemical breakdown of a compound, due to reaction with water. The rate of hydrolysis may be affected by the pH of the water and temperature.

5.2.9.2 Methods The substance is dissolved in aqueous buffer solutions at pH 4, 7 and 9 at a given temperature and the decrease in the concentration with time is followed. The logarithm of the concentration is plotted against time and, if the plot is a straight line, the firstorder rate constant for hydrolysis can be obtained from the slope and half-life calculated. When it is not practical to determine the rate constant and half-life directly at 25 ºC, it is possible to estimate it from the use of the Arrhenius relationship. This relationship makes use of the temperature dependence of the rate constant, such that from a plot of rate constant against temperature an extrapolation to the rate constant at 25 ºC can be made (Figure 5.3). A preliminary screening test is conducted in advance of the main test to determine if the substance has a half-life of less than one day or greater than one year. If the substance meets either of these criteria it is considered that no further testing is required, as it is considered that no further relevant stability information will be gleaned.

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Figure 5.3 A typical example of the graph generated in the estimation of rate constant at room temperature when making use of the Arrhenius relationship

The test is not applicable to substances that are readily biodegradable, insoluble in water or if a suitable analytical technique of sufficient accuracy cannot be developed. In addition, it is usually only used for single component substances rather than mixtures. The water solubility and biodegradability should be ascertained prior to the test being performed. In addition, information relating to oxidation, photolysis, vapour pressure and biodegradation are very useful to allow for adequate precautions to be taken to prevent non-hydrolytic losses of material.

5.2.9.3 Relevance of Results The result of the hydrolysis study will determine the persistence of a substance in the environment, where biodegradability is low. The method of preparation of the test solutions for use in ecotoxicology studies can be dependent upon the rate of hydrolysis. The hydrolysis result will influence the choice of static, semi-static or flow-through procedures which are used to maintain test concentrations at the required level or, will determine if the tests should be performed on either the parent substance or its degradation products, or a combination of both.

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Physico-Chemical Properties The use of water as a vehicle for dosing in toxicology tests, and the choice of test methods and interpretation of the results of the water solubility, surface tension, partition coefficient and adsorption coefficient tests should be questioned if the material is shown to hydrolyse. It may be that the results of these tests reflect the properties of the degradants rather than those of the parent substance. Where tests have been conducted, degradants may have to be identified, or at least predicted. Environmental fate and risk assessments are likely to be relevant for the degradants rather than the parent substance for rapidly hydrolysable substances, as discharge to water will lead to exposure to the degradants only. The rate of hydrolysis under physiologically relevant conditions and the identity of the degradants will impact prediction of toxicokinetic behaviour of the substance when ingested.

5.2.10 Dissociation Constant (OECD Test Guideline 112) [22]

5.2.10.1 Definition The dissociation constant is the constant that controls the reversible separation of a substance into two or more chemical products and is usually represented by Ka or pKa.

5.2.10.2 Methods There are three methods used to determine the dissociation constant of a substance; the titration method, spectrophotometric method and conductometric method. The nature and properties of the substance being tested determine the actual method used. The titration method involves titrating the test solution with an appropriate acid or base, measuring the pH after each addition. At least 10 incremental additions should be made before the equivalence point. A plot of pH versus volume of acid or base is obtained and the pKa calculated for the 10 measured points on the curve. The method is not suitable for substances that have low aqueous solubility. Spectrophotometric methods involve finding a suitable wavelength where the ionised and unionised forms of the substance have appreciably different extinction coefficients. The UV/VIS absorption spectrum is then obtained from a range of test solutions at various pHs where the substance is fully ionised and unionised and at several intermediate points. The pKa is then calculated using data from at least 5 pHs where the compound is at least 10% and less than 90% ionised. 103

Practical Guide to Chemical Safety Testing The method is only applicable to substances having appreciably different absorption spectra for the ionised and unionised forms. However, it may be used for low solubility substances and for non-acid/base dissociations. The conductometric method involves measuring the conductivities of a number of serial dilutions, with the concentrations being halved each time. The degree of dissociation is then calculated from the conductivity using the Onsager equation. In cases where the Onsager equation holds, the conductometric method may be used, even at moderately low concentrations and also for non-acid/base equilibria. For all three methods, the test is essentially only applicable to pure materials and the concentrations of substance used should not exceed 0.01M or half the water solubility value.

5.2.10.3 Relevance of Results The test is not applicable to water insoluble substances or where a suitable analytical method cannot be developed. Where dissociation will occur outside the environmentally relevant pH or there is no potential for dissociation, it may be that the test can be omitted, as no meaningful results will be obtained. Each component of a complex reaction mixture will theoretically have its own dissociation constant, so again the test may be omitted as irrelevant as a range of values will be obtained. The results of the test are used to determine how many of the other physico-chemical tests involving water are carried out. Physico-chemical tests should be conducted on non-dissociated forms of a substance where this is possible, within the environmentally relevant range (usually pH 5 to 9). The dissociation constant is used in determination of environmental fate. Where dissociation of a substance can occur at environmentally relevant pHs then the fate of the species produced by dissociation will be important.

5.2.11 Surface Tension (OECD Test Guideline 115) [23]

5.2.11.1 Definition The surface tension is defined as the free surface enthalpy per unit of surface area, at a given temperature and sample concentration. Values obtained are usually within the range of 30 to 72 mN/m, where 72 mN/m is the surface tension of pure water.

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5.2.11.2 Methods Although there are various methods available, the most commonly used by far, is the ring method. This method measures the minimum force required to separate a ring from the surface of a solution of the substance being examined. The maximum concentration used in the test is based on the water solubility of the material. For materials with a high water solubility the maximum concentration used should be 1 g/l, for other materials a 90% saturated solution is used. The test is, however, irrelevant for materials with a solubility of less than 1 mg/l, as only the surface tension of water will be measured. Information on the hydrolytic stability is useful prior to conducting the test. This allows an assessment of whether the result obtained is that of the parent substance, degradation products or a mixture of the two. The surface tension can be significantly affected by contamination from trace levels of various materials. For example, detergents, used in the cleaning of the associated glassware, vapours and fumes will interfere with the results. Thus, the instrument is always calibrated during the actual determination.

5.2.11.3 Relevance of the Result The value determines if a substance has surface-active properties, which will result in a lowering of the surface tension of water. A substance is usually considered surface active if the surface tension result is determined to be less than 60 mN/m. This can have implications for other physico-chemical tests such as partition coefficient and adsorption coefficient, which may be significantly affected by surfactant substances. Misleading results are sometimes obtained. For example where the structure is not that of a typical surfactant, the substance may not be a true surfactant. It is also advisable to ascertain if emulsification actually occurs when a solution of a substance is shaken with n-octanol and water. If the phases do not separate and an emulsion is formed this is good evidence to suggest that the substance exhibits surface-active properties. Generally speaking, true surfactants have typical surface tension results of less than 45 mN/m. Also, for substances with a low surface tension, there may be a suspicion that they are potentially bioaccumulative. A compound with a high surface activity may be able to pass through a membrane more easily than a low surface activity compound and so bioaccumulate. This could be due to disruption of active transfer systems, etc.

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5.2.12 Fat Solubility (OECD Test Guideline 116) [24]

5.2.12.1 Definition The fat solubility of a substance is defined as the saturation mass fraction of a substance which forms a homogenous phase with liquid fat without giving rise to chemical reactions, at a given temperature, normally 37 °C (body temperature).

5.2.12.2 Methods The method is only applicable to pure substances that are stable at 50 °C for at least 24 hours and do not have an appreciable vapour pressure at 50 °C. For substances that react with fat or for which a suitable analytical technique is not available, the method is not applicable. The basis of the method is to dissolve the substance in a standard liquefied fat by stirring, and continually adding further amounts of the substance until saturation is achieved. The test is conducted at 37 °C.

5.2.12.3 Relevance of Results The test is often conducted on substances for which a measurement of partition coefficient is not possible due to low water solubility, to give a measure of the potential for storage of the substance within fatty tissues in vivo. Therefore, the comments on relevance of partition coefficient (Section 5.2.5.3) are also relevant to the fat solubility, although migration of a substance into foodstuffs may also be indicated by the result.

5.3 Performance of the Polymer Specific Physico-Chemical Tests There are specific tests that are applicable to polymer substances, the main methods for which are discussed here. For details of the regulatory requirements for polymers refer to Chapter 12. The tests routinely performed for polymers are the number-average molecular weight and molecular weight distribution studies, and solution/extraction behaviour in water. These tests, together with information on the monomers, impurities, end-groups, reactive functionalities and biodegradability, are the starting point for hazard assessment of such

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Physico-Chemical Properties substances. It may be that the hazard assessment indicates that further polymer tests are required such as light stability and long-term extractivity testing.

5.3.1 Number-Average Molecular Weight and Molecular Weight Distribution of Polymers (OECD Test Guideline 118) [25] 5.3.1.1 Definition Polymers display a distribution of molecular weights. The number-average molecular weight (Mn) is the total weight of all the polymer molecules in a sample divided by the total number of polymer molecules in a sample.

5.3.1.2 Methods Gel permeation chromatography (GPC) is the preferred method for the determination of Mn, especially when a set of standards is available with structures comparable to the polymer structure. The technique involves a special type of liquid chromatography in which the substance is separated according to hydrodynamic volumes of the individual constituents. Separation is achieved by passing the substance through a column, which is filled with a porous gel based stationary phase. Detection by refractive index yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column with polymer standards of known molecular weight. These are usually polystyrene standards, but other standards are available such as polyethylene glycol, polymethyl methacrylate and polyacrylic acid. The Mn and molecular weight distribution are then calculated from the calibration data. Other methods are available to determine the Mn if GPC is not appropriate. Such methods make use of the colligative properties and end-group analysis of the polymer.

5.3.1.3 Relevance of the Results The test determines if a substance meets the criteria for definition as a polymer under the relevant regulatory scheme. It can be assumed that lower molecular weight polymers will be more bioavailable than those of higher molecular weight due to higher solubility and mobility. The content of species with a low Mn should, however, also be taken into account, together with solution/extraction behaviour as described below.

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5.3.2 Solution/Extraction Behaviour of Polymers in Water (OECD Test Guideline 120) [26]

5.3.2.1 Objective To quantify the total amount of the polymer that is extractable with water.

5.3.2.2 Methods The solution/extraction behaviour of polymers in an aqueous medium is determined based on the flask method as used for the water solubility method (Section 5.2.4.2), with slight modifications to the sample amounts, volumes taken and times shaken. The concentration of the polymer dissolved is determined by a suitable analytical technique such as GPC. However, if a suitable analytical technique is not available the total solubility/extractivity can be estimated by simple gravimetric techniques. It may be that water extractivity and extractivity with water at pHs 2 and 9 at 37 °C and extractivity with cyclohexane are required, in addition to a determination at neutral pH.

5.3.2.3 Relevance of the Results The test provides information on how the polymer behaves in solution under the relevant physiological and environmental conditions. Where leachates are formed a long-term extractivity test may be required to identify if they are present in significant quantities and hence if further toxicity testing is appropriate.

5.4 Performance of the Hazardous Physico-Chemical Tests The following tests are listed by EC test method, and contain a brief description of the techniques used in their determination. Again, the relevance of the results of each test is included in order to highlight how the results are used.

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5.4.1 Flash Point (EC Method A9) [27]

5.4.1.1 Definition The flash point is the lowest temperature, corrected to an atmospheric pressure of 101,325 Pa, at which a liquid evolves vapours in such an amount that a flammable vapour/air mixture is produced.

5.4.1.2 Methods An equilibrium method such as the closed cup method is most commonly used. The substance is injected into the apparatus and heated according to the test procedure. Ignition trials are carried out in order to ascertain whether or not the sample flashes at the test temperature. The test is applicable to substances that are liquids at room temperature. However, the test can be modified to accommodate low melting solids such as waxes or pastes where the alternative flammability tests are not practical.

5.4.1.3 Relevance of the Results The result is usually found in safety data sheets as an indication of fire hazard. For regulatory purposes the data is used, in conjunction with other tests such as boiling point and combustibility, to assess which class of danger or hazard label applies for flammable liquids. Note that a positive result in a test conducted to the EU method may not allow full classification under the UN Transport scheme.

5.4.2 Flammable Solids (EC Method A10) [28]

5.4.2.1 Definition Flammable solids are substances, which are easily ignited following brief contact with an ignition source, and if the resultant flame spreads rapidly.

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5.4.2.2 Methods The most common test used involves forming an unbroken strip or powder train of material to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation occurs within a specified time then the actual burning rate is measured in order to determine the degree of flammability. The standard test may not be applicable for low melting point solids, for paste type materials, and difficulties may be encountered with hygroscopic materials.

5.4.2.3 Relevance of the Results The data will always be required on test material data sheets for fire hazard. Classification of substances as flammable can result directly from the outcome of the test. Powders of metals or metal alloys may be considered to be highly flammable when they can be ignited and the flame or zone of reaction spreads over the whole sample in 10 minutes or less. Note that a positive result in a test conducted to the EU method will not allow full classification under the UN Transport scheme. Under the UN scheme, a further test is required to determine if the substance burns through a wetted zone. In addition a positive result in the flammability test, leading to classification under the EU scheme will remove the need to perform a test for oxidising potential, although this may not be true elsewhere.

5.4.3 Flammable Gases (EC Method A11), Flammable Substances on Contact with Water (EC Method A12) and Substances Liable to Spontaneous Combustion (EC Method A13) [29-31]

5.4.3.1 Definitions Flammable gases are those that are ignitable in air when in a given volume within air. On contact with water, flammable substances are liable to become spontaneously flammable or to give off flammable gases in dangerous quantities when they interact with water. Substances liable to spontaneous combustion are likely to catch fire following self-heating in contact with air.

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5.4.3.2 Methods Methods are not discussed here as these tests are rarely performed for regulatory purposes as they can usually be predicted negative on the basis of the chemical composition and structure of a substance, and experience of handling in use.

5.4.3.3 Relevance of the Results Any positive result will lead to a flammability classification. In addition, a positive result in a pyrophoric test usually removes the need for further flammability tests and would make any other tests extremely difficult to perform. For substances classed as flammable in contact with water, any further water-based tests are not possible, however, the corresponding toxicology and ecotoxicology tests may be possible on the reaction products.

5.4.4 Explosive Properties (EC Method A14) [32]

5.4.4.1 Definition Solid or liquid substances have explosive properties if they are capable of producing gases by chemical reaction at such a rate that they cause damage to the surroundings.

5.4.4.2 Method Prior to testing, information on the chemical composition is very useful as this can allow theoretical assessment on the potential for explosivity to be made. Such theoretical assessments can include: •

calculation of oxygen balance (stoichiometric composition),

•

a screen for the presence of explosophores (bond groupings known to confer explosivity) and auxoploses (explosion enhancing groups) and

•

measurement of exothermic decomposition energy from DSC determinations.

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Practical Guide to Chemical Safety Testing Where the results of these preliminary assessments show no possible explosivity potential for the substance no further testing may be necessary. Where testing is required, the method investigates whether a substance will present a danger of explosion due to heat sensitivity (flame test), mechanical sensitivity (shock test) and mechanical sensitivity (friction test).

5.4.4.3 Relevance of the Results Any positive result will lead to an explosivity classification. For EU classification purposes a positive result in the explosivity test removes the need for any other flammability or oxidising tests to be performed. The EU method is now partially harmonised with the UN method. However, additional testing is required in most cases for definitive UN classification.

5.4.5 Auto-ignition Temperature, Liquids and Gases (EC Method A15) [33] and Relative Self–ignition Temperature, Solids (EC Method A16) [34]

5.4.5.1 Definition The auto-ignition or self-ignition temperature is the temperature at which the rate of heat production from reaction of a substance with oxygen in the air exceeds the rate of heat loss to the surroundings. At this temperature, spontaneous combustion will occur.

5.4.5.2 Method For a liquid or gas, the standard test is conducted by injecting a known volume of material into a heated open neck flask. The contents are observed until ignition occurs or for a period of five minutes. The lowest temperature at which ignition occurs is taken to be the auto-ignition temperature. For a solid, a cube of fine wire stainless steel mesh of set dimensions is filled with the substance and suspended in the centre of an oven at room temperature. The temperature of the oven and sample are continuously recorded while the temperature of the oven is increased. When the material ignites, the sample thermocouple will show a very sharp temperature rise above that of the oven temperature.

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5.4.5.3 Relevance of Result The result is used as a physical property of the material that is included on test material data sheets. The result is also taken into account in risk assessment where it will warn of potential thermal hazards in manufacture, storage and use. The tests are run in conjunction with pyrophoric testing under the UN transport scheme and positive results can result in classification under the division of substances that are liable to spontaneous combustion.

5.4.6 Oxidising Properties (EC Method A17) [35]

5.4.6.1 Definition Substances which can cause or contribute to the combustion of other substances by exothermic reaction, whilst not necessarily combustible themselves.

5.4.6.2 Method Prior to starting testing, information on the chemical composition is very useful as this can allow theoretical assessment of the potential for oxidation to occur. Such an assessment should be based on the groups known to be oxidisers, such as nitrates, chlorates, bromates and perchlorates. Peroxides in particular are classified as oxidising without testing. Flammable and explosive substances should not be tested as the results will be meaningless. The study is performed by preparing mixtures of the substance and a standard combustible material (cellulose) in varying proportions. The mixtures are placed into a mould and ignited at one end. The time of burning over a set distance after the reaction zone has propagated an initial distance is recorded. The test is also performed with reference standard mixtures and the reaction times are compared in order to determine the degree of oxidation. Problems can occur with low melting solids that can lead to a false positive result being obtained. This is caused by the ‘wick-effect’ of the substance burning on the cellulose. In these circumstances the test is repeated with an inert substance such as kieselguhr, in place of the cellulose. This will confirm whether the reaction is a ‘true result’ or a ‘falsepositive’, since kieselguhr cannot be oxidised.

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5.4.6.3 Relevance of the Result Classification of a substance as oxidising for both EU and UN purposes will be based firstly on whether the mixture of substance and cellulose ignites and secondly on comparison of burning times with the reference standard mixtures used. Note that currently there is no EU guideline for testing the oxidising properties of liquids, however, Method A21 for liquids has recently been drafted.

5.5 Order in which Physico-Chemical Tests are Performed Where a full set of physico-chemical tests are undertaken, it is prudent to perform these in a prescribed order. Firstly, the safety of the scientist conducting the tests is paramount and secondly, the impact of results on the conduct of other tests should be considered. From experience the order described below is appropriate. The pyrophoric properties of the substance are determined first. Whilst this test is usually predicted as negative, a positive result would remove the need for further flammability tests and would make the majority of the other tests impossible to perform. A similar situation would be true following a positive result in a flammability (contact with water) test. This test is also usually predicted as negative, however, a positive result would render the conduct of any further water-based tests impossible. Hydrolysis and water solubility tests are dependent on each other and therefore are usually run simultaneously, followed by the dissociation constant test. It may be more appropriate to have the details of the water solubility value first, as the value is required to set the initial concentrations for the hydrolysis test. Surface tension, partition coefficient and adsorption coefficient tests take into account information on hydrolysis, water solubility and dissociation. If results of the surface tension study show potential for surface activity then the partition coefficient and adsorption coefficient studies may be invalid. Explosivity is usually predicted as negative but if a positive result is obtained then this usually negates the need for any further hazardous properties testing. Flammability and autoflammability tests can be performed in any order, although the physical form of a substance must be known to select the most appropriate test method. Oxidising potential can usually be predicted as negative from structure and is not usually performed on flammable or explosive substances.

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Physico-Chemical Properties The remaining physico-chemical tests are usually performed at any point given that they do not influence any of the others. However information related to particle size and vapour pressure is essential to decide relevant exposure routes in the mammalian toxicity tests.

5.6 Conclusion The determination of the physico-chemical properties of a substance are pivotal to the whole process of a regulatory submission. The endpoints measured dictate the test methods used, not only for other physico-chemical studies, but also in ecotoxicology and toxicology studies. The values obtained are used directly in assessment of environmental fate and the results serve to communicate chemical hazard and health and safety information in summary form. It is therefore essential that physico-chemical properties testing is performed both correctly and as accurately as possible, such that interpretation of the results will be scientifically valid.

References 1.

OECD, Guidelines for the Testing of Chemicals, OECD, Paris, France, 1993.

2.

OECD, Decision of the Council concerning the Minimum Pre-Marketing Set of Data in the Assessment of Chemicals, C(82)196/Final, 1982.

3.

Anon., 2000, Annex V of council Directive 67/548/EEC, as adopted to technical progress by Commission Directive 92/69/EEC of 31-7-92, Official Journal of the European Communities L383A. 29-12-92, Commission Directive 96/54/EC of 30-7-96, Official Journal of the European Communities L241, 30-9-96, Commission Directive 98/73/EC of 18-9-98, Official Journal of the European Communities L305, 16-11-98, Commission Directive 2000/32/EC of 19-5-00, Official Journal of the European Communities L136, 8-6-00 and Commission Directive 2000/33/EC of 25-4-00, Official Journal of the European Communities L136, 8-6-00.

4.

Recommendations on the Transport of Dangerous Goods (Model Regulations), United Nations, Geneva, Switzerland, 2001.

5.

P. Fisk, Inveresk Research Regulatory Affairs Bulletin, 2000, Number 81, 27.

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P.H. Howard and W.M. Meylan in Quantitative Structure-Activity Relationships in Environmental Sciences – VII, Eds., F. Chen and G. Schüürmann, SETAC Press, USA, Chapter 13, 1997.

7.

ECETOC, Technical Report No. 74, QSARs in the Assessment of the Environmental Fate and Effects of Chemicals, ECETOC, Brussels, Belgium, 1998.

8.

D.J. Knight, Speciality Chemicals Magazine, 2000, 20, Part 1, p.15 and Part 2, p.70.

9.

W.J. Lyman, W.F. Reehl and D.H. Rosenblatt, Handbook of Chemical Property Estimation Methods, McGraw-Hill, 1982.

10. OECD Guideline No. 102, Melting Point/Melting Range, 1995. 11. OECD Guideline No. 103, Boiling Point, 1995. 12. OECD Guideline No. 104, Vapour Pressure, 1995. 13. OECD Guideline No. 105, Water Solubility, 1995. 14. OECD Guideline No. 107, Partition Coefficient (n-octanol/water): Shake Flask Method, 1995. 15. OECD Guideline No. 117, Partition Coefficient (n-octanol/water), HPLC Method, 1989. 16. OECD Guideline No. 106, Adsorption – Desorption Using a Batch Equilibrium Method, 2000. 17. OECD Guideline No. 121, Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC), 2001. 18. OECD Guideline No. 109, Density of Liquids and Solids, 1995. 19. OECD Guideline No. 110, Particle Size Distribution/Fibre Length and Diameter Distributions, 1981. 20. Particle Size Distribution, Fibre Length and Diameter Distribution, European Commission Technical Guidance Document, Number 9, June 1996. 21. OECD Guideline No. 111, Hydrolysis as a Function of pH, 1981.

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Physico-Chemical Properties 22. OECD Guideline No. 112, Dissociation Constants in Water, 1981. 23. OECD Guideline No. 115, Surface Tension of Aqueous Solutions, 1995. 24. OECD Guideline No. 116, Fat Solubility of Solid and Liquid Substances, 1981. 25. OECD Guideline No. 118, Number-Average Molecular Weight and Molecular Weight Distribution of Polymers, 1996. 26. OECD Guideline No. 120, Solution/Extraction Behaviour of Polymers in Water, 2000. 27. EU, Method A9 of Commission Directive 92/69/EEC, Flash-Point, 1992. 28. EU, Method A10 of Commission Directive 92/69/EEC, Flammability (Solids), 1992. 29. EU, Method A11 of Commission Directive 92/69/EEC, Flammability (Gases), 1992. 30. EU, Method A12 of Commission Directive 92/69/EEC, Flammability (Contact with Water), 1992. 31. EU, Method A13 of Commission Directive 92/69/EEC, Pyrophoric Properties of Solids and Liquids, 1992. 32. EU, Method A14 of Commission Directive 92/69/EEC, Explosive Properties, 1992. 33. EU, Method A15 of Commission Directive 92/69/EEC, Auto-Ignition Temperature (Liquids and Gases), 1992. 34. EU, Method A16 of Commission Directive 92/69/EEC, Relative Self-Ignition Temperature for Solids, 1992. 35. EU Method A17 of Commission Directive 92/69/EEC, Oxidizing Properties (Solids), 1992.

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6

Alternatives to Animal Testing for Safety Evaluation Derek J. Knight and Damien Breheny

6.1 Introduction The toxicological properties of products ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals, including toiletries and cosmetics, have to be determined to make sure they are adequately safe (see Chapter 7). Such investigation of their toxicological properties has mainly been in experimental animals (see Chapter 2). The aim is to predict adverse effects in humans from these animal models. To provide safe products is undoubtedly of the utmost importance, but this aim has been brought into conflict with strong public opinion, especially in Europe, against animal testing. Hence, industry, academia and regulators have been working in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals and the severity of the tests using them. There is a long way to go before all the potential hazardous properties can be evaluated without any animal studies, but considerable progress has been made using a combination of in vitro (i.e., nonanimal) tests and prediction of properties based on chemical structure. The aim of this chapter is to describe these important and worthwhile developments in various areas of toxicology testing, with a focus on the European regulatory framework for general industrial and household chemicals. For a more comprehensive discussion of the areas covered in this chapter, the reader is referred to reviews by Knight and Breheny [1] and Fielder et al. [2]. In their 1959 book, ‘The Principles of Humane Experimental Technique’ [3], Russell and Burch defined a strategy for minimising animal use without compromising the quality of scientific work. This was to be achieved through the use of three different categories of alternative testing, which they termed the ‘Three Rs’: reduction, refinement and replacement (see Table 6.1). This approach has become a benchmark. Alternative tests are used in different contexts, from eliminating substances from further development on the basis of their potential toxicity to regulatory safety evaluation. This issue is further elaborated by the British Toxicology Society Working Group on in vitro toxicology in their report on the value of currently available in vitro studies in all aspects

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Table 6.1 Russell and Burch's ‘Three Rs’ as principles to reduce animal testing 1.

Refinement alternatives Methods that alleviate or minimise potential pain, suffering and distress, and which enhance animal well-being.

2.

Reduction alternatives Methods for obtaining comparable levels of information, or more, from the same number of animals.

3.

Replacement alternatives Methods that do not involve conducting experiments on whole living animals. Some of these approaches are only relative replacements, because they entail the humane killing of an animal to obtain cells, tissues or organs for in vitro studies. Others are absolute replacements that do not require any biological material from a fully developed, vertebrate animal.

of safety evaluation, which also made recommendations for future work [2]. There are great incentives to develop in vitro alternatives to supplement and even replace the existing animal toxicology methods. Firstly, safety assessment can be improved by better prediction of effects in humans, as the scientific understanding of the toxicological effect is improved. There are also commercial benefits in reducing the cost and increasing the speed of product development. The key consideration, though, is animal welfare (the ‘Three Rs’), underpinned in the EU by Council Directive 86/609/EEC [4], which requires that animal experiments should not be performed if another scientifically satisfactory way of obtaining the results sought is reasonably and practically available.

6.2 Validation of Alternative Methods Alternative tests, including in vitro tests, have to be validated scientifically and established as acceptable by industry and the regulators before being used in safety assessment. There are a number of international bodies which undertake this validation process (see Table 6.2). It is particularly important to obtain international agreement on the principles of validation, and the Organisation for Economic Co-operation and Development (OECD) have reported on recommendations for harmonizing the scientific validation and subsequent regulatory acceptance criteria for alternative test methods [6]. They define various key concepts (see Table 6.3). Fielder and co-workers [2] consider there is a

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ECVAM IVTIP ICCVAM Alternatives to Animal Testing for Safety Evaluation

Table 6.2 Organisations for validating alternative methods European Centre for the Validation of Alternative Methods (ECVAM) The European Commission strongly promotes the ‘Three Rs’ and undertakes research in this area. ECVAM was set up in 1991, to implement Article 23 of Council Directive 86/609/EEC on the protection of animals for use in experimental and other scientific procedures [4]. It is located at the Joint Research Centre (JRC) at Ispra in Italy. Its main aim is to promote the regulatory acceptance of alternative methods that comply with the ‘Three R’s’, while still providing useful data and information for the bioscience field. As part of its work, ECVAM holds workshops, and reports the recommendations of these [5]. Industrial Platform on In Vitro Testing (IVTIP) The IVTIP was set up in 1993 by European companies with activities in the pharmaceutical, chemical and cosmetic sectors with input from the European Commission. Its main objective is to maximise technology transfer from academia to industry. The US Interagency Co-ordinating Committee on The Validation of Alternative Methods (ICCVAM) ICCVAM is the US organisation comparable to the European ECVAM. ICCVAM undertakes scientific peer review on the validation of alternative test methods. The various US agencies then decide whether to adopt the methods validated by ICCVAM for their regulatory purposes. The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) also plays a leading role in the US validation process. Until non-animal test methods are validated and achieve regulatory acceptance, these methods cannot be relied on as alternatives to established test guideline studies for purposes of the High Production Volume (HPV) Challenge Program or any associated Test Rules. The Environmental Protection Agency (EPA) is a member of ICCVAM, however, and is working with other Federal agencies to identify, validate, and peer review potential alternative protocols, and to ensure the scientific and regulatory acceptability of the tests.

Table 6.3 Definitions of concepts for validation of alternative methods Validation: the process by which the reliability and the relevance of a procedure are established for a particular purpose. Regulatory acceptance: the process whereby a given test is considered suitable for risk assessment purposes aimed at the protection of human health and/or the environment. Reliability: the reproducibility of results from an assay within and between laboratories. Relevance: describing whether a test is meaningful and useful for a particular purpose.

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Practical Guide to Chemical Safety Testing pragmatic division between two types of validation, depending on the use of the studies. The first is validation of alternative procedures for use in non-regulatory studies or for mechanistic studies, perhaps to refine the safety evaluation, and normally require only ‘internal’ validation to ensure that the laboratory or company involved has confidence in the results. This type of informal validation may be often based on ‘in-house’ data on chemicals of a similar class, or products of similar formulation. In contrast, studies to be used as alternatives to existing test guidelines for routine safety evaluation require more extensive formal validation. Convincing evidence must be obtained from published validation studies (usually undertaken internationally) to support the wider, worldwide acceptance of the alternative method. Regulatory acceptance of new alternative methods follows the scientific validation process, and is in practice greatly facilitated if the regulatory authorities are involved in the validation process and design of the validation studies. ECVAM (see Table 6.2) has an independent ECVAM Scientific Advisory Committee (ESAC), who consider the outcome of a successful validation exercise. When ESAC publicly endorses a method as scientifically validated, ECVAM communicates this within the European Commission and to the US ICCVAM and other agencies, including the national Competent Authorities of the EU countries. The various services of the European Commission then consider the applicability of the scientifically-validated method in relation to their specific responsibilities, in consultation with their own advisers and the national Competent Authorities. Meanwhile, international discussions can lead to harmonization of the new test method as an OECD guideline.

6.3 Aspects of Human Toxicity Targeted By In Vitro Assays Due to the extreme complexity of the whole body system, it is impossible to model all the possible adverse responses to a product using only in vitro tests. Current tests are largely only capable of simulating one or two particular aspects of a toxicological response (e.g., individual steps in a mechanism of toxicity). A single in vitro test cannot serve as a complete replacement for most animal studies. Instead, a group of different in vitro assays (the ‘test battery’ approach) will provide the most accurate and comprehensive prediction of the toxic potential of a test material in man.

6.3.1 Systemic Toxicological Properties The status of alternative tests to evaluate the systemic toxicological properties of chemicals is summarised briefly in this section, as background information and to put into context the subsequent discussion in Section 6.3.2 of alternative tests for local toxicity, which are further along the line for regulatory acceptance.

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6.3.1.1 Mutagenicity and Carcinogenicity Genetic toxicology is covered in detail in Chapter 3. Many carcinogens are mutagenic (i.e., cause damage to DNA). Screening assays which determine if the test compound can cause mutations in vitro are accepted by regulatory authorities. An example of such a test is the classic Ames test, which utilises a strain of Salmonella bacterium. However, studies in animals are required to confirm if this is expressed as carcinogenicity in vivo. The major current concern is that many carcinogens are not mutagenic, and so cannot be detected by these tests. There are many mechanisms involved in these multi-stage processes, which makes the development of these tests quite difficult. Cell transformation assays, such as the Syrian hamster embryo (SHE) cell transformation assay [7], have been established and refined over many years to detect these ‘non-genotoxic’ carcinogens, as well as genotoxic carcinogens. Relatively comprehensive assessments of carcinogenic potential will be facilitated by the use of cell transformation assay results, in combination with genotoxicity data, computer-based prediction models, in vivo toxicity data and pharmacokinetics data.

6.3.1.2 Reproductive Toxicity Reproductive toxicity is another area that presents many problems for complete replacement tests, due to the high complexity of the reproductive system which provides a large number of potential targets for adverse toxicological reactions. There are also indirect effects, such as altered mating behaviour that can really only be investigated by using animals. The most likely use of in vitro assays in reproductive toxicity studies will be to provide mechanistic information on specific aspects of the toxicological response. For example, the use of somatic and germ cell co-cultures with specific markers for toxicological damage can be used to screen for potential spermatogenesis disruptors that can affect male fertility. Developmental toxicity (teratogenicity) is another area with potential for the widespread use of in vitro assays in the future. Currently existing mammalian systems such as whole embryo culture (WEC) [8] and rodent limb bud culture (micromass, or MM test) [9] still require a considerable number of experimental animals. The use of permanent mammalian cell lines, such as embryonic stem cell lines (embryonic stem cell test, or EST), is a promising way to establish an assay for teratogenicity testing in vitro with improved predictability to humans [10]. None of these tests could ever serve as a complete replacement due to the numerous potential mechanisms involved, as well as the indirect effects resulting from maternal toxicity. However, methods that detect direct effects would be of benefit in the identification of the most important teratogens (i.e., those that present a risk at maternally non-toxic doses).

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Practical Guide to Chemical Safety Testing Some reproductive disorders are thought to result from the disruptive effect of environmentally released chemicals on the endocrine systems of animals and humans. This is an area of growing concern among scientists and the public alike. Many of the screening tests being developed to detect these endocrine disruptors are conducted on in vitro systems. Such in vitro assays include simple competitive binding assays, which rely on the ability of the chemical to bind to the oestrogen receptor, as well as more sophisticated systems, in which the receptor is activated following binding of the chemical. Examples of the latter type of assay include yeast-based assays, which express either rainbow trout [11], or human [12] oestrogen receptors, vitellogenin gene expression in hepatocyte cultures [13], and MCF-7 (a human cancer cell line) proliferation [14]. It is generally accepted that a battery of in vitro assays, along with information from quantitative structure-activity relationship (QSAR; a computer prediction system) studies, should form the basis of a tiered testing strategy. The currently used in vitro assays are not without their problems, however [15-17], and the need to validate and to standardise these is vital, as inconsistencies between results obtained using the various assays abound in the literature.

6.3.1.3 Systemic Toxicity The investigation of systemic toxicity poses one of the greatest problems for in vitro assays. In theory, once the target organ is defined it would then be possible to evaluate toxicity by exposing the relevant cells in culture to the test substance. However, due to the large number of potential target tissues, it is not practical to expect such studies to be used to predict general systemic toxicity. Rather, they will most likely be used to investigate mechanisms of action in specific target tissues. This may be in conjunction with hepatocyte culture studies, which can be used to predict likely metabolites, allowing the direct effect of the metabolites on the target organ to be studied. The use of HepG2 transformants, a hepatocyte cell line that express a series of human cytochrome P450 subtypes, has recently been proposed as a means of investigating the metabolism and toxicology of chemicals [18]. An indication of systemic toxicity in humans may be facilitated by the coupling of a battery approach to QSAR-derived toxicokinetic parameters [19]. Before data from in vitro studies can be used to predict systemic toxicity, it is necessary to extrapolate the concentrations used in vitro to those that occur at the target site in vivo. Predictions of absorption via different routes are necessary for this to be possible. This can be facilitated through knowledge of the pysiochemical properties of the substance, as well as in vitro data (e.g., from in vitro skin penetration studies). Physiologicallybased pharmacokinetic (PB-PK) models can also be used to assess in vivo concentrations from in vitro studies.

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6.3.2 Validated Tests Currently in Use in the EU While efforts to develop in vitro tests capable of evaluating potential systemic toxicants has been hindered by the complexity of the systems involved, more progress has been made with assays that evaluate local effects. Some of these new alternative tests can already be used for regulatory purposes. After validation, and endorsement by ECVAM, alternative tests become part of Annex V of the EU Dangerous Substances Directive (DSD). The first alternative methods to go through this process are in the 27th Adaptation to Technical Progress (ATP) of the DSD [20], which officially came into force in October 2001. Method B40 is for evaluating skin corrosivity (the transcutaneous electrical resistance (TER) assay, and human skin model), while Method B41 is for phototoxicity (3T3 neutral red uptake phototoxicity test (3T3 NRU PT)). While ECVAM continues to evaluate and validate in vitro tests, alternatives not yet in Annex V may still in principle be used for EU notifications on a case-by-case basis. This can only happen if there is sufficient information to define the substance as ‘dangerous’, and if the information is adequate for risk assessment. This usually requires a positive test outcome, resulting in classification. If it is negative, the standard Annex V animal test is normally needed for confirmatory purposes. However, there is room for negotiation with the relevant Competent Authority, taking into account a ‘weight of evidence’ approach (i.e., considering the chemical structure and any data on analogue substances).

6.3.2.1 Corrosivity Skin corrosivity testing is a relatively simple procedure in biological terms. The endpoint, severe tissue destruction, is relatively easy to detect, and the application route is topical, with no problems of dilution or distribution. These two factors made the development of non-animal methods for the prediction of skin corrosion easier than for other toxic effects. However, only two in vitro assays (the TER assay and EPISKINTM) have been successfully validated to date, due to the fact that they alone met the agreed ECVAM criteria concerning acceptable underprediction and overprediction rates. These two assays can now be used as an alternative to the animal tests used to distinguish between corrosive and non-corrosive chemicals. The 27th ATP of the DSD adopted these two skin corrosivity tests as Method B40 of Annex V, and as noted, the Method can already be used [10]. The B40 corrosivity test is mainly for UN transportation classification, but it can be used as a screen before skin irritation testing using rabbits for notification of new substances. The rat skin TER test does not distinguish between UN packing groups or the EU classifications R35 (causes severe burns) and R34 (causes burns), whereas the human skin model does. If the corrosivity test is negative, a normal Method B4 skin irritation test would be needed for notification.

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Practical Guide to Chemical Safety Testing However, simpler assays such as CORROSITEXTM may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. This assay is based on the time it takes a substance to break through a biobarrier. Although not validated as an EU Annex V Method, the US ICCVAM recommended CORROSITEXTM as a non-animal test for skin corrosivity, and the US Department of Transport (DoT) now accepts the test for transportation classification. The second EU Method B40 test is referred to in Annex V under the non-proprietary name of the ‘human skin model assay’. This is the generic version of the ECVAM-validated EPISKINTM test. In the EPISKINTM assay, the test material is applied topically for up to 4 hours to a three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability below defined threshold levels at specified exposure periods. Other skin assays, such as EpiDermTM, are based on the same principle, i.e., the hypothesis that chemicals that are corrosive are those that are able to penetrate the stratum corneum (by diffusion or erosion) and are sufficiently cytotoxic to cause cell death in the underlying cell layers. However, they differ slightly with regard to the exposure times and classification criteria. Guidelines similar to those adopted in Annex V of the DSD were sent to the OECD Secretariat, and are now under consideration by OECD Member Countries. It is therefore likely that the new EU Methods will soon achieve worldwide acceptance through the OECD. However, there is an important difference between EU Annex V test Methods and OECD test guidelines, even though they are now almost totally harmonised, because the former are mandatory in the EU, whereas OECD guidelines are merely recommendations.

6.3.2.2 Phototoxicity Phototoxicity is a toxic response that is elicited after the first exposure of skin to certain chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic administration of a chemical. As mentioned earlier, the 27th ATP of the DSD also contains, as Method B41, an in vitro phototoxicity assay, 3T3 NRU PT [10]. This assay can be used for the evaluation of the phototoxic potential of a wide range of chemicals, including cosmetic ingredients. Thus, it is an important step forward in the EU initiative to stop all animal testing on new cosmetic ingredients and formulated products by 30 June 2002. Cosmetic ingredients, however, constitute only a small proportion of potential phototoxicants and it is important that any chemical capable of reaching the skin through systemic distribution (e.g., following ingestion or intravenous administration), or by topical application, should be tested. This test will also in due

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Alternatives to Animal Testing for Safety Evaluation course become an OECD guideline. The validation work, which took about seven years, was jointly undertaken by ECVAM and COLIPA, the European cosmetics industry association [21, 22]. The 3T3 NRU PT is a replacement test, based on a comparison of the cytotoxicity of a substance when tested in the presence and absence of a non-cytotoxic dose of ultraviolet (UV) light. Cytotoxicity in the test is expressed as a concentration dependent reduction in the uptake of vital dye neutral red, 24 hours after treatment with the test material and UV irradiation.

6.3.2.3 Skin Sensitisation The current standard bench mark animal study to evaluate skin sensitisation potential is the Magnusson and Kligman (M and K) guinea pig maximisation test of EU Method B6 [23]. This test is normally used for EU notification and classification. Non-adjuvant tests, such as the Buehler method [24], are used only if the M and K test is not technically feasible (i.e., the substance has a physical form which prevents injection). Nevertheless, such non-adjuvant tests are generally considered useful for risk assessment, because they are more relevant to normal dermal exposure. The local lymph node assay (LLNA), identifies contact allergens as a function of their ability to provoke T lymphocyte proliferative responses in draining lymph nodes. This is not an in vitro method, relying as it does on the measurement of lymph node cell responses stimulated by repeated topical exposure of mice to the test chemical. Nevertheless the LLNA does provide significant animal welfare benefits as fewer animals may be required and more importantly, the trauma to which animals are potentially subject is significantly reduced. The test has undergone extensive validation, and there is now an OECD guideline. Hence it is accepted instead of an M and K test for EU notifications. The LLNA has also recently been endorsed by the US ICCVAM [25, 26] as a stand-alone method to assess skin sensitising activity, providing various technical details in conducting the test are adhered to, notably that only female mice should be used until there has been a systematic comparison of results with male and female mice. The use of computerised expert systems based on the knowledge of chemical reactivity with proteins, together with consideration of skin absorption, offers promise as a first step in a hierarchical approach to identifying skin sensitisation and should be developed further. The only way at the moment to make a prediction of skin sensitisation hazard is on the basis of chemistry using data on skin permeation coupled with information on the presence of structural alerts. Nevertheless, predictions based on structural alerts for groups within a molecule often associated with skin sensitisation can be useful as a screening

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Practical Guide to Chemical Safety Testing tool before animal testing. DEREK (Deductive Estimation of Risk from Existing Knowledge) is an example of a system that can be used for this purpose [27].

6.3.2.4 Skin Irritation Despite the fact that numerous non-animal methods for predicting skin irritation have been developed, none has proved acceptable as a complete replacement for a regulatory guideline. This is largely due to the complexity of the endpoints in this tissue. Little is known about the mechanisms involved, and consequently most of the in vivo models are not based on knowledge of relevant mechanisms in humans. To varying extents, skin models such as EpiDermTM reflect dermal penetration of the chemical and its subsequent cytotoxicity. However, the precise roles of inflammatory mediators and other cellular signalling molecules in the skin irritation response are still insufficiently understood to develop an in vitro model for predicting human skin irritation effects accurately. Another complicating factor is the lack of reliable human and animal data against which to judge the predictive performance of novel in vitro tests. Progress has, however, been made in obviating the use of animals to assess severe effects by ‘screening out’ severe irritants using a hierarchial approach, and this is likely to improve with further use of structure-activity relationship (SAR) and mathematical modelling, as well as when improved in vitro methods for detecting severe irritants are developed.

6.3.2.5 Eye Irritation Much work has been done to assess ocular irritation with non-animal methods. The goal is to be able to predict eye irritation potential over the whole range of the response (i.e., from slight to severe) for any chemical substance. The information is needed to assess hazard (i.e., the intrinsic toxicity of the chemical), and to be able to protect users by classification and labelling of substances. The other issue is that, for finished products that may come into contact with the eye, such as ophthalmologic or cosmetic products, it is necessary to ensure that they are absolutely safe, i.e., non-irritant. On ethical grounds, the assessment of formulated cosmetic products can only be made using non-animal tests. Consequently, alternative methods have been chosen for their capacity to predict the ocular irritating potential of cosmetic products, whatever their formulation type (powder, emulsion, solution). Cosmetic companies develop their specific batteries of in vitro tests according to their product categories. A typical test battery might include some of the studies suggested in Table 6.4.

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neutral red release assay SkinEthicTM EpiOcularTM EpiDermTM rabbit enucleated eye test bovine cornea opacity and permeability assay fluorescein leakage assay Alternatives to Animal Testing for Safety Evaluation

Table 6.4 Typical eye irritation test battery •

The hen’s egg test-chorioallantoic membrane (HET-CAM) method [28], which can reflect specific eye irritation effects such as corneal opacity or vascular effects. It is one of the only in vitro methods that allows the blood vessels to be examined. Also, the same eggs examined morphologically in the HET-CAM test can also be used to provide more objective and quantitative information, by performing the chorioallantoic membrane-trypan blue staining (CAM-TB) method [29].

•

The neutral red release assay (PredisafeTM assay), which is a short-term monolayer culture system able to predict the cytotoxicity of cosmetic products.

•

Tests using reconstituted human epithelial cultures (e.g., SkinEthicTM, EpiOcularTM, and EpiDermTM models).

•

The rabbit enucleated eye test (REET) [30] and the bovine cornea opacity and permeability (BCOP) assay [31] both involve the use of eyes removed from recently killed animals. Corneal opacity and thickness, as well as fluoroscein dye uptake are assessed at set intervals following exposure to the test material. Considerable refinement and evaluation are required for both assays before they can be considered for regulatory purposes. The use of additional endpoints, such as those provided by histological examination can provide supplementary information that may improve the predictive ability of both assays.

•

The fluorescein leakage assay is a replacement test, which uses Madin-Darby canine kidney (MDCK) cell monolayers for the assessment of both ocular and dermal irritancy potential [32, 33]. The cells are grown to confluency on a porous filter, forming tight junctions similar to those found in the corneal epithelium. Chemicallyinduced loss of impermeability of the barrier is determined through the measurement of the leakage of a non-toxic dye, fluorescein, through the cellular layer following exposure to the test chemical.

6.4 Structure-Activity Relationships and Prediction of Properties Traditionally, the hazardous properties of chemicals are determined by testing using standardised procedures, normally with the studies conducted in compliance with Good Laboratory Practice. These laboratory studies are, in effect, a model for the effect that the chemical may have on the systems of primary concern, namely humans and the environment. There are, however, circumstances when adequate predictions of hazardous properties can be made without testing. In particular, in some cases the so-called ‘readacross’ approach can be used to predict properties of new substances by interpreting results from close analogues with similar physico-chemical properties and impurity

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Practical Guide to Chemical Safety Testing profiles. The principle is that similar biological properties are anticipated, since toxicokinetics (especially absorption and metabolism) would be comparable. It is preferable to see as many matching toxicological properties as possible to provide evidence in support of read-across for missing endpoints. The use of read-across, or other surrogate data, has to be negotiated in advance with the national Competent Authority. The typical physico-chemical properties testing programme to justify read-across for a notification is melting point, boiling point, density, surface tension, water solubility, partition coefficient and granulometry (measurement of particle size distribution). Assuming that a full set of standard GLP data are available for the first substance, it may be that only the tests which are considered pivotal in deciding the feasibility of readacross may have to be conducted according to GLP, notably water solubility, partition coefficient and granulometry. The most likely study to be acceptable for read-across is the 28-day repeat-dose oral rat toxicity study, as this uses a large number of animals, and fortuitously also offers a considerable cost saving. A basic toxicological package to confirm read-across would be acute oral toxicity, skin sensitisation (occasionally omitted) and an Ames test, with perhaps skin irritation and ready biodegradation tests. It may also be necessary to confirm there is similar acute toxicity to aquatic organisms by the basic ecotoxicological package of one acute study in fish, Daphnia or algae, chosen as the most sensitive species from the fully tested substance. Computerised determination of QSARs has been used for many years in the design of chemicals but it is only in recent years that these techniques have been used in toxicology. QSAR evaluations aim to use the known biological activity of a set of chemicals to establish mathematical relationships between the activity and the structure. An alternative, more mechanistically-based approach, using structure-activity relationships (SARs), uses computerised methods to identify fragments in molecules which are known to be associated with particular biological properties. QSARs can be used in certain circumstances for EU notification of new substances, in particular to refine the risk assessment rather than as surrogate data to replace the base set testing. It is the responsibility of the notifier to make the QSAR predictions, and establish that they are valid, although Competent Authorities may advise and offer expert scientific input. Chapter 4 of the Risk Assessment Technical Guidance Document [34] gives extensive regulatory and scientific information on the use of QSARs in the EU risk assessment of chemical substances. The emphasis, though, is on Competent Authorities using QSARs to support the risk assessment of the priority HPV chemicals selected for detailed review under the EU Existing Chemicals Regulation (ECR). When carrying out the risk assessment for humans and the environment, the exposure of the substance to humans and the environmental compartments is estimated. These exposure assessments will be based on available monitoring data and/or modelling. For

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Alternatives to Animal Testing for Safety Evaluation modelling exposure, several physico-chemical and environmental fate parameters are used. In the absence of experimental data, these parameters may be derived from QSARs. Depending on the exposure/effect ratio from the risk assessment, the decision is taken on whether the substance presents a risk to humans and/or the environment or whether further data are necessary to clarify a concern. When the Competent Authority considers the potential need for further data, QSARs may serve as a supporting tool in making this decision. Furthermore, if further testing is needed, QSARs may also be used to optimise the testing strategies. Validated QSARs are not currently available for toxicity endpoints. Instead, expert judgement is used based on close structural analogues and/or the presence of ‘structural alerts’ (i.e., fragments associated with effects). Nevertheless, such predictions may be useful for the risk assessment for human health, especially for endpoints without test results and they may be of value in indicating a potential hazard, toxicokinetic properties or the need for further testing. Since biological activity is a function of both partition and reactivity, a QSAR model must be capable of modelling both of these, so that a high correlation to the in vivo response can be expected. Furthermore, all of the chemicals covered by the relationship must have a dependent property that is elicited by a mechanism that is common both to the set of chemicals and to the dependent property [34]. Reliable QSAR estimates for fish, Daphnia and algal toxicity are available for chemicals with a non-specific mode of action. These estimates can be used to assist in data evaluation and/or to contribute to the decision making process on whether further testing is necessary to clarify an endpoint of concern and if so, to optimise the testing strategy. Also QSARs can be helpful in assessing long-term aquatic toxicity tests with very hydrophobic organic chemicals which are difficult to test. Another use of QSAR is in the selection of a set of chemicals for the validation of in vitro tests. In this context, QSAR can be used to select chemicals that differ greatly in the in vivo responses that they elicit. This approach was used in the recent ECVAM international validation study for skin corrosivity [35].

6.5 Strategies to Minimise Use of Animals The UK Health and Safety Executive (HSE) encourages the reduction of animal usage during chemicals testing, within the constraints of the EU and international regulatory systems in which the UK operates. For notification of new substances, the HSE makes particular efforts to bring together two or more intending notifiers of the same substance, so that they can share data and avoid duplication of animal testing. The HSE also encourages the use of methods (such as the fixed-dose procedure for acute oral toxicity) that offer reduced animal usage and/or reduced severity of the testing procedures. In general, the philosophy is to encourage thoughtful toxicology, rather than routine testing.

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Practical Guide to Chemical Safety Testing Hence, the aim is to promote a tiered approach to testing, with non-animal information being used in the initial assessments. The HSE also allows in vitro alternatives to be used for notifications, but these must be validated tests (ideally independently in several laboratories) and preferably by an OECD guideline, or at least be in development for the OECD. Generally, unless there is an EU Method (as for corrosivity and phototoxicity, see Sections 6.3.2.1 and 6.3.2.2 respectively), only positive in vitro tests are acceptable, and if the result is negative, the standard animal test would normally be required. In order to minimise testing for notification purposes, the German Competent Authority [36, 37] have developed stepwise assessment procedures, including structure-activity considerations, alternative methods (in vitro tests), and computerised SAR models. An electronic database was developed which contains physicochemical and toxicological data on approximately 1300 chemical substances. It is used for regulatory structureproperty relationship (SPR) and SAR considerations, and for the development of rules for a decision support system (DSS) for the introduction of alternative methods into local irritancy/corrosivity testing strategies. The information stored in the database is derived from proprietary data, so it is not possible to publish this directly. Therefore, the database is evaluated by regulators, and the information derived from the data is used for the development of scientific information about SARs. This information can be published, by means of tables correlating measured physico-chemical values and specific toxic effects caused by the measured chemical. This information is introduced to the public by means of a DSS that predicts local irritant/corrosive potential of a chemical by listing so-called exception rules. The DSS can predict whether a chemical produces: (a) corrosive effects (i.e., no testing is necessary); (b) might have corrosive effects (i.e., no animal testing, and instead in vitro tests are suitable); and (c) will produce no effects or only marginal effects (i.e., animal tests are necessary). In addition, the DSS provides reliable data for classification and labelling based on a specific result.

6.6 Future Developments and Conclusions An extremely important use of alternative tests is as a screen before animal testing, to eliminate obviously hazardous chemicals from further study. Alternative tests are also useful in the less prescriptive safety evaluation processes. Such studies, on their own or combined with property predictions based on chemical structure (by formal read-across

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Alternatives to Animal Testing for Safety Evaluation to tested analogues, QSAR or prediction by professional judgement) can be used for worker safety evaluation and safety data sheets (SDS). The other use is for risk assessments when there is no obligation to do new studies, i.e., to refine uncertain outcomes of risk assessment for some notifications and EU Existing Chemical Regulation (ECR) priority chemicals, and also for the less formal risk assessments for other product types, e.g., cosmetics. Other regulatory agencies outside the EU also accept alternative tests. There are cultural and regulatory differences. For example, in the US there is normally no obligation to do studies on new chemicals for the Toxic Substances Control Act or the Occupational Health and Safety Act, so available alternative tests can be used, whereas in Japan the testing for notification is highly prescriptive and stylised with no opportunity to use nonstandard tests. Alternative tests and other surrogate data to predict the hazardous properties of chemicals will be of key importance under the proposed EU Scheme for Registration, Authorisation and Evaluation of Chemicals (see Section 9.9), as discussed by ECVAM [38].

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R. Umansky, Developmental Biology, 1996, 13, 31.

10. H. Spielmann, I. Pohl, B. Doring, M. Liebsch and F. Moldenhauer, Toxicology in Vitro, 1997, 10, 1, 119. 11. F. Petit, P. le Goff, J.P. Cravedi, Y. Valotaire and F. Pakdel, Journal of Molecular Endocrinology, 1997, 19, 321. 12. E.J. Routledge and J.P. Sumpter, Environ. Toxicol. Chem., 1996, 15, 241. 13. S. Jobling and J.P. Sumpter, Aquatic Toxicology, 1993, 27, 361. 14. A.M.Soto, K.L. Chung and C. Sonnenschein, Environmental Health Perspectives, 1994, 102, 380. 15. T. Zacharewski, Environmental Health Perspectives, 1998, 106, 577. 16. N. Beresford, E.J. Routledge, C.A. Harris and J.P Sumpter, Toxicology and Applied Pharmacology, 2000, 162, 22. 17. R.D. Combes, Alternatives to Laboratory Animals, 2000, 28, 81. 18. S. Yoshitomi, K. Ikemoto, J. Takahashi, H. Miki, M. Namba and S. Asahi, Toxicology In Vitro, 2001, 15, 245. 19. B. Ekwall, C. Clemedson, B. Ekwall, P. Ring and L. Romert, Alternatives to Laboratory Animals (ATLA), 1999, 27, 339. 20. Anon., 2000, Annex V of council Directive 67/548/EEC, as adapted to technical progress by Commission Directive 92/69/EEC of 31-7-92, Official Journal of the European Communities L383A. 29-12-92, Commission Directive 96/54/EC of 30-7-96, Official Journal of the European Communities L241, 30-9-96, Commission Directive 98/73/EC of 18-9-98, Official Journal of the European Communities L305, 16-11-98, Commission Directive 2000/32/EC of 19-5-00, Official Journal of the European Communities L136, 8-6-00 and Commission Directive 1000/33/EC of 25-4-00, Official Journal of the European Communities L136, 8-6-00.

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Alternatives to Animal Testing for Safety Evaluation 21. H. Spielmann, M. Balls, M. Brand, B. Doring, H.G. Holzhutter, S. Kalweit, G. Klecak, H. L’Eplattenier, M. Liebsch, W.W. Lovell, T. Maurer, F. Moldenhauer, L. Moore, W.J.W. Pape, U. Pfannenbecker, J. Potthast, O. De Silva, W. Steiling and A. Willshaw, Toxicology in Vitro, 1994, 8, 793. 22. H. Spielmann, M. Balls, J. Dupuis, W.J.W. Pape, G. Pechovitch, O. de Silva, H.G. Holzhutter, R. Clothier, P. Desolle, F. Gerberick, M. Liebsch, W.W. Lovell, T. Maurer, U. Pfannenbecker, J.M. Potthast, M. Csato, D. Sladowski, W. Steiling, and P. Brantom, Toxicology in Vitro, 1998, 12, 305. 23. B. Magnusson and A.M. Kligman, Journal of Investigative Dermatology, 1969, 52, 268. 24. E.V. Buehler, Archives of Dermatology, 1965, 91, 171. 25. Interagency Coordinating Committee on the Validation of Alternative Methods, The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds, NIH Publication 994494, National Institute of Environmental Health Sciences. Research Triangle Park. NC, 1999. 26. D.M. Sailstad, D. Hattan, R.N Hill and W.S. Stokes, Regul. Toxicol. Pharmacol., 2001, 34, 249. 27. D.M. Sanderson and C.G. Earnshaw, Human Experimental Toxicology, 1991, 10, 261. 28. N.P. Luepke, Food and Chemical Toxicology, 1985, 23, 287. 29. S. Hagino, H. Itagaki, S. Kato, T. Kobayashi and M. Tanka, Toxicology in Vitro, 1991, 5, 301. 30. A.B.G. Burton, M. York and R.S. Lawrence, Food and Cosmetics Toxicology 1981, 19, 471. 31. P. Gautheron, M. Dukic, D. Alix and J.F. Sina, Fundamental and Applied Toxicology, 1992, 18, 442. 32. R. Tchao in Alternative Methods in Toxicology, Eds., A.M. Goldberg and Mary Ann Liebert, New York, 1988, 271. 33. A.J. Shaw, M. Balls, R.H. Clothier and N.O. Bateman, Toxicology in Vitro, 1991, 5, 569.

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Practical Guide to Chemical Safety Testing 34. Anon., Technical Guidance Document in Support of Commission Directive 93/ 67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No. 1488/94 on Risk Assessment for Existing Substances, European Commission, Luxembourg, 1996. 35. M.D. Barratt, P.G. Brantom, J.H. Fentem, I. Gerner, A.P. Walker and A.P. Worth, Toxicology in Vitro, 1998, 12, 471. 36. S. Zinke, I. Gerner, G. Graetschel and E. Schlede, Alternatives to Laboratory Animals (ATLA), 1999, 28, 29. 37. I. Gerner, G. Graetschel, J. Kahl and E. Schlede, Alternatives to Laboratory Animals (ATLA), 1999, 28, 11. 38. A. Worth and M. Balls, Alternative (non-animal) methods for chemicals testing: Current status and future prospects, European Centre for the Validation of Alternative Methods, Ispra, Italy, 2002, draft report for publication in Alternatives to Laboratory Animals.

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7

Toxicological Assessment Within A Risk Assessment Framework H. Paul A. Illing

7.1 Introduction In essence this chapter is a ‘bridge’. Earlier chapters are concerned with the conduct of tests (safety testing), and hence on the acquisition of physical, chemical and toxicity information relevant to toxicological risk assessment. Chapters in Part 2 of this book are concerned with the specific regulatory frameworks within which toxicological risk assessment is conducted. This chapter concentrates on the principles underlying the regulatory decisions concerning choice of test and the translation of test data into regulatory management decisions. It also examines how to carry out the translation from experimental results to regulatory action in respect of human health. Chapter 8 examines the corresponding requirements for environmental risk assessment. Although toxicity studies are necessary for the assessment and evaluation of risk, they are not sufficient. Exposure information is also required in order to conduct toxicological risk assessments. Thus another element of this chapter is an examination of exposure scenarios used in regulatory risk assessment for human health. Studies on other species and on ecosystems are examined in the next chapter. This chapter therefore has three aims. It will define and describe the concepts that lie behind toxicological risk assessment. It will also briefly examine exposure scenarios used in regulatory risk assessment for human health. Finally, it will set out the principles behind the judgements that underlie the regulatory decisions concerning choice of test and the translation from experimental results to regulatory action. Much of the material contained in this chapter is derived from a more extensive monograph [1].

7.2 Definitions and Concepts The first part of this chapter is concerned with definitions and concepts. The intention is to give the framework within which the detailed toxicological risk assessment is conducted. It therefore considers first risk, risk assessment, risk evaluation and risk management

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Practical Guide to Chemical Safety Testing with particular reference to managing toxicological risks. It then deals with how toxicology interfaces with risk assessment and risk management.

7.2.1 Risk

7.2.1.1 Hazard, Harm and Risk Although generally, people regard hazard and risk as interchangeable terms, in risk management they have distinct and different meanings. (1) Hazard is an inherent property of an agent or situation capable of having adverse effects on (harming) something [2]. Hence, it includes the substance, agent, source of energy or situation having that property. More succinctly, the US Presidential/Congressional Commission on Risk Assessment and Risk Management [3] called hazard a source of possible damage or injury. (2) Harm is therefore adverse effects, damage or injury, and hence, for human health purposes, ‘a deleterious perturbation of a biological system resulting in human illhealth’. (3) Risk is the probability of a specific outcome, generally adverse, given a particular set of conditions [3]. A more specifically toxicological definition is that risk is the probability of adverse effects caused under specified circumstances by an agent in an organism, population, or ecological system [2]. A short overall summary is that ‘risk is the possibility of suffering harm from a hazard’ [4].

7.2.1.2 Risk Assessment, Risk Evaluation and Risk Management

Risk Management The Royal Society Study Group [5] succinctly summarised risk management as: ‘The making of decisions concerning risk and their subsequent implementation.’ A much more thorough definition is that given in [2]:

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Toxicological Assessment Within A Risk Assessment Framework ‘Risk management: Decision making process involving the considerations of political, social, economic and technical factors with relevant risk assessment information relating to hazard so as to develop, analyse and compare regulatory and non-regulatory options and select and implement the optimal response for safety from that hazard.’ Before managing the risks it is necessary to find out what they are (risk assessment) and to evaluate their significance (risk evaluation). There is a distinct dichotomy in what is defined as the risk assessment, although this dichotomy can be reconciled. The Royal Society Study Group [5] defined risk assessment in relationship to source and release of agent. This definition is particularly suited to engineering risk and the risks associated with major hazards, both natural and industrial. The US National Research Council in its report (the ‘Red Book’) [6] defined risk in terms of ill-health outcome resulting from the chemical-receptor interaction. Almost all health risk assessors define risk in the manner given in the ‘Red Book’. The Royal Society divided risk assessment into risk estimation and risk evaluation, with risk estimation being the name given to the process equivalent to the Red Book’s risk assessment.

Risk Assessment A recent restatement [7] of the ‘Red Book’ approach defines risk assessment as: ‘The evaluation of the potential for adverse health effects in humans from exposure to toxic chemicals.’ The European Union [8] has identified that the risk assessment process consists of the following steps: (1) Assessment of effects, comprising: • hazard identification; identification of the adverse effects which a substance has an inherent capacity to cause; • dose (concentration)-response (effects) assessment: estimation of the relationship between dose, or level of exposure to a substance, and the incidence and severity of effect, where appropriate (hazard characterisation). (2) Exposure assessment: estimation of the concentrations/doses to which human populations (i.e., workers, consumers and man exposed indirectly via the environment) or environmental compartments (aquatic environment, terrestrial environment and air) are or may be exposed.

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Practical Guide to Chemical Safety Testing (3) Risk characterisation: estimation of the incidence and severity of the adverse effects likely to occur in a human population or environmental compartment due to actual or predicted exposure to a substance, and may include ‘risk estimation’, i.e., the quantification of that likelihood. Most recent restatements of the ‘Red Book’ approach call these steps hazard identification, hazard characterisation, exposure assessment and risk characterisation. The EU has grouped hazard identification and dose (concentration)-response (effects) assessment (also known as hazard characterisation) as assessment of effects. These two steps are also collectively known as hazard assessment. Whether it is called hazard assessment or assessment of effects, this is the gathering of data on physico-chemical and toxicological properties covered in Chapters 2 to 6. Exposure assessment is conducted as a separate activity, as is the risk characterisation. This chapter covers these two stages when the target is human health; Chapter 8 covers the equivalent material for the environment.

Risk Evaluation There is one further stage in the overall process of risk assessment and management covered in this chapter. Risk evaluation was defined by the Royal Society [5] as: ‘the complex process of determining the significance or value of the identified hazards and estimated risks to those concerned with or affected by the decision.’ The prediction has to be set against some societally derived criteria concerning the acceptability of the risk in the risk evaluation. It may include a study of risk perception and the trade-off between perceived risks and perceived benefits. Traditionally, the US National Research Council’s guidelines [6] did not include a formal risk evaluation stage. However, Lewalle [2] developed a very similar definition to that of the Royal Society for risk evaluation and found that there was a de facto risk evaluation stage bridging the Royal Society Study Group’s risk estimation (Red Book’s risk assessment) and the risk management phases of the process. However, they identified it as the first stage of risk management rather than the final stage of risk assessment. It is the bridge that links the two. Figure 7.1 summarises the relationships between the stages in the process of assessment. The outcome of a risk evaluation is the need for decisions on how to manage the risks. Companies, Governments or individuals may conduct risk evaluations. Society, through government, manages types of toxicological risks by setting up regulatory schemes (for

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Lewalle [2] is a report on an OECD/IPCS project aimed at harmonising hazard/risk assessment terminology. As the National Research Council did not distinguish the step of risk evaluation, in their terminology risk assessment covers the same ground as the Royal Society’s risk estimation. The source-release assessment (or release estimation) is a part of the exposure assessment relating particularly to major hazards from toxicity and ecotoxicity, and considered in Chapter 8.

Figure 7.1 Outline of the process of risk assessment Reproduced with permission from P. Iling, Toxicity and Risk: Context, Principles and Practice, Taylor and Francis, London, 2001

example for drugs, for medical devices or for chemicals leaching from food packaging into the diet). Suitable bodies are set up to assess, evaluate and manage the risks associated with particular chemicals, either generally or for specific purposes. These schemes often indicate what information is required in order to complete the risk assessment and may either (in the simpler cases) state the procedure by which a risk evaluation should be conducted, or (in more complex cases) set up suitable organisations to conduct the risk evaluation. Associated with the risk evaluation may be recommendations concerning how to manage the risks. The whole process is iterative, a risk evaluation needs regular revisiting in order to check that all available information (including new information on toxicity and re-interpretations of existing toxicity information) has been included in it and that the management procedures associated with it are still adequate. The different regulatory frameworks for assessing, evaluating and managing risks from chemicals are described in Chapters 9 to 15.

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7.2.1.3 Equity, Utility and Technology In risk evaluation there have to be criteria for reaching decisions. The criteria used by regulators in the health, safety and environmental field can be classified into three ‘pure’ criteria [9]. These are: (1) An equity-based criterion, which starts with the premise that all individuals have unconditional rights to certain levels of protection; (2) A utility-based criterion, which applies to the comparison between the incremental benefits of measures to prevent the risk of injury or detriment, and the cost of the measures; and (3) A technology-based criterion which essentially reflects the idea that a satisfactory level of risk prevention is attained when ‘state of the art’ control measures (technical, managerial, organisational) are employed to control risks whatever the circumstances. Regulators have either used these ‘pure’ criteria alone or as building blocks to create mixed criteria. An equity-based criterion leads to standards, applicable to all, held to be acceptable in normal life, or which refer to some other premise held to establish an expectation of protection. It converts to a fixed limit representing the maximum level of risk above which no individual may be exposed. It often requires taking of decisions based on worst case scenarios bearing little resemblance to reality, and hence it often over-estimates risks. Equity based criteria underlie many environmental standards for chemicals, such as environmental air quality standards, occupational exposure standards and acceptable/tolerable daily intakes for food additives/contaminants [10]. Utility based criteria compare, in monetary terms, the relevant benefits (statistical lives saved or life-years of extension of life) obtained by the adoption of the particular risk prevention measure with the net cost of introducing it, and require that a balance be struck. If the balance is deliberately skewed towards ensuring benefit it ensures that there is gross disproportion between cost and benefit. Utility based criteria tend to ignore ethical considerations. Schemes rarely use pure utility based criteria. Pure technology based criteria ignore the balance between cost and benefit. If there is ‘state of the art’ technology in one field it should be applied in all fields. The ‘clean technology’ used for manufacturing and packaging medicines would have to be applied to controlling wood dust during furniture manufacture - not a realistic proposition.

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Toxicological Assessment Within A Risk Assessment Framework The integration of all three criteria into a framework was first put forward by a Royal Society Study Group [5] and has been re-stated as the ‘tolerability of risk’ conceptual model developed by the Health and Safety Executive [9]. The Royal Society Study Group suggested a regulatory process and control strategy based on: •

an upper level of risk that should not be exceeded for any individual;

•

further control, so far as is reasonably practicable, making allowance if possible for aversions to higher levels of detriment; and

•

a cut-off in the deployment of resources below some level of exposure or detriment judged to be trivial.

Increasing individual risks and societal concerns

Figure 7.2 sets out the same idea in diagrammatic form.

Unacceptable risk

Tolerable risk

Broadly acceptable risk

Equity criterion for refusing permission/banning

Utility/technology criteria apply in this region Reqires management to ensure risk reduced to ‘safe’ or at least as low as reasonably possible. Management may be by choice of chemical, control of outlets or uses, requirements for protective equipment etc.

Equity criterion for ‘safe’

Figure 7.2 The Royal Society/Health and Safety Executive framework on the tolerability of risk

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Practical Guide to Chemical Safety Testing Many regulatory schemes make use of these mixed criteria, including those for air quality objectives and for workplace maximum exposure limits [10].

7.2.2 Toxicology

7.2.2.1 Definition of a Toxicant, and Hence Toxicology The most important definition in toxicology is that for a toxicant. A toxicant is an agent that, when applied to a biological system, causes a deleterious perturbation to that system [1]. The agent may be biological, chemical (natural or synthetic) or physical in origin. When dealing with human health the biological system is either the human being itself or humans in their environment. An interaction is being investigated – the interaction between the applied substance and people. Toxicology is concerned with ‘deleterious’ or harmful interactions. ‘Deleterious’ implies a value judgement. The same effect may be considered deleterious or beneficial in the same species, depending on the condition of the individual affected. Two examples illustrate this point. Herbal cannabis is proscribed in many countries. Society, through government, has decided that, when smoked, it causes deleterious effects, and legislates to discourage its use. There are sizeable minorities who take a different view – including most users. Antidiabetic agents reduce blood sugar levels – fine for the diabetic when high blood sugar levels are reduced to relatively normal blood sugar levels (a therapeutic effect), but not desirable when the blood sugar levels in a normal person are reduced to the point when they become severely hypoglycaemic and comatose. The value judgement of what is considered deleterious depends on who is making the judgement and the circumstances surrounding the exposure.

7.2.2.2 Dose-Effect and Dose-Response It is essential to distinguish between dose-effect and dose-response. Dose-effect is the relationship between the total amount of a substance administered, taken in or absorbed by a system and the magnitude of a specific, continuously graded change affecting it. The dose-effect curve relates dose to severity of effect – from minor disturbances to observations/clinical parameters, through mild and moderate pathological changes to, in the worst case, death. The ‘no observed adverse effect level’ is derived from the dose-effect curve; if the parameter measured is one relating to an adverse effect and not statistically significantly altered from the normal values, then there is no evidence

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Toxicological Assessment Within A Risk Assessment Framework that an effect has occurred. Non-adverse effects were defined as the absence of change in morphology, growth, development and life span in 1978 [11]. The Approved Guide to the EU New and Existing Chemicals Directives [12] prefers to define effects in terms of ‘serious damage to health’ and has suggested certain potentially adverse effects do not cause serious damage to health (Table 7.1). If dose-effect curves for individuals of the same species are similar, they can be aggregated to produce a dose-effect curve for the species.

Table 7.1 Evidence not indicative of serious damage to health on prolonged exposure but that need to be taken into account when determining a no-effect level. By implication, these effects are not seen as ‘serious’ [12] Clinical observations or changes in weight gain, food consumption or water intake which may have some (minor) toxicological significance Small changes in clinical biochemistry, haematology or urinalysis parameters that are of doubtful or minimal toxicological significance Changes in organ weight with no evidence of organ dysfunction Adaptive responses (e.g., marophage migration in the lung, liver hypertrophy and enzyme induction, hyperplastic responses to irritants) When a species specific mechanism of toxicity has been demonstrated

The dose-response curve is based on a fixed level of response. It may be applied to essentially all-or-nothing effects, such as cancers and birth defects (terata). It may also be applied in circumstances where dose-effect curves for individuals exist, but there is wide inter-individual variation – such as in the case of occupational asthmagens (‘pseudostochastic’ effects). In practice it can be derived for any effect, stochastic or non-stochastic. When the effect is not stochastic, the dose-response curve is derivable from the statistics of a population dose-effect curve (Figure 7.3). If a ‘no-effect level’ is taken from a doseresponse curve it depends on defining an (infrequent) frequency of occurrence of a given level of effect to be ‘no effect’. That level is usually dictated by the statistical power of the study to detect changes in the parameter being measured.

7.2.2.3 Risk of What? Input versus Intake/Uptake and the Concept of Source, Pathway and Receptor The interaction between chemical and receptor is the critical interaction for a toxic effect to take place. The chemical has to get to the receptor for this interaction to take place. It

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a

The letters A and B refer to the 95% confidence limits for the relationship between the dose and the given level of response. (from [1]; reproduced with permission from Taylor and Francis, London)

Figure 7.3a Dose-effect curve for a non-stochastic effect (the dotted lines represent the confidence limits associated with the line) Figure 7.3b Dose-response curve for the level of effect given by the line A to B Reproduced with permission from P. Iling, Toxicity and risk: context, principles and practice, Taylor and Francis, London, 2001

has to be released from a source, pass through a medium and, in the case of many effects, taken into the body before the interaction can take place. Figure 7.4 [1] outlines this process. Management can be undertaken at source, controlling the release into the medium, or at the interface between the external medium and the organism. The dichotomy between the Royal Society and the US National Academy is reflected here – the Royal Society was concerned with the risk of release of a chemical into a medium, the National Academy of Science was concerned with risk at the interface between the external medium and the organism. Regulatory judgements may be judgements concerning the acceptability of a particular chemical or use, or they may be about setting a maximum level of input from a source, intake from a pathway or uptake from all pathways regarded as acceptable (a standard). Decisions on acceptability of input include both the consequences of intended use (e.g., in manufacturing, as consumer products or as residues in foods from food contact

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Toxicological Assessment Within A Risk Assessment Framework PATHWAY

SOURCE Point

INPUT Emission limit Environment

Discharge limit

Diffuse

Maximum residue level/ limit (for individual food, or site)

INTAKE Exposure limit, (outdoor, indoor air, drinking water, occupational)

Environmental medium (air, water, soil)/ ecosystem Food basket (total diet) and drinking water

UPTAKE

RECEPTOR Biosphere (biodiversity)

Biological exposure limit

Ecosystem species

Daily/weekly/ annual intake Organism (e.g., humans)

Notes The input from a source maybe accidental or anticipated. For accidental releases the emphasis is on the identification of the size and duration of the potential release in order to determine the ways in which the likely end effects can be minimised or prevented. For anticipated releases the emphasis is to identify the likely end effects in order to determine the acceptable (or tolerable) pattern for releases. ‘Pathway’ may include dispersion pattern, transfer between environmental media, bioconcentration, etc., that may occur between the emission of the chemical from a source and the take up by the receptor. These are considered in detail in Chapter 8. Damage to the biosphere and to the environment is controlled mainly at source, through controlling emissions. Damage to human health may be prevented either by control measures based on intake/uptake or by control of inputs at source. For food safety, ‘pathway’ is the way in which different individual foods (and their contaminants, including leachates from packaging) are aggregated to obtain the total food intake.

Figure 7.4 The passage of a chemical from source to receptor and the standards that can be used when controlling exposure to that chemical Reproduced with permission from P. Iling, Toxicity and risk: context, principles and practice, Taylor and Francis, London, 2001

materials, or pesticides/veterinary medicines) and of waste disposal (to sewer, landfill or air). Decisions on intake are usually related to medium – air (workplace, indoor, outdoor), drinking water or the overall intake from food. Very occasionally standards are associated with total uptake into the body (e.g., for lead) and can be obtained from measurements on biological samples such as urine, exhaled air or blood.

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7.3 Exposure Scenarios Ideally, exposure assessment should include direct measurements of chemical in the appropriate medium. For uptake standards, exposure is measured in blood, urine or exhaled air as a surrogate for measurement at the site of action. In the UK, outside of the Control of Lead at Work Regulations, 2002 [13], implementing the EU Chemical Agents Directive [14], few regulations are based on uptake standards. Intake standards are more common. These require assumptions concerning the intake process and use simple models to describe the relationship between intake and uptake. Intake models may be for workers or consumers in general or they may involve classification of workers and consumers into different groups. One classification frequently used (e.g., in the Biocides Directive [15]), is to divide exposure into industrial and professional users and direct human exposure and indirect human exposure via the environment. The exposure may be measured or may be obtained using calculations based on assumptions and algorithms, or using knowledge based on general experience. Much more complex models are required when relating emission (or input) to toxic effects. These models are required to describe the passage of the chemical from the source through the medium (the environment or the food chain) to the receptor. These latter models include scenarios describing the movement through the medium (air, water, food) as well as models of intake. Whenever possible, measured exposure data should be used in order to establish the validity of the model.

7.3.1 Routes of Administration Although the most appropriate route of administration is the route by which exposure is likely to occur, there may be practical difficulties when carrying out the testing. This is particularly apparent when carrying out long-term repeated dose studies and studies on reproductive toxicity. In practice, oral administration is frequently preferred as it represents the best compromise between ease of administration, completeness of uptake and economics. In consequence it may be necessary to undertake route to route conversion of the dose-effect information, usually through conversion of the no observed adverse effect level (NOAEL) for the oral study into a predicted no observable adverse effects level (NOAEL) for the relevant route of exposure.

7.3.1.1 Inhalation Exposure – Relating Intake and Uptake Where possible, data from inhalation studies in animals should be used for examining the potential for human health effects from airborne exposure. As the dose is calculated 148

Toxicological Assessment Within A Risk Assessment Framework as mg/m3/duration of exposure and there are extrapolations for both lung surface area and body weight (or surface area), data from animal studies needs no further manipulation to allow for species differences. However, inhalation studies are often not available, since they are much more complex and expensive when compared with the corresponding oral study. In these circumstances, extrapolation from oral data can be undertaken as a first approximation. When only oral data are available the relationship between intake (in mg/m3) and uptake (in mg/kg; usually derived from data for oral exposure) is critical when dealing with airborne exposure and intake standards. That relationship depends on the amount of air inhaled (in m3) and hence on the type of population being examined and the likely amount of air being breathed. Air intake is increased during exercise. In addition, conversion to mg/kg involves an assumption concerning the body weights of the relevant population. The approach used in occupational hygiene is known as the ‘Stockinger-Woodward approach’. One statement of this approach assumes an 80 kg human breathes 10 m3 in a standard working day of eight hours for 5 days/week [16]. This air breathing volume assumes some manual work. It is assumed that all of the inhaled chemical is absorbed. The IPCS Environmental Health Criteria 170, Appendix 1 [11] uses a body weight of 64 kg and a daily inhalation volume of 22 m3. This is for a general population (i.e., including the elderly, the seriously ill and children as well as those in work). The procedure assumes 24-hour exposure and complete absorption from the inhaled air. The EU Scientific Committee for toxicity, ecotoxicity and the environment (CSTEE) [17] set out values for 1,4-dichlorobenzene exposure in its position paper on margins of safety in human health risk assessment. They use a body weight of 60 kg, a ventilation rate of 0.7 m3/h and assumed only 75% of the material in the air was being absorbed through the lung. They also acknowledged the lack of guidance in the Technical Guidance Document for the New Substances Directive and Existing Substances Regulation [8]. Table 7.2 summarises and compares these values. If data are available, the assumption of complete absorption following inhalation of highly volatile substances or respirable particles can be modified. A ratio may be derived by comparing the LC50 value in an acute inhalation toxicity study, after conversion to mg/kg body weight, with an LD50 value for acute oral exposure [8]. That ratio can then be applied to the oral NOAEL to obtain an inhalational NOAEL. In practice, this is applicable mainly to existing substances, as some of the newer test procedures for acute toxicity do not produce a specific LD50 value.

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Table 7.2 Comparison of parameters for comparing oral and inhalation exposure Source

Breathing rate (m3/h)

Total breathed in 8 h shift (m3)

Total breathed in 24 h day (m3)

Assumed weight of average human (kg)

Assumed % of inhaled material absorbed

Stockinger/ Woodward

1.25

10

N/A

80

100

IPCS

0.92

7.3

22

64

100

EU CSTEE

0.7

5.6

17

60

75

Footnote: The CSTEE values given are substance specific values for 1,4-dichlorobenzene, the other two sets of values are general, i.e., default values Bold numbers are those stated in the source, other numbers are derived values The data for the Stockinger/Woodward approach refer to the working population, the data for the other two entries refer to the general population

7.3.1.2 Dermal Exposure Unless there are data to the contrary, the oral NOAEL for repeated dose studies is assumed to be applicable to the dermal route [8]. Generally dermal absorption is slower than oral absorption and this extrapolation is claimed to err on the side of caution. More sophisticated models of dermal absorption can be described, but are rarely used. They may take into account the amount of the substance applied, the surface area over which it is applied, the penetration rate in vitro and the potential for reservoir formation.

7.3.1.3 Oral Exposure Generally, toxicity studies on industrial chemicals are conducted using the oral route where possible. The data are usually given in the form of mg/kg bodyweight/day to make allowances for the differences between the test species (usually rat) and the target species (humans) in body weight. If the substance is administered in the diet or in drinking water, the amount administered should be converted from ppm in diet or drinking water to mg/kg body weight/day using appropriate conversion factors. In the case of diet the factor is derived from food consumption and body weight data collected during the study.

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7.3.2 Exposure Prediction

7.3.2.1 Estimation and Assessment of Substance Exposure (EASE) (Workplace) EASE (Estimation and Assessment of Substance Exposure) is a system for describing workplace exposure, originally contained in a document intended for assessing notified new substances [18], but subsequently extended to existing substances [8]. It has also become the reserve information for use in the evaluation of existing chemicals under the OECD Existing Chemicals Schemes. It includes elements for inhalation exposure and for dermal exposure. The inhalation exposure part of EASE is a knowledge-based (or ‘expert’) system for assessing exposure. The computerised system includes a knowledge base, an inference engine and a user interface, contained within an expert system shell. The model makes use of measured exposure information contained in the UK National Exposure Database (NEDB) and 10 exposure bands. The dermal exposure part of EASE describes dermal exposure as none, incidental, intermittent and extensive, and is based on concepts developed by the US Environmental Protection Agency.

7.3.2.2 POEM (Pesticide Sprayers) The UK Predictive Occupational Exposure Model (POEM) is a system for predicting exposure, and hence absorption of pesticides by spray operators [19, 20]. It is therefore restricted to relatively small groups of workers. It is divided into two parts, one dealing with contamination from handling the concentrate (largely skin absorption) and the second concerned with contamination during application of the diluted spray (possible skin contamination and inhalation exposure). Product-specific and spraying techniquespecific input data are required to carry out assessments using this system. A European Union version of POEM is currently under development.

7.3.2.3 Consumer Exposure Assessment

Direct Exposure Possible consumer exposure scenarios are usually described using simple algorithms [8, 18, 21, 22], or are specific to separately managed uses (cosmetics, toys, food contact

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Practical Guide to Chemical Safety Testing materials, etc.). Separate algorithms are available for inhalation, dermal exposure and oral exposure. Information is required on the product (physical form, intended use, amount used/event, if appropriate - contact surface), the concentration of the chemical in the product and on ‘contact data’ for the product (how often and how long, and, for spraying/ inhalation the size of room and air exchange rate). An initial screening is used to indicate if the substance is used in/as a consumer product. If it is, then a qualitative assessment may be used to determine if the exposure is negligible. A full quantitative assessment need only be undertaken if exposure is likely to be significant.

Indirect Exposure via the Environment Indirect human exposure may occur via the environment. This may be the result of consumption of contaminated food and drinking water, inhalation of air, ingestion from soil and dermal contact. Essentially the assessment is carried out in three stages: •

assessment of the concentrations in intake media;

•

assessment of the intake rate from each medium; and

•

combination of the concentrations in the media with the intake of each medium (if necessary, using a factor to allow for bioavailability by that route).

It may be necessary to allow for bioconcentration (e.g., in fish and sea food) and transfer from air or soil to food (of plant or animal origin). These models are essentially the models used for ecotoxicological assessment, adapted for human exposure. Indirect exposure via the food supply is particularly important in terms of residues from biocides, plant protection products and veterinary products, and in terms of leaching from food packaging materials. Essentially the aim is to ensure that the maximum amount of residue likely to be found in a foodstuff is sufficiently low that the acceptable daily intake for the chemical is not exceeded. Generally this is based on residue studies or leachate studies, combined with the use of standard assumptions on the content of the food basket.

7.4 Judgements In an ideal world the risk characterisation (the ‘science’) for a toxic risk would be clearly separated from the risk evaluation. The latter includes social and political judgements as well as scientific judgements. Often, the procedures employed in risk management do not clearly differentiate between risk characterisation and risk evaluation.

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7.4.1 The ‘Precautionary Principle’ The UK Department of the Environment ‘Guide to Risk Assessment and Risk Management for Environmental Protection’ [23] stated the precautionary principle as: ‘Where there are significant risks of damage to the environment the Government will be prepared to take precautionary action to limit the use of potentially dangerous materials or the spread of potentially dangerous pollutants, even when scientific knowledge is not conclusive, if the likely balance of costs and benefits justifies it’. The UK Government’s ‘Sustainable Development Strategy’ argued that: ‘When potential damage to the environment is both uncertain and significant, it is necessary to act on the basis of the precautionary principle.’ This principle can easily be adapted to cover health effects. Taken a little more broadly, a ‘precautionary’ approach can also be applied to decide on whether there is a need for further testing. The nub of the principle is the balance of costs and benefits. A standard question in examining any chemical for possible health effects is, ‘Is the chemical a carcinogen?’ Application of a ‘hard’ precautionary approach would be to say ‘carcinogenicity is unproven, but unlikely – nevertheless, because there is a small benefit in reducing the uncertainty by conducting carcinogenicity assays in up to two species, even though it is expensive and involves use of animals, it should be undertaken’. On the other hand a much softer approach would be to say ‘we think the substance is unlikely to be a carcinogen and we think the expense (in terms of money and of use of animals in research) does not justify undertaking the study’. Softer decisions tend to be more likely when the same organisation judges the need for the test and funds the testing or when a chemical is already in use! Regulatory authorities concerned with granting licences (or some other form of permission) to use a chemical for some purposes, have the power to refuse a licence unless the intending licensee conducts (or funds) a test they request. The regulatory bodies undertaking these judgements on behalf of society are sometimes called ‘gate-keepers’. They are seeking to prevent failure to control the relevant use of the chemical adequately. Costs of failure to control are all too visible but benefits foregone through failure to licence can rarely be quantified, so a harder precautionary approach is preferred, with decisions skewed towards requiring further testing and/or refusal to grant the licence. As this is likely to be associated with the extreme scenarios associated with equity based criteria for judging risks, often the judgements associated with licensing systems are essentially ‘conservative’.

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7.4.2 What Test and When? One important question in examining human health risks is what tests should be conducted? The second question is when should a test be conducted? This depends on the intended use of the substance. Regulatory schemes will require different groupings of tests. Most have some form of phasing of the testing. For example, the requirements concerning the initial testing for new substances are outlined in Annex VII to Directive 67/548/EEC (Schedule 2 of [24]) and those for further testing are in Annex VIII of the Directive (Schedule 3 of [24]). These requirements depend on the type of chemical and the amount placed on the market. A more detailed rationale concerning when to conduct a test or group of tests is provided in the technical guidance [8]. Other regulatory schemes have different requirements and rationales. Phasing of testing for biocides, for example, is based, at least in part, on the stage of development. As detailed information for the different regulatory schemes is contained in Part 2 of this book, it will not be presented here. These are important questions as both ethics and costs are involved. Ethical considerations include those associated with the protection of human health as well as those concerning the testing of chemicals in animals. Also, if the costs are excessive when compared to the potential benefit (return to the Company), a company will be unable to develop the chemical for the intended use and society will have to forego the benefits that the chemical could have effected. Usually the individual regulatory scheme will specify at least an initial testing requirement and possible appropriate further testing and its timing. Individual schemes are examined in detail in Part 2 of this book.

7.4.3 The Interpretation of Toxicity Test Results for Classification and Labelling Purposes The criteria for classification and labelling substances and preparations for marketing in the European Union are becoming standardised across most of the regulatory schemes. The criteria are given in an Approved Code of Practice associated with the Chemicals (Hazard Information and Packaging for Supply) Regulations [12, 25]. It may be hazard identification alone or it may include some attempt at hazard characterisation. When only quality of evidence or a single trigger is used to classify the properties of a chemical then the classification is pure hazard identification. When the categorisation depends also on level of exposure, the classification includes some attempt at describing the dose (concentration)-effect relationship. In the case of acute and repeated dose toxicity data the classification into ‘harmful’, ‘toxic’ and ‘very toxic’ is on the basis of the dose levels at which serious effects or deaths occur. For irritancy there are two classifications, based on severity of effect. However, for skin sensitisation a single yes/no criterion is used, so the classification is based on hazard identification.

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7.4.4 Risk Assessment and Risk Evaluation – Interpretation of General Toxicity Essentially the two ways of handling risk assessment and evaluation for general toxicity are the ‘uncertainty factors’ approach used for limit setting and the ‘margin of exposure’ approach. The former is set out in a WHO document [11] and, more recently, in a UK document [7], both are described in [1]. Perhaps the most important piece of information used when determining a ‘safe’ level of risk for an equity based judgement is the no observed (observable) adverse effect level (NOAEL). Whichever approach is used there is a need to identify the critical effect and the pivotal study. In the uncertainty factors approach, the critical effect is the effect yielding the lowest value for the maximum acceptable exposure level (reference dose); in the margin of exposure approach, it is the value for the margin of exposure considered least acceptable. The pivotal study is the study from which the critical NOAEL or related measure is derived. In the uncertainty factors approach the maximum acceptable exposure (the reference dose or RfD) is calculated using the equation:

RfD =

NOAEL Uncertainty and modifying factors

If the RfD is higher than the predicted or measured exposure the exposure is acceptable. If the RfD is about the same as or lower than the predicted or measured exposure levels then decisions will be required. These may be measures to refine the data or to manage the risk (e.g., banning, restricted sale, limitation of exposure by process engineering, use of protective equipment, etc.). This procedure uses standardised factors for adjusting the NOAEL to a value considered the maximum exposure ‘without risk to health’. Uncertainty factors make allowance for interspecies variation (usually 10), inter-individual variation (usually 10), severity of effect (up to 10), duration of exposure (e.g., conversion of a NOAEL from a 28-day repeated dose study to one for a 90-day study; variable), lowest observable adverse effect level (LOAEL) to NOAEL (3, 5 or 10) and incomplete database. Modifying factors are introduced to cover the professional assessment of the scientific uncertainties of the study and the database, not explicitly included in the uncertainty factors. Judgement is required in deciding on the appropriate type and size of the factors employed when evaluating a particular chemical and use. The process is outlined in Figure 7.5. In this approach the risk characterisation and risk evaluation are not differentiated and the modifying factors

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Notes: 1. The IPCS divides interspecies and interindividual variation into variation due to toxicokinetics (what the body does to the chemical) and toxicodynamics (what the chemical does to the body). For each of inter-species and inter-individual variation, multiplication of the default factors for toxicokinetics and toxicodynamics together yields the anticipated default uncertainty factors (for a ‘safe’ level of exposure) of 10. If there is suitable information on the type of variation being encountered, it can be substituted for the default uncertainty factors. 2. It may be necessary to examine several effects successively, hence the inclusion of loops to allow for this possibility, either after having carried out an initial examination of the animal data or after having overviewed all the available data (based on information from IPCS, 1994).

Figure 7.5 Derivation of overall uncertainty factors

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Toxicological Assessment Within A Risk Assessment Framework include an assessment of the societal judgement concerning the acceptability of the standard. The second approach is the ‘margin of exposure’ or ‘margin of safety’ approach. The margin of exposure for a particular type of exposure is given by the equation:

Margin of exposure =

NOAEL (or LOAEL or BMD) Exposure level (measured or predicted)

(LOAEL and bench mark dose (BMD) are alternatives to the NOAEL. LOAEL is an alternative sometimes used when a NOAEL has not been determined; BMD is a measure based on the statistical confidence limits associated with the dose-response curve). This approach separates the risk characterisation (as represented by the calculation of the margin of exposure) from the risk evaluation (the decision concerning the adequacy of the margin of exposure). Whether the ‘margin of exposure’ is adequate can be addressed by examining if it is greater than or less than the appropriate combination of uncertainty factors. The best result is if the factor obtained as the ‘margin of exposure’ is greater than the uncertainty factors would have been. The traditional default value for the overall uncertainty factor (or margin of exposure/ safety) for a ‘safe’ exposure is 100 (10 for inter-species variation and 10 for inter-individual variation), based on good animal studies. Lehman and Fitzhugh [26] first applied it in 1954 for food chemicals risk assessment. It is widely used [7, 15, 17], although the uncertainty factors applicable to worker exposure are often lower (e.g., [11, 16, 27]). If one is dealing with utility/technology based criteria the uncertainty factor may be smaller, but there will be management measures aimed at reducing exposure to the ‘safe’ level, or at least ‘as low as is reasonably practicable’. This approach is closely related to the approach used for environmental risk assessment given in the next chapter. There the predicted exposure concentration (PEC) is divided by the predicted no effect concentration (PNEC) to find a risk characterisation ratio (RCR). The calculation of the PNEC includes allowance for ‘uncertainty factors’ and RCRs of greater than 1 are regarded as requiring further assessment.

7.4.5 Mutagenicity, Carcinogenicity and Reproductive Toxicity A mutagen is a genotoxic chemical, i.e., it causes damage to the genetic information in somatic cells or in germ cells. If the effect is on somatic cells, the possible consequence is cancer; if it occurs in a germ cell it may cause heritable genetic damage. No individual

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Practical Guide to Chemical Safety Testing assay is completely reliable so batteries of tests have been developed, together with rationales concerning which test to undertake and when. One example of this approach is that in the EC technical guidance document [8]. Classification as a mutagen is based on weight of evidence. Essentially, human evidence is required for the highest category (category 1); good evidence from testing is required for category 2 and less satisfactory evidence is required for category 3. Risk management depends on potential outcome; if the genotoxic event occurs in somatic cells this is part of the evidence that a substance could be a genotoxic carcinogen; if it occurs in germ cells it is part of the evidence that the substance is a reproductive toxin. Carcinogenic chemicals are identified and classified on the basis of the weight of evidence concerning whether the substance causes cancer or not. The quality and nature of the evidence determines the category of the classification, not potency (see e.g., [12]). Different bodies have subtly different definitions for their categorisations. Essentially, human evidence is required for the highest category (category 1); good animal evidence is required for category 2 and less satisfactory evidence is required for category 3. Potency is not formally examined, although establishing that a chemical is carcinogenic is likely to be easier for more potent carcinogens. The International Agency for Cancer Research (IARC) has also set up an important internationally accepted scheme for classification of chemicals as carcinogens. Once it has been decided that the substance is a carcinogen, there is a distinction between genotoxic and non-genotoxic that has also to be established. Thus, for risk management purposes, carcinogens can be divided into two categories: genotoxic and non genotoxic. A genotoxic carcinogen is one that is carcinogenic and that also gives positive results in genotoxicity tests and whose mechanism of carcinogenesis involves mutagenesis as a key initial event [28]. For these chemicals it is theoretically possible that ‘one hit’ in DNA may lead to clonal transformation of somatic (or germ) cells, eventually resulting in a malignant tumour. It is not possible, in practice, to identify a threshold level below which no effect would be expected. Thus, UK practice is to consider it prudent to assume that there is no threshold dose below which no carcinogenic effects occur. The policy generally adopted for this type of carcinogen is to eliminate exposure where possible, or, when this is not possible, to ensure that they are as low as is reasonably practicable. Thus UK risk management is based on hazard identification, with little, if any, consideration of potency. US Practice for risk assessment of genotoxic carcinogens does involve potency (see [1]). Mathematical models are used to extrapolate from the very high doses used in animal bioassays in order to estimate probable responses at low doses. Originally the ‘linearised multistage model’ was used, but, more recently, linear extrapolation has been preferred. In both cases, extrapolation is from the ‘upper bound’ 95% confidence limit. This

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Toxicological Assessment Within A Risk Assessment Framework extrapolation is used to estimate the likely risk of cancer at low exposure levels or to predict the level of exposure over a lifetime that would give rise to an increase in the incidence of cancer of one person in a million (106). In both UK and US practice, non-genotoxic carcinogens are believed to induce tumours as a secondary event following an effect that has a threshold, such as sustained cell proliferation, enzyme induction, etc. [28]. In US practice in particular, heavy emphasis is placed on understanding their mode of action. Risk assessment is based on the no-effect level for a key precursor event. These carcinogens are therefore dealt with in the same way as conventional toxic effects. However, the circumstances are such that the ten-fold additional factor for ‘serious effect’ is invoked and the uncertainty factor is based on 1000 rather than 100. Either a ‘safe’ dose is calculated using the uncertainty factors, or a judgement is made concerning the adequacy of the ‘margin of exposure’. Effects on reproduction can be divided into two parts: effects on fertility, which embraces gametogenesis, oestrous cyclicity, copulation and fertility and effects on the development of the offspring [12, 18]. The latter include embryotoxicity/foetotoxicity (reduced body weight, growth and developmental retardation), organ toxicity, death, abortion, structural defects (terata), functional defects, peri-postnatal defects and impaired postnatal mental or physical development up to and including puberty. The classification scheme for these effects is also based on weight of evidence, without formal consideration of potency. Generally toxic effects on reproduction are treated as effects amenable to use of the uncertainty factors approach. Toxicity to reproduction is viewed in relationship to general, non-specific toxicity. Unless there is evidence that terata (birth defects) are the consequence of mutagenic events, they are normally considered serious effects for which there is a threshold. Teratogens tend to be treated in the same way as non-genotoxic carcinogens. The uncertainty factors approach is considered acceptable and the serious nature of the effects means that the uncertainty factor is based on a value of 1000 rather than 100. If there is evidence of germ cell mutagenicity then the effects will be treated in the same way as genotoxic cancers.

7.5 Risk Management The intention of any risk assessment is to inform risk management. Management of the risks from individual substances and preparations can take several forms. Firstly there are the two extremes – complete banning, or unfettered sale and use. In between are a number of possibilities. These include substitution of the chemical, engineering control of the process and protective equipment for the operator during manufacture and use. They also include licensing of products, granting authority to purchase only to certain

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Practical Guide to Chemical Safety Testing groups of people or outlets, (and hence restricting use), and passing information to users so that they may either carry out their own risk assessments and take appropriate safety precautions or take specified safety precautions. Monitoring (that the risk management proposals are effective and are adhered to) and enforcement (taking sanctions when suppliers and/or users do not adhere to risk management procedures) are also required for successful risk management.

7.6 Final Word Toxicological risk assessment and management is never finished. Some form of monitoring (post marketing surveillance) and enforcement is always required. Also, science moves on and it may become necessary to re-interpret data in the light of new evidence and new understandings. The work is never finished!

References 1.

P. Illing, Toxicity and risk: Context, principles and practice, Taylor and Francis, London, 2001.

2.

P. Lewalle, Terminology, Standardisation and Harmonisation, 1999, 11, 1-28.

3.

Presidential/Congressional Commission on Risk Assessment and Risk Management, Framework for environmental health risk management. Final Report, Presidential/Congressional Commission on Risk Assessment and Risk Management, Washington, DC, 1997.

4.

J.J. Cohrssen, V.T. Covello, Risk analysis: A guide to the principles and methods for analysing health and environmental risks, Council on Environmental Quality, Executive Office of the President, Springfield, VA: National Technical Information Service, 1989.

5.

Royal Society Study Group, Risk Assessment, Royal Society, London, 1983.

6.

National Research Council, Risk assessment in the Federal Government: Managing the process, Report by the Committee on the Institutional Means for Assessment of Risks to Public Health, Commission on Life Sciences, National Academy Press, Washington, DC, 1983.

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Toxicological Assessment Within A Risk Assessment Framework 7.

Government/Research Council Initiative on Risk Assessment and Toxicology, Risk assessment approaches used by UK Government for evaluating human health effects of chemicals, Institute for Environment and Health, Leicester, 1999.

8.

European Commission, Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No. 1488/94 on Risk Assessment for Existing Substances. Part 1, Office for Official Publications of the European Communities, Luxembourg, 1996 and 2003 partial update and revision.

9.

Health and Safety Executive, Reducing risks, protecting people, HSE Books, Sudbury, Suffolk, 2001.

10. H.P.A. Illing, Regulatory Toxicol. Pharmacol., 1999, 29, 300-308. 11. IPCS, Assessing human health risks of chemicals: Derivation of guidance values for health based exposure limits, Environmental Health Criteria 170, World Health Organisation, Geneva, 1994. 12. Health and Safety Commission, Approved Guide to the Classification and Labelling of Substances and Preparations Dangerous for Supply. Fifth Edition. Sudbury, Suffolk: HSE Books, Sudbury, Suffolk, 2002. 13. Control of Lead at Work Regulations 2002, SI 2002/2677, The Stationary Office, London. 14. Council Directive 98/24/EC of 7 April 1998, Official Journal of the European Communities, 5.5.98, L131, 11. 15. Council Directive 98/8/EC of 16 February 1998, Official Journal of the European Communities, 24.4.98, L123, 1. 16. H.P.A. Illing, Ann. Occup. Hyg., 1991 35, 569. 17. EU Scientific Committee for Toxicity, Ecotoxicity and the Environment, Position paper on Margins of Safety (MOS) in human health risk assessment expressed at the 22nd CSTEE plenary meeting, Brussels, 6-7 March 2001. 18. Health and Safety Executive, Risk Assessment of Notified New Substances, HSE Books, Sudbury, Suffolk, 1994. 19. Pesticides Safety Directorate, UK Predictive Operator Exposure Model (POEM), Estimation of exposure and absorption of pesticides by spray operators, Pesticides Safety Directorate, York, 1986.

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Practical Guide to Chemical Safety Testing 20. Pesticides Safety Directorate, UK Predictive Operator Exposure Model (POEM), A user’s guide, Pesticides Safety Directorate, York, 1992. 21. ECETOC, Assessment of non-occupational exposure to chemicals, Technical Report No. 58, European Centre for Ecotoxicity and Toxicity, Brussels, 1994. 22. ECETOC, Exposure assessment in the context of the EU Technical Guidance Documents on risk assessment of substance, ECETOC Document No. 35, European Centre for Ecotoxicity and Toxicity, Brussels, 1997. 23. Department of the Environment, A guide to risk assessment and risk management for environmental protection, Her Majesty’s Stationary Office, London, 1985. 24. Notification of New Substances Regulations 1993, SI 1993/3050, The Stationary Office, London. 25. Chemicals (Hazardous Information and Packaging for Supply) Regulations 2002, SI 2002/1689, The Stationary Office, London. 26. A.J. Lehman, O.G. Fitzhugh, Q. Bull. Assoc. Food Drug Officials, 1954, 18, 33. 27. S. Fairhurst, Ann. Occup. Hyg., 1995, 39, 375. 28. Interdepartmental Group on Health Risks from Chemicals, Assessment of chemical carcinogens: Background to general principles of a weight of evidence approach, Institute for Environment and Health, Leicester, 2002.

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8

Environmental Risk Assessment Robert Diderich

8.1 Introduction Environmental risk assessment has become an integral part of regulatory decision making regarding the marketing of chemical substances. A brief overview of the environmental risk assessment methodology is outlined in the following sections. The current procedure being used in the European Union for decision making on new and existing industrial substances, as well as biocidal active substances is described to illustrate general principles. The main aspects of environmental risk assessment will be addressed. The decision scheme based on the risk assessment is briefly described. The principles of risk assessment are described in detail in Chapter 7, but reiterated to some extent in this section. The aim of legislative risk assessment schemes is to prevent unacceptable effects of chemical substances upon the environment. Under the prescribed use of a substance, the amount of the substance which is released to the environment must not have any adverse effects upon the ecosystems which constitute the environment. The process, by which the potential adverse effects upon the environment due to the use of a chemical substance are estimated, is called risk assessment. In more technical terms, the environmental risk assessment of chemical substances can be defined as the process comprising the following steps: hazard identification, effects assessment, exposure assessment and risk characterisation [1], [2]. The definitions listed in Table 8.1, as proposed by a joint working party of the International Program on Chemical Safety / Organisation for Economic Cooperation and Development (IPCS/OECD), apply to each step of the risk assessment process [3]. The risk assessment should ideally be carried out for all environmental target compartments, into which a substance is emitted or transported. In practice, many risk assessment schemes are limited to a number of protection goals, for example [2]: •

the aquatic ecosystem (including the sediment as well as marine ecosystems);

•

the terrestrial ecosystem;

•

top predators;

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Table 8.1 Definitions of the different steps (comprised) in an environmental risk assessment [3] Hazard identification consists of the determination of substances of concern, and the adverse effects they may have inherently on target systems under certain conditions of exposure, taking into account toxicity data. Dose (concentration) – response (effect) assessment is the analysis of the relationship between the total amount of a substance absorbed by a group of organisms and the changes developed in the group in reaction to the substance. Exposure assessment is the quantitative and qualitative analysis of the presence of a substance (including its derivatives) that may be present in a given environment. Risk characterisation is the qualitative and/or quantitative estimation, including attendant uncertainties, of the severity and probability of occurrence of known and potential adverse effects of a substance in a given population.

•

microorganisms in sewage treatment systems;

•

the atmosphere.

The general principles of environmental risk assessment apply to all types of chemical substances.

8.2 Exposure Assessment Ideally, the exposure assessment should be based on extensive and representative analytical monitoring results from all environmental compartments. In practice, however, sufficient monitoring results are very rarely available and usually the exposure assessment is performed with the help of estimation methods. Environmental exposure assessment consists of the following four steps: •

identification of the target compartments;

•

estimation of emissions or releases;

•

estimation of the environmental behaviour;

•

estimation of environmental concentrations.

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8.2.1 Identification of the Target Compartments All the possible releases to the environment resulting from the use of a substance need to be identified. This is done systematically for all life-cycle stages of the substance (cradle to grave approach). The relevant life-cycle stages are illustrated in Figure 8.1. After chemical synthesis of the substance it can be used – either directly or after formulation with other substances – in an industrial or private setting as a processing aid or for the treatment of articles. Furthermore releases to the environment are possible during the service life of the treated articles, as well as during elimination or recycling of the substance or the treated articles at the end of their life.

Figure 8.1 Schematic representation of the life cycle of a substance [2]

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Practical Guide to Chemical Safety Testing In Figure 8.2, a classical example for a local industrial setting is presented, which helps identify the release pathways and the receiving environmental compartments. According to the example in Figure 8.2, first the local releases of a substance to air and waste water are estimated or measured. Following its behaviour in the sewage treatment plant (STP), air, soil and surface water, the local concentrations can be estimated in surface water, sediment, air, soil and groundwater in the immediate vicinity of the industrial site. Soil (agricultural soil or prairie) is supposed to receive input from atmospheric deposition and sewage sludge applied as fertiliser. Exactly the same settings can be used for substances used on a private or domestic basis, by replacing the industrial site with a town releasing its waste water to a single STP. Other release and transport pathways can also be relevant.

Note: Prairie represents pasture and/or farmland

Figure 8.2 Release pathways and receiving environmental compartments from local releases of an industrial site [2]

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8.2.2 Estimation of Emissions or Releases In a second step, the releases to air, soil and waste water need to be quantified for all previously identified release pathways. The quantity of a substance released to an environmental compartment depends on its physico-chemical properties, but mainly on the technological process involved in the use of the substance. For specific technological processes, so-called ‘emission scenarios’ can be developed. An emission scenario can be defined as a set of conditions about sources and use patterns that quantify the emissions (or releases) of a chemical [4, 5]. These generic scenarios are meant to be representative for a certain operation and therefore applicable to every site (industrial or other) where this operation is performed. Since the processing conditions (for example the quantity of treated material) vary from site to site, they can be chosen to estimate an emission that: •

is likely to exceed actual emissions (a ‘bounding’ or ‘worst-case’ estimate),

•

is representative of the ‘high end’ of actual releases (a ‘reasonable worst-case’ estimate, the 90th percentile is often used),

•

is representative of ‘typical’ exposures, or

•

covers the complete set of actual release values resulting from those conditions.

The ‘representative high end’ or ‘reasonable worst-case’ approach is most often used in regulatory risk assessment. Many international activities aim at elaborating comprehensive and representative emission scenario documents. At OECD-level, a programme has been launched, based on national documents, to develop emission scenarios, which are as representative as possible regarding use patterns and technologies in OECD countries, and which can be used for internationally valid risk assessments. In the European Union, emission scenarios for industrial chemical substances as well as for biocidal substances are continuously developed and published in the Technical Guidance Document (TGD) for risk assessment of new and existing industrial chemicals as well as active biocidal substances [2]. A simplified example of an emission scenario is presented below. Cooling water circuits need to be treated periodically or continuously with anti-fouling products and corrosion inhibitors. For once-through systems (see Figure 8.3), the release to surface water can be estimated as shown in Table 8.2 and the following calculation (Equation 8.1). Model calculation: Elocalwater = Qcooling_water * Qactive * (1 – Fdeg)

(Eq 8.1)

The application rate of the injected substances is substance specific and will be known to the assessor from their marketing specifications. The flow rate of the cooling water is

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Table 8.2 Input and output parameters for a release scenario for biocidal substances used in once-through cooling systems Variable/parameter (unit)

Symbol

Input: Application rate of a substance

Qactive

kg.m-3

Flow of the cooling water

Qcooling_water

m3.d-1

Fraction of substance degraded during the treatment

Fdeg

Output: Local emission with the spent cooling water

Elocalwater

Unit

Default

-

0

kg.d-1

Figure 8.3 Schematic representation of a once-through cooling system [6]

site dependent. In order to choose a representative default value for this parameter, statistical data regarding the use of cooling water in industry would need to be available.

8.2.3 Distribution and Degradation in the Environment (Environmental Fate) Substances which reach the environment can transfer to other compartments or subcompartments of the initial target compartment. Different degradation mechanisms can occur in any of these compartments.

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8.2.3.1 Distribution or Transport in the Environment Substances can be distributed within an environmental compartment (intra-media transport) or be transported from one environmental medium to another (inter-media transport). The two main transport mechanisms are advection and dispersion which can be defined as: •

advection: transport of a substance from one place to another as a result of the flow of the medium in which it occurs (for example transport with infiltrating rain water from soil to groundwater);

•

dispersion: transport from one place to another as a result of differences between concentration gradients (for example, diffusion from surface water into sediment).

Dispersion also occurs within compartments if they consist of more than one phase (for example, soil or sediment). Substances tend to migrate from one phase to another if the phases are not in equilibrium. At equilibrium, the ratio between the concentrations in two phases is constant if the concentrations are sufficiently low. This ratio is called the ‘equilibrium partitioning coefficient’ between the two phases. If the concentration in one phase is known, the concentration in the other phase at equilibrium can be estimated with the corresponding equilibrium partitioning coefficient. A brief summary of the most important partition coefficients used in environmental risk assessment is presented in Table 8.3.

Table 8.3 Examples of equilibrium partitioning coefficients for modelling the environmental distribution of a substance Partition coefficient

Examples of use in an exposure assessment

Air/water

volatilisation rate from surface water stripping rate from sewage treatment plants wet atmospheric deposition

Solids/water

adsorption onto soil, sediment, suspended matter and sewage sludge leaching rate from soil to groundwater burial rate in sediment

Air/solids

adsorption onto aerosols in the atmosphere volatilisation rate from dry soil dry atmospheric deposition

Air/biota

accumulation in plants via air

Water/biota

accumulation in aquatic biota (fish, crustaceans) accumulation in roots of plants accumulation in terrestrial invertebrates

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Practical Guide to Chemical Safety Testing Some simple estimation methods are described below for some equilibrium partitioning coefficients. More detailed estimation methods can be found, for example in [2, 7].

Air:Water Partition Coefficient The air:water partition coefficient or the Henry’s law constant can be estimated from the respective water solubility and vapour pressure (Equations 8.2 and 8.3). HENRY =

VP ⋅ MW SOL

(Eq 8.2)

K air − water =

HENRY R⋅T

(Eq 8.3)

where: VP is vapour pressure [Pa] MW is molecular weight [g.mol-1] SOL is water solubility [mg.l-1] R is the gas constant [Pa.m3.mol-1.k-1] T is the temperature at the air-water interface [K] HENRY is the Henry’s law constant [Pa.m3.mol-1] Kair-water is the air:water partitioning coefficient Other estimation methods, for example based on the structure of the substance, can also be used [8, 9].

Solids:Water Partition Coefficient The estimation of the distribution between solids and water in the environment is often based on the organic carbon:water partitioning coefficient Koc. Internationally harmonised laboratory methods are available for measuring the Koc in soil and sediment [10]. The laboratory methods consist of batch techniques where amounts of soil or sediment are added to an aqueous solution containing the substance. After agitation and separation of the phases the concentration of the substance is measured in one or two phases. Using the Koc for estimating the solids:water partitioning coefficient is most appropriate for neutral, hydrophobic organic substances. Nevertheless, this approach has been applied

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Environmental Risk Assessment to a wide variety of substances as it works reasonably well. Based on the Koc, the solids:water partitioning coefficient can be estimated for different compartments (Equation 8.4). Kpcomp = foccomp * Koc

(Eq 8.4)

where: Kpcomp is the solids:water equilibrium partitioning coefficient [l.kg-1] and comp represents soil, sediment, suspended matter, sewage sludge foccomp is the organic carbon content of the medium Koc is the organic carbon:water equilibrium partitioning coefficient [l.kg-1] The Koc can also be estimated with quantitative structure-activity relationships [QSARs]. Most developed QSARs establish a relationship between the Koc and the n-octanol:water partitioning coefficient Pow. The experimental determination of the Pow is indeed required in most chemical substances assessment schemes. An overview of estimation methods can be found in [2, 7]. For example, regression models to estimate log Koc from log Pow, valid for a wide variety of substances as described in [11, 12], are presented below (Equations 8.5 and 8.6). Wide variety of substances

log Koc = 0.679 log Pow + 0.663

Non-hydrophobic substances log Koc = 0.52 log Pow + 1.02

(Eq 8.5) (Eq 8.6)

For specific classes of chemical substances, for example alcohols, amides and anilines, more appropriate relationships have been derived [7]. For substances interacting specifically with the solids, for example with the mineral fraction, a more specific correlation between the substance and its affinity to the solids would need to be derived. For example, for ionisable substances, a correlation has been developed to directly estimate the soil:water partitioning coefficient Kpsoil using log Pow, pKa, soil pH and the organic carbon content of the soil (focsoil) [13].

Water:Biota Partition Coefficient The most relevant biota:water partitioning coefficient is the water:fish partitioning coefficient or fish bioconcentration factor (BCF), the ratio of the substance concentration in an organism to the concentration in water. Based on the concentration in water, the concentration in fish can be estimated and hence the exposure to potential predators, as for example fish-eating birds as well as humans. Internationally agreed laboratory methods are available to experimentally determine the BCF in fish [14].

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Practical Guide to Chemical Safety Testing Many estimation methods based on QSARs assume that chemicals reach an equilibrium between the organisms and water and ignore dietary uptake. They establish a relationship between the lipophilicity of a substance (expressed as its Pow) and its accumulation in the fat of aquatic organisms. For substances with a log Pow < 6, similar linear regression models between log Pow and log BCF have been proposed by many authors [15-17]. The model recommended in [2] and developed by [15] on BCF data for fathead minnows (Pimephales promelas) is presented in Equation 8.7. log BCF = 0.85 log Pow – 0.7

(Eq 8.7)

For substances with a log Pow > 6, experimental BCF results indicate that linear regression models do not apply anymore as they tend to overestimate the BCF. This could on one hand be explained by experimental shortcomings, for example insufficiently long test duration as steady-state is achieved very slowly with very hydrophobic substances, which are eliminated very slowly from organisms. On the other hand, higher molecular weight and size of very hydrophobic substances could cause slow membrane permeation kinetics and thereby low BCFs. Bilinear, parabolic or polynomial regression models have been proposed by several authors [18-21].

8.2.3.2 Degradation Biodegradation, hydrolysis and direct or indirect photodegradation are the most relevant degradation processes for an exposure assessment. A substance is first degraded or transformed into primary degradation products or metabolites (primary degradation) which themselves can be further degraded. Some transient transformation products will appear for a very short duration only while others will be degraded much slower than the parent compound. Some chemical substances review schemes request that all relevant transformation products are identified in laboratory tests on hydrolysis, photodegradation as well as biodegradation simulation tests. In practice, the most stable breakdown products are assessed. The most relevant degradation rates to ascertain for the exposure assessment are the biodegradation rate in sewage treatment plants (STPs), surface water, sediment and soil. A tiered approach can be proposed. For example, a testing strategy for biodegradability elaborated for the implementation of the EU Biocidal Products Directive [22] is presented in Figure 8.4 [23]. Substances are usually first tested in screening assays on ‘ready biodegradation’. These are very stringent tests with a high substance/inoculum ratio and the test substance as sole carbon source. They provide limited opportunity for biodegradation and acclimatisation

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Notes (1) Kpsludge : solids:water equilibrium partitioning coefficient in sewage sludge (2) Kpsed : solids:water equilibrium partitioning coefficient in sediment

Figure 8.4 Testing strategy for establishing the biodegradation behaviour of biocidal active substances in different environmental compartments [22] to occur. Harmonised international test guidelines have been published by the OECD (OECD Guidelines 301 A – F [24]). A readily degradable substance will rapidly degrade to water and carbon dioxide in the environment and the probability of the formation of stable metabolites in the environment is very low. Half-lives in the different environmental compartments have been proposed in [2] for ‘readily biodegradable’ substances as shown in Table 8.4. For soil and sediment, the degradation rates are dependent on the partitioning behaviour as the bioavailability of the substance will be reduced due to adsorption onto solids. Comparable half-lives have been proposed by [25].

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Table 8.4 Half-lives of readily degradable substances in different compartments assuming first order kinetics, as proposed by [2] Compartment Sewage treatment plant Surface water Soil and aerobic sediment

Half-life (DT50) function of the adsorption coefficient Kpsoil 1.4 hours

-

15 days

-

30 days 300 days 3000 days

Kpsoil ≤ 100 1/kg 100 < Kpsoil ≤ 1000 1/kg 1000 < Kpsoil ≤ 10000 1/kg

Substances failing the stringent tests on ‘ready biodegradation’ can then be tested in screening assays on ‘inherent biodegradation’. These assays (for example OECD Guidelines 302 A-C [24]) operate under optimised conditions with higher inoculum concentrations and/or longer test durations. For substances which do not degrade in these systems (neither ultimate nor primary degradation), it can be assumed that they are not degraded in the environment either. No further testing is needed. If they are degraded to some extent in these test systems, it is very difficult to extrapolate the results towards environmental compartments. Furthermore they do not predict identification or quantification of intermediate transformation products. Therefore, for substances, which are not ‘readily biodegradable’, but which degrade to some extent in test systems on ‘inherent biodegradation’, it is necessary to perform biodegradation tests which simulate environmental conditions at test concentrations close to those which can be expected in the environment. The following simulation test methods could be recommended if a preliminary risk assessment indicates a high risk potential for a given compartment: •

Sewage treatment plant: OECD Guideline 303A, ‘Simulation test – aerobic sewage treatment’ [26].

•

Surface water: ISO/DIS Guideline, ‘Evaluation of the aerobic biodegradability of organic compounds at low concentrations’ [27] [28].

•

Soil: OECD Guideline 307, ‘Aerobic and anaerobic transformation in soil’ [29].

•

Sediment: Draft OECD Guideline, ‘Aerobic and anaerobic transformation in aquatic sediment’ [30].

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8.2.4 Predicted Environmental Concentrations In the last step of the exposure assessment, the concentrations in the receiving compartments are estimated.

8.2.4.1 Surface Water Based on the releases estimated to waste water (see Section 8.2.2), the concentration in sewage water can be calculated. For a generic risk assessment, a default sewage water flow rate needs to be set. In [2], a value of 2000 m3.d-1 is proposed, which corresponds to the quantity of sewage water produced by a small town of approximately 10,000 inhabitants. As shown in Section 8.2.3.2, simulation tests will permit estimation of the elimination of a substance in a full size STP. For some substances, measurements in full size STPs will even have been performed, which will allow a realistic estimation of the behaviour of a substance in any biological STP. In the absence of any information, mathematical models can be used. These kind of models have been proposed [31, 32]. Based on simple properties of the substance (organic carbon:water partition coefficient, Henry’s law constant, degradation rate constant), the elimination by degradation, stripping and adsorption on to sewage sludge can be estimated. The fraction of the concentration of the substance which is not eliminated in the STP will be released with the effluent into the receiving surface water (river, lake, estuary, marine coastal area). In the immediate vicinity of the point of release, the processes which have the biggest influence on the resulting local water concentration are dilution and adsorption onto suspended matter (see Figure 8.5). Assuming that volatilisation and degradation will have little influence upon the concentration in the vicinity of the point of release, especially in rivers, where the effluent is rapidly transported downstream, a local dissolved concentration in the receiving surface water can be estimated according to Equation 8.8. PEClocal water =

Clocal eff (1 + Kpsusp ⋅ SUSPwater ⋅ 10 −6 ) ⋅ DILUTION

(Eq 8.8)

where: Clocaleff is the concentration of the chemical substance in the STP-effluent (mg.l-1) Kpsusp is the solids:water partitioning coefficient of suspended matter (l.kg-1) SUSPwater is the concentration of suspended matter in the receiving water (mg.l-1)

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Volatilisation STP

Dilution Degradation Aqueous phase

Suspended

particles Sedimentation/suspension

Figure 8.5 Transport and degradation processes, which influence the local concentration in surface water [2] DILUTION is the dilution factor PEClocalwater is the local concentration in surface water (mg.l-1) The corresponding concentration in sediment can then be estimated with the sediment:water equilibrium partitioning coefficient. Furthermore, the concentration in fish can be estimated with the BCF and thereby the exposure to fish-eating birds and mammals.

8.2.4.2 Air The air compartment can receive direct emissions from industrial sites or households, as well as inputs due to stripping from STPs. The local concentration in air can be estimated with simple mathematical models, assuming a Gaussian diffusion of the effluent plume along the wind direction, as shown in Figure 8.6. The concentration in the plume at a certain distance from the source can be used to estimate the risks to humans or animals living in the vicinity of industrial sites. The atmospheric deposition to soil can also be determined by estimating the aerosol:air partition coefficient and the gaseous and aerosol deposition rates. Estimation methods are described for example in [33-35].

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Figure 8.6 Gaussian distribution of a plume from a point source [34]

8.2.4.3 Soil At a minimum, inputs into soil which need to be assessed routinely are atmospheric deposition and application of sewage sludge as fertiliser. A simple model for the estimation of the concentration in the upper soil layer has been proposed by [35]. For continuous releases to soil (for example through atmospheric deposition or input of leachate from treated wood), the equilibrium concentration in soil can be estimated according to Equation 8.9. Elimination processes from the upper soil level can include volatilisation, degradation and leaching to groundwater (see Figure 8.7). PEClocal soil =

DEPtotal ann DEPTH soil ⋅ RHOsoil ⋅ k

(Eq 8.9)

where: PEClocalsoil is the predicted environmental concentration in soil (mg.kg-1) DEPtotalann is the annual average total deposition flux (mg.m-2.d-1) DEPTHsoil is the mixing depth of soil (m) RHOsoil is the bulk density of wet soil (kg.m-3) k is the first order rate constant for removal from top soil (d-1) For repeated non-continuous emissions (for example the spreading of sewage sludge onto agricultural land over several years), the build-up over the years has to be taken

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AIR

Volatilisation Air Leaching

SOIL

Degradation GROUNDWATER

Leaching

Partitioning

Interstitial water Solid fraction

Figure 8.7 Possible fate processes in the soil compartment [2]

into account. A corresponding calculation method is proposed in [2]. The concentration of a substance in groundwater can, in a very preliminary approach, be assumed to be equal to the concentration in interstitial water in the upper soil layer. More sophisticated models have been developed in the context of the risk assessment of pesticides [36-38]. These models can be used for refinement of the soil exposure assessment (concentration in the upper soil layer as well as in groundwater).

8.3 Effects Assessment As shown in Table 8.1, effects assessment is a two-step process consisting of hazard identification followed by concentrations-effects assessment. In practice, the effects assessment for the environment consists of establishing a predicted no effect concentration (PNEC) for each relevant environmental compartment. According to [2], ‘a PNEC is regarded as a concentration below which an unacceptable effect will most likely not occur’. PNECs can either be estimated by applying uncertainty factors to test result values, or by extrapolating a value with a statistical method applied to the distribution of all available test results.

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8.3.1 Estimating PNECs by Applying Uncertainty Factors The uncertainty factor approach is used most commonly. It can be applied to substances for which the available pool of data is very limited. For a given compartment, the PNEC can be extrapolated from results of laboratory assays performed on single species from that compartment. This approach assumes that: •

the sensitivity of the ecosystem depends on the most sensitive species,

•

the protection of the structure of the ecosystem ensures the protection of the functioning of the ecosystem.

From the available data pool for a given substance, the test results for the most sensitive species are chosen to calculate a PNEC. By protecting this species, it is considered that the function and the structure of the whole ecosystem are protected. The extrapolation from the effects upon one species towards an ecosystem can be achieved with uncertainty factors. For the choice of the appropriate extrapolation factor, the following uncertainties need to be addressed: •

the variation within a given laboratory and between different laboratories performing the assays;

•

intraspecies variations due to the physiological state of the individuals of the same species;

•

interspecies variations, that is the differences of sensitivities towards a substance of different species belonging to the ecosystem;

•

the extrapolation of the acute or short-term toxicity towards long-term or chronic toxicity. Sublethal effects can appear after a long exposure duration and endanger a population, without being detected over the short term;

•

the extrapolation of laboratory results to the field.

In view of these uncertainties, the size of the factor depends on the quantity and quality of information available for the ecosystem. If test results on the ecotoxicity of a substance towards many species belonging to different taxonomic groups and trophic levels are available, the uncertainty factor will be lower compared to a substance for which only few test results are available. The approach described above is comparable to the ‘uncertainty factor’ approach used for human health risk assessment as opposed to the ‘margin of safety’ approach (see

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Practical Guide to Chemical Safety Testing Chapter 7). They both apply a factor to the test data values to estimate a value considered the maximum exposure ‘without risk’.

8.3.1.1 Surface Water In Table 8.5, the scheme for establishing a PNEC for the aquatic ecosystem as proposed in [2] is presented. The PNEC is calculated by dividing the L(E)C50 or NOEC from the species most sensitive to the substance with the appropriate uncertainty factor. Other schemes with similar uncertainty factors have been proposed [39, 40].

Table 8.5 Uncertainty factors for establishing a PNEC for the aquatic compartment according to [2] Available information

Uncertainty factor

At least one short-term L(E)C50 from each of three trophic levels (fish, invertebrates and algae)

1000

One long-term NOEC (either fish or invertebrates)

100

Two long-term NOECs from species representing two trophic levels (fish and/or invertebrates and/or algae)

50

Long-term NOECs from at least three species (normally fish, invertebrates and algae) representing three trophic levels

10

Field data or data from model ecosystem

Reviewed on a case by case basis

8.3.1.2 Sediment A similar approach as for aquatic organisms can be adopted for the sediment or benthic compartment. Some differences need to be taken into account though. As benthic organisms can be exposed via interstitial water as well as through the ingestion of sediment, the pathways through which the test organisms are exposed are very relevant. In addition to choosing test organisms of different benthic taxa and life stages, it is necessary to choose species representing different habitats and feeding strategies, which are exposed to sediment-bound substances by different exposure pathways. Furthermore, long-term

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Environmental Risk Assessment tests with sublethal endpoints like reproduction, growth, emergence, sediment avoidance and burrowing activities are regarded as most relevant. In Table 8.6, extrapolation factors as proposed by [41] are presented. For most substances though, no experimental results with benthic organisms are available. In these cases, a preliminary PNEC for the benthic ecosystem can be estimated by the equilibrium partitioning method. This method uses the PNEC derived for the aquatic ecosystem and the sediment:water partitioning coefficient [42] [43]. It assumes that: •

Benthic organisms and water column organisms are equally sensitive to the substance.

•

The concentration of the substance in sediment, interstitial water and benthic organisms are at a thermodynamic equilibrium.

Based on these assumptions, a PNEC for the benthic ecosystem can be estimated according to Equation 8.10.

PNECsed =

K sed − water ⋅ PNEC aqua RHOsed

(Eq 8.10)

where: PNECaqua is the predicted no effect concentration in water (mg.m-3) RHOsed is the bulk density of wet sediment (kg.m-3) Ksed-water is the sediment:water equilibrium partitioning coefficient (m3.m-3) PNECsed is the predicted no effect concentration in sediment (mg.kg-1)

Table 8.6 Uncertainty factors for establishing a PNEC for the benthic compartment according to [41] Available information

Uncertainty factor

One long-term NOEC

100

Two long-term NOECs with species representing different living and feeding conditions

50

Three long-term NOECs with species representing different living and feeding conditions

10

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Practical Guide to Chemical Safety Testing

8.3.1.3 Soil The same uncertainty factor approach is also used for the terrestrial compartment. Table 8.7 presents the scheme for establishing a PNEC for the soil ecosystem [2]. The equilibrium partitioning method as described above for sediment can also be used for a screening assessment if no experimental results with soil organisms are available (Equation 8.11).

PNECsoil =

K soil − water ⋅ PNEC aqua RHOsoil

(Eq 8.11)

where: PNECaqua is the predicted no effect concentration in water (mg.m-3) RHOsoil is the bulk density of wet soil (kg.m-3] Ksoil-water is the soil:water equilibrium partitioning coefficient (m3.m-3) PNECsoil is the predicted no effect concentration in soil (mg.kg-1)

Table 8.7 Uncertainty factors for establishing a PNEC for the soil compartment [2] Available information

Uncertainty factor

L(E)C50 from short-term toxicity tests (for example plants, earthworms or microorganisms)

1000

NOEC from one long-term toxicity test (for example earthworms)

100

NOECs for additional long-term toxicity tests of two trophic levels

50

NOECs for additional long-term toxicity tests for three species of three trophic levels

10

Field data or data from model ecosystem

Reviewed on a case by case basis

8.3.2 The Statistical Extrapolation Method The statistical extrapolation method can apply to substances with a large data pool [41, 44, 45, 46]. It can be used for each compartment provided that the database is sufficiently 182

Environmental Risk Assessment representative of the ecosystem of each compartment. The main advantage of this method is that it uses the whole sensitivity distribution of species in an ecosystem and not only the most sensitive species. The uncertainty factor system invariably proposes to use a fixed factor independent of the number of available NOECs, be it 3 or 30, while the uncertainty in extrapolating a PNEC decreases with the number of species tested. The statistical method assumes that the NOECs observed with different species follow a log-logistic or log-normal distribution (see Figure 8.8). Based on the distribution, the PNEC can then be chosen as the concentration for which the probability of finding a NOEC lower than the PNEC is, for example, 0.05. To use this method, it is necessary that a series of NOECs from different species is available. If several NOECs are available for the same species, the geometric mean of these values could be used to avoid an overrepresentation of that species in the distribution. The minimum number of NOECs for different species from an ecosystem which need to be available to apply this method varies among authors. Four NOECs would be sufficient according to [47], but as many as 10 covering at least 8 taxonomic groups are recommended and the use of a further uncertainty factor between 1 and 5 can be required to address further uncertainties according to [41].

Figure 8.8 The logistic density function and estimation of the concentration at which the NOEC of no more than p% of the species within an ecosystem is exceeded [48] Reproduced from C.J. van Leeuwen and J.L.M. Hermens, (Eds.), Risk Assessment of Chemicals: An Introduction, 1995, page 227, Figure 6.35, with kind permission from Kluwer Academic Publishers

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8.4 Risk Characterisation The last step of the risk assessment process is the risk characterisation. PEC/PNEC ratios (risk characterisation ratios, RCRs) are calculated for all exposed compartments for all relevant uses and life-cycle stages. If all RCRs are lower than 1, it can be considered that the risk for the environment is acceptable. If one or more RCRs are higher than 1, the underlying data of the assessment needs to be re-evaluated for its influence upon the RCR. If there is a high probability that further test results or further information could improve the reliability of the risk assessment and lower the RCRs, further information can be requested as for example: •

long-term toxicity tests with aquatic organisms allowing the use of a lower uncertainty factor in deriving a PNEC for the aquatic ecosystem;

•

screening toxicity tests with soil organisms leading to the derivation of a more realistic PNEC for the soil ecosystem compared to the PNEC estimated by the equilibrium partitioning method;

•

measuring the concentration of the substance in effluents of representative sites;

•

an experimentally determined BCF in fish allowing a more realistic exposure assessment for predators compared to an exposure assessment based on a BCF estimated by QSARs;

Most risk assessments, whether on a screening or comprehensive level, contain many estimation steps. As indicated in Section 8.2.2, most estimation methods used in risk assessment are chosen to represent a ‘realistic worst case’ situation. By combining several ‘realistic worst case’ estimations, the result could become overly conservative and therefore lead to unrealistically high RCRs. By replacing some of the estimations by experimentally determined results, the risk assessment will become more realistic and the RCRs will usually be lowered. If the probability of reducing the RCRs below 1 with additional information is low, it can be concluded that the substance represents an unacceptable risk to the environment and risk reduction measures should be considered. The risk characterisation process is summarised in Figure 8.9.

8.5 Conclusion As shown in this Chapter, quantitative environmental risk assessment is a very comprehensive way to allow authorities, as well as producers and users of chemical 184

Environmental Risk Assessment

Figure 8.9 Schematic representation of the risk characterisation process

substances, to take risk management decisions. The main inconvenience of the risk assessment process is that it is very resource consuming. The in-depth risk assessment for a given substance can take months to several years, and in the meantime any regulatory decisions are postponed. Given the large number of substances on the market, this is only acceptable for substances which are difficult to assess due to a large database or a complex use pattern. Furthermore some uncertainties inherent to the process are difficult to assess. For example, for highly lipophilic substances, the extent of transfer through the food chain towards higher trophic levels and towards top predators is difficult to estimate reliably. Authorities are therefore also looking for alternatives to comprehensive risk assessments. One option was recently established by the United Nations Environment Program. For substances which are highly persistent, bioaccumulative and toxic, worldwide emission reduction is envisioned. While other alternatives might be used in the near future, the improvement of risk assessment methodologies will ensure that comprehensive risk assessments remain the best basis for risk management decisions.

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Environmental Risk Assessment 43. D.M. Di Toro, C.S. Zarba, D.J. Hansen, W.J. Berry, R.C. Schwarz, C.E. Cowan, S.P. Pavlou, H.E. Allen, N.A. Thomas and P.R. Paquin, Environ. Toxicol. Chem., 1991, 10, 1541. 44. T. Aldenberg and W. Slob, Ecotoxicol. Environ. Safety, 1993, 25, 48. 45. C. Wagner and H. Lokke, Water Res., 1990, 25, 1237. 46. T. Aldenberg and J.S. Jaworska, Ecotoxicol. Environ. Safety, 2000, 46, 1. 47. J. de Bruijn, T. Crommentuijn, C. van Leeuwen, E. van der Plassche, D. Sijm and M. van der Weiden, Environmental risk limits in the Netherlands. Part I. Procedure, RIVM report no. 601640001, Bilthoven, The Netherlands, 2000. 48. C.J. van Leeuwen, Ecotoxicological effects, in Risk Assessment of Chemicals, An Introduction, Eds., C. J. van Leeuwen and J.L.M. Hermens, Kluwer Academic Publishers, The Netherlands, 1995, 227.

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PART 2:

REGULATORY FRAMEWORK

EU Chemical Legislation

9

EU Chemical Legislation John M. Hislop

9.1 EU Legislation within the European Economic Area and Europe There are 15 countries in the European Union (EU), and Austria, Finland and Sweden are the most recent members. All the European Free Trade Association (EFTA) countries except Switzerland entered into an agreement with the EU to form the European Economic Area (EEA). This arrangement required these EFTA countries to harmonise their environmental legislation with that of the EU, by a scheduled deadline of 1 January 1995. Meanwhile Austria, Finland and Sweden left EFTA to join the EU on 1 January 1995, and only Iceland, Liechtenstein and Norway are part of the EEA but not the EU. Hence, for the purposes of notification of new chemical substances and chemical hazard communication, the 18 countries of the EEA constitute a single market. Chemical control in the EU is based on a network of legislation for hazard communication and safety assessment. This EU legislation is brought into force in individual Member States by national laws, regulations and administrative procedures and hence, although chemical control is fundamentally harmonised, there can be minor differences between countries. The EU scheme for notification of new chemical substances under the ‘Seventh Amendment’ Council Directive 92/32/EEC [1] permits certain specific choices in national implementation. Also, individual Competent Authorities have different administrative practices. There are two primary routes in the EU for introducing measures to restrict or ban dangerous substances: the European Commission has the right of initiative to make proposals for restrictions; and individual Member States are able to develop proposals for national measures, which must be notified to the Commission. The legislative framework for such proposals is provided by Council Directive 76/769/EEC, the Marketing and Use Directive [2], which harmonises Community measures to control the marketing and use of dangerous substances. The restrictions made under the Directive are listed in an accompanying annex that is subject to amendments by subsequent Directives. Examples of substances controlled by this route are polychlorinated biphenyls (PCBs), polychlorinated terphenyls (PCTs), asbestos, cadmium and carcinogens.

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Practical Guide to Chemical Safety Testing As discussed in more detail in Chapter 11 (and Chapter 12 for polymers), chemical control legislation in Switzerland is different to that of the EU, although in practice new chemical substances can normally be notified and approved for supply in Switzerland using the available EU information or, with only minimal extra testing, although separate submissions to the Swiss regulatory authorities are necessary. It is anticipated that in due course chemical legislation in Switzerland will be harmonised with the EU, but there does not seem to be any immediate likelihood of Switzerland joining the EU, or even the EEA, although the country is still part of EFTA. Finally, the situation in the Central and Eastern European countries should be noted, and their tremendous challenges in transforming to market-based economies, including solving environmental problems. Generally chemicals are not currently well regulated in these states, although some countries have compulsory certification or registration procedures. Several countries have applied for EU membership, however, and have entered into Association Agreements which allow for existing chemical control measures to be harmonised with the EU.

9.2 Notification of New Substances Council Directive 92/32/EEC [1], which is the ‘Seventh Amendment’ of the ‘Dangerous Substances Directive’ (DSD), Council Directive 67/548/EC, requires pre-marketing notification of new chemical substances, and classification, packaging and labelling according to the degree of hazard. This notification ensures that sufficient information is available to enable the hazard to be assessed, the appropriate warning label to be assigned and any necessary control measures to be taken. Chemicals controlled under separate EU legislation, such as plant protection products, biocidal products and cosmetics, are exempt from notification under 92/32/EEC, as are ‘existing’ chemical substances. Existing substances are defined as those listed on the European Inventory of Existing Commercial Chemical Substances (EINECS) [3], a list of approximately 100,000 substances reported as being supplied during the reporting period of 1 January 1971 to 18 September 1981. Full notification is required 60 days before a substance is to be supplied in the EU at an amount of 1 tonne per annum (or 5 tonnes cumulative). Notification at this level is commonly referred to as the ‘Base-Set’ and the information required is specified in Annex VIIA of the Directive. Reduced notification applies at supply below 1 tonne per annum (or 5 tonnes cumulative), with Annex VIIB or Annex VIIC data needed for supply at above or below 100 kg per annum (or 500 kg cumulative) respectively.

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9.2.1 History of the Notification Process The EU-wide scheme for the notification of new substances was introduced as part of the Sixth Amendment to Council Directive 67/548/EEC (Directive 79/831/EEC) [4] concerned with the classification, packaging and labelling of dangerous substances. The Sixth Amendment was adopted by the Council of Ministers of the European Community in September 1979. After having more than 10 years’ experience in implementing the Sixth Amendment to the Directive, a Seventh Amendment (Directive 92/32/EEC) [1] was adopted by the Council of Ministers on 30 April 1992, and became effective as from the beginning of November 1993 in all Member States.

9.2.2 Data Sharing Once it has been established that a substance is notifiable in the EU, the first step is to check whether it has previously been notified. The prospective notifier may make a preliminary check of the European List of Notified Chemical Substances (ELINCS) [5], but since ELINCS is only updated every few years and as the identity of the previous notifier is not published, this does not provide a definitive answer. It is a legal requirement to make a formal data sharing enquiry to the Competent Authority (CA) in the Member State where it is planned to notify the substance before any animal testing can commence. The data sharing enquiry requires compositional and spectral data on the substance to allow the CA to establish if the substance has previously been notified in the EU. Brief details of the proposed use of the substance and the anticipated supply level should also be given to show that the notifier has a genuine intent to supply. If the outcome of the enquiry is positive (i.e., the substance has previously been notified) then the prospective new notifier is given the contact details of the first notifier. The two companies are strongly encouraged to come to a commercial arrangement to share the existing test data on the substance in order to avoid the need for unnecessary repeat animal testing. The Seventh Amendment permits national legislation to make data sharing obligatory, and several EU countries, notably Germany, have done this.

9.2.3 Base Set Studies for Full Notification The information required for full notification of a new chemical substance to enable it to be manufactured in or imported into the EU at 1 tonne per annum and above (or 5 tonnes

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Practical Guide to Chemical Safety Testing cumulative) is specified in Annex VIIA of the Seventh Amendment (see Table 9.1). The studies should be conducted according to the EU Methods of Annex V of the DSD [6], in compliance with Good Laboratory Practice (GLP). The particular Base Set studies required for a specific substance depend on the chemical structure and physical form, because some tests would be inappropriate or could have their results predicted. Note that some tests influence whether others are required. Existing studies may be acceptable providing they give adequate information for ‘dangerous’ classification and risk assessment, even if not conducted to the current EU methods or OECD guidelines [7]. It may also be possible to negotiate with the appropriate Competent Authority to predict the results for certain properties by ‘read across’ to a tested substance of close chemical structure.

9.2.4 Reduced Notification Studies The amount of information required for reduced notification (see Table 9.2) is substantially less than that in the Base Set for full notification, with that required for VIIC notification (< 100 kg per annum) being less than that for VIIB (> 100 kg per annum). Hence, in practice, the possibility of omission of studies by negotiation with the relevant CA is less than that for full notification. One important factor to note is that the Seventh Amendment permits EU Member States to decide on the need for vapour pressure and acute toxicity to Daphnia studies of Annex VIIB. In some countries, such as Germany, both of these studies are mandatory, whereas in others, such as the UK, the need for these is considered case-bycase, based on whether they are needed for adequate risk assessment.

9.2.5 Level 1 and Level 2 Notification Studies Further information must be submitted when the amount of substance supplied to the EU reaches the Level 1 and Level 2 trigger points. The possible studies are given in Annex VIII of the ‘Seventh Amendment’ Directive (92/32/EEC), although those required will depend on the particular substance. Level 1 testing may be required at 10 tonnes per annum (or 50 tonnes cumulative) but will definitely be required (if not already done) at 100 tonnes annually (or 500 tonnes cumulative). Level 2 testing will be required at 1,000 tonnes per annum or (5,000 tonnes cumulative). As at November 2000, 21 notified new substances had reached Level 2, 92 substances upper Level 1 (100 tonnes) and 259 lower Level 1 (10 tonnes). The full Level 1 testing of a notified new chemical substance at 100 tonnes per annum (or 500 tonnes total), is often referred to as Level 1b. Some of the tests may have already been requested at Level 1a, 10 tonnes per annum (or 50 tonnes total) or immediately after notification. The testing programmes are agreed at each stage with the Competent Authority who received the notification, and the substance can continue to be supplied at above the

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Table 9.1 Information for full notification of a new chemical substance in the EU Identity of the manufacturer and notifier: Identity of the substance:

Name, formula, purity, impurities, spectra, methods of analysis

Information on production and use, including safety recommendations: Physico-chemical Melting/freezing temperature properties: Boiling temperature (unless the melting temperature is > 360 °C) Relative density Vapour pressure Surface tension (unless water solubility < 1 mg/l) Water solubility Partition coefficient between n-octanol and water Flash point (only for liquids) Flammability (solids) Flammability (contact with water) Pyrophoric properties

}

these tests are normally predicted negative and omitted

Explosive properties (unless a negative result can be predicted from the chemical structure) Auto-ignition temperature (for liquids and low melting solids) Relative self-ignition temperature (solids)

}

one or both tests are required

Oxidising properties (not yet obligatory for liquids, and also omitted if a negative result can be predicted from the chemical structure) Granulometry (for solids)

various tonnage trigger levels while the testing is conducted. The selection of tests is based on the results of the Base Set studies, the use pattern of the substance and other factors as discussed in the Risk Assessment Technical Guidance Document [9]. The results of the Level 1a studies can influence which tests are required to complete the full Level 1

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Table 9.1 Continued Toxicological studies:

Acute oral toxicity (fixed-dose procedure, acute toxic class method or up and down procedure) Second acute toxicity study (dermal or inhalation, depending on the particle size distribution for non-waxy solids or the vapour pressure for potentially-volatile liquids) Skin irritation rabbit test Eye irritation rabbit test

}

unless predicted to be corrosive, e.g., from a highly acidic or alkali pH or from an in vitro test

Skin sensitisation study (normally the mouse local lymph node assay) 28-Day repeat-dose oral rat toxicity study Ames test In vitro mammalian cell test, normally a chromosome aberration test (or in vitro mouse lymphoma assay if the Ames test is positive) Assessment of toxicokinetic* behaviour (from the available information) Ecotoxicological studies:

Acute toxicity to fish study Acute toxicity to Daphnia study Algal growth inhibition study Ready biodegradation test Activated sludge respiration test (unless the substance is classed as ‘readily biodegradable’) Abiotic degradation by hydrolysis (unless the substance is classed as ‘readily biodegradable’ or is not potentially hydrolysable from the chemical structure) Adsorption/desorption screening test

Suggestions for rendering the substance harmless: * Toxicokinetics is an assessment of the absorption, distribution, metabolism and excretion of chemicals

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Table 9.2 Information for reduced notification of a new substance in the EU Identity of the manufacturer and notifier: Identity of the substance:

Name, formula, purity, impurities, spectra, methods of analysis

Information on production and use, including safety recommendations: Physico-chemical properties Flash point (for liquids) or flammability (for solids) for substances supplied at < 100 kg per annum: Physico-chemical properties Melting/freezing temperature for substances supplied Boiling temperature (unless the melting temperature ≥100 kg per annum: > 360 °C) Vapour pressure (if needed for risk assessment) Water solubility Partition coefficient between n-octanol and water Flash point (for liquids) or flammability (for solids) Toxicological studies for substances supplied at < 100 kg per annum:

Acute oral toxicity (or acute inhalation toxicity for gases)

Toxicological studies for substances supplied at ≥ 100 kg per annum:

Acute oral toxicity (or acute inhalation toxicity for gases) Skin irritation rabbit test Eye irritation rabbit test

}

unless predicted to be corrosive, e.g., from a highly acidic or alkali pH or from an in vitro test

Skin sensitisation study (normally the mouse local lymph node assay) Ames test Ecotoxicological studies:

None for supply at < 100 kg per annum

Ecotoxicological studies for substances supplied at ≥ 100 kg per annum:

Ready biodegradation Acute Daphnia toxicity (if needed for risk assessment)

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Practical Guide to Chemical Safety Testing programme. The selection of tests and the stage at which they are conducted are influenced by the outcome of the risk characterisation carried out by the Competent Authority. Certain Competent Authorities sometimes decide particular Level 1 studies are not essential to refine the risk assessment, in exceptional circumstances of low exposure, but others will probably need the full testing programme. The notifier must justify omitting any studies considered inappropriate or scientifically unnecessary. Level 2 testing is more flexible, in that the Competent Authority, in consultation with the notifier, draws up a testing programme covering the general aspects specified in Annex VIII of the Directive. If there are strong arguments supported by evidence that particular endpoints are not necessary, certain tests may be omitted. The Competent Authority specifies the time limit for conducting the Level 1 and 2 studies. The studies listed in Table 9.3 are likely as a maximum Level 1 testing programme (i.e., at 100 tonnes per annum) for a notified new chemical substance with a full Base Set.

Table 9.3 Likely maximum Level 1 testing programme Toxicological studies:

2-Generation rat fertility study by oral gavage exposure Rat teratology study by oral gavage exposure 90-Day repeat dose rat toxicity study by oral gavage exposure Basic toxicokinetic information (by expert assessment or an animal study) A further mammalian cell genotoxicity test, with confirmatory in vivo test(s) if any in vitro tests are positive

Ecotoxicological studies: 21-Day Daphnia reproduction study 28-Day fish growth test Fish bioaccumulation study with a depuration (purification) period (or an expert assessment of bioaccumulation potential) 14-Day earthworm toxicity study Sediment dwelling organism toxicity study (e.g., Chironomus riparius) Test on higher plants Further biodegradation test (unless the substance is ‘readily biodegradable’ or undergoes significant degradation)

Adsorption/desorption study (Tier 2 of the new OECD guideline 106) (if feasible due to low water solubility)

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EU Chemical Legislation Please note that it is unlikely that all of this testing would be required for a ‘safe’ chemical. It is not possible to define exactly what testing is required at Level 2, because this depends upon the Level 1 results and Risk Assessment at Base Set and Level 1.

9.2.6 The Notification Summary Form The notification summary form provides the main body of the regulatory submission. It is strongly recommended that notifiers use Data Entry Screens (DES) for Windows, available from the European Chemicals Bureau (ECB). The notification summary form is split into a total of nine sections, the information given in each is summarised in Table 9.4.

Table 9.4 Structure of the EU notification summary form Section 0

Details of the manufacturer and notifier (if different), plus the type of notification

Section 1

Composition of the substance, including purity, impurities, details of analytical methods 1

Section 2

Details of the manufacture (if made inside the EU) and use of the substance, including recommendations for safe handling and use

Section 3

Summary of the physico-chemical properties data

Section 4

Summary of the toxicological data

Section 5

Summary of the ecotoxicological data

Section 6

Possibility of rendering the substance harmless, including disposal recommendations

Section 7

Proposed risk assessment

Section 9

Declaration of unfavourable effects Classification and labelling Draft safety data sheet (for substances provisionally classified as dangerous) 2

2

Notes: Spectral and analytical data are also given as annexes 2 Safety data sheets and risk assessments are normally provided as separate documents in a suitable word processing package (e.g., Microsoft Word) as annexes, rather than being typed into the notification summary form. 3 Note that there is no Section 8 1

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Practical Guide to Chemical Safety Testing A Competent Authority will not accept a notification until all the required information has been provided and reviewed as being compliant. The substance can be supplied after the official waiting period has expired (60 days for a full notification, 15 or 30 days for a reduced notification).

9.2.7 The Sole-Representative Facility The sole-representative facility was introduced under the Seventh Amendment. Prior to this, each EU company wishing to import a notifiable substance directly from outside the EU had to make a new notification for the substance. Hence, several notifications would often be made for the same substance. This placed a considerable administrative burden on both the importers and the Competent Authorities. To obviate the need for multiple repeat notifications the sole-representative facility allows the non-EU manufacturer to appoint a sole-representative notifier, who makes a single notification, which lists all the EU importers. The full list of importers is given in the notification form and the sole-representative may add or delete importers as required by informing the Competent Authority. There can only be one sole-representative notifier per substance. The sole representative is obliged to report the total amount of substance imported into the EU on an annual basis to the Competent Authority.

9.2.8 Polymers Polymers are generally considered to be a special category of chemical substance which potentially pose less risk to health and the environment than other substances, on the basis of their inherent inertness and certain physical properties, and special provisions apply (see Chapter 12, Section 12).

9.2.9 Derogations/Exemptions from Notification There are four categories of new substance specified in Article 13, paragraph 2 of the Seventh Amendment as being ‘considered as having been notified’, and these are discussed below. All such new substances are subject to the normal classification, packaging and labelling requirements, and if the substance is known to be classified as very toxic, toxic, carcinogenic, toxic for reproduction or mutagenic on the basis of available information, the national CA has to be informed of the intended supply and provided with specified safety information, together with available acute toxicity data.

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EU Chemical Legislation As discussed (Sections 9.2.7 and Chapter 12), polymers are exempt from notification, providing they do not contain 2% or more in combined form of non-EINECS listed new substances and the OECD polymer definition is met. Substances supplied at below 10 kg per annum are exempt from notification, but individual Member States may choose to require appropriate technical and commercial data (not exceeding Annex VIIC Sections 1 and 2 of the Seventh Amendment) to be provided. Some Member States do not have any reporting obligations at this level of supply. The 10 kg per annum limit applies per manufacturer for substances supplied to the whole EU. It is strongly advised that the manufacturer (if made in the EU) or the importer keeps a written record of importation quantities in case of inspection by the national enforcement authority. Substances intended solely for scientific research and development (defined as scientific experimentation, analysis or chemical research carried out under controlled conditions: it includes the determination of intrinsic properties, performance and efficacy as well as scientific investigation related to product development) in quantities of less than 100 kg per year (per manufacturer) are exempt from notification. The national requirements vary, but normally the reporting requirements are the same as those for substances supplied at less than 10 kg per annum. Substances placed on the market for process-orientated research and development (PORD) with a limited number of customers registered with the notifier are exempt. There is no specific upper limit to the quantity of substance that can be used under a PORD, providing the applicant can justify it. Again, the information needed for a PORD varies between the Member States, but it will not exceed that needed for an Annex VIIB notification. PORD exemptions are only valid for 1 year but the applicant can request an extension of up to 1 further year if sufficient justification is available. In addition, substances intended exclusively for export to a country outside the EU, and those in transit through the EU under customs control and which do not undergo any treatment or processing in the EU, are exempt from notification.

9.2.10 Confidentiality Notifiers are permitted to claim certain information provided in support of the notification as confidential on the grounds of commercial secrecy. If the Competent Authority agrees to the request, the information will not be released to the general public, although it will be circulated to other Member State Competent Authorities and the European Commission.

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Practical Guide to Chemical Safety Testing However, in the interests of transparency and public right of access to information of relevance for environmental protection, notifiers are not allowed to claim the information specified in Table 9.5 as confidential.

Table 9.5 Information which cannot be claimed as confidential in an EU notification The trade name of the substance The name of the manufacturer and the notifier Physico-chemical data concerning the substance The possible ways of rendering the substance harmless The summary results of the toxicological and ecotoxicological tests If essential to classification and labelling, the degree of purity of the substance and the identity of impurities and/or additives which are known to be dangerous Recommended methods and precautions for handling, storage and transport of the substance as well as emergency measures in case of accidental spillage or injury to persons The information contained in the safety data sheet In the case of dangerous substances, analytical methods that make it possible to detect a dangerous substance when discharged into the environment as well as to determine the direct exposure of humans

9.3 Risk Assessment Risk assessment is pivotal to safety assessment of notified new substances (see Section 9.2) and priority existing substances (see Section 9.4) in the EU. The principles of risk assessment are the same for new and existing substances, although in practice the information available differs. The risk assessment principles are given in Commission Directive 93/67/EEC [8], and more details are given in a Technical Guidance Document [9]. The general philosophy of risk assessment is covered in Chapter 7. The risk assessment process involves hazard identification and dose (or concentration)response (effect) assessment and comparison with an exposure assessment to produce a risk characterisation for both human health and environmental effects, which are then combined in an overall integration of conclusions. Notifiers are encouraged to include a preliminary risk assessment with the notification.

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EU Chemical Legislation The Competent Authority sends a summary of the notification and risk assessment to the European Commission for distribution to the Competent Authorities of the other Member States. It may be necessary to refine the risk assessment by improving the exposure assessment, in which case additional studies may be requested to evaluate the hazard further. If necessary, risk management measures can be taken using existing EU provisions. The possible conclusions from the risk assessment for new and existing substances are given in Table 9.6.

Table 9.6 EU risk assessment conclusions New substances

(i)

The substance is of no immediate concern and need not be considered again until greater quantities are placed on the market.

(ii)

The substance is of concern and further information must be provided when supply levels reach a pre-determined level in the future.

(iii) The substance is of concern and further information is required immediately. (iv) The substance is of such concern that the assessor should propose measures for risk reduction. Existing substances

(i)

There is need for further information and/or testing.

(ii)

There is at present no need for further information and/or testing and no need for risk reduction measures beyond those which are being applied already.

(iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into account.

9.3.1 Human Health Risk Assessment

9.3.1.1 Principles Notified new substances and priority existing substances are subject to full risk characterisation, and at the first stage of hazard identification the effects and/or properties of concern are identified and the dangerous classification is reviewed in terms of all the available data. Full risk characterisation is required for notified new substances which are provisionally classified as ‘dangerous’ in terms of their toxicity or physico-chemical properties, or if there are other reasonable grounds of concern (e.g., positive in vitro

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Practical Guide to Chemical Safety Testing mutagenicity tests) for substances not classified on the basis of full notification data. If there are insufficient data for definitive classification following reduced notification, full risk characterisation is required only if there are reasonable grounds for concern, such as exposure considerations or indications of potential toxicity from structure-activity relationships (SAR). Further information on the general principles of human health risk assessment are given in Chapter 7.

9.3.1.2 Human Health Risk Assessment for Physico-Chemical Properties Effects The dangerous properties of explosivity, flammability and oxidisibility are evaluated in terms of potential adverse effects to workers, consumers and man exposed indirectly via the environment (although the latter is normally not applicable) based on the known or reasonably foreseeable conditions of use. Risk characterisation entails evaluation of the likelihood of an adverse effect. The substance is of no immediate concern if an adverse effect will not be caused, otherwise immediate risk management measures may be required.

9.3.1.3 Human Health Risk Assessment For Toxic Effects The dangerous properties of acute toxicity, irritation, corrosivity, sensitisation, repeateddose toxicity, mutagenicity, carcinogenicity and toxicity for reproduction are evaluated in terms of their potential toxic effects to workers, consumers and man exposed indirectly via the environment, based on the known or reasonably foreseeable conditions of use for each stage in the life-cycle of the substance from which exposure can occur. Risk characterisation is required for substances contained in preparations, if the preparation is classified as ‘dangerous’ because of the substance content, or if there are other reasonable grounds for concern. The objective of the exposure assessment is to make a quantitative or qualitative estimate of the dose/concentration of the substance to which a population is or may be exposed, taking account of spatial and temporal variations in the exposure pattern and the various additional factors given in Table 9.7.

9.3.2 Environment Risk Assessment

9.3.2.1 Principles Notified new substances and priority existing substances are subject to full risk characterisation, and at the first stage of hazard identification the effects and/or properties 204

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Table 9.7 Additional factors to take into account in human health exposure assessment Adequately-measured exposure data – a systematic study to measure the amount of the substance released during manufacture and/or use and with which workers and/or the public may come into contact The quantity of the substance – the amount manufactured or used at each stage during the life cycle at each site in the EU. (This is normally given as an average or typical assessment.) The form in which the substance is produced/imported and used (e.g., substance itself or as a component of a preparation) Use pattern and degree of containment Process data Physico-chemical properties of the substance including, where relevant, those conferred by the process (e.g., aerosol formation) Breakdown products and/or transformation products (for existing chemicals) Likely routes of exposure and potential for absorption Frequency and duration of exposure Type and size of specific exposed population(s) where such information is available

of concern are identified and the environmental classification is reviewed in terms of all the available data. Full risk characterisation is required for notified new substances which are provisionally classified as dangerous for the environment, or if there are reasonable grounds for concern in relation to environmental effects for substances not so classified (see Table 9.8) on the basis of full notification data, or if there are insufficient data for definitive classification following reduced notification: environmental risk assessment is discussed in more detail in Chapter 8. The object is to predict the concentration of the substance below which adverse effects in the environmental compartment of concern are not expected to occur, i.e., the predicted no effect concentration (PNEC). However, in some cases, it may not be possible to establish a PNEC, and a qualitative estimation of the dose (concentration)-response (effect) relation would have to be made instead. The PNEC may be calculated by applying an assessment factor to the values resulting from acute and/or long-term studies on aquatic organisms, i.e., EC50 and no observed effect level (NOEL) respectively. The assessment factor is an expression of the degree of uncertainty in extrapolation from test data on a limited

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Table 9.8 Reasonable grounds for concern necessitating environmental risk assessment for notified new substances Indications of bioaccumulation potential The shape of the toxicity/time curve in ecotoxicity testing Indications of other adverse effects on the basis of toxicity studies, e.g., classification as a mutagen, toxic or very toxic or harmful with the risk phrase R68 (Possible risk of irreversible effects) or R48 (Danger of serious damage to health by prolonged exposure) Data on structurally analogous substances

number of species to the real environment, and thus in general, the more extensive the data and the longer the duration of the tests, the smaller is the degree of uncertainty and the size of the assessment factor. The objective of the exposure assessment is to predict the concentration of the substance which is likely to be found in the environment, i.e., the predicted environmental concentration (PEC). However, in some cases, it may not be possible to establish a PEC and a qualitative estimation of exposure would have to be made. A PEC or qualitative estimation of exposure is only determined for the environmental compartments to which emissions, discharges, disposal or distributions are known or are reasonably foreseeable. It takes account of the factors listed in Table 9.9.

Table 9.9 Factors taken into account in environmental exposure assessment Adequately measured exposure data The quantity of the substance The form in which the substance is produced, imported and used (e.g., substance itself or as component of a preparation) Use pattern and degree of containment Process data Physico-chemical properties of the substance, in particular melting point, boiling point, vapour pressure, surface tension, water solubility and Pow Breakdown products and/or transformation products (for existing chemicals) Likely pathways to environmental compartments and potential for absorption/desorption and degradation Frequency and duration of exposure

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EU Chemical Legislation For new substances to be placed on the market in quantities at or below 10 tonnes per annum (or 50 tonnes cumulative), the PEC or qualitative estimation of exposure is usually determined for the generic local environment in which release of the substance may occur. Also, for existing substances with adequately-measured, representative exposure data, special consideration is given to these when conducting the exposure assessment. Where calculation methods are used for the estimation of exposure concentrations, adequate models have to be applied, and if appropriate, on a case-by-case basis, relevant monitoring data from substances with analogous use and exposure patterns or analogous properties may also be considered. For any given environmental compartment, the risk characterisation as far as possible entails comparison of the PEC with the PNEC, in the PEC/PNEC ratio. For notified new substances, if this ratio is equal to or less than 1, the substance is of no immediate concern (Table 9.6). If the ratio is above 1, the assessor decides on the basis of its value and the factors giving reasonable grounds for concern (Table 9.6) which other administrative conclusion applies. Similarly, for existing chemicals, if PEC/PNEC ≤1, no further information and/or testing and no new risk reduction measures are required, whereas if the ratio is above 1, the assessor may recommend such measures as appropriate. If it has not been possible to derive a PEC/PNEC ratio, the risk characterisation consists of a qualitative evaluation of the likelihood that an adverse effect will occur. The Base Set testing package for the notification of new chemical substances contains data relevant to the aquatic environment on biodegradation (biotic and abiotic) and acute toxicity to fish, Daphnia and algae. Little data of relevance to the atmospheric and terrestrial compartments are generated at this stage or for that matter at the Level 1 and 2 stages of notification. The decision to proceed with further testing of new substances should be made on a case-by-case basis with the objective to revise either the PEC and/or the PNEC. Resources should be geared to which of the PEC or PNEC would be more sensitive to revision from the result of additional testing, as well as the need to reduce the amount of testing with vertebrate animals. At higher tonnage levels the PEC may be revised on the basis of local or regional models dependent on the nature of the substance and its use pattern. The methods for environmental risk assessment are given in the Technical Guidance Document [9]. There is also a computer version of these models, called EUSES [10].

9.4 Existing Chemicals Regulation The Existing Chemicals Regulation (ECR), Council Regulation Number 793/93 on the evaluation and control of the risks of existing substances [11] entered into force directly in all EU countries on 4 June 1993.

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Practical Guide to Chemical Safety Testing The ECR foresees that the evaluation and control of the risks posed by existing chemicals will be carried out in four steps: Step I

Data collection

Step II Priority setting Step III Risk assessment Step IV Risk reduction The ECB is responsible for the scientific and technical support to the ECR regarding the first three steps of the Regulation.

9.4.1 Data Collection The ECR applies to all EU manufacturers or importers of existing chemical substances listed in EINECS [3]. Each manufacturer or importer had to report the available data specified in Annex III of the ECR (see Table 9.10) to the ECB in the first and second reporting phases for substances supplied at above 1,000 tonnes per annum during the period 23 March 1990 to 23 March 1994. The more limited data specified in Annex IV of the ECR (see Table 9.11) are to have been reported on all substances supplied at 10 to 1,000 tonnes per annum in the third reporting phase. There is an Annex II to the ECR which lists high-volume substances which are obviously non-hazardous to health or the environment and hence are not reportable unless separately requested. All manufacturers or importers who manufacture or import EINECS-listed substances at the relevant tonnages had to report at the appropriate time, including on substances not placed on the EU market, i.e., such as site-limited intermediates and export-only substances. The submitted information has to be updated when appropriate and late third phase submissions are still being made. Altogether 1,408 substances were reported for Phase I, and 1,209 in Phase II (i.e., a total of 2,617 high production volume chemicals, which were submitted in ca 20,000 HEDSET dossiers from ca 4,300 companies). Data had to be reported in summarised format using Harmonised Electronic Data Set (HEDSET) computer diskettes. To avoid language problems all text was codified. The ECB has collated all the HEDSET data into an EU database, now called International Uniform Chemicals Information Database (IUCLID) [12]. There is a public version on CD-ROM and also a confidential version containing information claimed as secret. Data reporters were encouraged to cooperate in submissions of general data, but separate submissions were needed for the information unique to the manufacturer or importer (on supply levels, etc.). The same HEDSET system was used for the Organization for Economic Cooperation and Development (OECD) Screening Information Data Sheet

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information Ecotoxicity ECB information Biodegradation Bioaccumulation EU Chemical Legislation

Table 9.10 Information to be submitted to the European Chemicals Bureau for Existing Substances supplied at above 1,000 tonnes per annum General information Identity of the substance:

Chemical name and structure, EINECS number, CAS number, synonyms

Specification:

Purity and impurities

Identity of the company reporting the substance: Quantity produced or imported: Classification and labelling: Use pattern: Physico-chemical Properties:

Melting point Boiling point Density Vapour pressure Partition coefficient Water solubility Flash point Auto flammability Flammability Explosive properties Oxidising properties Other data and remarks

Environmental fate properties:

Photodegradation Hydrolysis Stability in soil Environmental monitoring data

Transport and distribution in the environment:

Biodegradation Bioaccumulation

Ecotoxicity:

Toxicity to fish Toxicity to Daphnia and other aquatic invertebrates Toxicity to algae Toxicity to bacteria Toxicity to terrestrial organisms Toxicity to soil dwelling organisms Other remarks

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Table 9.10 Continued Toxicity:

Acute toxicity Acute oral toxicity Acute inhalation toxicity Acute dermal toxicity Acute toxicity (other routes of administration) Corrosiveness and irritation Skin irritation Eye irritation Sensitisation Repeated dose toxicity Genetic toxicity in vitro Genetic toxicity in vivo Carcinogenicity Toxicity to reproduction Experience with human exposure

Table 9.11 Information to be submitted to the European Chemicals Bureau for Existing Substances Supplied at 10 to 1,000 tonnes per annum General information Identity of the substance:

Chemical name and structure, EINECS number, CAS number, synonyms

Specification:

Purity and impurities

Identity of the company reporting the substance: Quantity produced or imported: Classification and labelling: Use pattern:

(SIDS) programme for evaluation of existing chemicals, but a revised version (also called IUCLID [13]) is now used for all high production volume chemical (HPV) programmes, including the US HPV initiative (see Chapter 11), the International Chemical Council Association (ICCA) programmes, and for biocides in the EU (Chapter 15).

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9.4.2 Priority Setting The ECR states that the European Commission, in consultation with the EU Member States, will regularly draw up lists of substances considered to be of particular concern to human health and/or the environment, utilising the information collected during Step I of the Regulation as a basis for selecting priority substances. In practice only about 50 chemicals can be subjected to full risk assessment each year. Therefore, reported chemicals are selected for full assessment in a priority setting scheme set up by the Informal working group on Priority Setting (IPS). The computerised IPS Priority Setting Method ranks chemicals according to relative risk from their reported properties, taking into account any absence of pertinent data. Quantitative structureactivity relationships (QSAR) can be used to enable some endpoints to be estimated to refine the ranking by replacing conservative default values for data gaps. Lists of priority chemicals are published after wide consultation of the draft version to allow expert judgement to be applied. The priority lists are published as Commission Regulations [14-17]. The first list of 42 chemicals selected on an ad hoc basis from Member States’ individual nominations was published in Commission Regulation No. 1179/94 [14]. The second list of 36 priority chemicals is given in Commission Regulation No. 2268/95 [15] and the third list of 32 chemicals in Commission Regulation No. 143/97 [16]. Further exposure data on 9 chemicals is required by Commission Regulation No. 142/97. The fourth list of 31 priority chemicals is given in Commission Regulation (EC) No. 2364/ 2000 [17]. Risk assessment reports from other international and national evaluation schemes are taken into account.

9.4.3 Risk Assessment A national rapporteur Competent Authority is selected to conduct the risk assessment on each priority chemical. Commission Regulation No. 1488/94 [18] sets out the principles for risk assessment and the Technical Guidance Document on risk assessment [9] advises on how to conduct the assessment, as discussed in Section 9.3. The summary data for the selected priority chemicals must be supplemented with full reports and other available existing data within 6 months of listing. A full EU Annex VIIA ‘Seventh Amendment’ Base Set of data (see Table 9.1) must be made available for risk assessment, and any necessary new studies to fill ‘data gaps’ have to be provided within 12 months of listing, unless the notifier applies for an extension of the time limits or derogations for data requirements on the grounds that the test is technically impossible or it is unnecessary for risk assessment. The rapporteur then evaluates this data package, and decides on whether any non-GLP or non-standard studies are adequate. The

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Practical Guide to Chemical Safety Testing rapporteur may request repeat testing or additional GLP-compliant studies if these are necessary for risk assessment, but this has to be approved at EU level first. Validated QSARs of appropriate reliability may be used in defined circumstances for the risk assessment. The manufacturers and importers who originally reported the substance are jointly responsible for providing these new studies, but data sharing by consortia formation is encouraged to prevent repeat animal testing. The rapporteur then produces a draft risk assessment regarding human health and the environment. The overall conclusion will be that the substance is of no concern, requires risk reduction or that further data are necessary to decide which conclusion applies (Table 9.6). Any necessary risk management strategies are for adoption at EU level using existing provisions, such as classification and labelling, occupational exposure limits or very occasionally, by restrictions on marketing and use under the provisions of the Marketing and Use Directive [2] and can be undertaken only after a risk/benefit evaluation on the chemical. The EU scheme is coordinated especially with the OECD programme, with EU evaluations being a direct contribution to the OECD scheme.

9.5 Chemical Hazard Communication

9.5.1 Classification and Labelling of Dangerous Substances All ‘dangerous’ chemical substances have to be classified, packaged and labelled according to the requirements of the DSD, and it is the responsibility of EU suppliers to ensure that these requirements are met. Substances officially classified as dangerous are listed in Annex I of Council Directive 67/548/EEC, after they have been evaluated by the appropriate Labelling Working Group of the European Commission and formally adopted by the Technical Progress Committee. A consolidated version of this list of classified substances is published by the UK Health and Safety Executive [19]. The criteria to enable substances to be classified and labelled are given in Annex VI of Council Directive 67/548/EEC, which is consolidated in Commission Directive 2001/59/EC [20]. The categories of danger are given in Table 9.12. Substances are classified for labelling by evaluation of their physical, toxicological and ecotoxicological properties. Existing data (e.g., from non-GLP and/or non-EU/OECD studies), can be used to classify existing EINECS-listed chemical substances, and there is no obligation for new testing if the existing information available is inadequate. For full classification, the Base Set of tests for notification of a new substance (Table 9.1) is effectively the minimum requirement, and some ‘existing’ substances which have not been officially classified and listed on Annex I of Council Directive 67/548/EEC are tested voluntarily by the EU suppliers to

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Table 9.12 Categories of danger for EU classification of dangerous chemcials Explosive Oxidising Extremely flammable Highly flammable Flammable Very toxic by acute exposure (by oral, dermal or inhalation) Toxic by acute exposure (by oral, dermal or inhalation) Toxic by prolonged exposure (by oral, dermal or inhalation) Harmful by acute exposure (by oral, dermal or inhalation) Harmful as an aspiration hazard Harmful by possible risk of irreversible effects from a single exposure Harmful by prolonged exposure (by oral, dermal or inhalation) Corrosive (causes burns or severe burns) Irritant to skin Irritant or severe irritant to eyes Irritant to the respiratory system Sensitiser by skin contact Sensitiser by inhalation exposure (and/or causes immunological contact urticaria) Carcinogenic (category 1, established carcinogenic in humans, category 2, to be regarded as carcinogenic in humans, or category 3, of concern as possibly carcinogenic) Mutagenic (category 1, 2 or 3, corresponding to carcinogens) Toxic to reproduction for fertility effects (categories 1, 2 or 3) Toxic to reproduction for developmental effects (categories 1, 2 or 3) Dangerous for the environment

enable adequate classification to be made. Labelling consists of a dangerous classification symbol, information on hazardous properties (R phrases) and advice on safety (S phrases).

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9.5.2 Classification and Labelling of Dangerous Preparations The EU scheme for classification and labelling of dangerous ‘preparations’ (i.e., products consisting of a mixture of substances) is covered by the Dangerous Preparations Directive (DPD). The original DPD, Council Directive 88/379/EEC [21], has been replaced by Directive 1999/45/EC [22], which comes into operation for most provisions from 30 July 2002. The classification and labelling of both new preparations and those currently supplied has to be determined by the manufacturer or importer, and a record kept of the evaluation for inspection by the regulatory authorities if requested. A single assessment is adequate for similar preparations. The physico-chemical properties of the preparation are determined using EU Methods [6]. Health hazards of preparations can be assessed either by studies using EU Methods or by evaluation from the dangerous components using the procedure given in the DPD. The required concentration limits for individual dangerous substances are given in Annex I of the DSD [19]. For dangerous substances not listed in Annex I, general limits for individual properties are given in the DPD for the estimation of health hazard. No account is taken of very toxic or toxic substances contained in preparations at below 0.1% or harmful, corrosive or irritant substances at below 1% (unless lower limits are specified for individual substances in Annex I of the DSD). Preparations containing substances which are carcinogenic, mutagenic or toxic for reproduction are classified only by the calculation method, and studies on the preparation are not appropriate. An important change in the new DPD is an animal welfare initiative requiring that no new animal studies be done on a preparation unless the supplier can demonstrate that the particular health hazardous property cannot be evaluated satisfactorily by either the calculation method or using existing data from animal studies on the preparation. The main change introduced by the new DPD is environmental classification and labelling for preparations. As for health effects, the intention is that most preparations will be evaluated for environmental hazardous properties by a calculation method, based on the environmental classification and labelling of the component substances and their percentage in the preparation. The calculation scheme is given in the new Directive, together with default values for the ‘weighting’ parameters. There is also the option to test the preparation for acute ecotoxic effects in aquatic organisms, and the results of such tests on the preparation take precedence over the calculation method. However, this option of testing the preparation does not apply to studies to investigate persistence in the environment (i.e., biodegradation testing) or bioaccumulation, because these are considered properties of individual substances, which are unaffected by formulation into a preparation. Ecotoxicity testing for preparations will probably be rare, because studies are needed in all three species for definitive environmental classification (i.e., fish, Daphnia

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EU Chemical Legislation and algae), unless the worst classification already results from testing in fewer species. GLP-compliant studies using the EU Methods [6] are required, which normally means solution analysis of all the components of the preparation must be undertaken to quantify the exposure of the test organisms to the potentially-toxic formulants. The ‘dangerous’ component substances of ‘dangerous’ preparations must normally be named in full on the label for the preparation, if present at above the appropriate concentration limit. However, suppliers can use an approved generic name for acutely harmful and irritant substances, providing that a Confidentiality Declaration is made. The third main feature introduced by the new DPD is to extend these hazard communication arrangements for general chemicals to plant protection products (PPPs) covered by Council Directive 91/414/EEC) [23] and Biocidal Products (see Chapter 15) covered by Directive 98/8/EC [24] as from 30 July 2004.

9.5.3 Safety Data Sheets Hazard communication for dangerous chemical substances and preparations is accomplished by classification and labelling of the key hazardous properties (as discussed in Sections 9.5.1 and 9.5.2) and by the obligation to provide a safety data sheet (SDS) to industrial users. Note that notifiers are required to provide a draft safety data sheet for notifications of new substances provisionally classified as ‘dangerous’. The safety data sheet should be in the standard 16 heading European format, with the specific information required under each heading being given in Table 9.13.

9.6 Transport Regulations 9.6.1 Introduction Classification and labelling of a chemical is an important way of communicating chemical hazards and associated health and safety information in a summarised form. Classification is necessary to establish or assess the hazard, and labelling then provides information reflecting this hazard. For users of chemicals, less severe and long-term hazards are as important as severe and immediate hazards. This is because people likely to be exposed to the chemical over a long period of time may be members of the public with limited hazard awareness, perhaps without suitable facilities or equipment to minimise risk. Hence the EU supply hazard communication scheme (see Section 9.5) takes into account all these hazardous properties.

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Table 9.13 Content of EU Safety Data Sheets 1.

Identification of the substance and of the notifier

2.

Composition/information on ingredients

3.

Hazards identification

4.

First aid measures

5.

Fire fighting measures

6.

Accidental release measures

7.

Handling and storage

8.

Exposure controls/personal protection

9.

Physical and chemical properties

10.

Stability and reactivity

11.

Toxicological information

12.

Ecological information

13.

Disposal considerations

14.

Transport information

15.

Regulatory information

16.

Other information

In contrast, classification and labelling for transport, which is based on United Nations (UN) Recommendations [25], is primarily concerned with the more severe and immediate risks. This is because chemical transportation is usually carried out in an industrial context, by trained personnel properly instructed and equipped, who, if the chemical is properly packaged or otherwise contained, are not likely to become exposed to it (and, if they are, through an accident, then exposure will generally be for a relatively short time). Also, some chemicals and articles containing them are dangerous for transportation for purely physical reasons, such as compressed gases. For this reason the system of classification, the criteria used and the labelling requirements differ greatly between supply and transportation. Furthermore, the requirements of four modes of transport, road, rail, sea and air, each have their own rules, all closely modelled on the UN Recommendations [25]. Finally, these rules apply to the transport of ‘dangerous goods’, for example pathogenic organisms, natural commodities (e.g., hay) and articles (e.g., self-inflating life jackets), and not just chemicals, although chemicals play a majority part in the Regulations.

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9.6.2 The United Nations Transportation Classification Scheme Chemicals are classified, packaged and labelled for safe transport according to the UN Recommendations on the Transport of Dangerous Goods [25]. Chemicals are classified in terms of the hazards most pertinent to transport, using available data and/or the results of standard UN tests interpreted according to the UN criteria and definitions. There are 9 Danger classes (Table 9.14). There is no category for ‘Environmentally Hazardous’. This category is presently covered by Class 9 ‘Miscellaneous Dangerous Goods’, but plans to include a Class 10 ‘Environmentally Hazardous’ are currently under consideration by the UN. Many of the classes are split into Divisions, which may consist of several Packing Groups depending on the degree of chemical hazard. The dangerous goods most commonly carried are listed in the UN Recommendations, and interested parties can now nominate new chemicals for inclusion, or request amendments, by submitting relevant classification data to the UN. The UN Recommendations enable national and international regulations to be made to ensure all modes of transport of chemicals operate safely.

Table 9.14 Danger classes for UN transport 1 to 1.6

Explosive

2.1

Flammable gas

2.2

Compressed gas

2.3

Toxic gas

3

Flammable liquid

4.1

Flammable solid

4.2

Spontaneously combustible

4.3

Dangerous when wet

5.1

Oxidising agent

5.2

Organic peroxide

6.1

Toxic substance

6.2

Infectious substance

7

Radioactive substance

8

Corrosive substance

9

Miscellaneous dangerous substances and articles

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Practical Guide to Chemical Safety Testing The UN scheme is the basis for a common approach to hazard identification and communication in international transport and is reflected in the rules and regulations of most countries. Substances are assigned UN Hazard Identification Numbers based on this system, and carriers then display the appropriate UN diamond, unless there are other national laws establishing other requirements. However, the national requirements closely parallel those established by the UN, so there is no significant difference between the UN approach and those national requirements that often exist in the developed nations. The four basic modes of transport, air, rail, road and sea (including inland waterways and lakes), each has its own labelling requirements and restrictions for international transport established under international agreements, based on the UN scheme (Table 9.15). These schemes are implemented by EU measures.

Table 9.15 International agreements on transport of dangerous goods Air:

IATA - International Air Transport Association ICAO - International Civil Aviation Organisation

Rail:

RID - International Regulations Concerning the Carriage of Dangerous Goods by Rail

Sea:

IMO - International Maritime Organisation IMCO - International Maritime Consultative Organisation

Road:

ADR - Road Transport Agreement

9.6.3 Transport of Marine Pollutants The International Convention for the Prevention of Pollution from Ships (MARPOL) was adopted in 1973 by the International Conference on Marine Pollution. MARPOL covers all the technical aspects of pollution that might be caused by discharges into the sea of substances from ships, except for the disposal of land-generated waste by dumping from ships, and the discharge of substances from exploration of sea bed mineral resources. MARPOL 73/78 is the adopted version for current use by The International Conference on Tanker Safety and Pollution Prevention of 1978. GESAMP (Joint Group of Experts on the Scientific Aspects of Marine Pollution) undertakes the ongoing task of evaluating the environmental hazards of additional substances proposed for carriage by ships which are not listed in the original MARPOL 73/78. A hazard evaluation procedure is used by GESAMP to develop hazard profiles [26], and this hazard rating system was included in the MARPOL 73/78 Convention. The hazard evaluation International Air Transport Association International Civil Aviation Organisation International Regulations Concerning the Carriage of Dangerous Goods by Rail 218 International Maritime Organisation International Maritime Consultative Organisation Road Transport Agreement

EU Chemical Legislation procedure is a stepwise process, and is based on four potential effects and targets: damage to living resources, hazards to human health, reduction of amenities and interference with other uses of the sea. Once it has been determined that a substance is carried by ships (step 1), and that it is not an oil (step 2), the third step considers substances which are likely to bioaccumulate, and also considers the possibility of tainting or colouring in cases where marine organisms are commercially exploited. Bioaccumulation should be measured by considering the rate of uptake and excretion of the substance, and the n-octanol:water partition coefficient. The fourth step considers dangers to marine organisms which are assessed by use of acute toxicity information. Wherever possible 96-hr LC50 tests should be conducted using a marine crustacean (e.g., Mysidopsis bahia) and a marine fish in sea water. The fifth step provides for ranking on the basis of acute toxicity, primary irritancy and longer term specific adverse health hazards to humans. These dangers are assessed by reviewing relevant published information. Finally, the sixth step makes provisions for protection of amenities. Other properties of the substance that are of interest in the environmental assessment are biodegradability, chemical oxygen demand (COD), biological oxygen demand (BOD), reactivity with sea water and air, lipid solubility and biotransformation. Any appropriate testing procedures can be used, such as those of the OECD. The International Maritime Organisation (IMO) has developed a questionnaire which Governments are expected to complete when proposing new substances for shipping regulations. The questionnaire covers physical and chemical properties of the substance as well as recommended guidelines for use and handling, in addition to the data mentioned previously. So far about 2,500 chemical products have been evaluated, and efforts are being made to store the relevant background data in a computerised system which would allow a rapid retrieval of information in case of emergencies. Major problems encountered by GESAMP are related to the deficiency of data, particularly in relation to effects on marine life. Other problems have arisen in cases where mixtures carried under trade or generic names had to be evaluated. The hazard profiles of bulk chemical products developed by GESAMP are used by expert groups of IMO in assigning for each product one of the four pollution categories (ranging from category A (most hazardous) to category D (least hazardous)), and ship type (chemical tanker 1, 2 or 3) with the objective of preventing pollution of the sea. Chemical products carried as packaged goods may be identified as marine pollutants or severe marine pollutants from the hazard profile. When carried by ship, marine pollutants have to be labelled as such, and carried under the provisions of MARPOL 73/73.

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9.7.1 National Product Registers National legislation has been implemented in some European countries, notably the Scandinavian states of Finland, Sweden, Denmark and Norway, in order to provide a record of chemicals available on the market place which are classified as dangerous or which may cause hazard (i.e., those for which a safety data sheet is required). Information on hazard-causing chemicals must be submitted by the company responsible for placing the substance on the market. The nature of the information to be provided is broadly similar to that provided on a standard EU-format safety data sheet but may also include identification codes, e.g., Chemical Abstract Service (CAS) numbers for all hazardous ingredients as well as the purpose of use and standard industrial classification of the preparation. Chemicals used exclusively for research and development purposes, and those supplied in very small quantities generally need not be registered. The national authorities should be informed when information on the chemical changes significantly. Such changes may concern the trade name, the identity of the manufacturer or importer, the composition of a preparation and any new information on the hazardous properties of the chemical. Product registers are intended for use by authorities, poison information centres and research institutes. Data is used when giving advice concerning treatment of poisoning, as a source of information for the preparation of legislation and as a tool of chemical control.

9.7.2 German Water Hazard Classification Scheme In Germany concerns over the contamination of potable and ground water supplies from handling, transport and storage of water-endangering materials resulted in a system for self-classification (‘Selbsteinstufung’) of substances and preparations by industry into water endangering classes (‘Wassergefährdungsklassen’, WGK). The classification scheme applies to chemical substances and preparations, but not to chemicals covered by separate specific legislation. The Water Hazard Classification System is described in a general administrative regulation to the Federal Water Act, i.e., the Administrative Regulation on the Classification of Substances Hazardous to Waters into Water Hazard Classes Administrative Regulation on the Classification of Substances Hazardous to Waters (English Version) (VwVwS). The revised VwVwS of 18 April 1996 entered into force on 1 May 1996 to enlarge the list of classified substances and introduce a scheme for classification of preparations. The VwVwS was further revised on 17 May 1999 [27] (which came into effect on 1 June 1999) to introduce a new WGK scheme based on a scoring system from the EU dangerous classification R-phrases. All substances and preparations which are stored and handled in Germany must be evaluated. The purpose

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Ready biodegradation Bioaccumulation EU Chemical Legislation of classification is to assess the important exposure pathways from accidents with chemicals which pose hazards to water. This enables graduated precautions to be taken in industrial plants, and the WGK scheme is used by the local German authorities in licensing and approval of plant procedures. Many common substances have been officially classified by the Commission for Classification of Substances Hazardous to Water (Geschaftsstelle der Kommission Bewertung Wassergefahrdender Stoffe) (KBwS). Those assessed as non-hazardous are listed in Appendix 1 of the VwVwS, and hazardous substances are given in Appendix 2. Such officially-assessed substances are described in the ‘Bewertung wassergefährdender Stoffe’. A substance not officially classified by KBwS and listed in Appendix 1 or 2 of VwVwS has to be self-classified by the German supplier. This classification has to be reported to the KBwS and the classifier has to inform the KBwS of any changes. Selfassessed substances are described as such by the supplier. All the WGK classifications are recorded by the KBwS, and a supplier can find out the status of a substance by contacting them or using the web site (www.umweltbundesamt.de). The extent of the hazard to water by a substance or mixture of substances depends on the local conditions, the amount of substance and the properties of the substance. Substances are classified either as non-hazardous to water or into a Water Hazard Class (WGK). The classification is based on the substance’s physical and chemical characteristics and on toxicological and ecotoxicological data. The new classification criteria for substances are given in Appendix 3 of the 17 May 1999 VwVwS. The EU R-phrases can be used directly for substances assessed at EU level and listed in Annex I of the DSD. The minimum ‘Basic Data Record’ (Basisdatensatz) used in ascertaining the danger to water from the properties of the substance are listed in Table 9.16. These studies are normally conducted to OECD guidelines or EU Methods in accordance with GLP, but equivalent test methods can be used where appropriate, and literature or data may be acceptable. The EU R-phrases or study results for the ‘Basic Data Record’ are used to generate ‘Rating Points’ (Bewerktungspunkten). In the absence of data, default

Table 9.16 the basic data record for German water hazard classification 1. Acute oral or dermal toxicity in the rat LD50 (tested at up to 2,000 mg/kg). 2. Acute toxicity to fish or Daphnia or algae growth inhibition (tested at up to 100 mg/l) 3. Ready (or inherent) biodegradation 4. Bioaccumulation potential (from the partition coefficient, either calculated or measured, or a fish bioaccumulation study)

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Table 9.17 German water hazard classification scoring scheme Total Score

WGK Number

WGK Description

0 to 4

1

weakly water polluting

5 to 8

2

water polluting

9 and over

3

strongly water polluting

It should be noted that, according to this scheme, substances which have not been tested are considered as strongly water polluting (WGK 3) as a precautionary measure. In order to be considered non-hazardous to water, a substance must have a total points score according to the VwVwS Appendix 3 criteria of 0 and be poorly water soluble (below 100 mg/l for gases and solids and below 10 mg/l for liquids), have no aquatic organism toxicity at up to the water solubility limit (with at least two species tested) and, for organic liquids, also be readily biodegradable. Preparations are classified according to the scheme of Appendix 4 of VwVwS. The usual means of classification is a calculation procedure based on the component substances, and this system corresponds broadly with the previous April 1996 VwVwS. All components present at quantities greater than 0.2% (or 0.1% for carcinogens) in the preparation must be taken into account, and the sum of components with the highest WGK value will determine the classification. Any component substance of unknown identity or classification is assumed to be WGK 3 as a precautionary measure. Under the latest 17 May 1999 VwVwS, the option was introduced to classify preparations based on studies on the preparation itself (acute toxicity in the rat and aquatic organism toxicity in at least two species). Test results take precedence over calculated WGK values. Note that testing for biodegradation and bioaccumulation potential of a preparation is not applicable.

9.8 Other EU Legislation for Specific Product Types There are several distinct classes of new chemical substance, which, due to their proposed use pattern and associated high levels of exposure to humans and/or the environment,

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EU Chemical Legislation are regulated under additional EU provisions. In most cases they are also subject to EU chemicals legislation on notification of new substances (see Section 9.2) and hazard communication (see Section 9.4). Such substances include cosmetic ingredients, food contact materials, i.e., food packaging (see Chapter 14), detergents and offshore chemicals.

9.8.1 Control of Cosmetics in the EU Cosmetics are controlled in the EU by Council Directive 76/768/EEC, and its subsequent amendments, notably the Sixth Amendment, Council Directive 93/35/EEC [28]. An indicative inventory of cosmetic ingredients in use in the EU has been developed, and the first version was published as Commission Decision 96/335/EC [29]. The labelling required for cosmetic products is specified. Also, the manufacturer or importer of the cosmetic product must keep a technical dossier for the product. The Directive also made provision for reducing animal tests on new cosmetic ingredients. The original intention was to prohibit (as from 30 June 2002) marketing of cosmetic products which contain ingredients which had been tested on animals. This deadline has already been postponed twice, and the Seventh Amendment to the Directive further postpones the ban. The reason is that there has not been sufficient progress in developing satisfactory methods to replace animal testing and the full range of necessary alternative methods of testing have not been scientifically validated. Two in vitro skin corrosion tests and an in vitro phototoxicity test have been validated, and are published as EU test Methods in Annex V of the DSD [6] (see Chapter 6). Cosmetics must not be harmful to health under normal conditions of use. Pre-marketing approval is not required, although manufacturers are responsible for compliance with the safety requirement. Notification of the composition of the formulated cosmetic product is not required under the EU legislation, but some countries may require notification of ingredients or product formulations. There is a list of prohibited cosmetic ingredients. The EU lists of permitted ingredients, usually with restrictions on incorporation levels and uses, cover colours, preservatives, ultraviolet filters and miscellaneous ingredients. New ingredients in these categories have to be approved at EU level and this requires the submission of a dossier including toxicological data on the substance concerned. Substances in categories not covered by the EU lists can be used under safe conditions, and the need for toxicological testing will depend on the proposed use and the animal and other safety data available on the substance, or close structural analogues from the supplier, or in the published literature. However, all cosmetic products have to be assessed for their safety in use by a qualified person, taking into account the use of the product,

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Practical Guide to Chemical Safety Testing any available toxicity data on the product and the known toxicological properties of all ingredients (irrespective of their function) in the product. The Scientific Committee for Cosmetology (SCC), since re-established as the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers (SCCNFP), have issued Notes of Guidance for the toxicity testing of cosmetic ingredients [30]. Generally the minimum information required for safety assessment of a new cosmetic ingredient is acute toxicity (if already available, not new studies), skin absorption, skin irritation, mucous membrane irritation, skin sensitisation, repeat-dose oral toxicity, mutagenicity (i.e., Ames test and in vitro chromosome aberration test), phytotoxicity and photomutagenicity for sunscreen ingredients, and available human data. Particular concern is paid to potential dermal absorption or if unavoidable oral exposure is anticipated, and further studies (toxicokinetics, metabolism, reproduction toxicity, further mutagenicity or carcinogenicity) may thus be needed. GLP studies conducted to EU Methods or OECD guidelines are preferred, with SCCNFP guidelines for studies without EU or OECD methods. Note that alternatives to animal studies are encouraged, and the SCCNFP Guidelines advise on non-animal alternatives. The SCCNFP also receive toxicity data from industry via the European Cosmetic Toiletry and Perfumer’s Association (COLIPA), and advise the European Commission on safety. Their opinions are considered by the EU Working Group on Cosmetics, which is comprised of national representatives. The SCCNFP Guidelines are also directly relevant to safety assessment of other cosmetic ingredients being used for the first time. A cosmetic ingredient which is a ‘new chemical’ under the EU chemicals legislation (i.e., not listed on EINECS) has to be tested and notified under the Seventh Amendment of the DSD (see Section 9.2) before it can be supplied for manufacture into cosmetic products, even if it is to be used solely for this purpose. However, non-EINECS listed substances supplied in the EU only in formulated cosmetic products are not notifiable.

9.8.2 Detergents Council Directive 73/404/EEC, as Amended, [31] regulates ‘detergents’ in the EU. Detergents are preparations which contain surfactants as essential constituents, and normally other components. Such detergent preparations can only be placed on the EU market if the average level of biodegradability of the surfactant components in them is at least 90%. The Directive is not concerned with other constituents such as phosphates. In addition, surfactants must not be harmful to human or animal health. It has been proposed to update the existing legislation on detergents by replacing tests based on primary biodegradability with those based on the more stringent ready

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EU Chemical Legislation biodegradability criterion. It is estimated that around 97% of existing surfactants could pass a ready biodegradability screen.

9.8.3 Offshore Chemical Notification Scheme: Oslo and Paris Convention for the Protection of the North East Atlantic The oil industry uses large quantities of chemicals in its offshore oil exploration and production activities in the North Sea, such as biocides, corrosion inhibitors, detergents and dispersants. Controls on chemical usage offshore have been relatively weak. For example, in the UK, the voluntary Offshore Chemical Notification Scheme (OCNS) was set up in 1979, administered by the then Department of Trade and Industry (DTI) with scientific input from the Ministry of Agriculture, Fisheries and Food (MAFF). Chemical suppliers agreed to provide details of the physical properties, nature and likely use of their products. The only environmental information was an acute toxicity test on a crustacean species, to rank products in one of five hazard categories. Offshore operators informed the DTI when discharges of a chemical at a particular installation were to exceed a specified tonnage in any one year. A mandatory regime for controlling the use of chemicals by the offshore oil and gas industry was, however, agreed at the annual meeting of the Oslo and Paris Commissions (OSPAR) in June 1996. The system requires companies to make applications for the use and discharge of chemicals in the form of a ‘harmonised offshore chemical notification format’ (HOCNF), and involves a progressive move to less hazardous substances following risk assessment. This revised UK OCNS operates in accordance with the OSPAR HOCNF. After a trial period, the HOCNF scheme was adopted into the Harmonised Mandatory Control System for the Use and Reduction of the Discharge of Offshore Chemicals (OSPAR Decision 2000/2 [32]), which came into force on 16 January 2001 (with the option of revision, if needed, following ongoing review, in 2004). Any use and discharge of offshore chemicals is regulated in four steps. Operators in the North Sea have to have adequate information on the chemicals they use from their suppliers to allow them to be assessed properly. Step I is to apply to the appropriate national authority for approval. Step II is pre-screening both to identify any chemicals in the preparation which are on the ‘OSPAR List of Substances/Preparations Used and Discharged Offshore, Which are Considered to Pose Little or No Risk to the Environment’ (PLONOR list) and can be used without further evaluation, but also to identify any component substances which are of concern and should be substituted if a less hazardous or non-hazardous substitute is available. Classification as a substance requiring substitution on this pre-screen results if any of these criteria are met: the substance is restricted by OSPAR or is of equivalent concern for the marine environment; it is inorganic

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Practical Guide to Chemical Safety Testing and highly toxic; it is persistent in the environment; or it has two of the characteristics of being not readily biodegradable, of high bioaccumulation potential or of high toxicity. Any chemical not in the PLONOR list has to be ranked as Step III, using a full HOCNF. The data needed are discussed below. Partition coefficient (by testing or calculation), is needed for all substances or components of a preparation to decide if the chemical is potentially bioaccumulative (i.e., log Pow > 3), in which case a fish bioaccumulation test can be conducted to clarify this concern. The chemical is treated as persistent, unless proven otherwise by a ready biodegradation test. Non-biodegradable chemicals, or those released daily, which are highly coloured are allocated a taint warning. Toxicity testing on marine algae and crustacea (copepod) are mandatory, and a chemical likely to associate with marine sediment also requires a sediment reworker test, otherwise a fish toxicity test is conducted instead. Standard GLP-compliant, OECD, International Standards Organization (ISO) or EU methods, as appropriate, should be used. Offshore chemicals are grouped A (very toxic) to E (innocuous), according to their toxicity, biodegradability and bioaccumulation potential. The simple grouping criteria may be modified for chemicals that are considered especially hazardous, such as those discharged in large quantities or containing heavy metals. The category group indicates at what tonnage the offshore operator has to consult the national authority, and an extra level of stringency has been added by requiring notification when the use of all chemicals in a particular hazard category group is likely to exceed the relevant tonnage threshold at a particular installation in any year, rather than for discharge of an individual chemical. Chemicals are evaluated for ranking using the Chemical Hazard Assessment and Risk Management (CHARM) model, which evaluates risk on the basis of the ratio between the predicted environmental concentration (PEC) and the no-effect concentration (NEC) in a standardised discharge situation, to give a ‘hazard quotient’ as a general PEC/PNEC ratio. Chemicals are ranked according to the potential risk they pose, and grouped into function categories. In Step IV national authorities make management decisions to decide whether individual chemicals can be authorised, substituted, or if a temporary permission (for up to 3 years) can be granted. In taking a decision over authorisations, national authorities must ensure that over time a shift is realised towards lower relative PEC/NEC ratios. If an operator wants to replace a chemical with one with a higher PEC/NEC ratio, permission must be obtained for temporary authorisation because of health, safety, technical or economic difficulties.

9.9 Summary and Future Developments Since ‘new’ chemical substances became notifiable in the EU approximately 3,000 substances have been notified. The notification scheme is generally regarded as

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EU Chemical Legislation achieving its main aims of generating safety data on new chemicals and preventing the entry of dangerous substances into commerce. There are, however, reservations about the suitability of the scheme. Firstly, European industry is concerned that the high cost of testing new chemicals puts European companies at a commercial disadvantage compared to manufacturers in countries where the regulations are less onerous, notably the USA. Secondly, new chemical substances still account for less than 1% by volume of all the chemicals on the market. It is this latter concern which has lead to plans for a complete overhaul of the chemical notification scheme in the EU. The European Commission’s review of legislation ‘Simple Legislation for the Internal Market’ (SLIM) has included the Dangerous Substances Directive (67/548/EC) in its Phase IV latest review [33]. The recommendations from this review include various practical improvements to the EU hazard communication and new substance notification schemes, which can be implemented fairly rapidly and easily without amending the EU Directives. The intended SLIM IV improvements to the existing EU chemicals regulation measures have to a large extent been superseded by the radical revisions proposed by the February 2001 White Paper ‘Strategy for a Future Chemicals Policy’ [34]. The White Paper proposed a wide-ranging fundamental overhaul of EU chemical control legislation, i.e., the Dangerous Substances Directive (DSD) (see Section 9.5.1) including the Seventh Amendment [1] new substance notification scheme (see Section 9.2), the Dangerous Preparations Directive [22] (see Section 9.5.2), the Existing Substances Regulation [11] (see Section 9.4) and the Marketing and Use Directive [2]. The main objective of the new EU Chemical Strategy is to ensure a high level of protection for human health and the environment, while maintaining an efficient internal market within the EU and stimulating innovation and competitiveness in the chemical industry. The White Paper also takes account of the need to increase transparency to decisionmaking and improve public access to information on chemicals. There will be integration with international initiatives. The Precautionary Principle will be applied, and substitution of substances with less dangerous alternatives will be encouraged. The new proposed scheme for assessing substances (both new and existing) is the Registration, Evaluation and Authorisation of Chemicals (REACH) system. The European Chemicals Bureau (ECB) would be expanded to administer the REACH system. All substances manufactured or imported in the EU at ≥1 tonne per annum will be registered with the ECB, who will forward the registration dossiers to Member State Competent Authorities and maintain a database of registered substances. Registration will be needed before new substances are manufactured or imported. Precise deadlines

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Practical Guide to Chemical Safety Testing will be established for registration of existing substances. Assuming rapid adoption of the new Chemicals Strategy, the White Paper proposed registration of existing substances produced at > 1,000 tonnes per annum by the end of 2005, those > 100 tonnes per annum by the end of 2008 and those > 1 tonne per annum by the end of 2012. Nevertheless, existing substances of concern may have to be registered earlier. These may be of concern as being used in consumer products or because of known or suspected hazardous properties. The studies to determine the hazardous properties of substances for registration will depend on the production volume: ≥ 1 to < 10 tonnes per annum: physico-chemical properties, in vitro tests ≥ 10 to < 100 tonnes per annum: Base Set tests (Annex VIIA of the DSD) ≥ 100 to < 1,000 tonnes per annum: Level 1 studies (Annex VIII of the DSD) ≥ 1,000 tonnes per annum: Level 2 studies (Annex VIII of the DSD) Nevertheless, waiving of testing may be accepted. The properties of the substance might be estimated by ‘read across’ to tested analogues, or particular properties might be calculated or otherwise estimated. Also, exposure-triggered testing will apply. Hence less data are needed for registration of chemical intermediates which are strictly controlled and rigorously contained. Conversely, further studies may be needed for certain highexposure uses. The registration dossier will include: •

Information on the identity of the substance;

•

The physico-chemical, toxicological and ecotoxicological properties (as described);

•

Intended uses, estimated human and environmental exposure;

•

Production quantity envisaged;

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Proposal for the classification and labelling of the substance;

•

Safety data sheet;

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Preliminary risk assessment covering the intended uses;

•

Proposed risk management measures.

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EU Chemical Legislation The ECB will undertake a computerised screening of registered substances, and also perform spot checks. It is anticipated that ca 30,000 substances will be registered, and that around 80% will not proceed to the next stage of evaluation. The information included in the registration of the ca 5,000 substances exceeding a production or import volume of 100 tonnes per annum will have to be evaluated. National Competent Authorities will be allocated substances to evaluate on behalf of the EU. When the volume reaches 100 tonnes per annum the manufacturer or importer will have to submit the available information and a proposal for a Level 1 testing programme. The rapporteur Competent Authority, in consultation with other Competent Authorities and the ECB, will agree the final testing programme, and evaluate the Level 1 studies when they are submitted. At 1,000 tonnes per annum an equivalent procedure is followed for Level 2 testing. For existing substances already exceeding the evaluation trigger values, a tiered approach is proposed with Level 2 testing to be completed by 2010 and Level 1 by 2012. Some substances supplied at below 100 tonnes per annum will also have to be evaluated on a case-by-case basis. These are substances suspected to be both persistent and bioaccumulative, those with certain hazardous properties (such as mutagenicity or high toxicity) and substances with chemical structures of concern. The rapporteur Competent Authority may request further information as the outcome of evaluation or recommend safety measures. Substances of very high concern will have to be authorised before being used for specific purposes, which have been demonstrated to present a negligible risk. It is estimated that ca 1,400 substances will be subject to authorisation. Step 1 is proposed to identify existing substances, or particular uses of substances, requiring authorisation, and to decide on a deadline for authorisation and any uses exempted from authorisation. As additional very high concern substances are identified, largely from testing for registration and evaluation, they will be fed into the authorisation system. Particular uses of very high concern substances will be authorised in Step 2 on the basis of a risk assessment covering all stages of the life-cycle for that particular use submitted by industry. The risk assessment will focus on exposure assessment for the use, and generally no new studies would be required. Uses will be authorised at EU level if they could affect the whole EU, or by national Competent Authorities if only one country is likely to be affected. There is also the option for conditional authorisation if this if justified on the grounds of socio-economic benefits. The majority of very high concern substances will be those classified as EU category 1 or 2 carcinogens, mutagens or toxic for reproduction (estimated as ca 850 currently with an additional ca 500 likely to be identified from future testing). The White Paper considers that most endocrine disruptors would require authorisation by being classified as carcinogenic or toxic for reproduction. The second type of substance requiring

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Practical Guide to Chemical Safety Testing authorisation are persistent organic pollutants (POPs), and the White Paper considers that further endocrine disruptors with adverse effects on wildlife (but not on human health) would be considered as POPs. The penultimate draft of the White Paper also contained options for authorisation of persistent, bioaccumulative and toxic (PBT) substances and very persistent and very bioaccumulative (vPvB) substances, but the final version requires such substances to be identified through further research and defers a decision on how to treat them. Finally, it should be noted that there may be general exemption from authorisation for uses of very high concern substances which do not give rise to concern (e.g., well-controlled industrial uses or in research laboratories). It is proposed to increase the 100 kg per annum exemption from notification of new substances used exclusively for scientific research and development to 1 tonne per annum. The current 1-year exemption for process-orientated research and development (extendable if justified for a further year) will be increased to 5 years (extendable for a further 5 years). The White Paper proposes to set up a working group to identify product categories (e.g., toys and textiles) and exposure situations which may require further controls. Under the current arrangements, notification of new substances and hazard communication for substances and preparations applies to chemicals placed on the EU market, and some products, especially those made outside the EU, may contain unassessed chemicals which have the potential for release in significant amounts during use, and disposal of the product containing them. The White Paper encourages research and validation to develop alternatives to animal tests and prediction methods and in risk assessment, especially exposure and use patterns. Accelerated risk management of substances which do not require authorisation will arise out of the REACH system. A key element in achieving this is targeted risk assessments, instead of the time-consuming detailed comprehensive risk assessments needed under the current arrangements. The precautionary principle will be applied. Downstream formulators and users of substances will have additional responsibilities under the White Paper proposals. The preliminary risk assessments in the registration dossiers submitted by the manufacturer or importer of each substance may not have covered thoroughly all the various downstream uses of the substance. Downstream users will have to inform the appropriate authority of any such use not included in the original preliminary risk assessment. They also have to perform an additional risk assessment covering all stages in the life-cycle for this new use. In order to do this information on exposure will have to be provided. The outcome may be that the authorities require additional test data from the downstream users.

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References 1.

Council Directive 92/32/EEC of 30 April 1992, Official Journal of the European Communities, 5:6:92, L154, 1.

2.

Council Directive 76/769/EEC of 27 July 1976, Official Journal of the European Communities, 27:9:76 L262, 201, as amended.

3.

EC Communication 90/C 146A/01 European inventory of existing commercial chemical substances, Official Journal of the European Communities, 15:6:90, C146A, 1.

4.

Council Directive 79/831/EEC of 18 September 1979, Official Journal of the European Communities, 15:10:79, L259, 10.

5.

Commission Directive 2000/C 72/01, Fifth Publication of the European List of Notified Chemical Substances, Official Journal of the European Communities, 11:3:2000, C72, 1.

6.

Annex V of Council Directive 67/548/EEC of 27 June 1967 as amended and adapted to technical progress (available at http://erb.jrc.it//testing-methods/).

7.

OECD Guidelines for the Testing of Chemicals, OECD, Paris, France, 1993, as updated.

8.

Commission Directive 93/67/EEC of 20 July 1997, Official Journal of the European Communities, 8:9:93, L227, 9.

9.

Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No. 1488/94 on Risk Assessment for Existing Substances, European Commission, Brussels, Belgium, 1996 and 2003 partial update and revision.

10. EUSES the European Union System for the Evaluation of Substances, European Commission European Chemicals Bureau Joint Research Centre, Ispra, Italy, 1997. 11. Council Regulation (EEC) No. 793/93 of 23 March 1993, Official Journal of the European Communities, 5:4:93, L84, 1. 12. IUCLID CD-ROM, European Commission Joint Research Centre European Chemicals Bureau, Ispra, Italy, 2000, second edition.

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Practical Guide to Chemical Safety Testing 13. International Uniform Chemical Information Database, European Commission Joint Research Centre European Chemicals Bureau, Ispra, Italy, latest version. 14. Commission Regulation (EC) No. 1179/94 of 25 May 1994, Official Journal of the European Communities, 26:5:94, L131, 3. 15. Commission Regulation (EC) No. 2268/95 of 27 September 1995, Official Journal of the European Communities, 28:9:95, L231, 18. 16. Commission Regulation (EC) No. 143/97 of 27 January 1997, Official Journal of the European Communities, 28:1:97, L25, 13. 17. Commission Regulation (EC) No. 2364/2000 of 25 October 2000, Official Journal of the European Communities, 26:10:2000, L273, 5. 18. Commission Regulation (EC) No. 1488/94 of 28 June 1994, Official Journal of the European Communities, 29:6:94, L161, 3. 19. Approved Supply List, Information approved for the classification and labelling of substances and preparations dangerous for supply, Health and Safety Commission HSE Books, London, England, latest edition. 20. Commission Directive 2001/59/EC of 6 August 2001, Official Journal of the European Communities, 21:8:01, L225, 1. 21. Council Directive 88/379/EEC of 7 June 1988, Official Journal of the European Communities, 16:7:88, L187, 14. 22. Directive 1999/45/EC of 31 May 1999, Official Journal of the European Communities, 30:7:99, L200, 1. 23. Council Directive 91/414/EEC of 15 July 1991, Official Journal of the European Communities, 19:8:91, L230, 1, as amended 24. Directive 98/8/EC of 16 February 1998, Official Journal of the European Communities, 24:4:98, L123, 1. 25. Recommendations on the Transport of Dangerous Goods Model Regulations, United Nations, Geneva, Switzerland, latest edition. 26. The Evaluation of the Hazards of Harmful Substances Carried by Ships Report No. 35, International Maritime Organisation, London, England, 1989.

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EU Chemical Legislation 27. Einstafung wassergefährdender Stoffe auf der Basis der Werwaltungsvorschrift wassengefährdende Stoffe (VwVwS) von 17:5:99 LtwS No. 10, Umweltbundesant, Berlin, Germany, 1999. 28. The rules governing cosmetic products in the European Union Volume 1 Cosmetics Legislation, European Commission, Brussels, Belgium, 1999. 29. Commission Directive 96/335/EC of 8 May 1996, Official Journal of the European Communities, 1:6:96, L132, 1, as adapted for the first time for Section II Parfume and Aromatic Raw Materials, Scientific Committee on Cosmetic Products and Non-food Products Intended for Consumers, Brussels, Belgium, report SCCNFD/0389/00 Final, 24 October 2000. 30. Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation, Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers, Brussels, Belgium, report SCCNFD/032/00 Final, 24 October 2000. 31. Directive 73/404/EEC of 22 November 1973, Official Journal of the European Communities, 17:12:73, L347, 51, as amended. 32. OSPAR Decision 2000/2 on a Harmonised Mandatory Control System for the Use and Reduction of the Discharge of Offshore Chemicals, OSPAR Convention for the Protection of the Marine Environment in the North-East Atlantic, London, England, June 2000. 33. Report from the Commission Results of the Fourth Phase of SLIM, European Commission, Brussels, Belgium, reference COM(2000) 56 final, February 2000. 34. White Paper Strategy for a Future Chemicals Policy, European Commission, Brussels, Belgium, reference COM(2001) 88 final, February 2001.

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10

Chemical Control in Japan Derek J. Knight

10.1 Introduction to the Japanese Regulatory Culture The regulatory systems governing the manufacture and supply of chemicals in Japan are relatively complicated being covered by various laws, which have developed over time and which are administered by different authorities. Moreover for successful notification of new chemicals in Japan it is necessary to grasp the social, business and legal culture in which these systems operate. Hence, for the non-Japanese supplier seeking to notify a new chemical in Japan it is advisable to use the services of an experienced local consultant or agent who will often be able to achieve this goal more easily. This is partly because a local expert would have a greater understanding of the philosophy and practice of the various schemes, and thus be able to make the most effective use of the available exemptions and derogations from notification of new substances. Another reason is because the outcome of any interaction with Japanese authorities is in practice often influenced by the manner and courtesy with which they are approached and dealt with. Differences in approach to dealing with regulatory requirements, approaching officials, attitudes on compliance, and indeed the entire framework and practice of chemical control measures, seem especially pronounced between North America or Europe and Japan. This in part explains why apparent misunderstandings and failures of communication occur, and why it is important for European and North American exporters of chemicals to understand the Japanese mentality and culture, as well as the details of the law and administrative practice. Although the Japanese test methods are, in principle, based on OECD guidelines, there are some differences in testing methods and interpretation of the test results. Thus it is always desirable to have a pre-consultation with each Japanese regulatory authority, especially if foreign study reports are to be submitted. The objective of the pre-consultation is to obtain each regulatory authority’s approval before submission. This procedure is especially important before finalising biodegradability or bioaccumulation study reports. Making the most of the pre-consultation system, particularly with the help of a local consultant or agent, can make the notification process much easier.

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Practical Guide to Chemical Safety Testing Chemical control in Japan has been described by Wellenreuther [1] and Knight [2], in papers which are still essentially current, and more recently by Cooke and Kirkham [3].

10.2 The Ministry of Economy, Trade and Industry and Ministry of Health, Labour and Welfare Chemical Substances Control Law

10.2.1 Introduction The Japanese Law Concerning the Examination and Regulation of Manufacture etc., of Chemical Substances [4] which was enacted on 16 October 1973 and came into force on 16 April 1974, was the first new chemicals notification scheme in the world. The legislation is commonly referred to as the Chemical Substances Control Law (CSCL). Until 2000, the CSCL was jointly administered by the Ministry of International Trade and Industry (MITI), who dealt with the environmental aspects, and the Ministry of Health and Welfare (MHW). The various Japanese ministries have since had changed responsibilities and names. From January 2001, the new Ministry of the Environment (MoE) has been involved in assessing notifications. Also MITI has been re-named the Ministry of Economy, Trade and Industry (METI). The MHW has amalgamated with the Ministry of Labour (MoL) to form the Ministry of Health, Labour and Welfare. To keep the distinction between the CSCL and the parallel workplace scheme (see Section 10.3), the abbreviation MHW will be applied to the section of the Ministry dealing with the CSCL. The CSCL regulates the use of industrial chemicals in general. The enactment of this legislation was largely triggered by the Minamata Bay incident in which hundreds of people were poisoned through consumption of fish contaminated with mercury derived from material which had been dumped in the bay. Thus, initially its main objective was to protect humans from exposure to hazardous substances via the environment, in particular via the food chain. Of major concern were detrimental health effects caused by persistent, accumulating and chronically-toxic substances such as polychlorinated biphenyls. Therefore, for the assessment of possible risks, special emphasis was put on biodegradability, persistency and bioaccumulation. Chemical substances found to be non-biodegradable, persistent and bioaccumulative, and with chronic toxicity, became subject to very stringent regulatory actions (classified as Specified or Designated substances, see Section 10.2.6). The law was revised in April 1987 to take account of adverse health effects from non-biodegradable but non-bioaccumulative environmental pollutants, which were evaluated as safe under the old scheme without a full evaluation of their chronic toxic effects. A further revision of the CSCL is in development to include evaluation of effects on the aquatic environment.

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Chemical Control in Japan The CSCL notification scheme is complicated to deal with because, in principle, it is a stepwise process (see Figure 10.1), beginning with a biodegradation study, which is central to the whole scheme, and there is a possibility of having to consult METI part way

Figure 10.1 Flow scheme for Japanese Chemical Substance Control Law Notification

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Practical Guide to Chemical Safety Testing through. Also, with CSCL notifications, regulatory decisions regarding the test material description are often relevant. In view of the stepwise nature of testing under the CSCL and the protracted review periods, it can easily take over two years for notification to be able to import or manufacture a new substance in Japan. Hence, in practice, the notifier may elect to take a calculated risk and assume that the parent substance (and not environmental degradants) needs to be tested and conduct the biodegradation, bioaccumulation and screening toxicity studies concurrently.

10.2.2 The Inventory of Existing Substances Chemical substances which do not fall into any of the exempted use categories discussed in Section 10.2.3 are potentially notifiable before they can be manufactured or imported into Japan, either neat or as a component of a mixture. There is a distinction between substances contained in a simple mixture or in a product (i.e., either an article with a particular form essential to its use or special formulations such as resin paints and photographic emulsions). Note also that new substances which are components of products are not notifiable, but any Specified or Designated substances in ‘products’ are subject to the normal reporting measures (see Section 10.2.6). Notifiable new substances are those chemicals not included in METI/MHW’s List of Existing Chemical Substances, METI/MHW’s published lists of notified new safe chemical substances under the old and new laws or METI/MHW’s published designated chemical substances. These various inventories are commonly referred to as the MITI List (presumably the term METI List will soon catch on). Although there is a separate inventory for the workplace notification scheme (see Section 10.3), in practice the MITI List is normally searched first, partly because an almost up to date version is available in English as the Handbook of Existing and New Chemical Substances, published by the Chemical Daily Co. [5]. Any substance included in the inventory is non-notifiable and is loosely referred to as an existing substance. Nevertheless, there is an administrative distinction between truly existing substances in commerce in Japan when the scheme came into force (as listed in the white pages of the Handbook) and the notified substances (as listed in the green pages). Each part of the Japan Chemical Daily Handbook is split into sections, based on type of chemical, and there is an index by Chemical Abstracts Services (CAS) number. However, there is a big practical difficulty in being confident that a substance apparently not present by CAS number is genuinely excluded from the inventory. This is because there are many entries, especially for existing substances, which have a very broad ‘generic’ description.

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Chemical Control in Japan Hence, there is not complete correspondence between CAS number and inventory entry. This means that computer searches using various systems, e.g., CAS and Ariel, can result in false negative results. The only way to deal with this problem is for a chemist to search the handbook by structural class. In practice though, for many complex reaction mixture substances it would be very difficult to give definitive advice that a substance is or is not present on the inventory, so METI/MHW may have to be consulted. It is noteworthy that Japanese industry tend to take a pragmatic and flexible interpretation of the generic inventory entries. Notified substances are listed on the inventory in due course. There is a period of uncertain and variable duration, which may be several years, after notification but before the official Notice is published. During this period only the original notifier can supply this new substance, and any competitors have to notify it again. There are no administrative arrangements for a potential notifier to establish whether their new substance has already been notified but not yet published, and no official means of finding out the identity of a first notifier in order to attempt to share the original data. Hence it is not uncommon for the same new substance to be tested twice. The Japan Chemical Daily Handbook is always somewhat out of date since notified substances are gazetted periodically. Hence the only way to be sure a substance has not been notified and listed on the ‘MITI List’ is to check the Japanese-language CD-ROM version of the inventory, which is updated more regularly, and for gazetted notices.

10.2.3 Exemptions from Notification Radioactive substances and specified poisons, stimulant drugs and their precursors, and narcotic and psychotropic drugs, which are regulated under separate legislation, are outside the scope of the CSCL, and none of its provisions apply. The notification requirements of the CSCL do not apply to the following substances, because they are subject to separate Japanese control procedures: food, direct food additives, food packaging (i.e., indirect food additives), detergents for cleaning food contact materials, toys, agricultural pesticides, fertilisers, animal feed, animal feed additives, human pharmaceuticals, medical devices and cosmetics (including ingredients imported for formulation into finished cosmetic products). Note that the other provisions of the CSCL still apply to these products even though they are exempt from notification. Site-limited chemical intermediates are not notifiable. This exemption also applies even when they are not strictly site-limited, in that the new substance can be made on one site and completely used up by the same company but on a different site.

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Practical Guide to Chemical Safety Testing Chemical intermediates for manufacture of pharmaceutical active ingredients are exempt from notification, even when not site-limited. This exemption must be interpreted carefully, however, because it only applies for intermediates of drugs already approved by the MHW under the Japanese Drugs, Cosmetics and Medical Instruments Act. Therefore the exemption strictly would not apply if the drug is still under development (i.e., not yet registered) or if it is registered only outside Japan. Indeed new pharmaceutical intermediates are often notified if they are exported for further processing outside Japan. This subject is discussed further below. New substances to be used solely for testing and research or as reagents are exempt from notification. The interpretation of the testing and research exemption seems in practice to be broad, and is used to cover many aspects of research into performance characteristics. The exemption is often considered to cover pharmaceutical intermediates for manufacture of unregistered drugs which are still under development. There is no volume or time restriction on this ‘testing and research’ exemption, although the substance cannot be sold to the user (i.e., it must be provided free of charge), and the customer has to certify that this is so. There are, however, no restrictions on the sale and use of products (e.g., drugs) resulting from the research carried out under this exemption. There is a special customs clearance scheme for import of new substances exempt from notification as pharmaceutical intermediates, reagents or for testing and research, and an appropriate customs form has to be completed. There is a low volume exemption (LVE) scheme for new substances manufactured or imported at below one tonne per annum. This upper limit applies to all suppliers combined, and hence, if there is more than one exemption in operation for the same substance, the permitted quantities would have to be shared out. Applications must be made annually, and there are specified 10-day submission periods (20 February to 1 March, 1 to 10 June, 1 to 10 September and 1 to 10 December) every three months for supply during the remainder of the year ending 31 March. Hence LVE applications made after the first submission period may not be approved if the one tonne supply limit has been reached already. Technical and administrative information and certain non-GLP physico-chemical data have to be provided. An important part of the LVE application is the justification for the International Union of Pure and Applied Chemistry (IUPAC) chemical name, based on a full explanation of the IUPAC rules used with the skeletons and sequence for naming of substituents. Note that the Japanese manufacturer or importer must make the LVE application, whereas a non-Japanese exporter can make a full notification.

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10.2.4 Standard Notification The philosophical approach of Japanese chemical control seems to be somewhat different to the rest of the developed world. For example, the fundamental aim of the CSCL notification scheme is to evaluate the potential human hazard from exposure to new chemicals through the environment. Therefore, a stepwise testing procedure is adopted, and the ecotoxicity and toxicity studies may have to be conducted on the environmental degradants, instead of on the parent substance. Another significant difference to other notification schemes is that the properties of the pure chemical substance are considered in Japan, whereas the standard technical grade substance (with its associated impurities) is evaluated in other notification schemes. Therefore, it may be necessary to test a purified sample of the substance for Japanese notification. Impurities contained at above 1% in a new chemical substance are regarded as components of a mixture. In principle, each such impurity should be tested separately for notification. One benefit of this approach is that the technical grade substance is considered to be a mixture of notified/existing substances, and consequently its composition can be varied freely if needed. One option is to use purified substances as the test material. Alternatively, the doses used for the three toxicity screening tests can be corrected to 100%, for a purity below 99%. Poorly defined reaction mixtures consisting of isomers and congeners can be tested and notified as the mixture. New substances must be notified three months before manufacture or import. For CSCL notification, the Japanese test methods are based on those of the OECD, although some are more stringent, and the standard EU studies have to be enlarged. Many of the studies have to be reported in a prescribed format with the data interpreted in a specific way. There is mutual acceptance of GLP between the EU Member States and Japan. Consequently, the Japanese regulatory authorities will accept foreign studies. In principle, existing GLP-compliant studies conducted to OECD/EU methods for nonJapanese notifiers are acceptable, but the results have to be suitable for interpretation by the Japanese authorities, which in practice can mean that only studies with results indicating that the substance is hazardous are accepted. The first stage in the testing programme is to evaluate the biodegradation potential of the substance. To pass the MITI ready biodegradability test, and hence be classified as a safe chemical substance under the CSCL scheme without further testing, virtually complete biodegradation is necessary to completely-mineralised products (i.e., carbon dioxide and water). The ready biodegradation test is technically demanding, and is often conducted at an experienced Japanese laboratory. The difficulty is that there has to be a virtually

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Practical Guide to Chemical Safety Testing 100% mass balance for a ready biodegradation study (i.e., all the test substance dosed has to be accounted for, as recovered parent substance, degradants or mineralisation products), and characterisation and quantification is required. If the substance undergoes a high degree of mineralisation in the MITI (I) ready biodegradation test, it may be that METI/MoE require a MITI (II) inherent biodegradation study to be conducted on the parent substance, before deciding what to test further. The CSCL scheme evaluates any degradation products produced from the notified substance, as well as the parent substance itself. Hence stable degradants may have to be further tested for bioaccumulation and toxicity, following discussion with the authorities. The next stage will be to evaluate the bioaccumulation potential of either the parent substance or its environmental degradant(s), assuming that the substance does not undergo complete mineralisation to be notified as a safe substance without further testing. For many chemicals, this evaluation can be done by measuring the n-octanol:water partition coefficient (Pow) using the OECD flask-shake method in compliance with GLP. Note that the high performance liquid chromatography (HPLC) method for estimating Pow is not applicable other than as a screen to decide whether to conduct the flask-shake method. The Pow is not considered to be a suitable indication of bioaccumulation potential for organometallic substances, surfaceactive substances or those which dissociate or associate in water. Hence, the pKa and a preliminary hydrolysis test are also required as part of the evaluation of bioaccumulation potential. For substances with ionisable groups, there are equations based on the measured pH of the aqueous phase in the Pow determination and the pKa to decide whether the substance is ionised. The criteria to decide if a substance with ionisable groups is dissociated are that pKa-pH < 1.7 for acids and pH-pKa< 1.7 for bases. If log Pow < 3, the substance can be considered unlikely to bioaccumulate, and further bioaccumulation testing is not needed. The definitive indication of bioaccumulation potential, for substances with a potential concern based on high Pow or for which Pow is not relevant, is a fish bioaccumulation study. Such a study is expensive and time consuming, especially if radiolabelled test material is needed. This may be the case if there are severe analytical difficulties in conducting such studies, because the test material has to be measured at very low concentrations in both water and fish. Two concentrations are used, based on the results of a 48-hour acute toxicity study. There are no published criteria for interpreting fish bioaccumulation study results, but based on experience with the notification scheme it is possible to make some generalisations. Certainly, if the bioconcentration factor (BCF) ≤ 100, the substance is defined as not bioaccumulative, and if 100 < BCF < ca 500 (and perhaps up to ca 1,000), the substance is normally regarded as not bioaccumulative. An elimination test is needed if the BCF is greater than 1,000, and METI reach a decision on whether a substance is bioaccumulative based on all of the measured parameters.

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Chemical Control in Japan Before conducting Pow or fish bioaccumulation studies, it is essential to consider the alternative of assessing bioaccumulation by analogy to chemically-similar compounds which have already been tested, either notified new substances or selected existing substances evaluated, and published by METI. If the tested analogue has log Pow < 3 or BCF < 100, the new substance is considered not to be bioaccumulative. Nevertheless, even if the chemical structures are not considered close enough for METI to agree to miss out the bioaccumulation study, they may agree to a shortened version of the study. Note that it may be the case that for notification of several closely related new substances in Japan, a bioaccumulation study may be needed on just one, and this is used as a basis for analogy for the others. Full toxicity testing would be required on a substance shown to bioaccumulate to establish whether it is a Class 1 specified or a safe substance. A full toxicity testing programme covers chronic toxicity, mutagenicity, carcinogenicity, reproduction toxicity (including developmental toxicity), toxicokinetics and pharmacology. Hence, bioaccumulative substances requiring full safety toxicity testing are not normally progressed. Assuming the substance (or its environmental metabolites) is not bioaccumulative, the final step is to undertake the screening toxicity studies. These are the 28-day repeat dose oral toxicity in rats, Ames test and in vitro chromosome aberration tests. Under the proposed amendment to the CSCL these screening toxicity tests would be needed only when the substance is to be supplied at 10 tonnes per annum or above. Additionally at this supply level, effects on the aquatic environment would have to be investigated by acute toxicity studies in fish and Daphnia and an algal test. The 28-day subacute toxicity study must include a satellite group (dosed at the maximum level of the main study) with a 14-day recovery period and a corresponding control group. Certain parameters additional to the standard OECD requirements are investigated, and all statisticallysignificant findings must be evaluated for toxicological significance. If the no-observed effect level (NOEL) is below ca 50 mg/kg/day, the substance may be classed as a Designated Chemical. Again the classification criteria are informal and changing. For example, prior to around the year 2000, it was generally accepted that the NOEL could be as low as 10 mg/kg/day without necessarily resulting in the substance being classed as Designated. The methods required for the Ames test and in vitro chromosome aberration test used to differ from the standard EU or OECD methods, but as from around 2001 MHW (and MoL, see Section 10.3) accepts the current OECD guidelines, although they do prefer Chinese hamster lung or ovary cell lines to be used rather than lymphocytes for the in vitro chromosome aberration test. The mutagenicity tests are evaluated, again according to unpublished informal criteria, to decide, with the 28-day repeat dose toxicity study results, if the substance is classed as Designated.

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Practical Guide to Chemical Safety Testing A notification consists of a notice of intended manufacture or import of the new substance, the so-called ‘Blue Card’ (New Chemical Substance Card), which provides the technical data and a summary of the studies, and multiple copies of the study reports. All the documents have to be in Japanese. Three copies of English-language study reports with original signature pages are also required, one for each Ministry. Notifications are reviewed in batches approximately monthly. METI, MoE and MHW each separately review the submission on various occasions over a period of about two months. Any questions must be answered within about a week to avoid postponement of the review to the next month’s meeting. At the beginning of the process the notifier or his representative attend hearings at METI, MoE and MHW to explain the study results. Experience is awaited on how the MoE will fit into the scheme. The notifier can obtain an unofficial result of the assessment at the end of the review period, but the official letter of judgement which permits import or manufacture of the new substance is sent only after the mandatory three-month waiting period.

10.2.5 Polymer Notification The OECD polymer definition does not officially apply in Japan. Under the CSCL, a polymer is considered to be a substance with a number-average molecular weight (Mn) of at least 1,000 (and typically 10,000) a molecular-weight distribution and physical characteristics typical of a polymer (see Table 10.1). The Japanese scheme for notification of polymers under the CSCL is discussed in detail in Section 12.3, so only a few practical aspects based on experience with the system are covered in this section. Non-notifiable polymers are included in the MITI List. A polymer not listed has to be notified, or an annual LVE applied for if applicable. Polymer notification of new polymers is summarised in Figure 12.2. Inert polymers may be notified without the full testing programme. In order to establish whether they are inert, the polymer flow scheme (PFS) is followed. In practice the PFS testing is always conducted by a Japanese laboratory. If a new polymer is established as stable by the prescribed physico-chemical testing of the PFS, no further studies are needed for the notification, otherwise the standard new chemical substance testing programme applies (Figure 10.1). Note that, in practice, it is likely that the Pow test method may not be applicable, and assuming bioaccumulation by analogy does not apply, a fish bioaccumulation study is normally needed. Expert judgement is essential, together with knowledge of the Japanese polymer scheme. It is useful to have some insight into whether a substance may pass the PFS. Any substance known or predicted from its structure to be water soluble, hydrolysable, soluble in solvents or to have a high oligomer content (i.e., greater than 1% of components with molecular 244

Chemical Control in Japan

Table 10.1 Characteristics of polymers for notification under the chemical substance control law Mn: generally > 10,000 (minimum > 1,000) Molecular weight distribution Polymers cannot be purified using techniques such as recrystallisation, distillation and sublimation Most polymers are not soluble in solvents, but some are readily soluble in specific solvents There is no clear solubility, and furthermore swelling occurs in crosslinked polymers There is no clear melting point Films or fibres are formed, or during processing crystallisation occurs Footnote: If it is not possible to measure the molecular weight, the substance can still be defined as a polymer for notification from the molecular design, molecular weight before crosslinking and information on the route of synthesis

weight less than 1,000) will not pass the PFS. A particular new polymer may be considered likely to pass the PFS water and solvent solubility and stability criteria, but likely to fail on the oligomer content criterion. In these circumstances there is an administrative option (known to be used by Japanese companies), to define the marketed product as a mixture of high molecular weight polymer as the main component with a minor component consisting of a complex reaction mixture of oligomers. The minor component can be regarded as a single substance and an LVE applied for on an annual basis (assuming supply is at less than one tonne per year). The major polymer component of the marketed product (excluding oligomers) is independently notified as a polymer. The PFS for testing would then be conducted on a sample of the notified polymer (i.e., excluding the LVE oligomer mixture). Hence, a specially purified sample will often be needed.

10.2.6 Class I and II Specified and Designated Substances Class I Specified substances are non-biodegradable, bioaccumulative and chronicallytoxic to human health. They correspond to the Persistent Organic Pollutants (POPs), which have become of concern worldwide, especially since the late 1990s. The intention of the CSCL is to identify such substances and control them. There are only around a dozen Class I Specified substances. These cannot be imported, manufactured or used without permission from METI. 245

Practical Guide to Chemical Safety Testing Class II Specified substances are non-biodegradable and chronically-toxic to human health but not bioaccumulative. There are around two dozen such substances. The manufacturer or importer has to notify METI of the planned supply amounts and also to provide guidance and advice to the users on recommendations to avoid environmental pollution. Designated substances are non-biodegradable and not bioaccumulative but suspected to be hazardous to human health on prolonged exposure. There are almost 200 such substances. Suppliers have to report the quantities imported or manufactured to METI, and also inform the users of the properties of the substance. METI undertake an exposure assessment by estimating the total amount in the environment to reach a decision on whether there is a potential for adverse health effects on humans. If so, in principle the suppliers can be requested to provide long-term toxicity tests to decide if the substance will be re-classified as safe or as Class II Specified. In practice this further testing rarely, if ever, is required.

10.3 The Ministry of Health, Labour and Welfare Industrial Safety and Health Law The Industrial Safety and Health Law (ISHL) [6] applies to chemicals used in the workplace, and hence those in domestic use are not covered. It applies to substances manufactured or used in the workplace (either neat or as a component of a mixture). The ISHL supplements the CSCL, is independent, and must be obeyed separately. The Ministry of Labour (MoL) administers the ISHL. Since 2000, the MoL has been part of the Ministry of Health, Labour and Welfare (MHW), but to facilitate the distinction between the ISHL and the CSCL, the abbreviation MoL will be used. The ISHL scheme came into operation on 30 June 1979. Substances already in use in Japan when the ISHL came into operation, and those notified to MoL since and added to the published list, are not notifiable. Hence notifiable ‘new’ substances are those not included in the MoL’s List of Existing Chemical Substances, MITI/MHW’s List of Existing Chemical Substances, MITI/MHW’s new chemical substances listed before 29 June 1979 or a new substance subsequently published as notified to MoL. Note that this separate ‘MoL inventory’ is not available in English, and in practice the MITI List is normally consulted first, since this is a good indication that the substance has also been notified under the ISHL scheme and will probably be on the MoL List. There are no exemptions from notification for pharmaceutical intermediates or cosmetic ingredients, and all site-limited intermediates are covered by the scheme since they are present in the workplace. Nevertheless there is an exemption for ‘research and development’ substances, but they must be used only in a research facility.

246

Chemical Control in Japan There is a LVE scheme to exempt substances from notification. A workplace user can request an exemption for use of up to 100 kg per annum per factory (i.e., the same company can have several exemptions, one for each site). The application can be made at any time, but must be 30 days before use, and it has to be renewed annually. Only technical and administrative information and non-GLP physico-chemical data have to be provided. There is also a polymer exemption. A ‘new’ polymer consisting of ‘existing’ monomers with a number-average molecular weight above 2,000 qualifies for exemption from full notification, by application to MoL without test data. However, this exemption does not apply for many categories of polymers (see Section 12.3.1.8). Under the MoL scheme it may be easier just to notify the new polymer (i.e., one Ames test only is needed, as discussed below). The main concern for MoL notification is to evaluate potential on mutagenicity and carcinogenicity. To judge these hazards the MoL require first an Ames test. If the Ames test is negative, or positive under 1,000 revertants/mg, notification normally proceeds without further requests (see Figure 10.1). However, if the results are near or above 1,000 revertants/mg, an in vitro chromosome aberration test is needed, and it is advisable to conduct an in vivo mouse micronucleus test as well. If these studies are negative, there is a good chance that no further testing will be needed. If one of these tests is positive, especially the mouse micronucleus test, the MoL normally require precautionary measures, such as informing employees of the possible mutagenicity risk and importers and/or distributors could be required to inform their workers accordingly by labelling. A positive outcome of the second mutagenicity test may result in further tests. If these are also positive, there will be stringent regulation of production and distribution. This process is illustrated in Figure 10.2. Once notified, the new substance is allocated a MoL registration number (which is different from the MITI registration number) and it can be imported, manufactured, or handled. Any labour protection measures are decided later as soon as a judgement on mutagenicity and carcinogenicity has been made by a committee of experts reporting to the MoL.

10.4 Hazard Communication and Product Liability Japanese labelling requirements are not as comprehensive as in the EU and the USA. Specified and designated chemical substances under the CSCL scheme should be labelled appropriately, as should dangerous substances under the ISHL and other legislation. Materials safety data sheet (MSDS) programmes were encouraged by Convention 170 of the International Labour Organisation (ILO), signed in June 1990, and Agenda 21 of the

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Practical Guide to Chemical Safety Testing

Figure 10.2 Japanese Industrial Safety and Health Law Notification Flow Scheme United Nations Conference on Environmental Development (UNCED), held in 1992. Thereafter in Japan there has been increasing acknowledgement that users of chemicals need adequate safety information. In July 1992 the MoL introduced an MSDS programme, and MITI/MHW issued a Public Notice in March 1993. Administrative guidance was provided in April 1993 for suitable recommended Japanese MSDSs, and the Japan Chemical Industry Association issued integrated ‘Guidelines for preparing MSDSs’ [7], consistent with the international ISO/EU format.

248

Chemical Control in Japan The 1994 Japanese Product Liability Law No.85 [8] came into force on 1 July 1995, and hence effective use of MSDSs is essential, because enforcement of the Law for chemicals is based on effective use of MSDSs. The Product Liability Law has the purpose of protecting consumers in case of injury from defective products. The new law shifts responsibility for defects from consumers to producers. The victim has to prove a defective product instead of a wilful act or negligence by the producer, which was previously the case if claims were made for damages against the producer of a defective product. In practice Japanese chemical companies have undertaken extra safety testing of chemicals since the Product Liability Law came into effect. The MSDS system for hazardous chemicals has been extended to become obligatory for certain chemicals and businesses from 1 January 2001 (although there was a grace period to 1 April 2001). The 1999 Japanese Law number 86 Concerning Reporting, etc., of Releases to the Environment of Specific Chemical Substances and Promoting Improvements in their Management [9] makes provisions for introducing this MSDS system to promote the management of substances by industry. This is linked with the Pollutants Release and Transfer Register (PRTR), to be discussed in Section 10.5. The MSDS/PRTR Law covers Class I and II Designated Chemical Substances. These are different to the Class I and II Specified and Designated Substances under the CSCL. Class I Designated Substances in the MSDS/PRTR scheme are substances which may be hazardous to health and/or impair the life and growth of flora and fauna, or which may transform to degradants with these properties, or which may damage the ozone layer. Class II Designated Substances have the same hazardous properties but are anticipated to have lower environmental exposure. The MSDS scheme applies to both Class I and II Designated Substances and to products containing them. The chemical substances and businesses to be covered by this PRTR/MSDS system are given in the March 2000 Enforcement Ordinance number 86 [10]. The content of the MSDS is officially specified in the METI Ordinance number 401 Pertaining to MSDS of 22 December 2000 [11] and there is a Japanese standard giving recommendations on MSDS preparation. The MSDS format is consistent with the international format of ISO 11014 [12]. Most of the text has to be in Japanese.

10.5 Other Chemical Legislation The CSCL and ISHL are of primary concern to non-Japanese manufacturers who export chemicals to Japan, but the following additional controls apply to particular chemicals: •

Poisonous and Deleterious Materials Control Law;

•

Fire Service Law;

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Practical Guide to Chemical Safety Testing •

High-Pressure Gases Control Law;

•

Law concerning the Protection of the Ozone Layer through the Regulation of Specified Substances and other Measures.

The purpose of the Poisonous and Deleterious Materials Control Law is to control acutelyhazardous chemicals. The substances classified by this law are listed in publications of MHW. The criteria for classification are given in Table 10.2. For production, import and sales of these substances, special licences are required. Labelling is also required. Labels must give the product name, poisonous ingredient and quantity, name of antidote (in special cases), and company name. Transport of these substances in quantities above one tonne must be accompanied by special instruction manuals for emergencies. The Fire Services Law regulates inflammable and oxidising substances and products. The classification is compatible with the UN scheme for transport of chemicals. Each substance falling under the law must be tested using specified methods to make a judgement whether it is safe according to the following hazard groups given in Table 10.3. The High-Pressure Gases Control Law regulates a number of gases, including combustible gases (lower explosion limit < 10% or range of explosion > 20%), toxic gases (TLV < 200 ppm), oxygen, and carbon dioxide. Special labelling is required. As described in Section 10.4, the Law Concerning Reporting, etc. of Releases to the Environment of Specific Chemical Substances and Promoting Improvements in Their Management [9] makes provisions for the introduction of PRTR and MSDS systems. The PRTR requires registration and publication of the volume of particular harmful substances released into the environment and the volume of such substances transferred as components of waste. The PRTR is intended to:

Table 10.2 Criteria for classification under the Japanese Poisonous and Deleterious Materials Control Law • Acute toxicity - Oral LD50 (mg/kg body weight) - Dermal LD50 (mg/kg body weight) - Inhalation LC50 (mg/kg body weight) • Skin and mucous membrane irritation • Experience by accidents • Other information

250

Poisonous substance

Deleterious substance

≤ 30 ≤ 100 ≤ 200

≤ 300 ≤ 1000 ≤ 2000

Chemical Control in Japan

Table 10.3 Japanese Fire Services Law Hazard Groups Group I

Oxidising solids

Group II

Combustible solids

Group III

Self-igniting and water-reactive substances

Group IV

Inflammable liquids

Group V

Self-reacting substances

Group VI

Oxidising liquids

•

provide information to set policies on chemicals;

•

promote voluntary management of chemicals;

•

provide basic information for environmental preservation;

•

inform the public;

•

gauge effectiveness of environmental preservation policies.

The first reports based on the PRTR system will identify the volume of pollutants released by individual businesses in the one-year period beginning April 2001. The reports are scheduled to be submitted after April 2002. The chemical substances and businesses to be covered by this system were determined in the March 2000 Ordinance [10]. Methods to estimate the volume of substances released and procedures for filing the reports will be defined by ministerial Ordinance. The PRTR system will operate as follows: •

Businesses determine the volumes of release and transfer of Class I Designated Chemical Substances and submit reports to prefectural governments, to be forwarded to the national government;

•

METI, MoE and industry jointly aggregate and publish the submitted data for each substance by type of business and by geographical region to be forwarded to the prefectural governors;

•

As a supplement to the reports received on the volumes of chemical substances released, MoE and METI jointly estimate, aggregate, and publish the estimated volumes released by households, agriculture, cars and other sources;

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Practical Guide to Chemical Safety Testing •

The national government upon request by the public discloses information relating to individual businesses;

•

Businesses are to abide by the Chemical Management Guidelines and aim to improve and strengthen their management of chemical substances. Furthermore, businesses are to keep the public better informed on release of such substances into the environment and their management.

Finally, it should be noted that Japanese industry, in partnership with the Japanese government, is actively participating in the International Council of Chemical Associations (ICCA) High-Production Volume (HPV) programme to evaluate existing substances.

10.6 Summary The philosophical basis of the Japanese notification schemes is fundamentally different to the rest of the world. There is also a drastic difference in culture between the Japanese and exporters from Europe and, perhaps especially, North America. These two factors mean that Japanese registration projects can be particularly difficult. Typically, the first stage in a project is to establish whether a substance is notifiable or not. Since there are two notification schemes, one firstly must establish if either or both apply. The MoL ISHL scheme covers substances, which are manufactured in Japan or used in the workplace. The METI/MoE/MHW (formerly MITI/MHW) CSCL scheme applies to substances manufactured in Japan or imported. There are various blanket exemptions to either or both schemes. The next stage is to check each of the inventories to identify if a substance has previously been notified or is an existing substance.

References 1. G. Wellenreuther, Toxic Substances Journal, 1988, 8, 45. 2. D.J. Knight, BIRA Journal, 1995, 14, 1, 26. 3. S. Kirkham and M. Cooke in Biocides Business Regulation, Safety and Applications, Eds., D.J. Knight and M. Cooke, Wiley-VCH, Weinheim, Germany, 2002, 104. 4. Japanese Law concerning the Examination and Regulation of Manufacture etc. of Chemical Substances (commonly referred to as the Chemical Substances Control Law), as amended.

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Chemical Control in Japan 5. Handbook of Existing and New Chemical Substances, Japan Chemical Daily, Tokyo, Japan, latest edition. 6. Japanese Industrial Safety and Health Law, 8 June 1972. 7. Guidelines for Preparing Material Safety Data Sheets, Japan Chemical Industry Association, 1992. 8. Japanese 1995 Product Liability Law No.85. 9. Japanese 1999 Law number 86 Concerning Reporting, etc. of Releases to the Environment of Specific Chemical Substances and Promoting Improvements in their Management. 10. Japanese Enforcement Ordinance number 86 of March 2000 Relative to the Law Concerning Reporting, etc. of Releases to the Environment of Specific Chemical Substances and Promoting Improvements in their Management. 11. Japanese METI Ordinance number 401 of 22 December 2000 Pertaining to Methods of Providing Information Concerning the Properties and Handling of Designated Substances etc. (commonly referred to as the Ministerial Ordinance Pertaining to MSDS). 12. International Standard Safety Data Sheet for Chemical Products, ISO 11014, International Organization for Standardisation, Geneva, Switzerland, 1994.

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Chemical Control in the US and the Rest of the World

11

Chemical Control in the US and the Rest of the World John M. Hislop, Derek J. Knight and Carlo Poncipe

11.1 Introduction Chemical control regulations in the EU and Japan have been covered in detail in the two preceding chapters. This chapter provides a summary of the remaining important chemical control schemes for notification of new substances and hazard communication that operate in the rest of the world, i.e., in the USA, Canada, Switzerland, Australia, Korea, the Philippines, China and New Zealand. This is followed by a brief summary of developing schemes in Mexico, Singapore, Malaysia, Thailand, Indonesia and Taiwan. It should be noted that other important markets, such as Russia, also have regulations affecting chemicals, and these are often being developed and extended. Evaluation of high production volume (HPV) chemicals has become increasingly important since the mid 1990s, so these schemes are covered in this chapter. Many of these HPV substances have been marketed for many years, but without the same degree of safety testing as required for new notifiable substances. The US HPV initiative, which is discussed in Section 11.2.3, in effect revitalised the OECD SIDS programme and the EU Existing Chemicals Regulation (Section 9.4) and resulted in the industry-lead ICCA HPV programme, as discussed in Section 11.18. Polymers generally warrant specific, sometimes simplified, control measures because of their chemical and physical properties, i.e., often they are chemically and biologically more inert than non-polymer substances. Therefore polymer notification worldwide is covered separately in Chapter 12. It is important to be aware that in addition to new substance notification, HPV programmes and hazard communication, there are other important regulations affecting the chemical industry and which control the manufacture and use of chemicals. For example there are environmental regulations, such as the Clean Air Act, the Clean Water Act, the Safe Drinking Water Act and the Comprehensive Environmental Resource Compensation and Liability Act (referred to as the Superfund) in the USA. There are also numerous physical safety laws, covering fire safety and major hazards and international regulations, such as the

255

Practical Guide to Chemical Safety Testing Chemical Weapons Convention and restrictions on substances which could be used to manufacture illicit narcotic and psychotropic drugs. Many countries have registration and licensing schemes and there are often additional state or provincial measures, especially in the USA. Finally some countries, such as the USA and Canada, control microorganisms as chemicals and require new ones to be notified. A key element in deciding whether to market new chemicals or support existing HPV chemicals is the cost of the safety testing. The studies are generally based on the OECD minimum premarketing data set (MPD), but vary between schemes. For notification of new substances, testing is generally supply driven, with less needed for supply typically below 1 tonne per annum (or 10 tonne per annum in some schemes). Additional testing may be required at higher supply levels, as in the EU. The standard testing programmes for the various schemes are given in Table 11.1, which includes the EU and Japan for comparison.

11.2 US Chemical Legislation: The Toxic Substances Control Act (TSCA)

11.2.1 Key Objectives of TSCA The Toxic Substances Control Act (TSCA) [1], which came into force on 1 January 1977, provides control over chemical hazards to human health and the environment that are not regulated by other legislation. TSCA requires pre-manufacturing notification of new chemical substances, testing of existing substances and regulation of substances that pose an unreasonable risk. The implementing regulations were revised as from 30 May 1995 to streamline the New Chemicals Program, encourage pollution prevention practices and to shift the focus to review of high risk chemicals. Substances controlled by other legislation are exempt from TSCA (i.e., pesticides, food or food additives, pharmaceuticals, cosmetics, nuclear material, ammunitions, tobacco and tobacco products). TSCA is administered by the Environmental Protection Agency (EPA). Useful TSCA guidance manuals are available [2, 3] and these detail subjects not covered in here, such as record keeping obligations, enforcement and penalties for non-compliance.

11.2.2 The TSCA Inventory The TSCA inventory is the list of substances that are manufactured or processed in the US. The most up-to-date version of the TSCA inventory is the TSCA Master Inventory File, which is maintained by the EPA and the Chemical Abstracts Service (CAS). It contains about 80,000 chemicals, of which approximately 66,000 are non-confidential (i.e., the chemical identity of the substance is disclosed) and approximately 14,000 are confidential

256

Table 11.1 Testing programmes for worldwide full notification of a new chemical substance Study

Requirement for supply in: EU/EEA (note a)

Switzerland (note b)

Spectra

✓

Melting point

USA (note c)

Canada (note d)

Australia (note e)

Japan (note f)

✓

✓

✓

note 1

✓

✓

✓

✓

✓} note g

note 1

✓

✓

✓

Boiling point

✓

✓

✓

✓}

note 1

✓

✓

✓

Relative density

✓

✓

✓

✓

note 1

✓

✓

✓*} note v

Vapour pressure

✓

✓

✓

✓

note 1

✓

✓

✓*

Surface tension

✓

✓

Water solubility

✓

✓

✓

✓

note 1

✓

✓

✓*

Partition coefficient

✓

✓

✓

✓

✓

✓

✓

✓*

✓ note h

✓

✓

✓

✓

note i

✓

✓

Dissociation constant Granulometry

✓

257

Henry’s Law

note i

Volatility from water

note i

Complex formation constants

note i

Stability

note i

Viscosity

note i

Permeability

note i

OECD MPD (note u) ✓

✓ note 1

✓* ✓

Chemical Control in the US and the Rest of the World

Fat solubility

South Korea Philippines (MoE and MoL)

Study

Requirement for supply in: EU/EEA (note a)

Switzerland (note b)

USA (note c)

Canada (note d)

Australia (note e)

Japan (note f)

South Korea Philippines (MoE and MoL)

Flash point (liquids)

✓

✓

✓

Flammability tests

✓

✓

✓

Explosivity

✓

✓

✓

Oxidising properties

✓

✓

Autoflammability

✓

✓

Acute oral toxicity

✓

note j

✓} note k

Acute inhalation toxicity

✓

OECD MPD (note u)

✓

✓ * note v

✓

✓

note j

✓} note k

✓} note k

✓}

note j

✓}

✓}

✓

✓*}

Skin irritation

✓

note j

✓

✓

✓

✓*}

Eye irritation

✓

note j

✓

✓

✓

Skin sensitisation

✓

note j

Subacute toxicity

✓

note j

Acute dermal toxicity

✓

✓

✓

✓

✓

✓ note p

✓

✓*} note k

✓ ✓

✓

Reproduction toxicity and developmental effects ✓

✓ note m

✓

Mouse lymphoma assay

note m

note j

Mouse micronucleus test

note n

note j

In vitro chromosome abberation test

✓* *

✓

Ames test

✓*

✓

✓

✓

✓

✓

✓

note t

✓*

✓

✓

✓

✓

note t

✓*

✓ note o

✓

note n

note n

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Table 11.1 Continued

Table 11.1 Continued Study

Requirement for supply in: EU/EEA (note a)

Switzerland (note b)

USA (note c)

Canada (note d)

Australia (note e)

Japan (note f)

Acute fish toxicity

✓

✓

✓

✓

✓

*

note t

✓*

Acute Daphnia toxicity

✓

✓

✓

✓

✓

*

note t

✓*

Algal growth inhibition

✓

note i

✓

✓

*

note t

✓*

note t

✓*

Fish bioaccumulation Ready biodegradation

✓

✓

✓

note i

note q

note q

✓

✓

✓

✓

✓

note l

note i

Activated sludge respiration inhibition

✓ note r

Abiotic degradation by hydrolysis

✓ note s

✓

✓

✓

✓

note i

✓

✓

Soil adsorption/desorption screening test Anaerobic biodegradation

note i

Soil biodegradtion

note i

Photolysis

note i

Transport and distribution between environmental compartments

OECD MPD (note u)

✓* ✓

note i

* *

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Daphnia reproduction study

South Korea Philippines (MoE and MoL)

NOTES: a. Chemical legislation is harmonised throughout the European Economic Area (EEA), so EU notification applies in Norway, Iceland and Liechtenstein. Note that the now discontinued Peoples' Republic of China chemical registration scheme corresponded to the EU notification data requirements, and it is reasonable to predict that the forthcoming Chinese notification scheme for new substances will be based on that of the EU. The testing requirements given are for full notification. b. These are the minimum data requirements for notification under the Swiss Ordinance on Environmentally Hazardous Substances. c. The Toxic Substances Control Act requires the available data to be submitted, but further data may be required for safety assessment of chemicals of structures corresponding to Categories of Concern. The core tests under the ‘exposure-based new chemicals testing strategy’ are listed for high-tonnage chemicals with significant human exposure and/or substantial environmental release. d. These are the data of Schedule III of the Canadian New Substances Notification Regulations to permit supply at above 10 tonnes per annum (or over 50 tonnes cumulative). e. These are for full notification for supply in Australia at 1 tonne per annum. f. Testing for Japanese Ministry of Economic Trade and Industry (METI)/Ministry of Health and Welfare (MHW) notification is stepwise, and the bioaccumulation potential and toxicity may be required on the environmental metabolite(s) identified from the ready biodegradation test. However, the mutagenicity studies for MoL notification are conducted on the parent compound. The Japanese new substance notification scheme is to be updated (see Chapter 10) with the ecotoxicity studies (denoted by *) required at a supply level of 10 tonnes per annum for persistent but non-bioaccumulative substances. g. It is adequate to determine either the melting point or the boiling point, whichever is most appropriate. h. Solubility in an organic solvent is adequate as an alternative to fat solubility. i. These additional studies may be required if the minimum data are inadequate for full environmental assessment. j. Available toxicity studies are evaluated for notification under the Swiss Ordinance on Environmentally Hazardous Substances and also under the Order relating to Toxic Substances. k. The choice of exposure route for the second acute toxicity study depends on the respirability of the substance evaluated from the granulometry test and the likely human exposure route. l. Certain physico-chemical properties data are required for Japanese METI/MHW notification, depending on the results of the biodegradation, bioaccumulation potential and toxicity examination. m. The mouse lymphoma assay is required as part of the EU Base Set instead of the in vitro chromosome aberration test if the Ames test is positive. Alternatively, a third in vitro study of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus test can be conducted. n. The mouse micronucleus test or an in vivo chromosome aberration test will normally be required immediately after notification if either of the in vitro mutagenicity tests are positive. o. The third mutagenicity study required for notification in Canada can be either the mouse micronucleus test or the in vivo chromosome aberration test. p. An acute toxicity study is required, which is normally by oral exposure, although volatile liquids or gases must be tested by inhalation exposure. q. A fish bioaccumulation study may be needed if the substance is not ‘ready biodegradable’ and has a high partition coefficient. r. The activated sludge respiration inhibition test is not needed for ‘readily biodegradable’ substances, and may sometimes be omitted on a case-by-case basis for substances which undergo significant biodegradation in the ready biodegradation test. s. Required for substances which are not ‘ready biodegradable’ and/or potentially hydrolysable, unless the study is technically impracticable because of low aqueous solubility. t. Appropriate ecotoxicology studies are required, with an evaluation of carcinogenic, mutagenic and teratogenic potential. u. Tests marked with a * are part of the OECD SIDS, see Section 11.18.1. v. Required as part of the OECD SIDS for inorganic substances only.

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Chemical Control in the US and the Rest of the World (i.e., a ‘generic’ name is used to disguise the chemical identity). The EPA will search the confidential section of the inventory if a bona fide enquiry of intent to manufacture or import the substance for a commercial purpose is submitted. The inventory provides the basis for distinguishing between ‘new’ and ‘existing’ chemicals. Substances listed on the inventory are identified as ‘existing’ and do not require notification.

11.2.3 Testing of Existing Substances Under Section 4 of TSCA the EPA has the authority to require testing of existing substances if it finds they present an unreasonable risk to health or the environment or which are produced in substantial amounts, have significant human or environmental exposure and there is insufficient data to allow evaluation. An Interagency Testing Committee advises the EPA on the priority for testing. The EPA issues Test Rules specifying the extra information required. A company subject to a Test Rule can perform the required tests, or apply for exemption for tests already performed and reimburse the owners of the data.

11.2.4 Manufacturing and Processing Notices Under Section 5 of TSCA a pre-manufacturing notice (PMN) must be submitted to the EPA at least 90 days before a chemical substance is manufactured or imported into the USA. This 90-day review period provides the EPA with an opportunity to evaluate risks posed by the new chemical. A Notice of Commencement of Manufacture or Import (NOC) must be filed with the EPA within 30 days of the first commercial import or manufacture of a notified substance. Eventually the substance is listed on the TSCA inventory and becomes an ‘existing’ chemical. Over 30,000 PMNs have been reviewed to date and EPA receives about 2,500 new applications annually.

11.2.5 PMN Requirements The information required for a PMN is summarised in the standard mandatory form: chemical identity, categories of use, amounts manufactured, by-products, employees exposed, the method of disposal and available test data on the effects of the chemical on health or the environment. The notifier is responsible for developing adequate test data to show that during its life cycle the chemical will not present an unreasonable risk of

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Practical Guide to Chemical Safety Testing injury to health or the environment. The EPA recommends the OECD MPD as a basis for PMNs, although it is not authorised to require specific tests unless the substance is subject to a Test Rule. If the information supplied is inadequate for risk assessment, or the substance is produced in large amounts, EPA can require extra data to be provided prior to manufacture or import. The EPA Office of Prevention, Pesticides and Toxic Substances’ (OPPTS) Harmonized Guidelines, which generally correspond with those of OECD, are used for tests specifically requested by the EPA, but other methods may be acceptable. The EPA uses guidelines to assist in identifying new chemicals that meet the TSCA Section 5e exposure-based finding. This enables sufficient data to be obtained for adequate risk assessment using the existing legislation. Core testing (see Table 11.1) is required if the production or import volume is above 100 tonnes per annum and there is either substantial or significant human exposure or substantial environmental release according to any one of the defined exposure-based criteria. Certain chemical structure types have been identified as ‘Categories of Concern’, and the EPA have specified the data needed for adequate hazard assessment for such new chemicals. There are currently over 45 such categories.

11.2.6 Significant New Use Rules (SNURs) The company that filed for the initial listing of a substance on the TSCA inventory and other companies submitting a PMN provide detailed use information. New uses may have a significant impact on the potential for human or environmental exposure to the substance. If EPA determines that a new use will have a significant impact, it will issue a significant new use rule (SNUR). Companies then have to submit a significant new use notice (SNUN) 90 days prior to the new use. The notice is submitted on the standard PMN form and contains the same information as a full PMN. Note that any new safety data have to be submitted to the EPA for a notified new substance even if not subject to a SNUR.

11.2.7 Exemptions from PMN

11.2.7.1 Low Volume Exemption (LVE) A Low Volume Exemption (LVE) is available to manufacturers or importers intending to manufacture or import a new substance at below 10 tonnes per annum. The standard PMN form is used for LVEs, including available test data.

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Chemical Control in the US and the Rest of the World If the EPA has taken no action following the 30-day review period, the applicant can consider the LVE application approved. If EPA determines that a substance presents an ‘unreasonable risk’ following the LVE review, the application will be denied. LVE substances are not added to the TSCA inventory and other suppliers are required to submit their own notifications.

11.2.7.2 LoREX Exemption Low release and exposure (LoREX) exemptions are available for new chemicals that are released to the environment at low levels and result in minimal human exposure during use, regardless of the production or import volume. Various stringent requirements must be met to satisfy this TSCA exemption standard. Applications for the LoREX exemption are made using the standard PMN form and the administrative procedures are much the same as for the LVE application. The EPA review period is 30 days. The manufacturer or importer of a LoREX substance may only supply it to customers who agree in writing not to distribute it further until it has been made into a physical form in which exposure to humans and to the environment at levels above the LoREX requirements will not occur.

11.2.7.3 Test Market Exemption New substances to be manufactured or imported solely for test marketing are exempt from notification under TSCA, providing this does not present an unreasonable risk to human health or the environment. The test market exemption (TME) allows the commercial viability of a new chemical to be assessed before filing a PMN. A TME can be submitted in letter format, although the PMN form is preferred by EPA. All the available safety data has to be included, together with a discussion of structural analogues, exposure estimates and the test marketing plan. EPA reviews TME applications in essentially the same way as a PMN. The review period is officially 45 days, but a TME applicant must await EPA approval prior to commencing marketing.

11.2.7.4 Self-Executing PMN Exemptions Several further categories of new chemical substances are exempt from notification. These are self-executing PMN exemptions, and there is no need to apply to EPA.

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11.2.7.5 Research and Development Exemption This covers: •

Chemical synthesis and physical/chemical properties testing in the laboratory.

•

Health and environmental effects testing.

•

Tests or demonstrations of equipment or production processes, which may involve full-scale commercial uses.

•

Efficacy and performance tests.

•

Consumer panel testing of the performance characteristics of a new chemical substance.

R&D substances are exempt from PMN and SNUR provisions providing that use of the substance is under the supervision of a technically qualified person and that workers using the substance are informed by the manufacturer or importer of any known health risks posed by the substance. Manufacturers or importers must maintain records of the risk evaluation carried out on each R&D substance, the nature and method of notification of any potential risk, the identity of any recipients and the amount of substance used for R&D.

11.2.7.6 Article Exemption New chemical substances imported into the United States as a component of an article are exempt from PMN and SNUR requirements. The EPA definition of ‘article’ is as follows: ‘A manufactured item (1) which is formed to a specific shape or design during manufacture, (2) which has end use function(s) dependent in whole or in part upon its shape or design during end use, and (3) which has either no change of chemical composition during its end use or only those changes of composition which have no commercial purpose separate from that of the article and that may occur as described in Section 720.36(g)(5) [of TSCA], except that fluids and particles are not considered articles regardless of shape or design.’

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11.2.7.7 Polymer Exemption This is discussed in detail in Chapter 12, Section 12.2.

11.2.7.8 ‘Polaroid’ Exemption This exemption relates to new chemical substances used in or for instant photographic and ‘peel-apart’ film articles.

11.2.7.9 Other Exemptions Other notable self-executing exemptions from the PMN and SNUR requirements are for the following classes of new chemical substances: impurities, by-products and non-isolated intermediates. The EPA definitions of each of these categories of substance is given below: Impurity: ‘A chemical substance which is unintentionally present with another chemical substance.’ By-product: ‘A chemical substance produced without a separate commercial intent during the manufacture, processing, use or disposal of another chemical substance or mixture.’ Non-isolated intermediate: ‘Any intermediate that is not intentionally removed from the equipment in which it is manufactured, including the reaction vessel in which it is manufactured, equipment which is ancillary to the reaction vessel, and any equipment through which the chemical substance passes during a continuous flow process, but not including tanks or other vessels in which the substance is stored after its manufacture.’ Finally, there are other exemptions, such as for ‘chemicals formed incidental to exposure to other chemicals’, ‘chemicals formed during the manufacture of an article’ and ‘polymer salts’.

11.3 US Occupational Safety and Health Act (OSHA) The Occupational Safety and Health Act (OSHA) [4] was enacted 29 December 1970 and became effective 28 April 1971. It provides the regulatory vehicle for enforcement of the standards developed under the Act, with the aim of assuring the safety and health of persons in the workplace. OSHA provides research, information, education and training

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Practical Guide to Chemical Safety Testing in the field of occupational health, with the physical working environment a priority. Situations that have the potential to induce acute or chronic health effects are covered by the Act. Under OSHA’s Hazard Communication Standard (HCS) [5] manufacturers and importers are required to ensure that the hazards of all chemicals produced or imported are evaluated, and that information concerning their hazards are transmitted to employees. This transmittal of information is to be accomplished by means of a comprehensive hazard communication programme, which includes container labelling and other forms of warning, material safety data sheets and employee training. American National Standards Institute (ANSI) standards apply for labelling [6] and preparation of material safety data sheets [7] under OSHA.

11.4 The US Chemical Right-to-Know Initiative for High Production Volume Chemicals

11.4.1 Voluntary Challenge Programme The US Chemical Industry, regulators and environmentalists have launched a ca $700 million voluntary programme to test chemicals for health and environmental effects. The Chemical Right-to-Know (ChemRTK) Initiative responds to an EPA study [8] that found that very little basic toxicity information was publicly available on most of the HPV commercial chemicals manufactured or imported and used in the US. The EPA’s analysis found that no basic toxicity information, i.e., neither human health nor environmental toxicity, was publicly available for 43% of the high volume chemicals manufactured in the US and that a full set of basic toxicity information was available for only 7% of these chemicals. Without basic information on hazardous properties, potential risks to humans and the environment cannot be assessed. The Initiative, which is scheduled to be completed by the end of 2004, tackles this problem by rapidly testing chemicals to provide the missing data. There are three aspects to the Initiative. Firstly, EPA invited US chemical manufacturers and importers to participate in a voluntary challenge programme to provide toxicity and ecotoxicity data on their HPV chemicals. As of 4 June 2002, voluntary commitments had been received for 2159 chemicals from 418 individual companies and 129 consortia. Of the 2159 chemicals, 728 are confirmed ICCA HPV commitments. Chemicals and their Sponsors can be tracked on these web sites: www.epa.gov/chemrtk/sumresp.htm

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Chemical Control in the US and the Rest of the World and www.hpvchallenge.com. EPA intends that HPV chemicals not adopted in this voluntary programme will be tested under subsequent HPV Test Rules under the TSCA. A proposed Test Rule for the first 37 chemicals was published on 26 December 2000 [9]. Secondly, EPA plans to obtain additional reporting of information on those chemicals that are persistent, bioaccumulative and toxic (PBT). Finally, HPV chemicals of particular concern to children’s health are the subject of more detailed and extensive testing in a separate Children’s Health Test Rule. The studies required are those for the OECD SIDS programme (see Table 11.1). Participants in the voluntary programme make commitments to particular chemicals they will sponsor for testing, and on which chemicals will be tested in each year of the programme. Sponsors of chemicals first collect and review all the existing data on the substance and prepare a Robust Summary for each key study which is available. A Test Plan (also referred to as a Work Plan) is submitted with the Robust Summaries during the ‘Start Year’ for the chemical, to indicate which SIDS endpoints are already filled and what new studies are planned. EPA post the Robust Summaries and Test Plans on the ChemRTK web site, and sponsors are encouraged also to put Test Plans on the industry US HPV Chemical Tracking System (US TS). There is a 120-day period from posting of the test plan for public and EPA comment before testing begins. After this period, the Test Plan is modified, if necessary, to take account of any further studies that have been made available, or in light of recommendations from the EPA or other interested parties. At present, the EPA has warned that it may not be able to meet the 120-day public comment period and will alert sponsors as appropriate if this is the case. As new studies are completed, Robust Summaries are posted to the web site. As of 4 June 2002, EPA has received 134 Test Plans covering 815 chemicals. Of these, 70 are category submissions and 64 are individual chemical submissions. The EPA encourages sponsors to prepare an OECD SIDS Initial Assessment Report (SIAR), so that this can subsequently be reviewed through the OECD SIDS program. Hence Sponsors are encouraged also to provide exposure information to help place the hazard information into context. In order to reduce testing, EPA will consider grouping similar chemicals together and testing only the group, but this applies only for volunteered chemicals. Testing of ‘categories’ of related chemicals was scheduled to take place in the early phases of the programme, to allow time for further studies if necessary. Single chemicals were scheduled to be tested after November 2001, to allow for possible developments in the use of nonanimal surrogate endpoints. Similarly, testing of closed-system intermediates is not to begin until 2003.

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Practical Guide to Chemical Safety Testing The US HPV chemicals were identified through information collected under the TSCA Inventory Update Rule (IUR) [10]. The HPV Challenge Programme Chemicals List consists of all the HPV chemicals reported during the 1990 IUR reporting year. As subsequent IUR reporting years identify additional HPV chemicals (including inorganic chemicals, once the corresponding reporting requirements have been added), EPA intends that testing of these extra chemicals will take place.

11.4.2 Persistent Bioaccumulative Toxic (PBT) Chemicals Persistent, bioaccumulative and toxic pollutants (PBTs) are highly toxic, long-lasting substances that can build up in the food chain to levels that are harmful to human and ecosystem health. They are associated with a range of adverse human health effects, including effects on the nervous system, reproductive and developmental problems, cancer and genetic impacts. The EPA’s challenge in reducing risks from PBTs stems from the pollutants’ ability to travel long distances, to transfer easily among air, water and land and to linger for generations in people and the environment. The four main elements of EPA’s strategy in reducing the risks to human health and the environment from existing and future exposure to priority PBT pollutants are [11]: 1. Develop and implement national action plans to reduce priority PBT pollutants: EPA is initially focusing action on the 12 high priority PBTs identified in the Canada-US Binational Toxics Strategy (BNS) [12]. EPA is developing action plans that will use the full range of its tools to prevent and reduce releases of these 12 (and later other) PBTs. 2. Continue to screen and select more priority PBT pollutants for action: Beyond the BNS Level 1 substances, EPA will select additional PBT pollutants for action. EPA will apply selection criteria in consultation with a technical panel. Candidate chemicals will be those highly scored by EPA’s Waste Minimization Prioritization Tool and other chemicals of high priority to EPA offices. 3. Prevent new PBTs from entering the marketplace: EPA is acting to prevent new PBT chemicals from entering commerce by: (a) proposing criteria for requiring testing/ restrictions on new chemicals, (b) developing a rule to control attempts to re-introduce out of use PBT chemicals into commerce, (c) developing incentives to reward the development of lower risk chemicals as alternatives to PBTs and (d) documenting how PBT-related screening criteria are taken into account for approval of new pesticides and re-registration of old pesticides.

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Chemical Control in the US and the Rest of the World 4. Measure progress of these actions against the Government Performance and Results Act (GPRA) goals and national commitments. EPA’s Community Right-to-Know program will add new PBTs and lower reporting thresholds for other PBTs on the Toxics Release Inventory (TRI) so that the public can readily identify local emissions of these pollutants. EPA is also taking action aimed at reducing certain PBTs in hazardous waste by targeting PBT chemicals found in hazardous waste for voluntary waste minimisation activities.

11.4.3 US Voluntary Children’s Chemical Evaluation Program EPA announced the Voluntary Children’s Chemical Evaluation Program (VCCEP) in the December 26, 2000 Federal Register [13]. The objective of the program is to generate chemical hazard and exposure information that can be evaluated to ensure that children are adequately protected from potential risks of industrial and commercial chemicals [14].

11.4.3.1 Background A key component of the Chemical Right-to-Know Initiative (announced on the eve of Earth Day 1998) is the identification and assessment of chemicals that may lead to high levels of exposure in children. After considering different comments offered by some of the stakeholders during public meetings or in comments submitted to the docket [15, 16], EPA decided to focus this program on chemicals found to be present as contaminants in: •

Human tissues or fluids

•

Food and water children may eat or drink

•

Air children may breath including residential or school air.

In order to identify chemicals to which children would have the highest likelihood of exposure, EPA selected chemicals that were found by biomonitoring data to be present in the human body and environmental data to be present in a person’s environment. If a chemical was listed in at least one biomonitoring database and at least one environmental database, it was identified as a candidate for the VCCEP. A list of the databases used is available in the Federal Register [13] and also on the EPA web site (www.epa.gov/opptintr/ chemrtk/vccepmth.htm).

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11.4.3.2 VCCEP Pilot Program EPA is running a pilot of the VCCEP to gain insight into how best to design and implement the VCCEP so that the Agency and public are provided with the information necessary to understand the potential health risks to children associated with exposure to these and other chemicals. The aim of the pilot is to identify efficiencies that can be applied to the subsequent implementation of the VCCEP and to test the performance of the Peer Consultation Process. The 23 chemicals chosen for the VCCEP pilot program are listed in the Federal Register [13] and can also be found on the EPA web site (www.epa.gov/ opptintr/chemrtk/ vccepmth.htm). These two sources, along with a third [17] include information on how the chemicals were selected. Each tier of the pilot VCCEP has 4 stages – the commitment to sponsor a chemical; development and submission of the required data; Peer Consultation to review the data and determine if further data are required; EPA review of the Peer Consultation results. Sponsor companies committing to submit Tier 1 data were due to commence gathering the necessary information no later than December 2001. After submission and assessment of the Tier 1 data, EPA will announce if further information is required to assess a chemical’s risk to children and indicate what information in Tier 2 should be provided. Companies will then be given the opportunity to sponsor chemicals through Tier 2, and the process repeated through Tier 3.

11.4.3.3 Hazard and Exposure Information Requested EPA is requesting information on both hazard (health effects) and exposure. The health effects information being requested at Tier 1 is the same as that requested in the HPV Challenge Program. The health effects tests in the VCCEP listed by tier are shown in Table 11.2.

11.5 Chemical Control Legislation in Canada 11.5.1 The Canadian Environmental Protection Act The Canadian Environmental Protection Act (CEPA) of 30 June 1988 has been revised by CEPA 1999 [18] which came into effect on 31 March 2000. CEPA requires new chemical substances to be notified before manufacture or import.

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Table 11.2 Health effects testing in the US Voluntary Children's Chemical Evaluation Program Tier 1

Tier 2

Tier 3

Acute toxicity

Subchronic toxicity

Neurotoxicity screening battery

Repeated dose toxicity with reproductive and developmental toxicity screens

Prenatal developmental toxicity Reproductive and fertility effects

Carcinogenicity Developmental neurotoxicity

Bacterial reverse mutation assay

Immunotoxicity

In vitro or in vivo In vivo chromosomal chromosomal aberrations or aberrations or in vivo in vivo micronucleus test micronucleus test

The Canadian notification scheme became effective on 1 July 1994, and the New Substances Notification Regulations (NSNR) [19] were published in the ‘Canada Gazette’ on 6 April 1994. ‘Guidelines for the Notification and Testing of New Substances: Chemicals and Polymers’ are available online (www.ec.gc.ca/substances/nsb/download/ cpg0901.pdf).

11.5.2 Inventories The Canadian Inventory is called the Domestic Substances List (DSL) and contains 23,600 substances in commerce in Canada at 100 kg per annum and above from 1 January 1984 to 31 December 1986. Any substance not present on the DSL requires notification prior to full commercialisation. The Non-Domestic Substances List (NDSL) consists of the US TSCA Inventory minus the substances on the DSL. The NDSL is updated annually with substances added to the TSCA inventory five years previously. Presently the NSDL contains 44,800 substances. NDSL-listed substances require notification under CEPA, however the data requirements are considerably less than for substances not on the NDSL. The DSL and NDSL contain confidential sections, which can be searched by Environment Canada if a bona fide intent to manufacture or import is established by submitting specified

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Practical Guide to Chemical Safety Testing data. Both the DSL and the NDSL are available online (www.ec.gc.ca/substances/nsb/ eng/Sub_e.htm). Canada has bilateral agreements with both the USA and Australia. The agreement between Canada and the USA is known as the ‘Four Corners Agreement’. It has been in place since 1996 and was established to facilitate the exchange of confidential new substance assessments between the US EPA and Environment Canada/Health Canada. The initial objective was to expedite the movement of substances to the NDSL and reduce the normal five-year lag. The Canada-Australia Bilateral Arrangement was formally established in 2002. The NSN regulatory requirements in both countries are very similar so it is relatively easy to make comparisons. The agreement also promotes the sharing of assessments between the government authorities. Most polymers are represented on the inventories in terms of the starting materials from which they are manufactured, and ‘products of biotechnology’ will be included subsequently if necessary when their regulatory controls have been finalised.

11.5.3 Environmental Assessment Regulations On 1 September 2001, it was announced that the Canadian government intends to introduce Environmental Assessment Regulations (EAR) to make environmental impact assessments for substances in products regulated under the Food and Drugs Act (F&DA), i.e., pharmaceuticals, biologics, food additives, novel foods, personal care products and cosmetics. Commencing September 13, 2001 all substances in these products are subject to the NSNR of CEPA. It is estimated that it will take five or more years to introduce the EAR under the F&DA, so in the meantime: •

The DSL becomes the reference inventory for F&DA substances and anything not listed is notifiable.

•

There will be an opportunity to add ‘grandfathered’ substances to the DSL, using the original DSL reference years 1984-1986.

•

New substances introduced as of September 13, 2001 must be notified through CEPA with an NSN.

•

Substances introduced between 1986 and September 12, 2001 are being held in a ‘parking lot’, pending the creation of the F&DA Environmental Assessment regulations.

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11.5.4 Data Requirements for Notification The information required for notification of new substances is listed in Schedules to the Regulations. Table 11.1 shows the Schedule III testing required for supply above 10 tonnes per annum (or 50 tonnes cumulative). Reduced testing is required for substances on the NDSL and also for substances used for product development, which are site-limited intermediates, or which are for export only. The waiting period for review by Environment Canada varies between 5 and 90 days depending on which Schedule is followed. Under the ‘Green Light Opportunities’ of CEPA 1999, the waiting period may be reduced in special circumstances. Polymers are covered by Part II of the Regulations. These are discussed in detail in Chapter 12, Section 12.2.2. Information submitted in a notification can be claimed as confidential business information (CBI). The degree of protection given to such data will be consistent with the provisions of the Access to Information Act. Hence confidentiality claims have to be substantiated with the supplementary information prescribed in the Confidential Information Disclosure Regulations. If the substance identity is claimed as confidential, a masked name has to be chosen for use in official publications such as the DSL.

11.5.5 Significant New Activity Notice A Canadian Significant New Activity (SNAc) notice broadly corresponds to a US SNUR (Section 11.2.6), and essentially triggers submission of information for specific new activities. The substance is listed on the DSL with a SNAc flag, to warn suppliers to submit the information specified in the SNAc notice for additional uses. Information is submitted in compliance with a SNAc notice and assessed to decide if the SNAc notice can be modified, or if different control measures are justified.

11.5.6 Administration Environment Canada and Health Canada administer the scheme. Evaluators from these agencies compare the predicted exposure to the substance with the anticipated hazardous effects of such exposure, based on the submitted data, for both human health and the environment, in order to determine the likely ‘toxicity’ of the substance. The definition of a ‘toxic’ substance in this regard is one that has, or may have an immediate or longterm harmful effect on the environment or constitutes, or may constitute, a danger to the environment on which human life depends, or to human life or health.

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Practical Guide to Chemical Safety Testing The purpose of the assessment and control process is to minimise the potential risk to human health and the environment from the commercial use of the substance. If a substance is assessed as ‘toxic’, control measures may be taken before the review period expires. Under CEPA 1999, Significant New Activity conditions (SNAc) may be applied to all industry for a substance not posing a concern when used as proposed by the notifier, but which could be if used otherwise (see Section 11.5.5). Following the 1995 NSNR review, Environment Canada recommended partial recovery of the cost of administering the scheme by charging notification fees. The proposed New Substances Fee Regulations (NSFR) were published on 30 June 2001, with a 60-day comment period, and will be brought into force in due course. Notified substances are listed in the DSL, as a supplement published in the ‘Canada Gazette’. Under CEPA 1999, there is a DSL update deadline of 120 days from the eligibility criteria being met. Notifiers with full data may choose whether to file a Notice of Commencement (NOC) on first manufacture or import to get immediate DSL listing, or to track the quantity and submit a Notice of Excess Quantity (NOEQ) for deferred DSL listing. After listing, substances can be manufactured or imported by other suppliers for unrestricted use. Substances notified with a reduced data set, because of limited use or exposure or with data waivers, are not listed on the DSL. Substances suspected of being ‘toxic’ can only be listed on the DSL after they are regulated under CEPA to ensure their safe use. The DSL has been re-organised to accommodate SNAc substances: the first one was published on 4 July 2001. Section 17 of CEPA requires Canadian importers or manufacturers to report information on chemicals to Environment Canada that reasonably supports the conclusion that the substance is ‘toxic’ or is capable of becoming ‘toxic’. Products exempt from notification because they are regulated separately may be reportable under Section 17 of CEPA if equivalent environmental information is not required by the separate legislation. Marketing information may be reportable, because the revised exposure estimate may indicate an unacceptable risk to an ecosystem or a sensitive population subgroup when combined with hazard data. A new requirement of CEPA 1999 (Section 71) is to ‘categorise’ DSL substances to determine whether they are ‘toxic’ or capable of becoming ‘toxic’. Categorisation identifies substances that have the greatest exposure potential or are persistent or bioaccumulative and inherently toxic. Such substances are subject to screening and risk assessment using published data, computer modelling and information collected from industry. The first ‘Section 71’ notice was published on 17 November 2001. This announced the 123 DSL chemicals for the ‘Pilot Phase’ of the categorisation and screening initiative. Canadian importers or manufacturers who exceeded prescribed value thresholds in 2000 had to

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Chemical Control in the US and the Rest of the World submit commercial and technical information and any available hazardous property data by 8 March 2002. The objective of the Pilot Phase is to develop a system to complete categorisation, and subsequent screening assessment, of the whole DSL.

11.5.7 Inspection, Enforcement and Penalties Environment Canada enforces CEPA. Enforcement Officers abide by the Enforcement and Compliance Policy which was established to ensure that the Act is applied throughout Canada in a manner that is fair, predictable and consistent. Violations can be dealt with by measures ranging from warnings to prosecution.

11.5.8 Future Changes Multi-stakeholder consultations on revising the NSNR were completed during 2001. Some of the expected changes are as follows: •

There will be a reduction in the number of schedules. Some of the special schedules will be eliminated to simplify and harmonise the requirements.

•

The notification threshold for new chemicals not on the NDSL will increase from 20 kg/year to 100 kg/year.

•

The need to track ‘accumulated’ and ‘in possession’ volumes of substances will disappear. Only per annum values will be used.

•

Generally, for R&D and site-limited substances, the notification requirements will be simplified with the removal of any volume limit, as long as the sole use remains within these categories. No test data will be specified.

•

The annual updating of the NDSL will move to a lag of only one year after substances are added to the US TSCA inventory, rather than the current 5 years.

•

High exposure NDSL substances will be subject to increased data requirements. Prior to achieving 50 tonnes per annum, they will require data similar to the TSCA High Exposure dataset.

•

Polymers of Low Concern (PLCs) remain notifiable, but mechanisms to make their notification and assessment more efficient are under development. The inventory status of their monomers will no longer affect PLCs.

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Certain categories of polymers will have exemptions from mammalian toxicity testing specified directly in the notification schedules.

•

The NSN Guidelines will be referenced in the new Regulations and will provide more extensive assistance to notifiers. One valuable upgrade will be the description of more bases on which waivers can be employed to avoid the need to supply the specified data.

By 2004 details will be available as to how the recommendations of these multi-stakeholder consultations will be incorporated into the next version of the NSNR of CEPA.

11.5.9 The Workplace Hazardous Materials Information System The Workplace Hazardous Materials Information System (WHMIS), which was established in 1988, aims to protect workers using chemicals by improved communication of hazards. This involves labelling and material safety data sheets, in English and French, and employee information and training programmes. There are recommended standard phrases.

11.6 Chemical Control Legislation in Switzerland

11.6.1 The Federal Law on Trade in Toxic Substances The 1969 Federal Law on Trade in Toxic Substances, which is implemented by the 1983 Order relating to Toxic Substances (OTS) [20], is concerned with protection of human health. It gives requirements for classification, labelling, listing and sale of ‘toxic’ substances and preparations (for public and commercial use). These are listed in the Toxic Substances Lists 1 to 3, respectively, which are referred to as ‘Giftliste’, and are updated annually. The Federal Office of Public Health (Bundesamt für Gesundheitswesen, BAG) classifies chemical substances based on the results of testing (see Table 11.1) and human exposure data. Substances are placed in one of five classes (Giftklasse) ranging from Category 1 (most hazardous) to Category 5 (least hazardous). All chemical substances subject to classification and preparations containing them are registered with BAG by the Swiss manufacturer or importer, and entered onto the Toxic Substances List on the basis of the data on the substance. Standard reporting forms are

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Chemical Control in the US and the Rest of the World available. It takes about 6 months for BAG to evaluate the classification of a new substance. The applicant is informed of the classification by BAG, and the decision is first published in the official ‘Amtsblatt’, then entered into the ‘Giftliste1’. Products containing new substances can normally only be registered when the new substance is listed in ‘Giftliste1’, although BAG may make a provisional classification in certain circumstances. Registered products are assigned a BAG-T number. The Otox was amended with effect from 1 December 1998 [21] to harmonise SDS requirements with the EU, and an EU-format SDS now has to be provided to all industrial users of ‘dangerous substances’. ‘Toxic’ substances can only be ‘traded’, which includes manufacture and import, if they are on the appropriate Toxic Substances List. Regulated toxic substances can only be traded by ‘authorised’ persons who hold the appropriate permit. However, there is no obligation to register a toxic substance which is to be traded exclusively for research, or as an intermediate for chemical production, or is contained in an article. Appropriate protective measures must be taken to protect human health when trading in toxic substances, which includes suitable packaging, labelling and user instructions.

11.6.2 The Federal Law on Environmental Protection

11.6.2.1 Main Provisions The 1983 Federal Law on Environmental Protection United States Government (USG), updated effective from 1 July 1997, deals mainly with the effect of chemicals on the environment and on humans via the environment. The Law is implemented through various Ordinances dealing with specific aspects of environmental protection, such as air and soil pollution, waste disposal, noise and radiation. The 1986 Ordinance on Environmentally Hazardous Substances (OEHS), as amended from 1 January 1996 [22], requires the measures discussed below. An importer or manufacturer may only supply a substance, product, or article if an assessment of its environment impact concludes that handling in accordance with its labelling and instructions for use does not present a hazard to the environment or to persons indirectly through the environment. The customer must receive appropriate information, in the form of labelling, instructions for use and SDSs. As from 1 December 1998, environmental classification and labelling in Switzerland has been fully harmonised with the EU. These provisions allow importers and manufacturers and their customers to comply with the general obligation to take due care required by the OEHS:

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Anyone who handles ‘substances’, ‘products’ or ‘articles’ must ensure that they cannot present a hazard to the environment nor to persons indirectly through the environment. This obligation also applies to the handling of any generated waste.

•

The protective measures given on the label and the instructions for use must be followed.

New substances must be notified to the Swiss Agency for the Environment, Forests and Landscape (SAEFL), abbreviated to BUWAL in German, before they can be supplied in Switzerland by manufacture or import in any quantity. Existing substances are defined as ones in EINECS (see Section 9.2), included in the 1985 Toxic Substances List 1 or marketed in Switzerland between 1975 and 1984 in a total quantity of at least 0.5 tonnes. The authorities can request notification of an existing substance. The categories of substances exempt from notification are as follows: •

Products regulated by separate legislation, such as pharmaceuticals, food additives, pesticides and wood preservatives.

•

Substances used exclusively as chemical intermediates.

•

Substances supplied for research and development to a small number of customers over a limited period in small quantities.

•

Polymers containing less than 2% of a new monomer substance in bound form, or which contain only carbon, hydrogen, oxygen and nitrogen.

The obligation to notify a new substance rests with the Swiss manufacturer or, for substances made outside Switzerland, with the Swiss importer. The SAEFL have recently introduced the concept of a ‘general importer’, however, to reduce the number of multiple notifications of the same substance imported by several Swiss companies. The OEHS requires new fertilisers and soil additives to be registered with the appropriate Swiss authority, and there are declaratory requirements for detergents and washing agents. Also, wood preservatives and plant treatment products can only be supplied when a marketing permit is granted.

11.6.2.2 Data Requirements for Notification The data requirements for the notification of new substances are based on the OECD MPD. The minimum information required is listed in Annex 2.1 of the OEHS (see Table 11.1). There are no official reduced data requirements for notification of substances

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Chemical Control in the US and the Rest of the World to be supplied only in low amounts. However SAEFL will negotiate on a case-by-case basis for certain of the standard tests to be omitted, especially if the substance is to be used in special applications or has special disposal methods that minimise environmental contamination. An Environmental Impact Report (Umweltverträglichlzeitsbericht) is also required for a notification. This is an assessment of the environmental compatibility made by the notifier, and is based on an evaluation of: •

degradation, accumulation and mobility in the environment

•

effects on microorganisms, plants, animals and ecosystems

•

long-term indirect effects on humans via the environment.

There is no official review period, thus the new substance can be imported or manufactured by the notifier as soon as SAEFL has received the notification. However, SAEFL can request further information necessary for full environmental assessment of the substance, or take regulatory action at any time after notification.

11.6.2.3 Existing Substances For existing substances, manufacturers and importers must, if possible, perform a similar evaluation to that for new substances. The authorities can demand a detailed environmental assessment for existing substances which are produced in large quantities, are poorly degradable, accumulate in the food chain, are harmful to plants or animals at low levels, or potentiate the environmental effects of other chemicals. For products and articles, manufacturers and importers must base their evaluation on the data provided by suppliers for each constituent (labels, user instructions and safety data sheets) and any other relevant information in their possession. The environmental impact of a substance, product or article must be re-evaluated when new uses or significantly larger volumes are introduced or the impurity profile changes.

11.6.2.4 Harmonisation with the EU The Swiss Federal Assembly has agreed the new Federal Chemicals Law on the Protection from Hazardous Substances and Preparations (Chemikaliengetz, ChemG) [23] of 15 December 2000. It is expected to enter into force in 2005, after the implementing Ordinances are developed. The 1969 Federal Law on Trade in Toxic Substances will be superseded and the 1983 Federal Law on Environmental Protection will be amended.

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Practical Guide to Chemical Safety Testing The new ChemG covers packaging, labelling and use responsibilities, notification of new substances, documentation and reporting requirements and provides for enforcement. Implementation will be by the cantons. The new law also applies to plant protection products and biocidal products. Harmonisation with the EU is anticipated, hence the existing scheme for plant protection product registration will have to be amended, and a new scheme to cover biocidal products developed.

11.7 Notification of New Chemical Substances in Australia

11.7.1 National Industrial Chemicals (Notification and Assessment) Scheme The Australian Industrial Chemicals (Notification and Assessment) Act 1989 [24] provides for a national scheme for the notification and assessment of industrial chemicals. The scheme, known as the National Industrial Chemicals Notification and Assessment Scheme (NICNAS), began operating in July 1990 and is administered by the National Occupational Health and Safety Commission (Worksafe Australia). A ‘Handbook for Notifiers’ is available from Worksafe Australia. The Act has been amended twice by The Industrial Chemicals (Notification and Assessment) Amendment Acts of 1992, which came into effect on 4 August 1992 and 1 March 1993. NICNAS has been further reformed to include total administrative cost recovery through fees, and these Amendments came into force from 1 July 1997. NICNAS applies to manufacturers and importers of new and selected existing industrial chemicals. NICNAS also provides for the secondary notification of new chemicals already assessed under the Scheme. Articles, formulated preparations of notified or existing substances and radioactive chemicals are not notifiable. Furthermore, agricultural chemicals, food additives, animal feed additives and veterinary and pharmaceutical products are exempt from the notification scheme, as they are controlled under separate legislation. There are also exemptions for confined reaction intermediates of transient existence, non-commercially produced by-products or impurities in another material. Under the latest NICNAS reforms, new chemicals that are supplied at below 10 kg pa and do not pose an unreasonable risk are exempt from notification, although suppliers are requested to inform Worksafe Australia. An application for this exemption for cosmetic ingredients must be approved before supply, and the exemption does not apply for cosmetic preservatives, colouring agents or ultraviolet filters or ingredients prohibited or restricted in the EU (Section 9.8.1). Also, if the ingredient will be present

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Chemical Control in the US and the Rest of the World in the formulated cosmetic product at 1% or more, the supplier must have information to indicate that the product is safe. Another new exemption is for new site limited research and development or analysis chemicals. To qualify for this exemption, information on the chemical and the intended research programme must be provided to Worksafe Australia.

11.7.2 Inventory The Australian Inventory of Chemical Substances (AICS) is the legal device that distinguishes between new and existing chemicals. It is a listing of all industrial chemicals in use in Australia between 1 January 1977 and 28 February 1990. In addition, it includes new assessed chemicals and corrections as required. AICS is a list of chemical identity data and does not contain information on toxicity, use, manufacturers or importers. Any chemical not included in AICS is regarded as a new industrial chemical, unless it is outside the scope of the Act or is otherwise exempt from notification and must be notified and assessed before import or manufacture in Australia. AICS has a confidential section and a public (non-confidential) section. The public section may be searched by either submitting a request to NICNAS or using the AICS CD-ROM available from NICNAS. To determine if a chemical is in the confidential section, the applicant must write to the Director of NICNAS including a declaration of bona fide intent to introduce the chemical into Australia. Notified new substances are added to AICS after 5 years and can be kept in the confidential section of AICS for periods of 5 years thereafter.

11.7.3 Data Requirements for Notification The information required for a full notification essentially corresponds to the OECD MPD, and is detailed in Parts A, B and C of the Schedule to the Act (Table 11.1). The review period is 90 days, and there is now an Early Introduction Permit option in operation to allow a safe new chemical to be supplied while the notification is being reviewed by Worksafe Australia. Further information may be required when the substance is supplied at 10, 100, 1000 or 10,000 tonnes annually, or before the 90-day assessment period begins if this is essential for adequate evaluation of the hazard of the substance. NICNAS provides for confidentiality and flexibility of data requirements in a notification; for example, it may be agreed that certain matters required in a notification are irrelevant, unnecessary or economically prohibitive for the assessment of the chemical.

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Practical Guide to Chemical Safety Testing Limited notification is available for small volume chemicals (i.e., less than 1 tonne per annum), site-limited chemicals (i.e., not confined and transient reaction intermediates) up to 10 tonnes per annum and substances used only for research and development for supply at 50 kg to 1 tonne per annum. The review period is 90 days, but, as for full notification, there is the option to apply for an Early Introduction Permit. The information required for a limited notification is Parts A and B of the Schedule: identity, physicochemical properties, amount, use and potential hazards, including a material safety data sheet (MSDS) [25] and labelling. There is also the option to apply for a Commercial Evaluation Permit, which has data requirements consisting mainly of administrative information, for new chemicals to be imported or manufactured solely for this purpose. The maximum quantity permitted is 2 tonnes for 2 years, but amounts above 1 tonne and times longer than 1 year have to be justified. There is no legislative timeframe for Commercial Evaluation Chemicals (CEC) applications but they are usually processed within 14 days. Application can be made under the Low Volume Chemicals Category for chemicals which will be introduced in quantities of 100 kg or less per year. The permit is valid for three years, which is renewable on application. Applications will be processed within 20 days. Polymer notification is discussed in Section 12.3.2. For each notification, the assessment report, the full public report (i.e., a non-confidential version) and the summary report (for subsequent publication in the ‘Chemical Gazette’) will be sent to the notifier for comment. The assessment certificate is given to the notifier within 7 days of publication of the assessment report.

11.7.4 Existing Substances NICNAS provides a mechanism for evaluating AICS-listed existing chemicals that are declared as priority existing chemicals (PECs). Interested parties can nominate chemicals for consideration as a PEC. Declared PECs are assessed for the risks they represent to human health or the environment, and appropriate recommendations made on their use. Evaluation of existing chemicals is now funded from a new Company Registration Scheme.

11.7.5 Hazard Communication Road and rail transport of chemicals in Australia is covered by the Australian Dangerous Goods Code (ADG Code), which corresponds with the United Nations transportation

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Chemical Control in the US and the Rest of the World recommendations [26]. The criteria used for the Australian user classification and labelling scheme closely parallel those of the EU, except for corrosives and physico-chemical hazards, which are in accordance with the ADG Code. However, there is as yet no classification of ‘dangerous for the environment’. There are National Codes of Practice for classification and labelling of workplace hazardous substances [27] and the preparation of material safety data sheets [25].

11.8 Chemical Control in Korea

11.8.1 The Toxic Chemicals Control Law and Ministry of Environment Notification The Toxic Chemicals Control Law (TCCL) [28] was enacted on 1 August 1990 and has been enforced since 8 February 1991. All new chemical substances have to be notified to the Ministry of Environment (MoE) before being imported or manufactured, to enable the National Institute of Environmental Research (NIER) to conduct a toxicity examination to decide if action is needed to prevent harmful effects from this substance. Existing chemical substances can also be re-evaluated to decide if their use should be controlled as a Toxic Chemical. The Korean government is considering significant amendments to the TCCL. The TCCL applies to all chemical substances except those regulated by other legislation: i.e., radioactive materials, pharmaceuticals (both formulated products and active ingredients, but not pharmaceutical intermediates), cosmetics (including ingredients), plant protection products (but not biocides and disinfectants or plant protection product active ingredients), fertilisers, direct and indirect food additives, animal feed additives, toxic gases covered by the High Pressure Gas Safety Control Act, explosives and ozonedepleting substances. However, naturally occurring substances and articles (i.e., accompanying machinery or equipment, such as toner cartridges, or in solid finished form for use by consumers, including paints and paper chemicals) are excluded from the provisions of the TCCL.

11.8.1.1 Inventory The ‘Korean Existing Chemicals Inventory’ (KECI) lists existing substances (manufactured or imported prior to 2 February 1991). The KECI contains approximately 35,000 substances and is divided into six categories by chemical structure, but there are

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Practical Guide to Chemical Safety Testing indices of Korean-language chemical names and CAS numbers. Online and CD-ROM versions of the inventory have been developed by CAS. The Korean Chemical Management Association (KCMA) has produced a new master inventory of ca 36,000 existing substances, consolidating the MoE and Ministry of Labour (MoL) inventories. Substances in commerce in Korea before 2 February 1991 but not nominated for the KECI by 31 March 1994 can be supplied in Korea without notification on application for exemption via the KCMA.

11.8.1.2 Data Requirements for Notification A new chemical has to be notified to the MoE before it is first manufactured or imported at 100 kg per annum or above. The review period for both NIER and MoL is 45 to 60 days, which is extendable to 3 monthly by NIER. Technical and commercial information is included in the notification, including details of the proposed use and disposal. Test data are required as shown in Table 11.1. The MoE can request additional studies necessary for the toxicity examination. Surrogate data (e.g., information on a chemical analogue) are accepted on a case-by-case basis. There are simplified notification requirements for substances that are in the inventories (published before 2 February 1991) of two countries with equivalent chemical legislation. The results of tests for melting point, boiling point, solubility in water or organic solvents, vapour pressure and Pow are needed, and one of the following toxicity examination testing programmes: •

Acute oral toxicity study (or dermal or inhalation if more appropriate) and Ames test (plus mouse lymphoma assay if the Ames test is positive and a mouse micronucleus test if the mouse lymphoma assay is positive).

•

Acute oral toxicity and chromosome aberration test in vitro (plus mouse micronucleus test if this is positive).

•

Ready biodegradation test (or hydrolysis study if abiotic degradation is rapid).

Non-GLP studies are accepted for simplified notifications and only study summaries are essential. Notification is not required for substances manufactured or imported at below 100 kg per annum (Low Volume Exemption), used in small amounts for R&D only by qualified researchers or for small packages of chemical reagents. Application for exemption must be made to the MoE at least 3 days prior to first import or manufacture.

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Chemical Control in the US and the Rest of the World Separate notification requirements apply for those polymers complying with the OECD definition of ‘polymer’ (see Section 12.3.4.1). Alternatively if these polymer-specific data are not available, the simplified notification requirements can be applied instead.

11.8.1.3 Evaluation of Notifications The NIER use the information in the notification to decide whether the substance is potentially harmful to human health, gives rise to environmental pollution, requires special handling precautions or whether it has been banned or restricted in foreign countries or by any international organisation. If the risk assessment establishes that there is a potential to cause serious harm the MoE may restrict the substance’s use, require annual reporting of the amount supplied or designate it as a Toxic Chemical or Observational Chemical. The result of the NIER toxicity examination is reported on a special form to the notifier and published in the Official Gazette on an ad hoc basis. Once notified, new substances do not have to be notified by other suppliers. A prospective notifier may submit a Bona Fide Notice to MoE to establish whether a substance has been notified previously. A registration procedure applies for customs clearance. Toxic Chemicals are substances designated by a Prime Minister’s Decree of the TCCL as being harmful to public health or the environment. They are included in the KECI, and are exempt from notification. Specific Toxic Chemicals may be banned or have their use restricted. There are currently 520 Toxic Chemicals (which includes at least 59 Banned or Restricted Toxic Chemicals) and 8 Observational Chemicals, either listed as single chemical substances or in chemical classes (e.g., compounds and salts) in the KECI. Importers of Toxic Chemicals have to register them annually. The MSDS from the foreign manufacturer of imported substances can be used to confirm that the technical information submitted to the MoE is correct. There is a 100 kg per annum low-volume exception from annual reporting of Toxic Chemicals, but none for Observational Chemicals. Any organisation engaged in the manufacture, import, sale, storage, transport or use above a specified amount of Toxic Chemicals has to be registered as a Toxic Chemical Business. The MoE can order such a business to be relocated if there is considered to be a high risk to local residents from accidents involving Toxic Chemicals.

11.8.2 The Industrial Safety and Health Law and Ministry of Labour Toxicity Examination There is also a notification scheme in operation in Korea under the Industrial Safety and Health Law (ISHL) for all substances used in the workplace. The NIER reviews the

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Practical Guide to Chemical Safety Testing toxicity of notified new substances and reports to the Ministry of Labour (MoL). The MoL accepts the same studies as MoE. It may be that the amendments to the TCCL will result in a single notification scheme overseen by the MoE. Existing chemicals (in use in Korea before 30 June 1991) qualify for inclusion in the MoL Existing Chemicals List. The MoL and MoE inventories are combined, as previously discussed, under the management of the MoE, and the consolidated KECI was published in January 1997. The chemical name and degree of toxicity is published in the ‘Official Gazette’ or a daily newspaper. Reported new chemicals are added to the MoL inventory. The applicant can request the notified chemical is listed only by Trade Name for 3 years, which can be extended by a further 3 years on request. The chemical name can only be claimed as confidential if the substance is not listed in any foreign inventory or any public document, such as a CAS entry. The MoL can designate new chemicals as exempt from toxicity examination on the grounds that they are non-hazardous or there is no risk of worker exposure. New chemicals are also exempt if they are only for research and development, used by consumers without being processed by industry in Korea, or if supplied at below 100 kg per annum.

11.8.3 Hazard Communication The MoL has introduced a MSDS scheme, in the EU/US 16-heading format. There is a Standard for MSDS Preparation and Provision, which is consistent with the ISO 11014-1 format [29]. The MSDS has to be in Korean, except for reagents for which English is acceptable. Korean-language labelling is obligatory, although English labelling (featuring the EU risk and safety phrases) according to the UN/International Maritime Dangerous Goods (IMDG) Code is adequate to allow import and transport to the first destination in Korean, but Korean labelling then applies for storage. The full chemical name can be kept secret in the MSDS and product label. In general the classification and labelling criteria are similar to the EU, although in particular with differences for environmental classification.

11.9 Chemical Control in the Philippines 11.9.1 The Toxic Substances and Hazardous and Nuclear Wastes Control Act Chemical control in the Philippines is covered by Republic Act 6969 (RA 6969), also known as The Toxic Substances and Hazardous and Nuclear Wastes Control Act, 1990

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Chemical Control in the US and the Rest of the World [30]. RA 6969 does not cover chemicals controlled by other Philippine legislation, such as radiochemicals, pesticides, agricultural and veterinary products, pharmaceuticals and food additives. The Act is administered by the Department of Environment and Natural Resources (DENR), using DENR Administrative Order No. 29 [31] which outlines the implementing rules and regulations. Industry participation in policy development is welcomed, and ‘round table’ discussions take place every 6 months.

11.9.2 Inventory The Philippine Inventory of Chemicals and Chemical Substances (PICCS) of ca 24,000 existing chemical substances was published in April 1996, and a supplement was produced in 1999 (available from the PICCS web site). Components of mixtures are included as separate substances in the PICCS. Trade names and the identity of the nominators do not appear in the public version of the PICCS. DENR gives special consideration to chemicals whose listing by chemical name would be commercially undesirable to the nominator, and there will be a confidential section of the finalised PICCS. The PICCS will be updated every 5 years. Small-scale distributors and manufacturers, but not importers are exempt from the updating requirement. Also, chemicals manufactured or distributed for market testing or manufactured, distributed or imported for non-commercial research and development, both at below 1 tonne per annum, are not included in the PICCS update reporting. As from 31 December 1993, any ‘new chemical’ not on PICCS in principle became notifiable between 90 days and 180 days before it is imported, manufactured, used, stored, transported or processed, although small-scale business (except importers) are exempt. The notification scheme only began operating in 1999, and previously new chemical substances could be imported under amnesty provisions on completion of a form to obtain an Interim Import Clearance Certificate (ICC) with data corresponding to PICCS reporting. Notifications still take some time for review, so DENR and industry have reached a voluntary agreement to operate an Interim Status Permit (ISP) Scheme, to ensure safe use of a new substance while the notification is being evaluated. The ISP is valid for one year, and is renewed 90 days before expiry with the notification form.

11.9.3 Data Requirements for Notification Importers or manufacturers of new substances have to submit a pre-manufacturing and pre-importation notification (PMPIN) to DENR between 90 and 180 days prior to supply. There are two types of PMPIN forms for notification. The Abbreviated Form is used when the chemical is being used with no controls in a country with a similar chemical

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Practical Guide to Chemical Safety Testing review process to the Philippines, and sufficient information is submitted by the notifier that clearly exhibits that the chemical will not pose an unreasonable risk. The Detailed Form is used when the notifier cannot adequately document the safety of the new chemical or when DENR determines that the information submitted does not contain sufficient documentation to enable DENR to determine the safety of the new chemical. Technical and commercial information are required in the notification, including details of the proposed use, supply level, exposure limits, plus test data as shown in Table 11.1. Technical dossiers are considered to be public documents, but the notifier can claim certain information as confidential, providing this request is justified. The submission can be made by an appointed agent in the Philippines, the importer or Filipino manufacturer.

11.9.4 Administration The Chemical Review Committee within the Environmental Management Bureau (EMB) reviews and assesses the chemical based on hazard identification, exposure and dose response assessment, risk characterisation and management. If the information submitted is incomplete or not adequate to assess accurately the risks posed by the chemical, the notification will be returned to the submitter. If the information is adequate and the chemical does not pose an unreasonable risk, DENR issues a clearance to import or manufacture and the chemical is added to PICCS (public or confidential version) after the Notice of Commencing Import and Manufacture has been submitted. If the information is adequate and the chemical poses an unreasonable risk, the chemical is added to the Priority Chemicals List (PCL) and DENR will determine whether a clearance to import or manufacture should be granted. DENR may issue a Chemical Control Order (CCO) for a Priority Chemical that DENR determines should be regulated, phased out or banned because of the serious risks it poses to public health, workplace and the environment. DENR may impose a regulation, phase out plan or ban on a chemical at any time when it determines such action is necessary. Any CCO is published in the Official Gazette or other national newspaper. DENR may designate a particular use of a notified new chemical as a significant new use, based on the anticipated human or environmental exposure or other considerations, and a separate notification is required for this use. Joint notifications by more than one manufacturer or importer are permitted. New chemicals are listed on the PICCS 5 years

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Chemical Control in the US and the Rest of the World after being notified for the first time. Until listed on PICCS, subsequent suppliers require a repeat notification, and DENR can refer to the original data only with permission from the first notifier. DENR allow the supply of 1000 kg/year of new substances without notification for justified Research & Development supplies. As well as the R&D exemption, there is a process to allow the supply or manufacture of more than 1,000 kg/year before a new substance has completed the review period. It is possible to obtain an Interim Status Permit (ISP) for a 6 month period to import or manufacture an agreed quantity of the new substance. The permit has to be renewed every 6 months until the PMPIN is approved. Although there are at present no volume restrictions, the ISP will only be issued to manufacturers or importers if they have: •

Completed and submitted the hazardous waste registration form to EMB

•

Obtained an EMB ID number

•

Completed and submitted the PMPIN form

•

Reached a voluntary control agreement with EMB to ensure safe handling of the new chemical until such time that the EMB provides clearance.

•

If the PMPIN information submitted to EMB was not sufficient for EMB to issue a clearance, but special circumstances exist to justify an ISP being issued.

Although useful, the ISP is an onerous process that should only be used for notifications that have been justifiably delayed by EMB. There is an exemption from the PMPIN scheme for new substances placed on the market in quantities of < 1,000 kg/year. A declaration of intent in the form of a letter containing details such as the chemical name, CAS number, safety data sheet and the volumes, has to be submitted prior to supply. If successful, a certificate is issued which is valid for 1 year. It is recommended to provide details only on the substance, and once an allowance has been given, the permit will allow that substance to be supplied in accordance with the volume stated either on its own or in formulation with other PICCS listed chemicals. Export-only substances are reported to DENR with a Notice of Export. Non-isolated intermediates are not notifiable, and site-limited intermediates manufactured at below 5 tonnes per annum are also exempt. Polymer notification is discussed in Section 12.3.5.

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11.9.5 Priority Chemicals List (PCL) The initial PCL contains chemicals on the draft PICCS which are of concern in foreign priority chemical review schemes. A listing can be found on the Internet at http:// www.emb.gov.ph/chemicals.html. Further existing chemicals can be added to the PCL if new information becomes available. Manufacturers, distributors, users and importers of chemicals on the PCL must obtain a DENR ID number for hazardous wastes disposal and report biannually (between 1 September and 31 December beginning in 1996) to DENR on quantities and use. CFCs are subject to immediate regulation and phased-out withdrawal, in accordance with the Philippines commitment as a party to the Montreal Protocol.

11.10 Chemical Control in The People’s Republic of China

11.10.1 Latest Developments On 8 October 2002, China’s State Economic Trade Commission (SETC) adopted the following three laws on dangerous (i.e., hazardous) chemicals covering registration, licensing, packaging and classification: •

The Regulations on the Registration of Dangerous Chemicals

•

The Regulations on Licensing for the Business and Sale of Dangerous Chemicals

•

The Regulation on the Manufacturing of Packaging and Containers for Dangerous Chemicals.

These new laws, which became effective on 15 November 2002, will require dangerous chemicals to be registered and dangerous chemical operations licensed. These laws serve as key implementing measures under the State Council ‘Safe Management Regulation for Dangerous Chemicals’, which was issued on 26 January 2002 and came into effect on 15 March 2002. The details of the new schemes will be given in implementing measures and guidance documents which are being developed. A notification scheme for new substances will be introduced, possibly based on the EU scheme, and an inventory of existing substances not requiring notification is in development, as discussed below. There will be a system to control the import of hazardous substances, with lists of prohibited substances, controlled substances (which must be registered before import) and automatically-permitted substances (which require a customs notice before import).

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11.10.2 First Import and Toxic Chemicals Regulations The ‘Regulations for Environmental Management on the First Import of Chemicals and the Import and Export of Toxic Chemicals’ (abbreviated to EMFIC) [32] which came into force on 1 May 1994, regulated imports until 8 October 2002, and hence are described below. The Regulations did not cover pharmaceuticals, veterinary products, cosmetics, food additives or radioactive materials, articles or chemicals in transit which have not passed through Chinese Customs. One purpose of the Regulations is to implement the provisions of the ‘London Guidelines for the Exchange of Information on Chemicals in International Trade (1989 Amendment)’ [33] which require ‘prior-informed consent’ (PIC) from the participating national regulatory authorities before specified toxic chemicals are imported or exported. There are 27 restricted industrial chemicals and pesticide active ingredients in the ‘List of Toxic Chemicals Banned or Severely Restricted in the People’s Republic of China’. Foreign exporters and Chinese importers of these listed toxic chemicals have to apply to the State Environmental Protection Administration (SEPA), which was formerly called the National Environmental Protection Agency (NEPA), for a ‘Clearance Notification’ for each shipment, which must be used for the Customs declaration within its validity period. The second purpose of the Regulations was to require foreign exporters to register all chemicals before first import, either neat or as a component of a mixture. The registration scheme did not apply to domestic manufacturers. Chemicals imported solely for experimental purposes at below 50 kg per annum were also exempt from registration, as were plastics or rubber in the form of finished product articles. Chemicals were reported to SEPA using a standard application form. The information required in principle corresponded to that for EU notification (Section 9.2). The study methods were specified by SEPA, based on OECD Guidelines. In practice, however, a summary of the available studies from an existing SDS was usually adequate.

11.10.3 Inventory SEPA are developing an inventory of existing chemical substances, commonly referred to as the Provisional Inventory of the People’s Republic of China (PRC). The intention is to manage listed substances as existing chemicals, whilst new substances will require notification. The first version of the Provisional Inventory covered substances imported into the PRC during 1992 to 1994. The inventory has been updated a number of times, with the latest nomination period ending 30 April 2003.

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11.10.4 Hazard Communication Classification, packaging, labelling and transport of imported and exported chemicals have to be in accordance with the United Nations or national requirements for transport of dangerous commodities. Suppliers of chemicals are responsible for labelling and must provide a SDS. Employers have to ensure SDSs are available to workers and that chemicals are used safely. There is a SDS Regulation for international compliance with the ISO Standard 11014-1 [29], which came into effect from 1 January 2001 (although formal adoption into law is awaited). There are no provisions for confidentiality of chemical names. There are also provisions for classification and labelling based on the UN scheme and classification and labelling for common chemicals is specified. The EU classification and labelling (translated using simplified characters and pictograms) and EU SDSs (translated and giving the emergency number) may also be acceptable.

11.11 Chemical Control in New Zealand

11.11.1 Toxic Substances Act The 1979 Toxic Substances Act (TSA) and the 1983 Toxic Substances Regulations required new toxic substances to be notified to the Ministry of Health (MoH) before manufacture or import. The information required was the name, composition and uses, although more could be requested. The MoH maintains a registry of ca 215,000 substances by product name and component substances. The definition of toxic in the Act is broad and in practice virtually all substances and preparations were notifiable. However, substances controlled by other legislation were not covered, such as pesticides, food additives, and human and veterinary pharmaceuticals.

11.11.2 Resource Management Act The Resource Management Act came into force on 1 October 1991. It consolidates over 50 existing statutes governing air, land and water resources. Included are laws on coastal issues, town and country planning, mining, pollution, water and soil management. One of the key aspects of this Act was the provision for the establishment of an Environmental Risk Management Authority (ERMA) for the management and control of hazardous substances and new organisms.

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11.11.3 Hazardous Substances and New Organisms Act The Hazardous Substances and New Organisms (HSNO) Act [34] came fully into force on 2 July 2001, with the implementation of regulations for chemicals. An extensive range of ERMA guidance documents is available on the ERMA web site (www.ermanz.govt.nz). Ministry for the Environment (MfE) publications are available on their web site (www.mfe.govt.nz). In association with the MfE, ERMA has developed a web site for HSNO (www.hsno.govt.nz). The existing measures continue in force until the HSNO Act is applied to the various product types regulated under existing schemes. In particular, products covered by TSA registrations benefit from the transitional period of the HSNO (3 years, extendible to 5 years). Substances notified under the TSA, but which have not been assessed under the HSNO scheme (excluding those that are already scheduled toxic substances) are known as NOTS. Current work by ERMA for the MoH on NOTS is focussed on receiving and acknowledging these notifications. The new scheme is a product registration scheme for hazardous chemicals, whether substances or formulated preparations. There will be a public register of evaluated products on the ERMA web site, and any product not listed must be registered, but there will be no inventory of existing substances. Non-hazardous chemicals, small amounts of substances for scientific investigation, teaching and research and development, and substances in manufactured articles (such as printer cartridges, but not products such as glues or paint) are all exempt from reporting. Approval by ERMA will take about 80 days. The evaluation is complex, and involves extensive consultation of all stakeholders. Risks relative to seven statutory categories have to be considered, and for any risks, which cannot be controlled, a detailed costbenefit evaluation is necessary. The first stage in deciding whether a chemical needs an approval under the HSNO scheme is to decide if it is covered by any of the exemptions referred to above. If not, it may be covered by an existing approval. Until the ERMA public register is established, a first step in finding out if the substance is already approved is to check if it is one of the toxic substances specifically listed by the MoH in Schedules to the TSA, an authorised explosive listed on the Explosives Authorisation Order 1994, on the registered pesticides database, on the licensed animal remedies list or dangerous goods covered in the HSNO Act. In addition to these lists, ERMA has a confidential database of toxic substances notified under the TSA, which can be searched on application to ERMA, for which a fee may be charged. Finally, it is possible that the new chemical may not be hazardous. A substance

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Practical Guide to Chemical Safety Testing is hazardous if it has the potential to harm people or the environment, i.e., it is explosive, flammable, oxidising, toxic, corrosive or ecotoxic according to the threshold levels of the HSNO Act. ERMA have issued a User Guide to Thresholds and Classification, which is also available in summary form, to assist in deciding if a chemical is hazardous. This guide includes classification of mixtures. The supplier of the chemical can make the determination, use a consultant to advise, or request ERMA to issue a formal determination on the status of the substance (a service which incurs fees, and is limited by ERMA resources). Manufacturers or importers of a hazardous substance which is not exempt or covered by an existing approval have to apply to ERMA for HSNO approval. There are various types of application for hazardous substances. The standard application is for a substance for release where rapid application is not applicable, which requires public notification. Rapid assessment, without public notification, applies for low hazard substances or for a substance very similar to one already approved (but note it will take some time for existing products to be transferred to the HSNO Act as ‘deemed approvals’ or new substances to be approved under the HSNO scheme). Substances that will remain in secure containment to prevent escape (i.e., restricted to limited manufacturing or trial situations) qualify for a containment application, again without public notification. The other types of application are for use in emergency and transhipment of hazardous substances imported temporarily solely for re-export (i.e., if they are in New Zealand for more than 20 working days).

11.11.4 Data Requirements for Notification Applications have to be made using the appropriate ERMA application form. There is detailed guidance published by ERMA, but applicants should seek advice before submitting the application. The information required depends on the scale and significance of the risks, costs and benefits of the substance, and the ERMA has adopted three categories for an initial judgement on the information for an application. Only limited information is needed for Category A substances of low hazard and low risk (A1) or moderate to high hazard but very low exposure (A2). Information Category B is for substances with managed risks (B1) or, probably a common situation, for substances approved in another jurisdiction (B2), providing there are no unique risks for use in New Zealand. Category C is for high potential risk substances, with a comprehensive information package required. Ideally the physico-chemical properties of appearance, pH, density, vapour pressure, boiling/ melting temperature, water solubility and Pow are provided. Hazardous properties information also has to be provided, considering each of the six potential hazardous

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Chemical Control in the US and the Rest of the World properties, and if possible, with a determination of which of the properties are triggered. Ideally for mixtures information on the mixture itself is used, but if this is not available, information on the hazardous properties of each hazardous component is provided instead. It is recognised that a full set of test data may not be available, and predictions may be acceptable. Confidential information can be claimed as secret, but certain information cannot be claimed as confidential without good justification. After release and emergency applications have been verified by ERMA, they have to be publicly notified and posted on the web site for public comment. There is also a User Guide to HSNO Control Regulations to help interpret the performance standards.

11.11.5 Hazard Communication The 1992 Health and Safety in Employment Act administered by the Department of Labour (DoL), requires that safety data sheets are made available in a specified 4 or 12 section format, although the standard ISO 16-section format is acceptable in practice [29]. Labelling according to international standards is necessary. Data sheets must contain a statement of hazardous nature. Non-hazardous components can be claimed as confidential at any concentration. Hazardous components can only be claimed as confidential if below the lowest relevant concentration cut-off.

11.12 Mexico

11.12.1 Legislation Toxic substances are regulated primarily by the Inter-Secretarial Commission for Control of Pesticides, Fertilisers and Toxic Substances, although Mexico does not have an extensive system for screening new chemicals. As Mexico is part of the North American Free Trade Association (NAFTA) with the US and Canada, chemical control measures consistent with those of the other members are anticipated. Mexico has accepted the 13 OECD decisions on Environmental Matters, related to chemical substances and hazardous wastes. Mexico has a law called ‘The General Law of Ecological Equilibrium and Environmental Protection’ [35] and under it provisions are made for:

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testing of ‘existing chemicals’

•

record keeping and reporting of exposure information

•

some import and export controls

•

labelling requirements for ‘hazardous materials’.

Eventually it is likely ‘chemical notification’ requirements will be added as an amendment. Mexico publishes official lists of chemicals subject to special handling, labelling and reporting requirements. There is also a list of hazardous chemicals that are banned from use in Mexico. Generally Mexico follows international guidelines in determining whether industrial chemicals will be allowed to be used or imported and it relies on information from the country of origin and from international organisations regarding health, safety and environmental problems of chemicals.

11.12.2 Safety Data Sheets Mexican SDSs are basically similar to those under US OSHA (Section 11.3), but with some differences. No specific format is required, but the specification of data elements is more detailed than for OSHA. In particular, SDSs must provide a ‘degree of risk’ and an Immediately Dangerous to Life or Health (IDLH) value for each hazardous component, an indication of mutagenicity and also information on ecology. SDSs must also be written in Spanish.

11.13 Singapore The Pollution Control Department (PCD) of the Ministry of the Environment controls toxic and environmentally hazardous chemicals under The Environmental Pollution Control Act (EPCA) and The Environmental Pollution Control (Hazardous Substances) Regulations. The EPCA incorporates part of the earlier Poisons Act of 1953 (most recently amended 1991), specifically the Poisons List, Part II, which is concerned with chemicals considered to be a hazard to the public or the environment. Hazardous Substances controlled by the PCD are generally those that may pose a mass-disaster potential, are highly toxic and pollutive and/or generate toxic wastes that could only be disposed of with great difficulty. In order to import, sell or export any hazardous substance controlled under the EPCA, a Hazardous Substances Licence has to be obtained. Similarly, to purchase, store and/or

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Chemical Control in the US and the Rest of the World use any hazardous substance controlled under the Environmental Pollution Control (Hazardous Substances) Regulations, a Hazardous Substances Permit and a Transport Approval must be obtained.

11.14 Malaysia There are a number of different acts in force (and pending) that regulate chemicals in Malaysia. The controlling authority is the Department of Occupational Safety and Health (DOSH) and the two main pieces of legislation are the Occupational Safety and Health Act, 1994 and the Occupational Safety and Health (Classification, Packaging and Labelling of Hazardous Chemicals) Regulations, 1997. These are supported by various Guidelines, notably for Classification, Labelling and for the Formulation of Chemical Safety Data Sheets.

11.15 Thailand In Thailand the control of industrial chemicals is governed mainly by the Hazardous Substance Act B.E. 2535 (1992), which is part of a national master plan on chemical safety. A number of different Ministries are involved – Industry, Defence, Agriculture and Co-operatives, Public Health and others. All producers, importers, carriers and persons in possession of hazardous substances must comply with B.E. 2535 and anybody seeking to import or use a toxic substance must register with the Office of Hazardous Substances of the Department of Industrial Works. The technical information required is related to the chemicals’ life cycle, disposal and safety issues.

11.16 Indonesia The Department of Industry regulates handling of toxic substances in hazardous waste used in industry. The Indonesian Ministry of Labour Department specifies a minimum set of information on SDSs.

11.17 Taiwan The Toxic Chemical Substances Control Act of 1986 (amended December 1999) administered by the Environmental Protection Administration lists 21 kinds of toxic

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Practical Guide to Chemical Safety Testing substances. In 1994 the list was increased to 85 chemical substances classified into 58 varieties. Substances are currently banned or supervised under a system of licenses. Future regulations may be based on a version of this system by adding chemicals to the ‘toxic’ list and having permitted and prohibited uses for those chemicals. The Taiwan EPA has not defined a clear process for adding toxic chemicals to their ‘inventory’, but proposed hazard criteria are open for comment. There remains discussion of the development of a new chemical assessment scheme. A draft inventory has been established, not from industry nominations but from import records of the Ministry of Economics. It reportedly contains about 20,000 entries – a collection of chemical substances, mixtures, products by chemical name and Trade Name.

11.18 HPV Programmes

11.18.1 OECD In 1987 the OECD member countries decided to undertake a systematic investigation of the safety of existing chemicals [36]. Through a 1990 OECD Council Decision [37] Member countries decided to undertake the investigation of HPV chemicals in a cooperative way, based on the assumption that production volume is a surrogate for data on occupational, consumer and environmental exposure. The HPV chemicals include all chemicals reported to be produced or imported at levels greater than 1,000 tonnes per year in at least one Member country or in the European Union region. The OECD List of HPV Chemicals serves as the overall priority list from which chemicals are selected for SIDS data gathering and testing and initial hazard assessment. The most recent OECD HPV Chemicals List was compiled in 2000, contains 5,235 substances and is based on submissions of nine national inventories and that of the EU. The next list will be compiled in 2003. The data elements needed for screening HPV chemicals were agreed by OECD and called the SIDS. The SIDS comprises characterisation and effects data similar to the OECD Minimum Pre-marketing set of Data (MPD) [38] for new chemicals (See Table 11.1), as well as readily available information on exposure. It is regarded as the minimum information needed to assess an HPV chemical to determine whether any further investigation should be carried out or not. However, all available data are used to make the assessment, which is then reviewed and agreed by the SIDS Initial Assessment Meeting (SIAM).

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Chemical Control in the US and the Rest of the World In 1998 the OECD HPV Chemicals Programme was refocused in order to increase the output significantly. The overall Programme was divided into six segments, each with distinct outputs and a clearly defined mechanism for oversight. The refocused Programme comprises the following segments: (1) maintenance and improvement of the consolidated OECD HPV List; (2) improvement of tools to select chemicals from the HPV List for investigation; (3) enhancement of the SIDS testing programme; (4) streamlining of SIDS initial assessments to focus on hazard; (5) coordination of post-SIDS work; and (6) pilot projects on joint IPCS/OECD detailed international risk assessments. Since 1999 the work in OECD is concentrating on the first four segments. Detailed exposure information gathering and assessment is carried out in follow-up at the national (or regional) level as appropriate, but is no longer part of the SIDS initial assessment. Detailed international assessment of risks to human health and/or the environment is also no longer carried out under the SIDS initial assessment, but rather will be undertaken jointly by OECD and the International Programme on Chemical Safety (IPCS) for appropriate pilot cases.

11.18.2 International Council of Chemical Associations Global Initiative Progress with the OECD HPV Program has been relatively slow. Only ca 100 chemicals have so far been evaluated, with ca 275 under testing. As a result, the ICCA has begun a Global Initiative on HPV Chemicals to speed up the OECD SIDS programme. ICCA has made a commitment to complete SIDS testing on ca 1000 priority chemicals drawn from the OECD list of 4100 by the end of 2004. The responsibility for acting under this initiative lies with all companies producing HPV chemicals. They are responsible for the collection of hazard information and, where necessary, the generation of tests needed to supplement the available data. Companies are encouraged to join together to form consortia to undertake this work and to share the burden. The information that is required under the Initiative is based on the OECD Screening Information Data Set (SIDS) and covers acute toxicity, repeat dose toxicity, reproductive/ developmental toxicity, genetic toxicity, ecotoxicity and environmental fate. In addition, basic exposure/use information is required for the chemical in the sponsor country. The ICCA initiative is only concerned with the collection of data to allow a Hazard Assessment to be made. This is because experience under the OECD program has shown

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Practical Guide to Chemical Safety Testing that the collection of exposure information needed for global and even regional Risk Assessment is such a time-consuming and difficult process, that progress is too slow. ICCA decided to concentrate on the collection of hazard information first, so that the information would be available for Risk Assessment and prioritisation of chemicals at a later date. It is believed that in most cases risk Assessment will take place at national or regional level due to different use situations, regulatory requirements or risk perceptions. The data collected are reported in the OECD SIDS Dossier format. The key information from the dossier is then used to produce a hazard assessment in the form of a SIDS Initial Assessment Report (SIAR). The draft SIAR is reviewed by the sponsor country and, once approved, forwarded to the OECD for consideration and appropriate action at a SIAM. Within Europe the hazard assessment data will be used as a scientific basis for selecting candidates for further risk-based investigation through the Existing Substances Regulation (793/93) [39].

11.19 Useful Web Sites See Table 11.3.

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Table 11.3 Useful web sites Canada

USA

Australia

China

Environment Canada

http://www.ec.gc.ca/envhome.html

Health Canada

http://www.hc-sc.gc.ca/english/index.htm

Environment Canada – New Substances Search Engine for Chemicals and Polymers

http://www.ec.gc.ca/substances/ese/eng/ cas_e.htm

Environment Canada – Existing Substances

http://www.ec.gc.ca/substances/ese/eng/esehome.cfm

Workplace Hazardous Materials Information System (WHMIS): What You Need to Know

http://www.utoronto.ca/safety/whmis1. htm

Environment Protection Agency (EPA) Homepage

http://www.epa.gov/

EPA – Office of Pollution Prevention and Toxics

http://www.epa.gov/opptintr/

Chemicals in the Environment: OPPT Chemical Fact Sheets

http://www.epa.gov/docs/chemfact/

Department of Labour – Occupational Safety & Health Administration

http://www.osha.gov/

State Department – Policy – Oceans, International Environmental/Scientific Affairs

http://www.state.gov/www/global/oes/index.html

Department of Commerce Homepage

http://www.doc.gov

Environment Australia – Chemicals in the Environment Branch

http://www.environment.gov.au/epg/chemicals.html

NICNAS

http://www.nicnas.gov.au

Chemical Registration Center of Environmental Protection Chinese State Administration (SEPA)

http://www.crc-sepa.org.cn/home.english.html

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Table 11.3 continued Hong Kong

Environmental Protection Department

http://www.info.gov.hk/epd/

India

Ministry of the Environment

http://envfor.nic.in

Indonesia

Environmental Impact Management Agency (BAPEDAL) Environment Ministry (not in English)

http://www.bapedal.go.id/

Japan

Ministry of Health, Labour and Welfare (MHLW)

http://www.mhlw.go.jp/english/index.html

Ministry of the Environment

http://www.env.go.jp/en/index.html

Ministry of Economy, Trade and Industry (METI)

http://www.meti.go.jp/english/index.html

Korea

Ministry of the Environment

http://www.me.go.kr/english/

Malaysia

Ministry of Human Resources (Labour)

http://www.jaring.my/kwm

Department of the Environment

http://www.jas.sains.my/

Malaysian Government/Prime Minister Site

http://www.smpke.jpm.my/

Department of Labour

http://www.dol.govt.nz/

Department of Labour – Occupational Safety & Health Service

http://www.osh.dol.govt.nz/

NZ Government Home Page

http://www.govt.nz

Ministry for the Environment

http://www.mfe.govt.nz/

Environmental Risk Management Authority

http://www.ermanz.govt.nz/

New Zealand

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Table 11.3 continued Philippines

Singapore

Taiwan

Thailand

Department of Environment http://www.psdn.org.ph/emb and Natural Resources – Environmental Management Bureau Department of Health

http://www.doh.gov.ph/

Government Home Page

http://www.gov.sg/

Ministry of the Environment

http://www.gov.sg/env/

Ministry of Health

http://www.gov.sg/moh/

Environment Protection Administration

http://www.epa.gov.tw/english/

Environment Policy Monthly – Journal

http://www.epa.gov.tw/english/EPM

Ministry of Science, Technology and Environment

http://www.moste.go.th/eng/index.htm

National Institute for the Improvement of Working Conditions and Environment (NICE)

http://www.inet.co.th/org/nice/abnice.htm

Acknowledgement The authors are grateful for general editorial advice and specific comments on the US, Canadian and Korean notification schemes from Dr George Dominguez of Regulatory Assistance Corporation.

References 1.

US Toxic Substances Control Act, 15 U.S.C. § 2601-2629 (1994 & Supp. I 1995).

2.

J.R. Wheeler, Toxic Substances Control Act, Compliance Guide and Service, Environment Books Inc, Plano, Texas, USA, 1988, as updated.

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Practical Guide to Chemical Safety Testing 3.

McKenna & Cuneo, L.L.P., TSCA Handbook, 3rd Ed., Government Institutes Inc., Rockville, Maryland, USA, 1997.

4.

Public Law 91-596, 91st Congress, S.2193, December 29, 1970.

5.

OSHA, Hazard Communication Standard; 29 CFR 1910.1200, 2002.

6.

American National Standard for Hazardous Industrial Chemicals- Precautionary Labelling, ANSI Z129.1-1988.

7.

American National Standard for Hazardous Industrial Chemicals-Material Safety Data Sheets – Preparation, ANSI Z400.1-1993.

8.

EPA, OPPT, Chemical Hazard Data Availability Study: What Do We Really Know About the Safety of High Production Volume Chemicals?, April 1998.

9.

EPA, Testing of Certain High Production Volume Chemicals; Data Collection and Development on High Production Volume (HPV) Chemicals; Proposed Rule and Notice, Federal Register (40 CFR 799, December 26, 2000).

10. EPA Inventory Update Rule, 40 CFR 710, 1990. 11. EPA Persistent, Bioaccumulative and Toxic Pollutants (PBT) Plenary Group and EPA Office Directors Multimedia and Pollution Prevention Forum. A multimedia Strategy for Priority Persistent, Bioaccumulative, and Toxic (PBT) Pollutants, November 16, 1998. 12. The Great Lakes Binational Toxics Strategy: Canada-US Strategy for the Virtual Elimination of Persistent Toxic Substances in the Great Lakes, 1997. 13. EPA, Voluntary Children’s Chemical Evaluation Program; Notice, Federal Register, Part V, Vol. 65, No. 248, December 26, 2000. 14. Regulatory Affairs Bulletin, June 2000, Number 79, 34. 15. CMA, Letter with 3 attachments from Sandra Tirey to James Aidala and Susan Wayland, USEPA, Office of Prevention, Pesticides and Toxics, Washington DC. September 21, 1999. 16. American Public Health Association (APHA), Children’s Environmental Health Network, Environmental Defense, National Environmental Trust, Physicians for Social Responsibility. Comments on the Environmental Protection Agency’s Framework for a Voluntary Children’s Chemical Evaluation Program, April 12, 2000. 304

Chemical Control in the US and the Rest of the World 17. USEPA, Methodology for Selecting Chemicals for the Voluntary Children’s Chemical Evaluation Program (VCCEP) Pilot, December 5, 2000. 18. The Canadian Environmental Act, 1999 (CEPA, 1999), Canada Gazette Part III, Vol. 22, No. 3, Chapter 33, November 4, 1999. 19. New Substances Notification Regulations, Canada Gazette Part II, April 6, 1994; Available online at www.ec.gc.ca/Substances/nsb/download/NSNR.pdf 20. Ordonnance sur les toxiques (Otox) du 19 septembre 1983, RO 1983 1387. 21. Ordonnance du 9 novembre 1998 sur les fiches de donées de sécurité relatives aux toxiques et aux substances dangereuses pour l’environnement, RO 1999 28. 22. Ordonnance sur les substances dangereuses pour l’environnement, Modification du 29 novembre 1995, RO 1995 890. 23. Loi fédérale sur la protection contre les substances et les préparations dangereuses du 15 décembre 2000. 24. Industrial Chemicals (Notification and Assessment) Act 1989 (Cwlth). 25. NOHSC National Code of Practice for Preparation of Material Safety Datasheets [NOHSC: 2011 (1994)]. 26. Federal Office of Road Safety, Australian Code for the Transport of Dangerous Goods by Road and Rail, 5th Edition, AGPS, Canberra, September 1992. 27. NOHSC National Code of Practice for the Labelling of Workplace Substances [NOHSC: 2012 (1994)]. 28. Toxic Chemicals Control Law, 30 December 1996, as amended. 29. ISO 11014-1, Safety data Sheet for Chemical Products – Part 1: Content and Order of Sections, 1994. 30. Toxic Substances and Hazardous and Nuclear Wastes Control Act, 1990. 31. Implementing Republic Act 6969, DENR Administrative Order No. 29, Series 1992. 32. Chinese National Environmental Protection Agency (NEPA), Regulations for Environmental Management on the First Import of Chemicals and the Import and Export of Toxic Chemicals, 15 April 1994.

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Practical Guide to Chemical Safety Testing 33. UNEP, London Guidelines for the Exchange of Information on Chemicals in International Trade (1989 Amendment). (Available online at www.chem.unep.ch/ ethics/english/longuien.htm). 34. Hazardous Substances and New Organisms (HSNO) Act, 1996. 35. The Federal General Law of Ecological Equilibrium and Environmental Protection Act, 1988. 36. OECD, Decision-Recommendation of the Council on the Systematic Investigation of Existing Chemicals [C(87)90(Final)], 1987. 37. OECD, Decision-Recommendation of the Council on the Co-operative Investigation and Risk Reduction of Existing Chemicals [C(90)163(Final)], 1991. 38. OECD, Decision of the Council concerning the Minimum Pre-Marketing Set of Data in the Assessment of Chemicals [C(82)196(Final)], 1982. 39. Council Regulation (EEC) No. 793/93, Official Journal of the EC, 1993, L84.

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12

Notification of Polymers Worldwide John Moore

12.1 Introduction To date, nine regions of the world have implemented regulatory control measures for the supply, importation, and in some cases, the manufacture of industrial chemicals. The regions are: US, Japan, EU, Australia, Canada, Korea, Switzerland, Philippines and New Zealand, whilst China (People’s Republic) is currently completing its chemical inventory. These all distinguish between new and existing chemical substances, in that new substances, whether polymeric, or non-polymeric have to be formally notified before they are placed on the market, unless they are specifically made exempt by the authorities. Many of these regions have adopted the formal OECD polymer definition: ‘‘Polymer’ means a substance consisting of molecules characterised by the sequence of one or more types of monomer units and comprising a simple weight majority of molecules containing at least three monomer units which are covalently bound to at least one other monomer unit or other reactant and consists of less than a simple weight majority of molecules of the same molecular weight. Such molecules must be distributed over a range of molecular weights wherein differences in the molecular weight are primarily attributable to differences in the number of monomer units. In the context of this definition a ‘monomer unit’ means the reacted form of a monomer in a polymer.’ This requires: •

> 50 percent of molecules containing at least three monomer units covalently bound to at least one other monomer unit or reactant.

•

Molecules must be distributed over a range of molecular weights.

•

No single molecular weight molecule can be > 50 percent (w/w) of total molecules.

Although these regions make a clear distinction between polymers and non-polymeric substances, the strategies employed by the regions and the data requirements vary significantly. These differences range from what constitutes a new and existing polymer, 307

Practical Guide to Chemical Safety Testing to the actual data required for their notification. This chapter reviews, summarises and contrasts the requirements for the worldwide notification of polymers building on the description of chemical control measures in the EU (Chapter 9), Japan (Chapter 10) and the USA and elsewhere (Chapter 11).

12.2 North America 12.2.1 USA As discussed in more detail in Chapter 11, Section 5 of the Toxic Substances Control Act (TSCA) [1] requires a producer, or importer of a new chemical substance (i.e., one which is not listed on the TSCA Inventory) to file a Pre-manufacture Notice (PMN) to the Environmental Protection Agency (EPA), 90 days prior to manufacture or import. Although the US TSCA does not specifically define a polymer, this requirement applies to polymers, unless they are exempt under the 1995 low risk polymer rule, the exemption criteria for which are detailed in the next section. Besides the exemption for low risk polymers, all the other TSCA facilities which apply for industrial chemicals are also relevant to polymers. These include research and development (R&D), low volume chemicals (< 10 tonne/annum limit for all manufacturers/importers), manufacture for export only and the fairly recent Low Environmental Release and Low Human Exposure (LoREX) exemption. When searching the TSCA Inventory to determine whether or not a certain polymer is listed, monomers, or reactants, which form part of the polymer backbone at < 2% may be ignored if a precise listing for the polymer cannot be located. This concept is widely known as the 2% monomer exemption rule and has been adopted by other regions.

12.2.1.1 PMN Exemption Rules for Polymers The 1995 rules for claiming exemption for polymers are fairly complex. Although only a brief overview is given here, comprehensive details are provided in the EPA’s ‘Polymer Exemption Guidance Manual’ [2]. There are basically three types of polymers exempt from PMN: •

Polymers with a number average molecular weight (Mn) equal to or greater than 10,000. Candidates must contain less than 5% of polymer with a molecular weight (MW) of less than 1,000 and less than 2% of polymer with MW less than 500.

•

Polymers with an Mn equal to or greater than 1,000 and less than 10,000. These must contain less than 25% polymer with MW less than 1,000 and less than 10% of polymer with a MW less than 500.

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Polyester polymers made only from those reactants specifically listed in the polymer exemption regulations. There are no limits on the amounts of oligomers with MW less than 1,000.

It is important to note that only polymers which comply with the OECD definitions and produced from TSCA Inventory listed reactants at greater than 2% qualify for the exemption. Other aspects to consider include ionic nature, elemental content, presence and types of functional groups, water solubility/dispersibility/adsorbivity and degradation potential. Manufacturers and importers of polymers which qualify for the exemption and wish to take advantage of it have to provide an annual report to the EPA and produce and maintain records which include: •

Identity of all reactants

•

Structural representation of the polymer

•

Analytical data to show compliance with the exemption

•

Annual submission of a notice to the EPA.

It should also be noted that in the absence of a PMN, the polymer will not be listed on the inventory. This means that each manufacturer, or importer must keep the polymer exemption records and file a notice with EPA. Many users therefore prefer to have the polymer listed on the inventory. However, if the polymer is manufactured in the US, then it is not as much of a problem since only the manufacturer is required to keep records. Other considerations are that the assessment of criteria for low risk polymers is fairly complicated, the data to prove exemption is significant, and the penalties for noncompliance are massive. Due to these factors, many companies feel that it is prudent to file a PMN and submit a Notice of Commencement (NOC) so that the polymer is added to the inventory, rather than use the polymer derogation facility. The advantage of the exemption is that the 90-day review period is not imposed and the EPA fee is waived.

12.2.1.2 PMN Information Requirements for ‘Non-Exempt’ Polymers There are no mandatory testing requirements imposed by the EPA. PMNs can be filed using surrogate test data, or even no data at all. The regulations, however, specify that notifiers must submit all the data in their possession and provide the following information: 309

Practical Guide to Chemical Safety Testing •

Chemical identity

•

Use, exposure and disposal information

•

Production/import volume

•

Information on by-products

•

Available health/environmental effects test data.

The EPA are experts on structure-activity relationships and whilst test data is not an essential element of the scheme, they expect notifiers to examine any potential areas of concern. For polymers, information on molecular weight, monomer content and their associated hazards are useful parameters in determining the need to generate test data on the actual polymer. It should also be noted that the EPA are more likely to impose more onerous testing requirements and issue restrictions on marketing and use if no test data is provided in-line with their concerns. If notifiers are in any doubt about the best policy to adopt, the agency can be requested to provide guidance. Following the completion of a PMN and the filing of a NOC, the polymer is added to the inventory using Chemical Abstracts Service (CAS) nomenclature. If the polymer contains monomers in combined form at < 2%, notifiers have the choice of whether or not to include these in the chemical description.

12.2.1.3 Review Period Following submission of a PMN, the agency imposes a statutory 90-day review period. A Notice of Commencement has to be filed 30 days prior to first commercial import or manufacture to enable the polymer to be added to the inventory. Failure to do this will result in an act of non-compliance and result in substantial penalties.

12.2.1.4 Conclusions on the US Scheme With the polymer exemption facility and no mandatory test requirements, the US PMN scheme often provides an easily administered process for the majority of polymers. However it should be emphasised that the EPA are experts on the use of structure-activity relationships and have built a comprehensive database. Therefore it is essential that notifiers supply relevant information to cover any areas of concern and remember to submit their NOC.

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12.2.2 Canada Canada, in-line with other regions, has adopted the OECD polymer definition. Polymers feature on both the main inventory, the Domestic Substances List (DSL) and the subsidiary Non-Domestic Substances List (NDSL). Once listed on the DSL, the polymer can be manufactured or imported without the need for notification. When searching the DSL for suitable listings the 2% monomer exemption rule can be employed. For polymers which are not listed on the DSL, the notification test data required depends on criteria such as: NDSL status of the polymer; the distinction between commercial and R&D activities; DSL and NDSL status of monomers and reactants; the annual amount of the polymer being placed on the market and whether or not the polymer meets the low concern criteria. Another consideration in determining the test data requirements, is whether the polymer meets the Canadian Environmental Protection Act (CEPA) definition, which is virtually the same as the OECD definition and transitional status. Transitional polymers are defined as those not listed on the DSL, but imported or manufactured in quantities > 20 kg, after 1st January 1987 and before the implementation of the CEPA. Further information on notification of new substances in Canada is given in Chapter 11.

12.2.2.1 Polymers of Low Concern Full details of the Polymer of Low Concern (PLC) criteria are provided in the Canadian Environmental Protection Act (CEPA) Guidelines [3]. In summary, polymers of low concern are those which do not possess structural concerns in items 1,2,3, or 4 of Schedule IX of the CEPA Guidelines and meet the following criteria: •

Mn > 10,000

•

< 2% species of MW < 500

•

< 5% species MW < 1,000

Also if not described in any part of Schedule IX and •

Mn > 1,000

•

< 10% species of MW < 500

•

< 25% species MW < 1,000

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Practical Guide to Chemical Safety Testing For polyesters, these have to be manufactured solely from DSL listed reactants and meet the CEPA Schedule X requirements.

12.2.2.2 Notification Test Data Requirements Polymers being commercialised (i.e., excluding R&D, site specific, or export only) are assessed using the scheme in Figure 12.1. This leads notifiers to the appropriate testing schedules, which are one of schedules VI, VII, or VIII. A brief summary of the data required under each of these features in Table 12.1. Polymers being supplied for R&D and other types of development are regulated under schedules XI or XII. These have no mandatory toxicological or environmental test requirements.

12.2.2.3 Review Period Following submission, the review period for Schedule VI and VII is 45 days and 90 for Schedule VIII. Notifiers should check the status of schedule VIII notified polymers for DSL inclusion and note that until this happens only the notifier can import or manufacture, unless a repeat notification is first completed.

12.2.2.4 Conclusions on the Canadian Scheme The Canadian scheme provides notifiers with easily administered schemes for R&D and site specific polymers. When the PLC criteria is met, no toxicological data are required therefore registration is both simple and cost effective. However, non-PLC polymers supplied in quantities of > 10 tonne per annum which do not qualify for any of the other reduced testing criteria, have to be tested in a similar way to non-polymeric materials. Many polymers, however comply with the reduced testing requirements of Schedules VI and VII.

12.3 Asia Pacific

12.3.1 Japan There are two separate approval schemes in Japan which have to be satisfied to allow new substances to be placed on the market (see Chapter 10). Firstly, the Japanese Chemical

312

313

Figure 12.1 Canada: Polymer Notification Strategy Decision Diagram

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Footnotes: The Schedules of CEPA [3] specify the data requirements acc = accumulated total

Practical Guide to Chemical Safety Testing

Table 12.1 Canada: Overview and Summary of Polymer Test Data Requirements Test

Schedule VI

VII

VIII

Chemical name

✓

✓

✓

Trade name

✓

✓

✓

CAS number

✓

✓

✓

Molecular formula

✓

✓

✓

Structure

✓

✓

✓

Polymer composition

✓

✓

✓

MSDS

✓

✓

✓

Number average molecular weight

✓

✓

✓

Low molecular weight species

✓

✓

✓

Water solubility

x

✓

✓

Octanol solubility / Log P

x

✓

✓

Hydrolysis as a function of pH

x

x

✓

Acute aquatic toxicity (fish / daphnia)

x

✓*

✓*

Biodegradation

x

✓*

✓*

Acute toxicity green algae

x

✓*

✓*

Acute oral toxicity

x

✓

✓

Skin irritation

x

x

✓

Skin sensitisation

x

x

✓

28-day sub-acute

x

x

✓

Ames

x

x

✓

Chromosome aberration

x

x

✓

Use profile

✓

✓

✓

Footnotes: ✓Test required x Test not required The Schedules of CEPA [3] specify the data requirements * The need for the aquatic tests depends on water solubility and ionic characteristics

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Notification of Polymers Worldwide Substances Control Law (CSCL), aims to prevent adverse human health effects arising from environmental contamination. The law is administered by the Ministry of Economy, Trade and Industry (METI), and the Ministry of Health, Labour and Welfare (MHLW) and the Ministry of Environment (MOE). Secondly, the Industrial Safety and Hygiene Law (ISHL) administered by the MHLW, aims to protect worker’s health from workplace exposure, being primarily concerned with occupational cancer. There are therefore two separate regulations and three separate Ministries to consider, before placing a new chemical on the Japanese Market. The inventory, known as the Handbook of Existing and New Chemical Substances (ENCS) [4], has become the focal point of the CSCL. The latest edition contains over 22,000 entries, covering over 49,000 individual chemical substances of which some 3,200 have been notified. Substances listed on the ENCS Inventory are termed existing and can be freely manufactured, imported and supplied without the need for notification. Those not listed, are termed new and require notification before they can be placed on the market. The CSCL is concerned with both manufacture and import and ISHL for manufacture only. The Japanese definition of placing on the market is therefore one of supply, importation and manufacture, although for the latter, exemptions are available even under the CSCL for certain conditions.

12.3.1.1 Existing Polymers The first recommended action is to check whether the polymer is covered by any of the listings in Sections 6 and 7 of the existing substances inventory, or on the list of newly notified substances. If an appropriate listing can be found, then the polymer is deemed to be existing and no notification is required. Japan has not adopted the 2% monomer exemption rule, however, when searching the inventory for suitable candidates, monomers present in combined form at < 1% can be ignored. If a listing cannot be found, then the polymer is new and requires notification before being placed on the market. When searching the inventory it is worthwhile noting two special cases which may allay the need for notification: •

When the unit polymers constituting a block polymer, are all existing chemical substances, the block polymer is not treated as a new chemical substance.

•

When the stem and branch polymers constituting a graft polymer are all existing chemical substances, the graft polymer is not treated as a new chemical substance.

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12.3.1.2 New Polymers All new industrial chemicals can be supplied for purposes of R&D in justifiable amounts without being registered, but notification is required to each of the three Ministries for purposes of commercialisation. If this is limited to < 1 tonne per annum, a simplified approach with a minimum data set suffices. Alternatively, if the polymer is manufactured and consumed on-site, only a notification under ISHL is required. The majority of polymers however, need to be fully commercialised.

12.3.1.3 Biodegradable New Polymers An important point to note is that polymers which biodegrade according to the CSCL criteria of complete mineralisation can be notified without needing to conduct toxicological and environmental data.

12.3.1.4 Non-Biodegradable New Polymers Non-biodegradable polymers are tested either, exactly the same as non-polymeric substances, or by reduced testing if they meet certain physico-chemical criteria. Therefore for new, non-biodegradable polymers it is necessary to study certain physico-chemical end points for both the laws to establish if any exemptions can be obtained, especially from the toxicological and environmental testing demands of the CSCL, which are far more onerous than those required under ISHL.

12.3.1.5 Japanese Chemical Substance Control Law Concept for New NonBiodegradable Polymers A polymer is defined under the CSCL as a substance generally with a number average molecular weight of greater than 10,000 and physico-chemical properties characteristic of a polymer, such as molecular weight distribution and no clear melting point or water solubility. Those substances of Mn 1,000 to 10,000 are usually considered as oligomers, but providing the Mn value is above 1,000, an exemption from the CSCL toxicological and environmental screening tests applies to those non-biodegradable polymeric materials which fulfil certain criteria. The process which decides whether or not this testing is required is outlined by the flow diagram in Figure 12.2.

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Figure 12.2 Japanese Chemical Substances Control Law Evaluation Flow Scheme for Non-Biodegradable Polymers

As indicated in Figure 12.2 there are three key elements to consider: •

stability

•

solubility

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Polymer light stability Polymer solubility Practical Guide to Chemical Safety Testing •

number average molecular weight.

These are described more fully in Table 12.2.

Table 12.2 Key elements in Evaluation of Polymers for Stability for the Japanese Chemical Substances Control Law Stability • Light Both photo and fluorescent stability are examined. • pH Stability is examined at pH 1.2 for 20 hours at 40 °C and at pHs 4, 7 and 9 at 40 °C for 2 weeks. These are selected to represent accidental ingestion and environmental release. To conclude a polymer as stable for all the tests, there must be < 2% weight loss, no change to the infrared spectrum and molecular weight distribution (some relaxation at ± 15% for a wide distribution and ± 5% for a narrow distribution, may be allowed on a case by case basis). Additionally, for the pH tests the total organic content in the filtrate must be < 5 ppm. Solubility The solvents specified for the testing are split into 3 categories. • Water Distilled water has to be employed. • Aliphatic n-heptane, 1-octanol are specified. • Common solvents The common solvents for testing are: Tetrahydrofuran (THF), toluene, 1,2-dichloroethane; 2-propanol, methyl isobutyl ketone and dimethyl formamide (DMF). According to Figure 12.2, there are two possible routes for solubility. For those polymers which are concluded as insoluble and for which a chemical structure can be drawn, the outcome is that no toxicological or environmental testing should be imposed. If a structure cannot be assigned, then the solubility under both acid and alkaline conditions has to be examined. In order to declare the polymer as ‘insoluble’, tests in all 9 media have to be examined. For those polymers for which insolubility is not claimed, this includes water soluble polymer, tests can be confined to water, one aliphatic and one common solvent. Molecular Weight Properties The low concern criteria is supported if low molecular weight species (Mn < 1,000) are present at < 1%. The molecular weight assessment is not required for polymers which are insoluble in all 9 solvents.

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12.3.1.6 Structural Characteristics Certain stable polymers of low concern may have to be tested in some or all of the standard toxicological and environmental tests, if they are based on structures, or contain chemical fragments which are of concern. Examples include ionic and isocyanate containing polymers. If crosslinking or crystallinity is considered as a structural characteristic of the polymer, a physico-chemical constant (i.e., degree of swelling, deformation temperature) which verifies these characteristics may be required. It is also possible that the crosslinked structure could be clarified from design of polymer or production process.

12.3.1.7 Japanese Chemical Substances Control Law Polymer Notification Test Data Requirements Those non-biodegradable polymers which fail to gain exemptions from the toxicological and environmental tests imposed by the CSCL have to be tested in exactly the same way as a non-polymeric substance (see Chapter 10, especially Section 10.2.1 and Figure 10.1).

12.3.1.8 Japanese Industrial Safety and Hygiene Law Notification of New Polymers Certain stable, high molecular weight materials are exempt from having to undergo full ISHL notification, if the following criteria are satisfied: •

MW is > 2,000

•

the polymer is synthesised from existing (inventory listed) monomers and does not have any of the following features: - a positive electronic charge - a carbon content of < 32% - covalent bonds with elements other than S, Si, O, H, C, N - ionic bonds with metal (or metal complex) ions other than Al, K, Ca, Na or Mg - a synthetic route which involves extraction or isolation from living organs, or derived from such polymers by chemical reactions. This concept also extends to structurally similar analogues. - halogen or cyano or reactive functional groups.

319

Practical Guide to Chemical Safety Testing Reactive functional groups are defined as: isocyanic and acrylic acid groups, branched acrylic acid derivatives, epoxy groups, acid anhydrides, acid halides, aldehydes, amines, phenols, thiophenols, sulphur acid containing groups and their derivatives, aziridines, isocyanic acid derivatives, vinylsulfones, halosilanes, alkoxysilanes and lactones with three (or four)-membered rings. Polymers which fail to meet these criteria have to be tested initially in an Ames study. If this produces a positive result, a chromosome aberration test is also required, as discussed in Chapter 10.

12.3.1.9 Completion of the Notification Process The reports for the toxicity, genotoxicity and environmental studies have to be translated into Japanese. These, together with the physico-chemical results are submitted to each of the three Ministries. Under the CSCL, a fragmented type review process then begins. This involves various hearings, committee meetings and judgements until finally a clearance letter is provided. This can take six months or even longer. By comparison, the ISHL notification is completed as soon as the Ames test data is submitted. Notifiers should carefully monitor publication of the notified polymer, because until this appears in the public domain only the notifier is able to place the polymer on the market.

12.3.1.10 Conclusions on the Japanese Scheme It is both time consuming to assess and difficult to achieve reduced testing for polymers. Many polymers have to undergo the tests which are required for any non-biodegradable, non bioaccumulative, non-polymeric substances. The process involving the selection of a test sample of adequate purity, devising a suitable testing strategy, all the way through to the final submission and negotiations with the Japanese Ministries can be full of uncertainties. In order to increase the chances of success, it is essential to engage the support of a respected, qualified, resident Japanese speaking representative.

12.3.2 Australia Polymers listed on the Australian Inventory of Chemical Substances (AICS) can be placed on the market by virtue of their import or manufacture, without the need for notification. The Australian Authorities have adopted the OECD polymer definition and allow the 2% monomer exemption rule to be applied.

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Notification of Polymers Worldwide New polymers require a formal notification dossier to be prepared and submitted, before the polymer itself, or any product containing it, is placed on the market. Further information on notification of new substances in Australia is given in Chapter 11 and in the official guidance manual [5].

12.3.2.1 Polymers of Low Concern The Polymer of Low Concern (PLC) category was developed in 1993 to encourage industry to introduce less hazardous polymers into Australia. To be categorised as a polymer of low concern, the polymer must meet all the following criteria: •

Mn > 1,000. Less than 5% by mass of molecules should have a MW < 1,000 and less than 2% < 500.

•

A low charge density. The polymer should not be polycationic. Furthermore it should not be cationic or anionic in the pH range of 4.9.

•

Low residual monomer level. The polymer must not be classified due to presence of a monomer.

•

Water solubility < 1 mg/1.

•

A particle size distribution such that < 1% particles have an aerodynamic diameter of < 70 µm.

•

Stable under conditions of use.

•

Must not contain reactive functional groups which may undergo further reaction.

12.3.2.2 Notification Test Data Requirements For new polymers, various registration schemes are available. These include full and low volume notification schemes and special provisions for R&D and site limited polymers. For commercialisation at > 1 tonne per annum, there are schedules available for three categories of polymer: PLC, Mn > 1,000 and Mn < 1,000. A brief outline of the testing requirements for all of these is given in Table 12.3.

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Safety data sheet Practical Guide to Chemical Safety Testing

Table 12.3 Australia: Testing Requirements for Notification of Polymers POLYMERS OF LOW CONCERN Chemical Name CAS Number Justification opposite PLC criteria Molecular Formula Structural Formula Spectral Data and Identification Number Average Molecular Weight Weight Percentage of Polymer Species with MW < 1,000 and MW < 500 Charge Density Polymer Constituents Residual Monomers and Impurities Water Solubility Particle Size Distribution Polymer Stability Reactive Functional Groups Intended Use Appearance of the Polymer

Manufacture or Import Volume Site of Manufacture or Reformulation Melting Point Density Flammability Auto-ignition Temperature Explosive Properties Reactivity Occupational Health Environmental Impact Public Health Label Material Safety Data Sheet (MSDS)

POLYMERS OF Mn > 1,000 Identity of the Substance Chemical Name Use Appearance CAS Registry Number Molecular Formula Structural Formula Molecular Weight Spectral Data and Identification Purity Identity of Impurities Melting Point/Boiling Point Specific Gravity, Density Vapour Pressure Water Solubility Hydrolysis as a Function of pH Partition Coefficient Adsorption, Desorption Dissociation Constant (pKa) Particle Size Flash Point Flammability Auto Ignition

322

Explosive Properties Oxidising Properties Reactivity Exposure Data; Health Monitoring; Risk Assessment Label Safety Data Sheet Emergency Procedures Identity and Composition of the Polymer Manufacturing Information: Weight Percentage of each Monomer and Reactant Number Average Molecular Weight Content of Residual Monomer(s) and Reactant(s) Molecular Weight Distribution Content of Polymer of Molecular Weight < 1,000 and 500 Details of: Decomposition/Degradation/Depolymerisation products Information on Loss of Monomers Additives, Impurities

Notification of Polymers Worldwide

Table 12.3 Continued POLYMERS OF Mn < 1,000 Data requirements as for polymers of Mn > 1,000 plus: Acute Oral Toxicity Acute Dermal Toxicity *Acute Inhalation Toxicity Skin Irritation Eye Irritation Skin Sensitisation Ames * IVC and/or in vitro Chromatid Exchange Assay

Dominant Lethal (or in vivo study, e.g., mouse micronucleus) 28-Day Sub-acute study in the Rat Acute Fish Toxicity Daphnia Acute Toxicity and Reproduction Algal Growth Inhibition Ready Biodegradation Evaluation of Bioaccumulation Potential

Footnotes: * Only relevant for gases, volatile substances or aerosol/small particulate molecules The above represents an overview. Please refer to the relevant legislation for precise details [6]

12.3.2.3 Review Period Following submission, a review period of 50 days is imposed on PLC and 90 days for standard or reduced notification. Notifiers should carefully monitor publication of the notified polymer on the AICS, because until this appears in the public domain only the notifier is able to place the polymer on the market.

12.3.2.4 Future Changes In response to infrequent use of the PLC facility, the Australian Authorities, in consultation with the chemical industry and other government agencies, have proposed that the criteria for defining PLC be revised. The revision has been based in part on the current polymer criteria of the US EPA. A Focus Group composed of experts in polymer science has assisted in the scientific aspects of development of the proposed criteria, which should make PLC status easier to attain.

12.3.2.5 Conclusions on the Australian Scheme Reduced testing schemes are available for just about every situation. For full commercial situations, significant toxicological and environmental testing only applies for low

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Practical Guide to Chemical Safety Testing molecular weight materials. The proposed new criteria should make PLC status easier to achieve in the future.

12.3.3 New Zealand The Toxic Substance Act (TSA) issued in 1983 by the Department of Health, has now been replaced by the Hazardous Substance & New Organism Bill (HSNO) [6]. HSNO, administered by the New Zealand Environmental Risk Management Authority (ERMA) was implemented on 2 July 2001. Therefore so far there is insufficient experience and information to provide a comprehensive review of polymers under the HSNO. Nevertheless, a few key points to mention are that: •

Substances previously registered under TSA automatically become HSNO approved

•

The HSNO definition of ‘substance’ includes, polymers, products and mixtures

•

Only hazardous substances need to be registered

•

Data requirements for HSNO approval are in line with the OECD recommended minimum package.

With respect to polymers, early indications are that there are no distinctions made between substances, polymers, or mixtures which contain them. Indeed ERMA requests data on the hazardous products being commercialised, rather than discrete substances or polymers, that is unless the product being placed on the market is a discrete substance such as a homo- or co-polymer. Under the HSNO Act, companies wishing to manufacture or import a hazardous substance into New Zealand for the first time need to obtain an approval for the substance. However, there are a number of circumstances under which an approval is not required: •

substance is non-hazardous

•

substance is exempt or excluded from the provisions of the HSNO Act

•

substance was legally existing in New Zealand before 2 July 2001.

Substances that are imported into, or manufactured in New Zealand in small amounts for purposes of Research & Development are exempt from the HSNO Act, providing they are made or kept in laboratories that meet the requirements of the Hazardous Substances (Exempt Laboratories) Regulations [7].

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Notification of Polymers Worldwide

12.3.3.1 Classification of Hazard If the substance does not fit into any of the exclusion or exemption categories, prospective notifiers need to formally determine whether or not it is hazardous, which determines the need to submit an application. In HSNO terms, a substance is considered hazardous if it triggers any one of the threshold levels defined in the Hazardous Substances (Minimum Degrees of Hazard) Regulations [8] for any of the hazardous properties – explosiveness, flammability, oxidising capacity, corrosiveness, toxicity or ecotoxicity. The criteria for hazard classification can be found on the ERMA web site which is given at the end of this section. Alternatively, an application can be made to the authority to determine if the substance is hazardous. For this the following information is required: •

Substance identity, chemical name

•

Full composition, purity profile

•

Chemical name

•

CAS Registry Number

•

Chemical and physical properties

•

Hazard information

For polymeric mixtures, e.g., polymer emulsions, information on the mixture is preferred, but if not available information on the components will suffice. Further details on the developing scheme and how to make an application have already been published and are regularly being updated on the ERMA web site (www.ermanz.govt.nz).

12.3.4 Korea Polymers feature on the Korea Existing Chemicals Inventory (ECI) and can still be nominated if they were in commerce (by virtue of their importation or manufacture) prior to 8 February 1991. When searching the inventory, monomers or reactants present in combined form at < 2% can be ignored. Those not listed have to be notified to both the Ministry of Environment (MoE) and the Ministry of Labour (MoL). Polymers, like non-polymeric substances, are exempt from notification if they are placed on the market in quantities of < 100 kg per annum, or for R&D in justifiable amounts.

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Practical Guide to Chemical Safety Testing Chemical control in Korea is covered in Chapter 11. The key legislation is published in English [9].

12.3.4.1 Notification Schemes for Polymers In Korea there are four schemes for notifying new chemicals. Besides the standard Korean base set, the other three are simplified schemes. Two are for materials which are listed on two or more foreign inventories, one being specific to non-polymeric substances and the other applies to both polymers and non-polymers. Irrespective of the class of new substance (polymer or non-polymer) the mandatory information contained in Table 12.4 is required. The MOE has adopted the OECD polymer definition. Any polymer not listed on the inventory and not generally exempt has to be notified prior to being placed on the market unless exempt according to the National Institute of Environmental Research (NIER) Public Notice No. 1998-34: amended Nov. 12 1998 [9]. The exemption criteria are as follows: •

Block copolymer in which all blocks are listed in the ECL

•

Graft polymer in which the stem and all branches are listed in the ECL

•

Polymers which contain new monomers at ≤ 2%

As previously mentioned there are two schemes for notifying new polymers in Korea. The polymer may be notified with Polymer Specific Data (Table 12.4). In essence, there are no toxicity or ecotoxicity tests required. However, any results which indicate bioavailability could result in the request for any of the standard notification tests (Chapter 11). Instead of the polymer specific data, in principle notifiers are able to use one of the simplified schemes also outlined in Table 12.4, i.e., the one based on: •

Acute oral toxicity and an Ames test (or in vitro chromosome aberration test), or

•

Ready biodegradation test

If this approach is used then the mandatory information also has to be included. The authorities have also confirmed a preference to receive data on number average molecular weight and now appear to insist on receiving data on the stability in acid and alkaline media.

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Table 12.4 Korea: Data Requirements for Simplified Notification of Polymers Mandatory data required for all substances (polymers and non-polymers) using detailed or simplified schemes Information about the chemical identity: chemical name, structure, CAS Number (if available) and empirical formula (when appropriate). Proposed uses and use category. Estimate of amounts to be placed on the market per annum for the next three years. Purity profile. Physico-chemical properties: nature of the substance, melting/boiling point, vapour pressure, water solubility, solubility in common organic solvents, octanol-water partition coefficient. Release pathway and consequential environmental impact Polymer Specific Tests Number average molecular weight Molecular weight distribution GPC data Monomer identity Residual monomer content % monomer ratio % of molecular weight species < 1,000 Stability in acid and alkaline conditions (OECD 111, or under certain conditions OECD 120) Simplified Notification Specific Test The mandatory identification data plus Ready biodegradation test or Ames test and acute oral toxicity study or In vitro chromosome aberration test and acute oral toxicity study

12.3.4.2 Review Period For all the schemes the formal review period is 60 days, but in practice this is often reduced if there are no issues.

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12.3.4.3 Conclusions on the Korean Scheme For the majority of polymers only basic physico-chemical data is required. Even in those instances where the Ministries request more data, the requirements are unlikely to be more than is required for a good quality safety data sheet.

12.3.5 Philippines Polymers are defined in the Philippines Republic Act, RA 6969 Law as follows: ‘Substances consisting of large molecules built up by the sequence of repetition of one or more types of monomer units in a linear or branched fashion. Such substances comprise a simple weight majority of molecules containing at least three monomer units that are covalently bound to at least one other monomer unit or reactant. Such molecules must be distributed over a range of molecular weights wherein differences in molecular weight are primarily attributable to differences in the number of monomer units. The reactants (monomers, crosslinking agents, chain transfer agents and post-polymerisation reactants, i.e., neutralising agents)) are included in the definition of the polymers, if they are present at levels equal to or above two percent by weight of the polymer.’ Any substance which may appear to be polymeric in nature, but which does not meet the formal definition, is treated as a non-polymer. Polymers are included in the Philippines Inventory of Chemicals and Chemical Substances (PICCS). Notification is required before a new polymer is placed on the market (manufacture or importation). There are no special tests for the notification of polymers, i.e., new polymers have to be tested in the same way as non-polymeric substances. Therefore if they do not fulfil the criteria for an abbreviated notification, the process can be quite onerous compared to other regulatory controlled regions. It is therefore important for notifiers to be aware of the exemptions that are available from the Pre-manufacture Pre-importation Notification (PMPIN) scheme: •

Polymers listed on PICCS. All monomers and other reactants including crosslinking, chain transfer agents and post polymerisation reactants added at quantities of less than 2% (by weight) can be ignored when searching the inventory.

•

Polymer for which all monomers present at ≥ 2% are listed on PICCS.

•

New polymers in which 2 or more of the top (top by weight) monomers are included in the definition of another polymer listed on PICCS.

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Notification of Polymers Worldwide New polymers can be supplied prior to notification in quantities of < 1,000 kg/annum for purposes of R&D and there is also an exemption from both Abbreviated and Detailed PMPIN for new substances commercially placed on the market in quantities of < 1,000 kg/annum. A declaration of intent in the form of a letter containing details such as the chemical name, CAS Number, Safety Data Sheet and the volumes planned has to be submitted, prior to supply. Polymers which are existing in other regulatory controlled regions can be registered via the Abbreviated Notification Scheme.

12.3.5.1 Abbreviated Pre-Manufacturing Pre-Importation Notification This scheme allows substances which are approved (i.e., listed on the inventory, or notified in one other regulatory region) to be notified by a simplified process which demands no mandatory data, but requires notifiers to complete the Abbreviated Pre-manufacturing Pre-importation Notification (PMPIN) form and summarise the available data. A safety data sheet is a suitable format for the summary, which should include the source of data (e.g., testing the actual substance, a formulation, SAR, QSAR) and the protocol adopted.

12.3.5.2 Standard Pre-Manufacturing Pre-Importation Notification Although polymer specific testing schemes have been discussed, none have been published to date. Consequently, notifiable polymers are subject to the same testing schedules as non-polymers. These requirements are outlined in Table 12.5.

12.3.5.3 Review Period The review period for all substances whether being assessed under the Detailed or Abbreviated scheme is 90 days. It is understood that a notice of commencement to import or manufacture has to be submitted prior to commercial activity. An Interim Certificate for Commercial Import/Manufacture can be obtained for reviews which cannot be completed within the 90 day period.

12.3.5.4 Conclusions on the Philippines Scheme The test data requirements, as currently stipulated are not too onerous, but possibly inappropriate for certain types of polymers. Further developments are awaited.

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Table 12.5 Philippines: Notification Data Requirements for Polymers and Non-Polymers 1.

Chemical name

2.

Trade name(s)

3.

Chemical structure and molecular formula

4.

CAS number

5.

RTECS number, if available

6.

UN number, if available

7.

UN class, subsidiary risk category if applicable

8.

Physical characteristics (a) boiling point (b) melting point (c) specific gravity (d) vapour pressure (e) appearance (f) odour (g) purity (h) octanol-water partition coefficient

9.

Chemical properties (a) water solubility (b) solubility in organic solvents

10.

Toxicological data (a) measured lethal dose (median) in two species* (b) measured lethal concentration (median) in two species* (c) acute skin irritation (d) acute eye irritation (e) short-term sublethal toxicity test

11.

Recommended Time-Weighted Average (TWA)

12.

Flash point (closed cup)

13.

Explosivity: Upper Explosion Limit (UEL) and Lower Explosion Limit (LEL)

14.

Stability and incompatibilities

15.

Carcinogenic, teratogenic, mutagenic potential

16

Name and address of nominating person

17

Anticipated volume per annum to be placed on the market

Footnote: *One species in practice may suffice. The above represents an overview

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12.3.6 China The draft People’s Republic of China (PRC) Inventory was re-opened until April 2003 for nominations of non-listed chemicals, including polymers which were produced and imported into PRC before 30 April 2003. For polymers, processes are being requested to nominate monomers as well as polymers. The polymer name can include all monomers or reactants or only those contained ≥ 2%. Although no plans for future notification have been announced the guidelines provide some clues.

12.4 Europe

12.4.1 EU The EU requirements are largely controlled by the decisions made under the Sixth Amendment of the Dangerous Substances Directive (79/831/EEC) [10] and the subsequent changes made for the Seventh Amendment (92/32/EEC) [11]. Under the Sixth Amendment, polymers were not formally defined and during the compilation of the EU Existing Substances Inventory (EINECS) [12], polymeric materials were exempt from reporting. Hence, there are no polymers listed on EINECS. At that time, in order to place a polymer on the market (by virtue of supply or importation) without notification, its monomers contained at ≥2% had to be listed on EINECS. The chemical incorporation of nonEINECS listed starting substances (even if they had been fully notified) into the polymer at a total level of ≥2% (by weight) requires the polymer to be notified in the same way as any non-polymeric industrial chemical. In practice, suppliers were able to obtain exemptions from some of the tests as they were not scientifically practical for polymers. When the Seventh Amendment to the Dangerous Substances Directive came into force, the OECD definition was adopted and the 2% rule extended to include reactants as well as monomers. Presently, existing (non-notifiable) polymers not only have to conform to the OECD definition, but they still need to be produced from EINECS listed monomers and reactants, if the latter are incorporated at ≥2%. Unlike other inventories, EINECS is static and monomers which have been notified are not added. Instead, notified substances are added to the European List of Notified Chemical Substances (ELINCS) [13]. When considering the monomers to ascertain whether or not a polymer needs to be notified ELINCS does not equal EINECS, i.e., if a polymer contains ≥2% of an ELINCS listed monomer, or reactant the polymer is notifiable. When the wealth of data which generally accompanies

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Practical Guide to Chemical Safety Testing a fully notified substances is compared to a substance listed on EINECS, there appears to be little justification that polymers, which are derived from fully notified monomers (present at ≥ 2% in combined form) do not qualify for exemption from notification, i.e., in the same way as those derived from EINECS listed materials. The inclusion of the polymer definition into Directive 92/32/EEC meant that there were materials in commerce that were no longer considered to be polymers, but which were not listed on the EINECS inventory. To avoid the need for retrospective notifications, these so termed ‘no longer polymers’ were exempt from notification, providing they were on the EU market from September 18, 1981 to October 31, 1993 (date of implementation of the Seventh Amendment) and all ‘monomers’/reactants used in their synthesis at ≥2% were listed on EINECS. Industry was given the opportunity to submit details of its ‘no longer polymers’ to an EU ‘No Longer Polymers List’ [14], which effectively has the same status as EINECS. Many companies, however, chose to simply make their own assessments, rather than make them publicly available. The Seventh Amendment also brought about four new concepts: standard test package polymers (STP), reduced test package polymers (RTP), the grouping approach to testing and the polymer specific testing requirements of Annex VIID of the Seventh Amendment [15]. The latter being the routine type data which helps differentiate between polymers, non-polymers and RTP/STP categories. The distinction between RTP and STP is extremely important as it defines the testing requirements for new polymers and determines whether or not expensive, time-consuming, toxicological and ecotoxicological tests need to be performed. Extensive data guidance on these parameters are covered in the Polymer Guidance Document for the Implementation of Annex VIID [16], which is available from all the Competent Authorities.

12.4.1.1 Reduced Test Package Polymer Notification There are two routes by which a polymer can obtain RTP status. In the traditional method, the basic requirement is for new polymers to meet all of the following criteria: •

High number average molecular weight

•

Water extractivity < 10 mg/l per 10 g sample

•

Low molecular weight species with molecular weight of < 1,000 to be < 1%.

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Notification of Polymers Worldwide Although ‘high’ number average molecular weight is not legally defined, the third criterion requires it to be significantly higher than 1,000, and 10,000 is generally regarded to be the qualifying figure. The third requirement is not obligatory for polymers being placed on the EU market in quantities of < 1 tonne per annum. A flow diagram explaining this process is outlined in Figure 12.3.

Are all polymer reactants present at ≥ 2% on EINECS?

Does the polymer meet all of the following criteria of Annex VIID, para.C.2 [16] Mn≥ 1,000 - 10,000 % of oligomers with Mw < 1000 is ≤ 1% water extractivity < 10 mg/l

Figure 12.3 EU: Polymer Notification Decision for Status as Reduced or Standard Test Package

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Practical Guide to Chemical Safety Testing The Polymer Guidance Document [16] provides an alternative route to attain RTP status. This is shown in Figure 12.4. In summary, this requires the polymer to show suitable levels (but not all) of the following properties: •

Monomers/reactants not classified as very toxic, toxic or corrosive. The polymer must also possess an aquatic toxicity EC50 > 1 mg/l.

•

Monomers/reactants not classified as harmful, irritant or not having a positive mutagenicity test and possessing aquatic toxicity EC50 1 to 10 mg/l.

•

Polymers not classified as skin or respiratory sensitisers, or mutagenic. The Daphnia toxicity value must also be > 10 mg/l.

•

Water extractivity < 10 mg/l.

•

Polymer to contain < 10% species with molecular weight < 1,000. This is compared to 1% in the traditional RTP scheme.

A couple of features of the scheme which are worthy of special mention are that the low molecular weight cut off point of 10% is compared to 1% in the traditional RTP scheme and a polymer can attain RTP status and seemingly meet RTP without having to be examined in the water extractivity test. New polymers which meet RTP status by either method, may ultimately be disqualified from reduced testing, if they possess any of the following features: •

Presence of reactive functional groups (as defined in the US EPA Polymer Exemption Guidance Manual [2])

•

Cationic or anionic density charge

•

Respirable, due to aerodynamic size

•

Unstable.

12.4.1.2 Grouping of Polymers The authorities recognise that industry may often wish to manufacture a range of polymers from the same monomers, i.e., varying molecular weights, or relative proportion of the monomers either within a narrow, or wide range. The narrow range leads to the ‘polymer substance’ concept, requiring one notification, whereas the wider range facilitates the

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Notification of Polymers Worldwide

Footnotes: * RTP route cannot be applied to polymers containing certain reactive groups as specified in Annex 2 to the polymer guidance document [16] nor to respirable/high molecular weight polymeric dusts. ** The polymer qualifies for RTP testing providing the Mn > 1,000 and it can be demonstrated that, if swallowed, the polymer would only decompose to these non-classified starting substances or their equivalent. *** The usual caveats as to possible post-notification testing [15] apply. **** Tests for STP may be omitted if all the monomers and substances in the polymer do not satisfy or are not thought likely to satisfy the criteria for EU classification for a particular end point. The evidence may be that derived from reliable literature test data on close structural analogues or in-house worker protection tests. The notifier has to provide this information.

Figure 12.4 EU: polymer notification, alternate routes to Reduced Test Package Status

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Practical Guide to Chemical Safety Testing ‘family approach’. A notification at both ends of the family range, one a formal notification, the other usually a nominal notification suffices to register the entire group.

12.4.1.3 Notification Test Data Requirements RTP qualifying polymers are notified by conducting the appropriate Annex VIID tests. The final selection of tests from the RTP scheme is based on annual amounts being placed on the market and their accumulative total. See Table 12.6 for a brief summary. On the other hand, STP polymers have to be tested by employing both VIID and the appropriate non-polymer tests given in Annex VII of the Seventh Amendment [11]. The selection of the relevant Annex VII schedule again depends on the annual and accumulative amounts of the polymer being placed on the market. For the majority of polymers, the VIIA base set will be the most likely outcome. An outline of the Annex VIID STP and the Annex VIIA, B and C tests are shown in Table 12.7. When the annual amount of the polymer placed on the market reaches pre-defined annual or accumulative quantities, the need to carry out additional tests has to be considered. The annual tonnage triggers in question, together with the accumulation levels in brackets are as follows: 10 tonnes (50 tonnes) or 100 tonnes (500 tonnes) for Level 1 and 1,000 tonnes (5,000 tonnes) for Level 2. RTP polymers are evaluated using a cascade system which generally delays Annex VIIA Base Set testing until 100 tonnes (Level 1) is reached. The standard Level 1 and 2 testing of Annex VIII of the Seventh Amendment (see Chapter 9) applies to STP polymers.

12.4.1.4 Review Period The review period of a full notification dossier to enable supplies of ≥1 tonne is 60 days.

12.4.1.5 Supplies for Research and Development and Process Development Supplies of new polymers for R&D are limited to 100 kg/annum per supplier. Supplies for Process Orientated R&D (PORD) can be made on a case by case basis. A formal application has to be made and data is often required.

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Table 12.6 Testing requirements for EU Notification of Reduced Test Package Polymers Annex VIID tests

Annual sales*

≥ 1 tonne

< 1 tonne

Chemical Name

✓

✓

Molecular and Structural formula

✓

✓

Number Average Molecular Weight

✓

✓

Molecular Weight Distribution

✓

✓

Identity and Concentration of Monomers

✓

✓

Identity and Frequency of Reactive Functional Groups

✓

✓

Composition of Polymer (purity, residual monomers, impurities)

✓

✓

Melting Range

✓

✓

Density

✓

x

Water Extractivity

✓

✓

Flammability

✓

✓

Explosive Properties

✓

x

Auto Flammability

✓

x

Particle Size

✓

x

Thermal Stability

✓

x

Extractivity - Water pH 2 & 9 - Cyclohexane

✓ ✓

x x

Footnotes: ✓ = test required x = test not required * There are also corresponding cumulative supply triggers of 5 times the annual limit. The above represents an overview. Please refer to the relevant legislation for precise details, i.e., Annex VIIB of the Seventh Amendment [15]

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Table 12.7 Testing Requirements for EU Standard Test Package Polymer Notification Annual sales *

≥ 1 tonne

≥ 100 kg to < 1 tonne

< 100 kg

Number Average Molecular Weight

✓

✓

✓

Molecular Weight Distribution

✓

✓

✓

Identity and Concentration of Monomers

✓

✓

✓

Identity of end groups/Identity & Frequency of reactive functional groups

✓

✓

✓

Identity and % of non-reacted monomers

✓

✓

✓

Water extractivity

✓

✓

x

Light stability *

✓

x

x

Long-term extractivity (leachate test) *

✓

x

x

Characterisation / identification / purity assessment spectral data

✓

✓

✓

Melting point

✓

✓

x

Boiling point

✓

✓

x

Relative density

✓

x

x

Surface tension

✓

x

x

Water solubility

✓

✓

x

Partition coefficient

✓

✓

x

Particle size distribution

✓

x

x

Hydrolysis as a function of pH

✓

x

x

Vapour pressure

✓

x

x

Flash point

✓

✓

✓

Flammability

✓

✓

✓

SPECIFIC POLYMER TESTS

PHYSICO-CHEMICAL STUDIES

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Notification of Polymers Worldwide

Table 12.7 Continued Annual sales * ≥ 1 tonne

≥ 100 kg to < 1 tonne

< 100 kg

PHYSICO-CHEMICAL STUDIES Continued Explosivity

✓

x

x

Oxidising properties

✓

x

x

Self ignition temperature

✓

x

x

Acute oral

✓

(Choose)

(Choose)

Acute dermal or inhalation

✓

( one )

( one

Skin irritation

✓

✓

x

Eye irritation

✓

✓

x

Skin sensitisation

✓

✓

x

28-day subacute toxicity

✓

x

x

Ames

✓

✓

x

In vitro cytogenetics/mouse lymphoma

✓

x

x

Toxicokinetic assessment

✓

x

x

Acute toxicity to fish

✓

x )**

x

Acute toxicity to daphnia

✓

x)

x

Algae growth inhibition

✓

x

x

Ready biodegradation

✓

✓

x

Absorption desorption test

✓

x

x

TOXICOLOGICAL STUDIES

)

ENVIRONMENTAL STUDIES

Footnotes: ✓ = Required x = Test not usually required *There are also corresponding cumulative supply triggers of 5 times the annual limit ** One of the acute ecotox end points may be requested post base set The above represents an overview. Please refer to the relevant legislation for precise details, i.e., the Seventh Amendment Annex VIIB [15] and Annex VIIA, B and C [11]

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12.4.1.6 Future Considerations During 2000 and 2001, industry has been working with a European Commission appointed Polymer Working Group to develop additional flexible, scientifically sound procedures for testing STP polymers. Although it is too early to predict whether or not these will be adopted, or even provide ideas for dealing with polymers in the new EU Chemicals Policy, one suggestion has been named the Modified Test Procedure (MTP), which seeks to adopt an EU Dangerous Preparations Directive (1999/45/EC) [17] approach to provide a worst case classification of the polymer based on the content of low molecular weight species of MW < 1,000. The idea is to test the STP polymer against these and any other end points of concern, as an alternative to the entire Annex VIIA and VIID tests as portrayed in Table 12.7. The success of such an approach depends on notifiers having sufficient good quality data on the monomers and reactants which form the polymer structure. This requirement will be supported by the new EU Chemicals Policy in which all chemicals produced and imported in quantities of > 1,000 kg will have to be registered, and be accompanied by an approved level of data. It must be emphasised that MTP is being suggested as an alternative to traditional STP which may be a better option if sufficient data is not available on the monomers or reactants.

12.4.1.7 Conclusion Although the ‘EINECS listed monomers’ concept allows polymers to be placed on the EU market without need for notification, it is disappointing that fully tested and notified monomers do not have the same status. Because of this, innovation for the polymer industry often leads to notification of both the monomers and resulting polymers. The family approach also provides opportunities for some flexibility. However, another crucial element that cannot be ignored is that polymers which narrowly fail to meet the RTP criteria may have to undergo both VIIA and VIID tests, i.e., they are tested more comprehensively than non-polymeric substances. Despite recent attempts to make the RTP less onerous, it is still currently difficult to meet, although some encouraging work, e.g., the MTP concept is ongoing, which could provide future alternatives.

12.4.2 Switzerland As discussed in Chapter 11, any new substance being supplied in Switzerland has to be notified under the 1986 Ordinance on Environmentally Hazardous Substances (OEHS) [18] to the Swiss Agency for Environment, Forestry and Landscape (SAEFL) before being marketed. The aim of SAEFL is to ensure that only new substances, which present no

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Notification of Polymers Worldwide threat either to the environment, or indirectly to humans are allowed to be placed on the market. A separate registration procedure also exists to the Federal Office of Public Health (FOPH), for the assessment of user and health protection. All substances and products are affected by this procedure, i.e., no distinction is made between new and existing substances. Although not a member of the EU, some of the SAEFL notification procedures are very similar to those adopted by the EU. EINECS for example, is used as the basis for distinguishing between new and existing substances, although substances contained in the 1985 Second Edition of the FOPH Toxic Substances List 1 and those supplied between 1975 and 1984 in quantities of greater than 500 kg, are also defined as non-notifiable existing substances. For polymers: •

Switzerland uses both its own and the OECD polymer definition

•

OECD conforming polymers derived from EINECS listed monomers and reactants are exempt from SAEFL notification, as are those made up exclusively from carbon, hydrogen, oxygen and nitrogen.

•

Monomers present in combined form at < 2% can be ignored when assessing whether a polymer is existing or new.

There are differences however between the Swiss and EU schemes. The Swiss Authorities offer no reduced testing schedules for new substances being on the market in quantities of < 1 tonne per annum. Also, the same tests are required for the notification of both new polymers and non-polymer substances. The tests required are very similar to those in the EU Seventh Amendment Annex VIIA base set [11], although exemptions from certain tests can be negotiated in certain cases (e.g., low market volumes and exposure). Other specific polymer exemptions are outlined in Article 20 of the regulation. The information required to enable the Agency to assess the potential environmental impact of new substances is outlined in Table 12.8. No formal review period is imposed, however a delay may occur if issues arise.

12.5 Overall Comparison of the Nine Polymer Notification Schemes It is fairly difficult to compare nine current schemes which are quite different from each other, however, for industry, a difficult new substance notification scheme is one which

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Table 12.8 Switzerland: Swiss Agency of Environment, Forestry and Landscape Notification Requirements Chemical Name Structural Formula CAS No. Empirical Formula Spectra and analytical method Manufacture and Use Water Solubility Melting Point Boiling Point Density Solubility in Organic Solvents Octanol:water partition coefficient Dissociation Constant Surface Tension Biodegradation Hydrolysis Test Fish Toxicity Daphnia immobilisation and reproduction Mutagenicity, bacterial and non-bacterial tests Footnote: The above represents an overview. Please refer to the relevant legislation for precise details [18]

delays marketing for six months or more, requires test data above and beyond safety data sheet requirements and which consequently requires a significant additional investment compared to existing substances. In order to achieve unrestricted sales of a new polymer, a personal view is as follows:

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Notification of Polymers Worldwide •

The EU is the most demanding, in terms of data requirements, if RTP status cannot be attained and the family concept inappropriate. In this respect, the EU scheme is followed by Australia, Canada, Japan and Switzerland. (Not listed here in any specific order.)

•

The US scheme is the most unforgiving if you get it wrong.

•

Japan, in the absence of an exemption, is the most difficult scheme to successfully complete and without doubt takes the longest.

•

At the present time, Korea is the easiest scheme to consistently achieve successful polymer notification.

References 1.

USA Toxic Substances Control Act, Public Law 94 – 469, 1976.

2.

Polymer Exemption Guidance Manual, US EPA Office of Pollution Prevention and Toxics, Washington, USA, reference 744-B-97-001, June 1997.

3.

Guidelines for the Notification and Testing of New Substances: Chemicals and Polymers Persuant to the New Substances Notification Regulations of the Canadian Environmental Protection Act, Environment Canada and Health and Welfare Canada, Ottowa, Canada, March 1993.

4.

Handbook of Existing and New Chemical Substances, Japan Chemical Daily, Tokyo, Japan, latest edition.

5.

Handbook for Notifiers A Users Guide for the Notification and Assessment of Industrial Chemicals in Australia, National Industrial Chemicals and Assessment Scheme, Sydney, Australia, latest edition.

6.

User Guide to the HSNO Control Regulations, Environmental Risk Management Authority New Zealand, Wellington, New Zealand, November 2001.

7.

Hazardous Substances (Exempt Laboratories) Regulations 2001, 2001/115, 2001.

8.

Hazardous Substances (Minimum Degrees of Hazard) Regulations 2001, 2001/ 112, 2001.

9.

Laws and Regulations on Chemicals in Korea, Second Edition, Kim and Chang, Seoul, Korea, June 1999.

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Practical Guide to Chemical Safety Testing 10. Council Directive 79/831/EC of 18 September 1979, Official Journal of the European Communities, 15:10:79, L259, 10. 11. Council Directive 92/32/EEC of 30 April 1992, Official Journal of the European Communities, 5:6:92, L154, 1. 12. EC Communication 90/C 146 A/01 European Inventory of Existing Commercial Chemical Substances, Official Journal of the European Communities, 15:6:90, C146A, 1. 13. Commission Communication 2000/C 72/01 Fifth Publication of the European List of Notified Chemical Substances, Official Journal of the European Communities, 11:3:2000, C72, 1. 14. No Longer Polymer List, European Commission, Brussels, Belgium, September 1996. 15. Commission Directive 93/105/EC of 25 November 1993, Official Journal of the European Communities, 30:11:98 L294, 21. 16. Manual of Decisions for Implementation of the Sixth and Seventh Amendments of Directive 67/548/EEC on Dangerous Substances (Directives 79/831/EEC and 92/32/EEC), European Commission, 23:1:02, NOTIF/3/2001, 70. 17. Directive 1999/45/EC of 31 May 1999, Official Journal of the European Communities, 30:7:99, L200, 1. 18. Ordinance relating to Environmentally Hazardous Substances, SR814.013, 9 June 1986, revised version.

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Medical Device Regulation

13

Medical Device Regulation Sandra Costigan and Jeremy Tinkler

13.1 Introduction Medical devices range from crutches, stethoscopes, and X-ray machines, via sutures and adhesive dressings to highly complicated, electronic implants. This wide variety of products means that the regulatory systems set up to deal with these products, have to be flexible to allow for both the existing variety and future developments. On top of this, regulation for medical devices has in many cases developed later than that for medicinal products or indeed, is still being developed in some regions. This provides a greater opportunity for international harmonisation, both within geographic regions (viz. European Economic Area (EEA)) and globally. Many regions adopt the use of international standards, a selection of these are listed in Table 13.1, and all incorporate risk evaluations in the regulatory approval processes, which allow for less stringent regulation for lower risk devices. In Europe the risk evaluation is the responsibility of the manufacturer, rather than the regulatory authority.

13.2 European Economic Area

13.2.1 Background The European Medical Devices Directive (93/42/EEC) defines a medical device as: “any instrument, apparatus, appliance, material or other article, whether used alone or in combination, including the software necessary for its proper application, intended by the manufacturer to be used on human beings for the purpose of: •

diagnosis, prevention, monitoring, treatment or alleviation of disease,

•

diagnosis, monitoring, treatment, or alleviation of or compensation for an injury or handicap,

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Table 13.1 Examples of international standards for medical devices Standard

Year

Title

ISO 10993-1

1997

Biological evaluation of medical devices - Part 1: Evaluation and testing

ISO 10993-2

1992

Biological evaluation of medical devices - Part 2: Animal welfare requirements

ISO 10993-3

1992

Biological evaluation of medical devices - Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity

ISO 10993-4

2002

Biological evaluation of medical devices - Part 4: Selection of tests for interactions with blood

ISO 10993-5

1999

Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity

ISO 10993-6

1994

Biological evaluation of medical devices - Part 6: Tests for local effects after implantation

ISO 10993-7

1995

Biological evaluation of medical devices - Part 7: Ethylene oxide sterilization residuals

ISO 10993-8

2000

Biological evaluation of medical devices - Part 8: Selection and qualification of reference materials for biological tests

ISO 10993-9

1999

Biological evaluation of medical devices - Part 9: Framework for identification and quantification of potential degradation products

ISO 10993-10

2002

Biological evaluation of medical devices - Part 10: Tests for irritation and delayed-type hypersensitivity

ISO 10993-11

1993

Biological evaluation of medical devices - Part 11: Tests for systemic toxicity

ISO 10993-12

2002

Biological evaluation of medical devices - Part 12: Sample preparation and reference materials

ISO 10993-13

1998

Biological evaluation of medical devices - Part 13: Identification and quantification of degradation products from polymeric medical devices

ISO 10993-14

2001

Biological evaluation of medical devices - Part 14: Identification and quantification of degradation products from ceramics

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Table 13.1 Continued Standard

Year

Title

ISO 10993-15 2000

Biological evaluation of medical devices – Part 15: Identification and quantification of degradation products from metals and alloys

ISO 10993-16 1997

Biological evaluation of medical devices – Part 16: Toxicokinetic study design for degradation products and leachables

ISO 10993-17 2002

Biological evaluation of medical devices – Part 17: Establishment of allowable limits for leachable substances

ISO 14155-1

2003

Clinical investigation of medical devices for human subjects – Part 1: General requirements

EN 1441

1998

Medical devices – Risk analysis

ISO 14971

2001

Medical devices – Application of risk management to medical devices

ISO 16054

2002

Implants for surgery – Minimum data sets for surgical implants

International Standards in development Standard

Title

ISO/DIS 10993-18

Biological evaluation of medical devices – Part 18: Chemical characterization of materials

ISO/AWI 10993-19

Biological evaluation of medical devices – Part 19: Physicochemical, mechanical and morphological characterization

ISO/AWI TS 1099320

Biological evaluation of medical devices – Part 20: Principles and methods for immunotoxicology testing of medical devices

ISO/FDIS 14155-2

Clinical investigation of medical devices for human subjects – Part 2: Clinical investigation plans

•

investigation, replacement or modification of the anatomy or of a physiological process,

•

control of conception

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Practical Guide to Chemical Safety Testing and which does not achieve its principal intended action in or on the human body by pharmacological, immunological or metabolic means, but which may be assisted in its function by such means.” To cope with the plethora of medical devices, the EEA currently has three Directives in place. These Directives benefit manufacturers by harmonising controls within a single system and avoid them having to comply with a multitude of different, national sets of rules. The Directives work by setting out Essential Requirements that the products must meet. These requirements primarily protect and benefit patients and users. They specify that devices must not compromise the health or safety of the patient, user or any other person, and that any risks associated with the device are compatible with patient health and protection. Any side effects must be acceptable when weighed against the intended performance of a device. The first Directive came into effect from 1 January 1993 and covers all powered implants or partial implants that are left in the human body: the Active Implantable Medical Devices Directive (90/358/EEC). The full text of the Directive can be downloaded from the European Commission’s web site. The second Directive, the Medical Devices Directive (93/42/EEC) came into force on 1 January 1995. It covers most other medical devices such as, for example, tongue depressors, heart valves and wheelchairs. The transitional period, during which manufacturers could choose whether to follow the existing national controls in force as at 31 December 1994 or the requirements of the Directive as implemented by new national regulations, ended on 15 June 1998. The most recent is the In Vitro Diagnostic Medical Devices Directive (98/79/EC), which came into force on 7 June 2000, with a transitional period until 7 December 2003. It covers any medical device that is intended for use in vitro for the examination of specimens, including blood and tissue donations, derived from the human body. Examples of such devices are pregnancy kits and Hepatitis B test kits. Directives on medical devices incorporating stable derivatives of human blood or plasma (2000/70/EC and 2001/104/EC) have recently been developed and are at the time of writing being incorporated into national legislation across Europe. Manufacturers must certify that their devices comply with the relevant Essential Requirements in order to affix the standard, so-called CE-mark, which informs users that the medical device can be marketed in the EU. The final responsibility for product safety and performance thus lies with the manufacturer. Once CE-marked, the product can be placed on the market anywhere in the European Economic Area without further control, barring some specific additional national regulations. The Competent Authority acts on behalf of the government of a Member State to ensure that the requirements of the Medical Devices Directives are implemented in that particular country. The responsibilities of a Competent Authority include (but are not limited to):

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Medical Device Regulation •

enforcing the Regulations

•

resolving disputes such as those between manufacturers and Notified Bodies

•

undertaking appropriate measures to withdraw unsafe devices from, or prohibit or restrict their being placed on, the market

•

maintaining registers of Class I devices, custom-made devices and systems or procedure packs and in vitro devices (IVDs)

•

receiving notifications of clinical investigations and reviewing these ensuring public health and safety

•

designating and auditing Notified Bodies within their country

•

operating a vigilance system for the reporting of adverse incidents in that country.

It is worth emphasising the distinction between a medical device and a medicinal product (such as drugs). Medicinal products are regulated under the Medicinal Products Directive (65/65/EEC) which long predates the European Medical Devices Directive. Whether any new product is regulated under the medical devices or the medicinal products directive is dependent on the intended purpose of the product and the main mode of action. If the pharmacological, immunological or metabolic performance is auxiliary to the original device effect, the product is considered a medical device. If the drug effect is the main aim of the therapy, the product is a medicinal product. Besides complying with the Medical Devices Directive, the safety, quality and usefulness of the medicinal substance incorporated in a medical device must be verified by analogy with the methods in Directive 75/318/EEC concerning the testing of proprietary medicinal products. Devices that are used to administer medicinal products (such as syringes) are covered by the Medical Devices Directive if they are sold empty. If sold pre-filled, they are subject to the Medicinal Products Directive and do not bear the CE-marking, but in addition must comply with the relevant Essential Requirements in Annex I of the Devices Directive with respect to safety and performance related features of the device. There is a growing trend for medical devices now to incorporate drugs, e.g., in drug releasing cardiovascular stents. Obviously the criteria described above can then present ambiguity and this has led to different classifications of nominally similar products. Currently, guidance as to which Directive applies is provided upon request on a case-by-case basis by individual Competent Authorities. An EU guidance document on the matter is available [1]. The medical devices directives are ‘New Approach’ directives. Removing restrictions to free movement of products is one of the aims of the EEA. Such restrictions can only be avoided or eliminated through technical harmonization on Community level. However,

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Practical Guide to Chemical Safety Testing this harmonization was first rather slow for two reasons. Firstly, the legislation became highly technical, as it had the objective of meeting the individual requirements of each product category. Secondly, the adoption of technical harmonization directives was based on unanimity in the Council. New Approach directives were introduced as a regulatory technique to address these problems. This reduced the control of public authorities prior to a product being placed on the market, and integrated quality assurance and other modern conformity assessment techniques. In practice this means that legislative harmonization is limited to Essential Requirements which provide only a raft of general technical requirements that products placed on the Community market must meet. Harmonized European standards address the Essential Requirements in more detail, providing both objective definitions of what the necessary requirements are for particular products, and practical means for manufacturers to show that their products comply with the Essential Requirements. Once European standards are accepted as addressing specific Essential Requirements in a Directive, they are listed in the Official Journal of the European Communities and are thereby ‘harmonized’. Products manufactured in line with harmonized standards will be presumed to be in compliance with the Essential Requirements that those standards address. Often standards that have been developed at a wider international level, such as International Standards Organization standards, will be adopted as harmonized European standards. There are three types of harmonized standards: 1. Horizontal standards – covering areas common to many types of devices, such as quality management systems, risk analysis, biological safety assessments and sterilisation methods. 2. Semi-horizontal standards – covering requirements for a related family of devices, e.g., particular requirements for joint replacement implants. 3. Product standards with requirements for a specific type of medical device such as infusion pumps, syringes, etc. It is not obligatory to comply with the harmonized standards, but it is generally perceived as the easiest way to show compliance with the Essential Requirements. Alternative standards such as other international, national or in-house standards or Pharmacopoeia Requirements may also be used. Indeed, the Medical Devices Directive notes that several European Pharmacopoeia monographs may be considered equal to harmonized standards. However, such monographs are generally designed for use with products with a specific purpose in mind and well-defined circumstances of use. This does not cater for the diversity found in material applications in medical devices. Thus it can be expected that more explanation will then be needed to demonstrate that work performed according to these

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Medical Device Regulation documents is relevant in meeting the Essential Requirements. Indeed, where a harmonized standard has not been employed, the manufacturer must document the solutions adopted to demonstrate an equivalent level of safety and performance, i.e., how the Essential Requirements have been met. Lists of harmonized standards are provided on the European Commission’s web site (http://europa.eu.int/comm/index_en.htm). English language guidance on many aspects of the Directives can be obtained from the medical devices area of the European Commission web site, and the web site of the Medical Devices Agency, the UK Competent Authority (http://www.medicaldevices.gov.uk).

13.2.2 Before Marketing

13.2.2.1 The ‘Licence’ to Market - Conformity Assessment Requirements The rules on classification are set out in Annex IX of the Medical Devices Directive. Broadly speaking Class I contains most of the non-invasive devices. Class IIa contains some of the higher risk non-invasive devices and generally the short-term invasive devices. Class IIb generally contains the long-term invasive and implantable devices. Class III contains devices such as those in direct contact with the heart, the central circulatory system, or the central nervous system, those which incorporate a drug or those which are intended to have a biological effect or to be absorbed or undergo chemical change in the body. The In Vitro Diagnostic Medical Device Directive does not require a toxicological assessment and will thus not be discussed here. The Active Implantable Medical Device Directive also covers implanted passive parts of active devices such as pacemaker leads and adapters and external parts that are an essential part of the system, e.g., pacemaker programmers. In all cases, the manufacturer is responsible for ensuring that the product complies with all the relevant Essential Requirements. For Class I devices, the manufacturer self-certifies this by drawing up a declaration of conformity. In addition, for Class I devices placed on the market in a sterile condition or those with a measuring function, the manufacturer must apply to a Notified Body (see below) for certification of the aspects of manufacture relating to sterility or metrology. Then it is only necessary to register the product with the Competent Authority where the manufacturer (or Authorised Representative) has its place of business and subsequently affix the CE-mark.

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Practical Guide to Chemical Safety Testing For all other classes of medical devices, the Notified Body has to be involved in confirming that the product and/or its manufacturing processes complies with the Requirements of the Directive, i.e., perform a conformity assessment. In those cases, the Notified Body also has to confirm the manufacturer’s classification before embarking on the conformity assessment procedures. Once certification from the Notified Body has been received, the manufacturer may CE mark the product. There is no need to register the product with the Competent Authority. Notified Bodies are third-party certification organisations that have been designated by the national Competent Authority. Such a designation may be restricted to specified types of devices and/or specific methods of conformity assessment, e.g., product testing or QA assessment. It is unlikely that any single Notified Body will have sufficiently broad knowledge and expertise to assess conformity for all the medical devices the Directives cover. Manufacturers can apply to any Notified Body designated to carry out the desired conformity assessment, regardless of the country in which that Notified Body is located. Currently all Notified Bodies are located in EU countries, but a validation process is ongoing to ensure that organisations in the US achieve an equivalent to Notified Body status, and some organisations in Europe will perform US regulatory tasks. The original target date for the start of the operational phase had been December 2001 but the ‘confidence building period’ of this Mutual Recognition Agreement (MRA) has been extended by a further two years. Similar agreements are also being negotiated between the EU and Canada and Japan. An MRA already exists between the EU and Australia. The conformity assessment necessary will vary with the classification of the device, becoming more rigorous with higher Classes, intended to represent higher risk. For the highest risk (Class III devices), the Notified Body will always have to approve the design dossier of the device.

13.2.2.2 Biological Safety Requirements Several of the Essential Requirements in the Medical Devices Directive and the Active Implantable Medical Devices Directive refer to, or also apply to, the biological safety of the device [2]. They describe which aspects should be taken into account in the design and manufacture of a medical device. The main safety aim is that the device will not compromise the clinical condition or safety of the patient or user or other persons, “provided that any risks which may be associated with their use constitute acceptable risks when weighed against the benefits to the patient…”. To be able to determine what are acceptable risks, qualified personnel are needed to perform a state of the art risk benefit analysis. If such expertise is not present within the company, external advice

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Medical Device Regulation from suitably qualified sources should be sought. The Essential Requirements provide some detail as to what aspects of biological safety should be considered, namely: 1. The choice of materials used as regards toxicity (Requirement 7.1) 2. The compatibility between the materials used and biological tissues, cells and body fluids, taking account of the intended purpose of the device (also Requirement 7.1) 3. Minimisation of risk from contaminants and residuals, where particular attention must be paid to the tissues exposed and the duration and frequency of exposure (Requirement 7.2). The concept of minimisation rather than acceptable risk is used here because residuals and contaminants are expected to only bring risk and no benefits 4. Safe use of the device with the substances it will contact during its normal use (Requirement 7.3) 5. Risks from substances leaking from the device must be minimised (Requirement 7.5) 6. Risks from unintentional ingress from substances into the device must be minimised (Requirement 7.6) 7. Risks from ageing of the materials used in situations where the device cannot be maintained or calibrated (such as implants) should be minimised (Requirement 9.2). From a toxicity point of view this means that breakdown products of the materials used should be considered in the risk assessment of long-term devices. Where risks cannot be eliminated or reduced to negligible levels, appropriate protection measures should be taken. If there still remain residual risks, the user has to be informed of them. Thus there is a need for a biological risk assessment of the device. This assessment should include consideration of all toxic end points. In most cases an exposure assessment will already provide sufficient justification for limiting the number of toxic end points that need to be addressed in more detail. Similarly, consideration of the physical and chemical characteristics of the device, including the potential for leaching and degradation of substances, will further dictate where safety data might be needed. Safety data can comprise different elements such as documented safe history of use of similar devices or materials in similar applications, existing data, published literature, in vitro and in vivo data and prospective or retrospective clinical trials. Basically the Essential Requirements dictate that a scientifically sound biological safety assessment should have been made for use in a risk benefit analysis.

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Practical Guide to Chemical Safety Testing The most relevant standard giving more detailed guidance on what to consider in such a risk assessment is ISO 10993 [3]: Biological evaluation of medical devices. Part 1 of this standard explains the general principles governing the biological evaluation of medical devices. It broadly categorises devices based on the nature and duration of their contact with the body and it gives guidance, based on accepted toxicological considerations, on selecting the most appropriate tests. It includes two matrices summarising the generic advice on what toxic end points to address. These matrices are titled “….tests for consideration” and specifically include the note: “This table is a framework for the development of an assessment programme and is not a checklist”. Unfortunately it is still often used as a checklist, whereas what is needed is a qualified individual developing an evaluation programme based on the specifics of the device. As explained in the section on the general principles applying to the risk evaluation, it might well be that data is already available from other sources that allow evaluation of the hazard without the need for further tests. Similarly, individual evaluation of the device might identify a need to assess toxic end points over and above the ones listed in the table. For example, a device intended for long term implantation in contact with the reproductive organs, should be evaluated on its reproductive toxicity even though this is not indicated in the table in the current version of the standard. Other parts of the standard give guidance on: •

specific test methods to address different toxic end points

•

animal welfare requirements

•

methods to assess degradation products

•

allowable limits of ethylene oxide residues

•

standard reference materials and sample preparation

•

toxicological risk assessment to determine allowable limits of residues of a compound

•

physical and materials characterisation.

The guidance on the specific tests only discusses the test and sample methods, they do not specify pass/fail criteria. It is not possible to comply with the Essential Requirements by listing the tests done according to 10993 [3] and indicating a ‘pass’ for each test. The results of the tests should be interpreted in the context of the overall risk assessment to know whether a specific outcome indicates acceptable risk or not. This once again emphasises the need for an overall, scientifically valid risk assessment. A document that will put all the aspects addressed in the different 10993 parts into the larger context of a biological safety evaluation is currently being prepared. It will include

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Medical Device Regulation matters such as the acceptability of data for use, evaluation and interpretation of test results in terms of performance criteria and clinical relevance. There is a widespread misconception that ‘medical grade’ materials, such as polyvinyl chloride, exist. In the EU there is no regulation or standard that defines what a medical grade material would be. Presumably the original designation had been intended to mean that the material met the requirements of a specific Pharmacopoeia, or that exactly the same material was already used in marketed medical devices. Compliance of a material with a Pharmacopoeia requirement confers some reassurance insofar as that is a wellrecognised starting point.

13.2.2.3 Clinical Investigation In order to demonstrate that a new device, or a new purpose for an existing device complies with the relevant regulations, it will usually be necessary to provide clinical data. The need for clinical data will normally be established as a result of technical and safety assessments performed as part of the risk analysis (see also Chapter 7). Annex X indicates that, as a general rule, pre-market clinical data are expected in particular in the case of implantable and Class III devices. This should be read as amplifying the need for clinical data in these groups of devices; i.e., it is likely that clinical data will be required to demonstrate compliance with the Essential Requirements. Such clinical data could be either a compilation of relevant scientific literature, if appropriate with a written report containing a critical evaluation of the compilation, if enough such data exist. Otherwise it could be the results and conclusions of a specifically designed clinical investigation. A clinical investigation will probably be required in the following circumstances: •

the introduction of a completely new concept of device into clinical practice where components, features and/or methods of action, are previously unknown;

•

where an existing device is modified in such a way that it contains an important novel feature;

•

where a device incorporates materials that have previously not come into contact with the intended location in the body;

•

where an existing device, either CE-marked or not, is proposed for a new purpose or function.

Such an investigation must be designed to demonstrate the performance claimed, and that the risk:benefit ratio of the device is acceptable. A clinical investigation will only be

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Practical Guide to Chemical Safety Testing permitted if the information sought cannot be obtained by any other means, e.g., laboratory testing. If the purpose of a clinical investigation is, for example, user handling or preference studies, then it can only be performed on CE-marked devices. Methodology and ethical considerations relating to clinical investigations are set out in Annex X of the Medical Devices Directive. Additionally, the principles of such investigations are set out in the recently revised harmonized European Standard ISO 14155 (formerly EN 540) [4]. One of the requirements of a clinical investigation plan is that it must include sufficient devices and human subjects to reflect the aims of the investigation, taking into account the potential risk of the device. It is recognised that for a number of devices, for example orthopaedic implants, the majority of associated adverse incidents may not become manifest for a number of years and the clinical investigation will only demonstrate an absence of major safety problems. It is intended that long-term safety problems are identified by post-market surveillance. Requirements for reactive surveillance are set out in Article 10 of the Medical Devices Directive. Depending on the risk assessment, proactive post market studies may also be necessary to address long-term safety. A clinical investigation for CE-marking purposes must be notified in writing to the Competent Authority of each country in which the trial is to take place. If within a period of 60 days of receipt of the notification that Competent Authority has not given written notice of objection, the clinical investigation may proceed. The Competent Authority may authorise commencement of the investigation before the 60-day period has expired. Besides a lack of objection from the Competent Authority, approval from a relevant Local Research Ethics Committee (or equivalent) is required. Some EU Competent Authorities require the Ethics Committee’s opinion to be submitted with the clinical investigation application, others allow it to be submitted later, but before the commencement of the trial. All changes to protocol must be notified to the Competent Authority and not implemented until agreement has been obtained from the Competent Authority. No time limit is set for a response to a request for change in protocol. If the Competent Authority considers that the changes proposed effectively constitute a new investigation, it may request a new clinical investigation notification. If the investigation involves a multi-centre international trial, all the Competent Authorities involved must be notified. If it involves centres both in the EU and in other regions, such as the US, it is recognised that the study may include wider objectives than those required by the EU Medical Devices Regulations. A Competent Authority may consider such a study acceptable provided it entails no additional risk to the subjects. Any serious adverse events involving a medical device under clinical investigation in the EU should be reported to the Competent Authority. Similar requirements are not present in the Active Implantable Medical Device Directive, but most Competent Authorities do

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Medical Device Regulation strongly encourage reporting of serious adverse incidents during clinical investigation of active implantable medical devices.

13.2.2.4 Custom-Made and Humanitarian Devices Certain devices are manufactured specifically in accordance with a written prescription of a duly qualified healthcare professional to conform to the special needs of their patients (e.g., certain dental devices and specially designed orthopaedic footwear) are considered custom-made devices. Such devices are exempt from carrying the CE-mark. However, they must still conform to all the relevant requirements of the Medical Devices Directive. As with Class I devices, confirmation of this is left up to the manufacturer. No submission is made to Notified Bodies but the manufacturer must register with the Competent Authority for the Member State in which he has his registered place of business. In exceptional circumstances devices which have not undergone the relevant conformity assessment procedures may be put into service for humanitarian reasons. Approval may be given where it can be demonstrated that no alternative CE marked device is available and use of the device is in the interests of health of a particular named patient. In such a case the device may not be used until an application to use it for that specific case has been submitted to and granted by the Competent Authority.

13.2.3 After Marketing Manufacturers are legally bound to report any serious incidents involving the devices they produce or sell to the Competent Authority of the country where the incident took place. In Europe this is dictated by the Vigilance requirements. This includes any malfunction of or deterioration in the characteristics and performance of a device, as well as any inaccuracies in instruction leaflets, which might lead to, or might have led to, the death of a patient or to serious deterioration in his/her health. Incidents which did not result in any serious harm, but might have done so, so-called near-incidents, are also reportable. A ‘serious deterioration in health’ is difficult to define and will sometimes be interpreted differently by manufacturers and regulatory authorities. However, events that are life threatening, result in permanent impairment of a body function or permanent damage to a body structure, or any condition necessitating medical or surgical intervention to prevent such permanent impairment/damage, will certainly be considered reportable. The time limit between a manufacturer first being informed of the incident and when it must be reported to the relevant Competent Authority is a maximum of 10 days for incidents, and 30 days for near-incidents. In many cases the actual cause of the incident will not have been identified in that time and the cause could well impact on the decision

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Practical Guide to Chemical Safety Testing as to whether the incident was reportable in the first place. In such a case, a report should be filed if the incident could be reportable, and the final report can then later clarify the case. Generally the manufacturer will carry out the investigation while the Competent Authority monitors progress. The manufacturer is obliged to institute and keep up to date a systematic procedure to review experience gained from devices in the post-production phase and to implement appropriate means to apply any necessary corrective action. In other words, it is not enough to file those incidents that are spontaneously reported, there is also an obligation to ensure that incidents can be reported in the first place, and to regularly review them for trends. If a manufacturer systematically recalls a particular type of device for a technical or medical reason or issues an advisory notice, this must also be reported to the Competent Authority. Guidance produced by an organised group representing designated Notified Bodies states that “the requirements of the PMS (post-market surveillance) should be in direct proportion to the risk associated with the device based on its intended use”. It is primarily for the manufacturer, possibly in conjunction with the Notified Body, to establish appropriate PMS procedures. However, it is possible that a CE-mark may be placed on medium to high risk devices that are intended for long-term use, before the medium and long-term clinical performance of that device is known. In such cases, PMS should generally include properly structured post-market clinical studies designed to confirm the medium and long-term performance of the device and to ensure that any claims made regarding its performance are justified. Post-market changes to the device that could affect conformity with the Essential Requirements of the Directive or with the conditions prescribed for use of the product, mean conformity must be reassessed. This includes changes in the materials used, in the design of the product or in the indications for use. They may need approval from the Notified Body that provided the original certification. In all cases the risk assessment in the technical file kept by the manufacturer should be updated with the change and any potentially existing Declaration of Conformity reviewed. What has been set out above reflects the requirements of the Medical Devices Directive and the Active Implantable Directive. However, the Directives allow for a degree of flexibility on the part of the Member State to determine the terms of and procedures for any notification of a clinical investigation taking place on their territory. Whilst a review of national implementing legislation is always recommended for products falling within the scope of European Directives, it is particularly important to consider local requirements of Member States in which a clinical investigation is to be carried out.

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Medical Device Regulation

13.3 United States of America 13.3.1 Background The Center for Devices and Radiological Health (CDRH) is the part of the FDA that deals with devices. For US regulations a device is defined as any healthcare product that does not achieve its principle intended purposes by chemical action in or on the body or by being metabolised. Products that do work by such chemical or metabolic action are regulated as drugs. The term ‘devices’ also includes components, parts, or accessories of devices, diagnostic aids such as reagents, antibiotic sensitivity discs, and test kits for in vitro (outside the body) diagnosis of disease (e.g., diabetes), and other conditions (e.g., pregnancy). In some aspects this is thus a slightly broader definition than the one used in the EEA. It should however, not be confused with ‘biologics’ (biological and related products including blood, vaccines, tissue, allergenics and biological therapeutics), which is regulated by the part of the FDA called the Center for Biologics Evaluation and Research (CBER). Biologics, in contrast to drugs that are chemically synthesised, are derived from living sources (such as humans, animals, and microorganisms). Most biologics are complex mixtures that are not easily identified or characterised, and many biologics are manufactured using biotechnology. The original device requirements in the US date back to the 1906 Federal Food, Drug, and Cosmetic Act, which was significantly revised by the Medical Device Amendments of 1976. As a result pre-1976 devices are regulated slightly differently than more recent devices. Further amendments were put in place in 1990 and 1992 enhancing pre- and post-market controls and providing for additional regulatory authority. The current relevant regulation is the Federal Food, Drug and Cosmetics Act as Amended by the FDA Modernization Act of 1997, where the device related requirements are found in sections of Chapter V (Drugs and Devices).

13.3.2 Before Marketing

13.3.2.1 The ‘Licence’ to Market Unless exempted by regulation, all establishments engaged in manufacturing a medical device intended for human use must be registered with the FDA, and their products listed with the FDA (Section 510). This requirement also applies to any repacker, relabeller, and initial distributor of imported devices. Foreign manufacturers of devices are not required to register, but are encouraged to do so. However, they are required to list their devices with the FDA. 359

Practical Guide to Chemical Safety Testing Devices that were already marketed in the US prior to 1976 have been divided by the Food and Drug Administration (FDA) into three classes of regulatory control. Class I products are subject only to the general controls such as registration of manufacturers, record keeping requirements, labelling requirements, and compliance with Good Manufacturing Practice Regulations. Class II devices require additional product specific special controls. This could for example include compliance with specific performance standards or measures such as post-market surveillance or patient registries. Class III devices additionally require pre-market approval. Devices marketed after the 1976 amendments are firstly judged on their similarity to pre-1976 devices. If they are substantially the same, they are placed in the same class as the parent device, if they are judged to be substantially different, they are automatically class III (regardless of their inherent risk levels) and pre-market approval is thus needed. If a manufacturer wants a new version of an existing device assessed, a so-called ‘metoo’ device, he needs to submit a pre-market notification (a ‘510(K)’) to the FDA 90 days before marketing. A 510(K) needs to be submitted only when a change from an approved device, or the sum of the incremental changes, exceeds the CFR21, Volume 8, Part 807.81(a)(3) threshold, i.e., the change “could significantly affect the safety or effectiveness of the device.” The FDA then decides within that time limit whether the device is or is not substantially equivalent to a pre-1976 device. A device may not be marketed until the firm receives a notice from the Agency that their device is substantially equivalent to a device that does not require pre-amendment approval. If the device is judged to be class III, a pre-market approval (PMA) application will need to be submitted to and approved by the FDA demonstrating the device is safe and effective before the device may be marketed. In response to complaints from industry that FDA reviews of PMAs can take up quite some time, a possibility for expedited review has been implemented. Expedited review will generally be considered when a device offers a potential for clinically meaningful benefit as compared to the existing alternatives, or when the new medical device promises to provide a revolutionary advance over currently available alternative modalities. Additionally, FDA is working on the backlog of devices which were already on the market prior to 1976 and which were originally classified as requiring PMAs. To date, FDA has called for or proposed calling for PMAs in a limited number of device categories, including heart valves, breast implants and certain dental implants. FDA has also initiated the retrospective review of these pre-1976 products under Section 515(i) of the Act, which is likely to result in down-classification of a significant number of them. Nevertheless, it is likely that a number of devices will still require retrospective PMA submissions.

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Medical Device Regulation In the US, both licensing and enforcement authority lie with the FDA. The FDA may initiate an enforcement action (seizure, injunction, prosecution, and/or civil penalties) to protect the public from adulterated or misbranded devices.

13.3.2.2 Biological Safety Requirements The PMA application must contain sufficient information to reasonably assure FDA of the safety and effectiveness of the device. This requires valid scientific data to demonstrate that the device is safe and effective for its intended use. In most cases, this includes wellcontrolled clinical studies; full reports of safety and effectiveness and data regarding the manufacturing of the device. Detailed guidance on what is expected in a PMA submission can be found in the FDAs ‘Blue Book Memo’ of 1995 [5]. It instructs reviewers to use a modified version of ISO 10993-1 and emphasises the scientific basis of an assessment. However, the two matrices in the standard are modified to suggest more tests to bring them more in line with the earlier FDA guidance. Unfortunately, the Blue Book Memo omits the important notes referred to earlier, stating that the matrices are an aid and not a checklist. Also, a part of clause 6 of the standard is omitted. In the standard, this clause indicates that an evaluation may include both a study of relevant experience and actual testing and may result in the conclusion that no testing is needed. Thus it is possible that more testing can be needed to market a product in the US than in Europe.

13.3.2.3 Clinical Investigations Devices used for trials in the US to conduct investigations of their safety and effectiveness, are considered ‘investigational devices’ (Section 520(g)). Exemptions from certain requirements of the Federal Food, Drug, and Cosmetic Act that would otherwise impede these studies, need to be obtained by filing an application for an Investigational Device Exemption (IDE). The FDA regulations to protect the subjects of research on investigational devices are found in 21 CFR 812 (general) and 813 (intraocular lenses). Any serious adverse events involving a device under clinical investigation in the US should be reported to the FDA.

13.3.2.4 Custom-Made and Humanitarian Devices Custom made devices are exempt from registration in the US and from otherwise applicable performance standards or pre-market approval requirements (Section 520(b)). The exemption applies only to devices not generally available to or used by other health

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Practical Guide to Chemical Safety Testing professionals. US custom devices are not exempt from other provisions of the Federal Food, Drug and Cosmetic Act and regulations. The FDA may grant an exemption from the effectiveness requirements of Sections 514 (performance standards) and 515 (pre-market approval) of the Federal Food, Drug, and Cosmetic Act for humanitarian use. Requirements that the device needs to comply with are: •

it is designed to treat or diagnose a disease or condition that affects fewer than 4,000 individuals in the United States.

•

it is not available otherwise, and there is no comparable device available to treat or diagnose the disease or condition.

•

it will not expose patients to unreasonable or significant risk of illness or injury, and the benefits to health from the use outweighs the risks.

Devices granted an exemption may only be used at facilities that have an established institutional review committee, and the humanitarian use must be approved by the committee before studies begin (Section 520(m)).

13.3.3 After Marketing The reporting of any adverse events resulting in serious injury or serious illness with devices is captured under the ‘Medical Device Reporting’ (MDR) system. Serious injury or serious illness have been defined as those that are life threatening, result in permanent impairment of a body function or permanent damage to a body structure, or any condition necessitating medical or surgical intervention to prevent such permanent impairment/ damage. Reports on events that require remedial action to prevent an unreasonable risk of substantial harm to the public health (and some other types of events designated by FDA) need to be reported within 5 working days of the company becoming aware of them. Deaths, serious injuries and malfunctions must be reported within 30 calendar days. Recent changes that have come into effect in the US since March 2000 are: 1. Medical device manufacturers, importers, and distributors are no longer required to submit an annual certification statement. 2. Domestic distributors no longer have to submit MDR reports (importers do), but they must continue to maintain records of adverse events.

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Medical Device Regulation In the US, for class II or III devices, it is the FDA’s decision whether to require PMS for a specific device or not. They may require a manufacturer to conduct post market surveillance: 1. Where failure of the device would be reasonably likely to have serious adverse health consequences; or 2. If the device is intended to be implanted for more than a year; or 3. If the device is intended to be a life sustaining/supporting device used outside a device user facility. If any changes are introduced to the device or production process after marketing, PMA supplements are required for all those changes that affect safety or effectiveness. However, if such change involves modifications to manufacturing procedures or method of manufacture, changes might only require a 30-day Notice or, where FDA finds such notice inadequate, a 135-day PMA supplement. Guidance is available (http://www.fda.gov/ cdrh/modact/daypmasp.html) describing what changes generally will qualify for the 30-day Notice and what changes generally will not. Questions concerning US device regulation should be directed to the Food and Drug Administration, Center for Devices and Radiological Health, Division of Small Manufacturers Assistance (HFZ-220), 1350 Piccard Drive, Rockville, MD 20850, USA (telephone: 800-638-2041, toll free or 301-443-7491, http://www.fda.gov/cdrh).

13.4 Japan

13.4.1 Background In Japan medical devices are regulated under the same law as medicinal products, cosmetics and quasi-drugs. This is the Pharmaceutical Affairs Law which is enforced by the Japanese Ministry of Health, Labour and Welfare (MHLW). However, scientific and technical matters regarding examination for approval are delegated to the Pharmaceutical and Medical Devices Evaluation Centre within the National Institute of Health Sciences (NIHS). The definition of medical devices in Japan is somewhat different to that in Europe and the US, whereas it does include devices used for animals, other products such as wheelchairs, artificial legs, etc., are not included. Specifically medical devices are defined as “instruments and apparatus intended for use in the diagnosis, cure or prevention of

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Practical Guide to Chemical Safety Testing diseases in man or other animals; intended to affect the structure or functions of the body of man or other animals”.

13.4.2 Before Marketing

13.4.2.1 The ‘Licence’ to Market There are two types of governmental authorisations relevant to selling a Medical Device in Japan [6]: 1. A manufacture or import licence that should be obtained from a prefectural governor for each manufacturing plant or importing office. 2. A device specific approval (Shonin) from the Ministry of Health, Labour and Welfare (MHLW) to ascertain the safety and effectiveness of the device. This licence is not needed for a range of predetermined devices that are listed in Article 18 of the Enforcement Regulations Law such as tongue depressors, stethoscopes, etc. The licence is given based on personnel and facility conditions and the quality assurance system of the factory or office. The approval or Shonin is based on product specifications, quality and performance of the product. Shonin can be obtained either directly by the manufacturer himself, or by his importer. In the latter case the approval will be granted in the importer’s name which can restrict the manufacturer’s freedom to change importers. Following discussions within the Global Harmonisation Task Force (GHTF), the current classification system in Japan is based on the perceived risk of the device which has been divided into 4 classes (class I presenting the lowest, class IV the highest risk). Classification is dependent on the extent of patient contact (i.e., the part of the body in contact with the device and the duration of exposure) and whether a device malfunction would result in a serious adverse event. For most products the approval process starts with a check for equivalency to existing products. If equivalency is established, the MHLW decides whether the device requires further evaluation, or whether approval can be given without further work being done. If a pre-market approval is required, this has to include a scientifically sound biological safety assessment. Additionally, for some devices compliance with mandatory standards still exists in Japan, but MHLW has pursued an active policy of decreasing their number in more recent years.

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13.4.2.2 Biological Safety Requirements Japanese guidelines on the general principles and selection of biological tests have been published [7] which show great resemblance to the ISO-10993 series [3] of standards discussed earlier. These guidelines allow for the fact that a medical device is usually comprised of several materials of which only a few need be new. As long as materials do not change during manufacture or processing, it is more practical to carry out tests on the raw materials and therefore a large quantity of applicable data will already exist for devices using known materials. Identification of toxicological hazards and doserelationships are recognised as the essential steps in the risk assessment of a device. Therefore positive results in biological tests do not necessarily suggest a deficiency in the material or device. A scientifically sound assessment, putting all of the data into perspective and extrapolating to the human situation is crucial. Though it is theoretically possible to omit certain tests that are suggested in the matrix in ISO 10993-1, in practise very good reasons are required before the Japanese authorities will accept this. The Japanese National Institute of Health Sciences (NIHS) has a broad experience in biological tests and can provide specific advice on test methods that, in many cases, goes beyond that in ISO 10993.

13.4.2.3 Clinical Investigations Clinical trials used to have to be performed on Japanese patients to take into account racial and environmental differences encountered in global trials. However, in the interest of global harmonisation, Japan has been accepting data gathered in other geographical regions.

13.4.3 After Marketing Japan maintains a medical device adverse events reporting system through which hospitals are expected to report information about device problems. Manufacturers are also expected to carry out post-market studies of new devices. Depending on how much of the device was new and innovative, re-examination of the medical device will be required 3 to 7 years after approval. This rule has been introduced because, especially for new technologies, the state of the art changes rapidly and confirmation that the product is still effective and safe based upon the existing scientific knowledge is needed. There are eight specific medical devices which require ‘tracking’ after implantation, these are implantable cardiac pacemakers and defibrillators and their lead wires, cardiac valve prostheses, annuloplasty rings, vascular grafts and artificial heart (assist) devices. This

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Practical Guide to Chemical Safety Testing means that the manufacturer should retain data regarding the patient’s identity and location and the medical institutions where the devices were implanted.

13.5 Conclusion The process of global harmonisation of medical device regulations is an ongoing process. Significant international recognition of basic principles and standards has already been achieved, which means that most of the work done to demonstrate safety and efficacy for one geographic region, will be equally valuable in obtaining approval in another region. There are several elements that are common to the three larger economic regions (US, Europe and Japan): pre-market approval/registration requirements based on exposure and risk assessments, in market monitoring via adverse incident reporting, and the need for post-market studies for higher risk devices. This makes sense as, more and more, regulations aim to reflect sound science rather than national administrative needs.

References 1.

European Commission, DG Enterprise, MEDDEV2.1/3rev2, Medical devices guidance document, July 2002.

2.

J.J.B. Tinkler, Biological Safety and European Medical Device Regulations, Quality First International Press, London, 2000.

3.

ISO EN 10993, Biological evaluation of medical devices, 1992-2002.

4.

ISO/FDIS 141555, Clinical investigation of medical devices for human subjects, 2002.

5.

FDA; General Program Memorandum (Blue Book Memo) G95-1, July 1995.

6.

Hirokazu Hasegawa, The Regulatory Affairs Journal (Devices), August 2000, 194.

7.

MHLW, Guidelines for basic biological tests for medical devices and materials, Notification No 99; Medical device Division, Japanese Ministry of Health, Labour and Welfare, 1995.

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14

Regulation of Food Packaging in the EU and US Lesley A. Creighton and Derek J. Knight

14.1 Introduction Food packaging and other articles and materials, which come into contact with food during storage, preparation, cooking and serving, are a potential source of contamination. Chemicals could leach from packaging into the food, and these might cause health effects from long-term exposure, especially in sensitive consumers. There is an enormous variety of food packing used, ranging from plastic and paper and board, which may contain recycled waste paper, to ceramics and metals. Food packaging is often surface treated with resins and inks, which may or may not be in direct contact with the food. There is also a huge range of equipment which comes into contact with food during production, and again, in principle, such incidental food contact materials could adulterate food. Given the obvious importance of producing safe and wholesome food, with adequate shelf life, it is not surprising that the food industry is heavily regulated. Normally the onus is on the manufacturer and supplier to provide good quality safe food, with adequate nutritional and storage labelling for the consumer. Packaging is essential in achieving this aim, and as new foods and cooking methods are introduced, new packaging is needed. Regulation of food packaging is one aspect of this general scheme to ensure food safety. The approaches vary, depending partly on the type of packaging and food involved, but even more on the particular country or region of supply. Control measures for food packaging seem to have developed gradually, perhaps as safety issues have arisen, based on, or at least within the framework of, previous national food legislation. These measures differ significantly between Europe and the US, and indeed controls on many types of packaging differ between countries within the EU; although there is a strong harmonising influence from the Council of Europe as well as the European Commission. Food packaging regulations are continually under revision, as the work planned to deal with existing products progresses but also as the need arises to deal with new types of packaging. There is also a struggle to target limited regulatory resources effectively, by dealing with the highest potential risk situations and avoiding over regulating products which clearly have minimal risk.

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Practical Guide to Chemical Safety Testing This chapter covers food packaging regulation in the EU, the general framework Directive and the main ‘daughter’ Directive on plastics, followed by a brief description of the national legislation relating to food packaging for most of the important European countries. Safety of food packaging in the absence of EU or national regulation is largely assessed from Council of Europe Recommendations, or draft Recommendations, other European national approvals (especially German approval) or US approval. Hence, the Council of Europe work on food packaging is described followed by the complex regulatory US system, with its various approval and certification schemes.

14.2 Control of Food Packaging in the EU

14.2.1 EU Framework Directive The EU Single Market, which came into effect on 1 January 1993, establishes an area with free movement of goods, people, services and capital. For this initiative, the European Commission developed a new approach strategy on the free movement of foodstuffs within the EU, and part of this involved developing rules governing materials and articles intended to come into contact with foodstuffs. Most EU countries already had national legislation or voluntary codes of practice in force in this field, but these varied, sometimes significantly. Furthermore, there is considerable public concern about the safety of food packaging. Therefore, the aim of the EU legislation being developed is to remove ‘technical’ barriers to trade within the EU and to protect the health of consumers from the migration of harmful substances from packaging into foodstuffs. It has proven to be difficult to develop EU legislation, both because of the complexity of the subject, worsened in many cases by the limited technical and toxicological data available, and because of political problems in harmonising different laws in all EU Member States. Hence the European Commission decided to develop EU legislation in a stepwise manner, dealing with the more important and urgent aspects first. Consequently, the regulation of food contact materials in the EU is currently in a state of development, with various aspects still subject to national provisions until the European Commission has completed the harmonisation process. The ‘framework directive’ (Council Directive 89/109/EEC) [1] provides the legal basis for subsequent EU regulation of food contact materials and sets out general principles applicable to all materials and articles: •

Food contact materials must be inert and not transfer any of their constituents into food in quantities which could endanger human health and affect the purity of the

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Regulation of Food Packaging in the EU and US food by causing an unacceptable change in its composition or deterioration in its organoleptic characteristics. •

Food contact materials must be labelled as being suitable for this use, either with a statement or the standard symbol specified in Commission Directive 80/590/EEC [2], together with advice on restrictions on their use.

•

The European Commission is eventually to develop EU rules covering the product sectors of plastics, including varnishes and coatings, regenerated cellulose, elastomers and rubber, paper and board, ceramics, glass, metals and alloys, wood including cork, textiles and paraffin and microcrystalline waxes.

•

Before specific Commission Directives on particular product sectors are adopted by the European Commission Standing Committee for Foodstuffs, rigorous health criteria are ensured by the European Commission consulting the Scientific Committee for Food (SCF), an independent advisory body of experts set up in 1974 (which is to be replaced by an ad hoc panel of the European Food Safety Authority).

14.2.2 Food Contact Plastics in the EU The European Commission began developing EU regulations for plastics for use in food contact materials in 1980. This complex and economically important product sector required harmonisation of a wide range of existing national provisions. The process is further complicated by the comparatively limited data available on migration and toxicity of the substances used to manufacture plastics. Whilst many chemicals migrate in only negligible quantities from packaging into foods, it is apparent that others can migrate in higher quantities, in some cases even exceeding the amounts of other chemicals present in foods from their deliberate use as food additives. Thus, food contact materials warrant careful toxicological appraisal with a need for appropriate toxicity testing to support their use. The EU regulation of plastics is based on the following principles: •

There will be EU approved lists of substances authorised for use in the manufacture of plastics for starting substances and additives.

•

An overall migration limit into food for all components (10 mg/dm2 of plastic, or 60 mg/kg of foodstuff) applies to all food contact plastics. This in effect objectively defines the principle of maintaining food purity which is required by the framework directive, avoids establishing restrictions such as specific migration limits (SMLs) for substances regarded as non-hazardous at above the value of the overall migration limit and reduces the health risk from migrating substances not yet evaluated. However,

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Practical Guide to Chemical Safety Testing if there are technical reasons for claiming analytical methods are inadequate, the measured overall migration limit can be replaced by a calculated value of the sum of the SMLs of the plastic ingredients. •

There are restrictions on the use of certain approved plastic ingredients, in the form of a SML (into foodstuffs or food simulant liquids), ‘maximum quantity of residual substance in the finished product’ (QM) or expressed as mg per 6 dm2 of surface content with foodstuffs (QMA). The SCF consider the technical information on the substance, especially the migration properties and toxicological studies, in setting these restrictions.

•

Standard test conditions are to be used to measure the overall migration limit SMLs or QMs. The framework for both types of migration testing is established by Council Directive 82/711/EEC [3], as amended by Commission Directive 93/8/EEC [4], which specifies the testing conditions (food simulant liquid, contact times with the plastic and temperature) for various intended use conditions for plastics intended to come into contact with all foods. The migration testing is modified, in particular regarding fatty foods, by Commission Directive 97/48/EC [5]. Provision is made for departing from these standard test conditions if they are inadequate. Less testing is required if the plastic is to be restricted for use with only certain foods, and the appropriate food simulants are specified in Council Directive 85/572/EEC [6]. Only the general criteria for testing methods are specified by the CEC, and European standards on analytical methods are prepared by the European Committee for Standardisation (CEN).

Commission Directive 2002/72/EC [7], which consolidates and replaces Commission Directive 90/128/EEC, as amended, specifies the overall migration level for food contact plastics and lists the monomers and other starting substances in Annex II and a partial list of additives in Annex III, together with any restrictions in their use, permitted for manufacture of food contact plastics for use in the EU. The European Commission, assisted by the ad hoc Working Party on Packaging Materials of the SCF, has classified the monomers and starting substances and additives already in use in at least one EU country (i.e., appearing on the national positive lists or used in practice in the absence of national law) into ten categories (SCF Lists 0 to 9), as detailed in Table 14.1. Monomers and other starting substances with adequate data for the SCF to evaluate their safety (i.e., SCF Lists 0 to 4) appear in Section A of Annex II of Commission Directive 90/128/ EEC, as amended, and the evaluated additives appear in Annex III and are authorised for use at EU level. Substances on SCF Lists 6 to 9 cannot be evaluated by the SCF until additional information is available. Such monomers and other starting substances can continue to be used while a decision is made and are in Section B of Annex II of Commission Directive 90/128/EEC, as amended. If the requested information is not provided by the deadlines specified by the CEC, the substance is removed from Section B

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Table 14.1 EU scientific committee for food categories for food contact plastic starting substances and additives List 0

Substances are considered to be safe. They are food ingredients or normal products of intermediate metabolism in man. Apart from the evidence for their classification into foods or food ingredients, no toxicological data are required. Migration data are needed though, to ensure that the use of the plastic packaging does not violate any existing legislative requirements applicable to the packaged food.

List 1

Substances are largely those which are also used as direct food additives, for which a full or temporary acceptable daily intake (ADI) has been set by the SCF or by the World Health Organisation (WHO)/ Food and Agriculture Organisation (FAO) Joint Expert Committee on Food Additives (JECFA). This list also includes a few substances present naturally in food, but for which intakes need to be limited, and for which JECFA has set Maximum Tolerated Daily or Weekly Intakes. These food additive substances will be automatically added to List 1, and no further data on toxicology will be required. However, migration data are needed, because for some food additives there are restrictions on use.

List 2

Comprises substances which are not naturally present in foods or in the body and are not direct food additives, but for which the SCF has been able to set a tolerable daily intake (TDI) on the basis of the available toxicity data.

List 3

Comprises substances for which the toxicity data are insufficient to set an ADI or TDI but which are acceptable for use, for the following reasons: • They are unlikely to be present in food other than in very small quantities because they possess properties which render levels in food self-limiting. For example, they may have high volatility, be reactive gases or have strong flavour and/or smell. • They are known to be inert. • They are of low or very low migration, and toxicity data are adequate to establish that their use is acceptable. A SML or QM for the plastic material is set to ensure that they cannot subsequently be used in ways that would give rise to higher migration than the maximum level the toxicity data will support. Should other uses resulting in higher migration levels be proposed, then further toxicity data must be submitted.

List 4

Contains substances for which an ADI or TDI cannot be set, but whose use is acceptable. There can be no detectable residues in food of starting substances, as determined by an agreed sensitive method. For additives the residue in food is reduced as much as possible.

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Table 14.1 EU scientific committee for food categories for food contact plastic starting substances and additives List 5

For substances with adequate data for the SCF to conclude that they should not be used as ingredients of food contact plastics. The list includes compounds which can form carcinogenic derivatives in vivo or which are highly bioaccumulative.

List 6

Contains existing substances for which data are insufficient or absent but serious toxicity is suspected. This suspicion may emerge from preliminary toxicity data, indicating possible genotoxicity, or may derive from the fact that the substance is closely related in structure to other chemicals with known serious toxicity, such as genotoxicity, carcinogenicity, teratogenicity or neurotoxicity. The possibility of serious toxicity cannot be eliminated at this stage of the evaluation, so restrictions on the use of these substances are applied.

List 6A

For existing substances suspected to have carcinogenic properties, normally based on structural alerts. These substances should not be detectable in foods or in food simulants by an appropriate sensitive method.

List 6B For existing substances suspected to have toxic properties other than carcinogenicity, again often based on structural alerts. Restrictions on their use may apply. List 7

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For existing substances for which some toxicity data exist, but insufficient to set a TDI. At this stage of the evaluation there are no serious toxicity alerts, but the SCF require further specified data to be submitted, to allow classification into Lists 0 to 4. The SCF may use hydrolysis data for List 7 substances in order to reduce the amount of toxicological testing. This may arise when the chemical structure of compounds such as monoesters suggests ready hydrolysis into substances which are toxicologically acceptable and already in List 0, 1, 2 or 3. Demonstration of hydrolysis may be carried out in foods or food simulants, representing the range of foods with which the substance may come into contact. Alternatively, or in cases where hydrolysis in food does not occur, hydrolysis can be evaluated in simulated saliva and/or gastrointestinal fluids. However, toxicity data may render the request for hydrolysis data superfluous. If the substance does hydrolyse, toxicity testing will not be needed. The level of hydrolysis demonstrated, however, needs to be fairly substantial, of the order of 95% or more, to give reassurance that the parent compound does not need to be tested further.

Regulation of Food Packaging in the EU and US of Annex II in a subsequent amendment to Commission Directive 90/128/EEC. Member States may approve substances listed in Section B of Annex II for use nationally until 31 December 2004. For some additives, the specific migration limits cannot be applied in all circumstances. These are listed in Section B of Annex III of the Directive, again with a deadline of 31 December 2004 for implementing the limits. SCF List 5 is for banned substances. There is also a waiting list (SCF List W) for new substances with inadequate data for full evaluation and an SCF List P for substances whose evaluation by the SCF has been postponed. It is planned that the list of additives becomes a positive list from 1 January 2005 (i.e., only those additives can be used).

Table 14.1 Continued List 8

For existing substances with little or no relevant toxicity data. The information already available may be inadequate in that it is not specifically required for a core or reduced dossier according to the SCF guidelines, or because the studies were not in conformity with EU/OECD guidelines and/or are not GLP compliant.

List 9

Comprises existing ingredients which are groups of substances or individual substances with inadequate chemical descriptions to enable them to be properly identified. They must be adequately specified before the SCF can evaluate them further.

List W

Waiting list substances not yet included in the EU lists, as they should be considered ‘new’ substances, i.e., substances never approved at national level. These substances cannot be included in the EU lists, as they lack the data requested by the SCF.

List W7

Substances for which some toxicological data exists, but for which an ADI or a TDI could not be established, and additional information is required.

List W8

Substances for which no or only limited and inadequate data were available.

List W9

Substances and groups of substances which could not be evaluated due to lack of specifications (substances) or to lack of an adequate description (groups of substances).

The SCF Working Party meet regularly and their activities are summarised in SCF Reports. The SCF lists, and the data required by the SCF together with the submission deadlines, are contained in a ‘Synoptic Document’ [8]. The SCF has also issued a ‘Practical Guide’ [9] and ‘Note for Guidance’ [10] to answer various questions that have arisen frequently and to specify the data required for the technical dossier and its mode of transmission to the European Commission, SCF and national authorities.

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Practical Guide to Chemical Safety Testing Any substance not listed in Annex II or III ‘as appropriate’ of the consolidated Commission Directive 90/128/EEC, as amended, has to be authorised at EU level before it can be used to manufacture food contact plastics for use in the EU; although national authorisations can be made in anticipation of EU approval. The technical data required by the SCF, as specified in the ‘Guidelines for presentation of an application for safety assessment of a substance’ [11] are information on chemical identity, physical, chemical and other properties, the intended uses, migration studies and toxicological studies. The migration test can be omitted if it is assumed all the substance migrates from the plastic into food. Also, results of hydrolysis studies carried out in food or food simulants, or alternatively in simulated saliva and/or gastrointestinal fluids especially if the substance is stable in food, can reduce the toxicological testing required for adequate safety evaluation. These studies may show that hydrolysis to degradants, which are established as safe will occur during normal use conditions. The SCF guidelines [11] define a core set of tests which should generally be sufficient to identify any main targets of toxicity. These tests are required for any substance migrating in excess of 5 mg/kg, up to the overall migration limit maximum permitted value of 60 mg/kg of food or food simulant. If it is assumed as a worst case that 1 kg of food wrapped in a particular type of packaging may be consumed by an individual in any one day, the maximum possible intake of a single substance by a consumer is 1 mg/kg bodyweight/day. The core set of tests enable potential toxicity to be identified and adequate safety margins applied by the SCF in deciding if the substance can be used safely at this maximum potential human exposure level. This core set of toxicological studies consists of 90-day oral toxicity studies in 2 species, 3 mutagenicity tests (Ames, in vitro chromosome aberration and 1 for gene mutation in cultured mammalian cells such as the mouse lymphoma assay), long-term toxicity and/or carcinogenicity studies (normally in 2 species), reproduction (1 species) and developmental toxicity studies (normally in 2 species), and absorption, distribution, metabolism and excretion (ADME) testing. The toxicological studies have to be conducted to EU methods [12] or OECD guidelines [13] in compliance with GLP. This technical dossier also applies to monomers and starting substances on Section B of Annex II of Commission Directive 90/128/EEC, as amended. For non-genotoxic substances a dosage level causing no observed adverse effects in laboratory animals (NOAEL) is usually determinable. A ‘tolerable daily intake’ (TDI) for man, expressed in mg/kg bodyweight can be calculated by applying a safety factor which is sufficiently large to allow for: •

Possible differences between animals and man in their reaction to chemicals.

•

Possible differences between individuals in any population in their sensitivities to chemicals.

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Uncertainties involved in assessing the safe level in animals.

•

Uncertainties due to difficulties in carrying out adequate monitoring of human populations to detect unexpected adverse effects in man.

In principle, if the data are considered adequate, a value of 100 for the safety factor is applied. Since the available toxicological data are often less extensive than in the case of food additives, a larger safety factor than usual is chosen, and the concept of a TDI is used instead of an ADI (acceptable daily intake). The SML can be derived from the ADI/TDI by multiplication by 60, because it is assumed that an average person weighs 60 kg and consumes 1 kg of food per day which has been packed in the plastic of concern. If the level of migration is 0.05 ≤ SML < 5 mg/kg, a ‘reduced dossier’ containing at least the following data can be submitted to the SCF instead of the full core set of tests: •

Data demonstrating the absence of potential bioaccumulation in animals (e.g., n–octanol:water partition coefficient, log Pow > 3).

•

The three mutagenicity tests as required for core testing.

•

90-Day oral toxicity study in the rat.

In this case, the SCF normally proposes a restriction less than or equal to 5 mg/kg. The rationale for this reduced set of tests is that for this migration range, intake from food would not exceed 0.1 mg/kg bodyweight/day, and at this low level of exposure, long-term, reproductive or teratogenic effects are extremely unlikely to occur. There are very few long-term effects other than carcinogenicity which are not detected in a thorough shortterm, repeat dosing study. Thus, provided the mutagenicity tests are all clearly ‘negative’, i.e., not mutagenic to bacterial cells in the Ames test and non-clastogenic in the in vitro mammalian chromosome aberration and gene mutation assays, reduced testing is acceptable. This is because the possibility of the substance being a genotoxic carcinogen is virtually ruled out and non-genotoxic carcinogens are generally only active at relatively high, sustained exposures. Developmental and reproduction effects have generally been established in humans only at above 0.1 mg/kg/day, except for substances which bioaccumulate, so the latter are excluded from approval with this reduced testing. Furthermore, the theoretical extreme intake calculated from migration data are worst-case situations and so for the vast majority of consumers, there is an additional safety factor due to a considerably lower actual intake. Nevertheless, this reduced data set is regarded as a minimum only and hence the SCF can request further tests on substances falling within the lower range of migration. For example, if comparison with structurally-related substances suggests that the substance might be toxic, then further tests would be required.

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Practical Guide to Chemical Safety Testing If SML < 0.05 mg/kg, normally only the 3 mutagenicity tests are required. In this case, the SCF normally propose a restriction of less than or equal to 0.05 mg/kg. The maximum possible intake is only 1 µg/kg bodyweight/day and, in practice, likely to be much less than this for the average consumer. For these substances, the only tests required are the mutagenicity tests to establish that they are free of genotoxic potential. However, if there are any grounds for concern as a result of these tests, additional studies could be requested. Polymers which are used as starting substances to prepare food contact plastics do not have to be evaluated and listed, providing the monomers and other starting substances used to manufacture them are approved. In contrast, polymers used as additives for food contact plastics that will not be further polymerised during use, and hence may migrate into food, have to be evaluated by the SCF. The molecular weight (MW) distribution curve is especially important, because if the monomers or starting substances are on lists 0, 1, 2, 3 and 4, polymer additives with all components of MW above 1,000 daltons are toxicologically acceptable and classified in list 3 with the indication ‘polymer’ without specific individual evaluation. Alternatively, if the monomers or starting substances are on lists 6, 7, 8 or 9 or not yet evaluated, data to enable them to be evaluated must be provided. Depending on the circumstances, the SCF may require toxicity data for polymers used as additives with part of their MW distribution below or equal to 1,000 daltons. The distinction between these categories is based on the following considerations: •

The absorption by the gastrointestinal tract is negligible when the MW exceeds 1,000.

•

The migration from plastic materials is very low for the higher MW substances.

•

The purification of polymeric additives and the removal of residual monomers is often easier for the lower MW compounds.

14.2.3 Future Developments for Food Plastics in the EU The European Commission is working towards updating the Plastics Directive: •

To update the positive list of monomers and other starting substances to include new ones and take account of SCF evaluations of ones already listed.

•

To complete the list of additives, and update this as necessary.

•

To extend the positive lists for monomers and additives to apply also to food contact products currently excluded.

•

To extend the scope of the regulations to cover multi-layer food contact materials containing at least one non-plastic layer.

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Regulation of Food Packaging in the EU and US The European Commission are also considering introducing consumer-related reduction factors, which would be used to modify SMLs to take account of the probable overestimate of dietary exposure because their calculation is based on the assumption that a person consumes 1 kg of packaged food daily. Note that consumer-related reduction factors should not be confused with food consumption factors used in the USA (see Section 14.5) and which has a different meaning. The European Commission is also considering whether or not to introduce a ‘threshold of regulation’ concept, as in the US (see Section 14.5.4). This would mean that a food contaminant present at very low concentrations in the diet, and which is not detectable by an agreed sensitive analytical method, can generally be used without specific approval. However, to base an acceptable level of safe use on the sensitivity of an analytical method is fraught with problems on whether a method is unnecessarily too sensitive or not sufficiently sensitive. Furthermore, advances in analytical techniques may require constant review of approved methodologies. However, as specified in the SCF Guidelines [11], it is possible for a petitioner to justify the limited risk associated with a new substance without submitting extensive toxicology data. A further initiative by industry to reduce the number of SMLs is to introduce a fat consumption factor (FCF), which is based on the assumption that an average person consumes approximately 200 g of fat per day. This approach is currently being considered by SCF. Directive 2002/72/EC [7] allows the possibility of using mathematical modelling to predict potential migration. The concept may be used to assess compliance with SMLs without conducting migration levels experimentally. The obvious advantage to this approach is gained where analytical procedures used for measuring the migrant are complex, time consuming and expensive. Migration modelling assumes a relationship between the partitioning of the migrant and food simulants, and in most cases follows Fick’s laws of diffusion. Migrants shown to be in non-compliance using migration modelling must be verified experimentally.

14.2.4 Other EU Food Packaging Measures Food contact materials made from regenerated cellulose film are regulated by separate EU legislation, i.e., Commission Directive 93/10/EEC as amended [14]. Ceramic articles are regulated by Council Directive 84/500/EEC [15]. The migration from PVC food contact materials containing free vinyl chloride monomer is controlled by Council Directive 78/142/EEC [16], with the analytical methods specified in Commission Directives 80/766/EEC and 81/432/EEC [17]. Commission Directive 93/11/EEC [18] regulates the

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Practical Guide to Chemical Safety Testing release of N-nitrosamines and substances capable of being converted into these from teats and soothers made of elastomer or rubber, and products which do not comply with the specified migration limits cannot be used in the EU. Finally, under Commission Directive 2002/16/EC [19] which came into force on 28 February 2003, restrictions are placed on the use of certain epoxy derivatives, i.e., 2,2-bis (4–hydroxyphenyl) propane bis (2,3-epoxypropyl)ether (BADGE), bis (hydroxyphenyl) methane bis (2,3-epoxypropyl) ethers (BFDGE) and novolac glycidyl ethers (NOGE) in food contact materials, articles, surface coatings and adhesives.

14.2.5 Strategy for Food Contact Plastic Approval in the EU The consolidated Directive 90/128/EEC [7] contains an exclusive list of monomers/ starting substances. These are the only permissible starting materials that can be used to make a food contact plastic. There is also a list of additives, some with restrictions on their use. These are recommendations only and alternative additives may be used if they meet the general provisions of the Framework Directive [1]. Hence, if a substance falls into one of these categories then approval at EU level must be gained before the material can be used in food contact applications. Substances not falling within one of these function categories must comply with the general Framework Directive, which places the responsibility on the user of food contact materials to ensure the substance is safe for use. Alternatively there may be national legislation, which may be applicable for a particular use. Some European countries have national ‘positive lists’ (see Section 14.3). Alternatively, there may be national recommendations, such as Codes of Practice, as in the UK, or other general safety measures. The references to EU and national food packaging legislation [20] have been published by the European Commission. There is also a useful text book on EU food packaging legislation and migration studies by Ashby and co-workers [21]. If a particular substance is not yet registered at EU level and is not covered on a country’s ‘positive list’, a general reassurance of its safe use may be taken if it has been reviewed by the SCF and published in the latest Synoptic Document [8], prior to being formally accepted at EU level. In addition to this a Council of Europe Recommendation (Section 14.4) and a US FDA (Section 14.5) approval may be sufficient to give reassurance of a particular substance’s safety, assuming the use conditions are comparable. As a guide for assessing whether a particular substance is suitable for use in food contact plastics in those countries which do not have positive listings, or for substances which have not yet been reviewed by the SCF, it is recommended that the guidelines given for EU registration are followed. However, note that some Member States may have additional criteria for testing requirements such as the ‘Hemmhoftest’ for preservatives used in paper/board in Germany.

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Regulation of Food Packaging in the EU and US It can take several years to gain approval at EU level and hence, it is acceptable to submit a request for a national approval in those countries that operate an approval system. Once the substance is approved at EU level the national approvals become void. The pathways to marketing of substances used to produce food packaging for use in the EU are summarised in Figure 14.1.

14.3 National Controls on Food Packaging in EU Countries Seven of the fifteen EU Member States (the United Kingdom, Germany, France, the Netherlands, Belgium, Italy and Spain) have some form of national ‘positive list’ of permissible substances for use in manufacturing food contact materials beyond the required legislation implementing the EU Directives (see Section 14.2). In Germany and the United Kingdom, however, these positive lists are not binding and other factors can be used to demonstrate that a given compound is safe. The most influential and important countries with national positive lists for food packaging additives are covered briefly in this section. Austria, Finland, Greece and Sweden have some additional national measures, whereas Denmark, Ireland, Luxembourg and Portugal only have EU provisions. A useful listing of national food packaging legislation has been compiled for the European Commission [20] and more detailed information on particular countries is given in the publication referenced in [21], Chapter 7.

14.3.1 Germany Germany regulates food contact materials pursuant to its Law of 15 August 1974 on Trade with Foodstuffs, Tobacco Products, Cosmetic Agents and Other Articles, Lebensmittel und Bedarfsgegenständegesetz (LMBG). Sections 30 and 31 of this law generally mirror the basic safety requirement set forth in the EU’s framework Directive for food contact materials. The Regulation of 10 April 1992 on Food Contact Materials implements the Monomers Directive. One way for a manufacturer to ensure products that are not covered by the Regulation of 10 April 1992 on Food Contact Materials meet the LMBC’s general safety requirements is to consider guidance contained in Kunststoffe im Lebensmittelverkehr of the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinarmedizin (BgVV), also known as the BgVV Recommendations. These define specific positive lists of starting substances and additives, including reaction control agents that are permitted for use in individual food packaging applications. Although they are not legally binding, the BgVV Recommendations are widely respected in Germany, and German manufacturers often

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Figure 14.1 Pathways to approval of substances for use in food packaging in the EU

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Regulation of Food Packaging in the EU and US insist that materials meet existing BgVV recommendations. However, products whose safety can be demonstrated by other means are also equally compliant with German law.

14.3.2 France Food contact materials in France are regulated under a series of laws, decrees, arrêtés and circulars. Decree 73-128 of 12 February 1973 (the ‘1973 Decree’) and a series of subsequent arrêtés and circulars, as reproduced in the Recueil 1227 of the French Official Journal, provide, among other things, several positive lists of those starting substances and additives that are permitted for specified uses in food contact materials. France’s Decree No. 92-631 of 8 July 1992 and Order of 14 September 1992 implement into French Law the EU Framework Directive and the EU Plastics Directive respectively. Additional circulars, decrees, and arrêtés apply to other types of food contact materials, including aids to polymerisation; however, these additional circulars, decrees, and arrêtés are not organised according to the specific types of food contact materials they address. Consequently, they must all be reviewed to determine whether a specific material is permitted for a particular use.

14.3.3 The Netherlands Food packaging materials are regulated in the Netherlands by the Decree of 1 October 1979 on Packaging and Articles of Daily Use (‘Verpakkingen en Gebruikartikelen-besluit (Warenwet)’). This decree is implemented by the Ministerial Regulation of 25 January 1980 (the ‘Regeling verpakkingen en gebruiksartikelen (Warenwet),’ as amended). These regulations are essentially a compilation of ‘positive lists’ for different types of substances, including plastics, that are permitted in the Netherlands for use in manufacturing food packaging materials. The Warenwet Regulations are structured in ten Chapters: I. Plastics; II. Paper and Board; III. Rubber; IV. Metals; V. Glass; VI. Ceramics; VII. Textiles; VIII. Regenerated cellulose; IX. Wood and cork; and X. Coatings. Chapter I on plastics applies to monomers, additives, and aids to polymerisation used in the production of food contact plastics.

14.3.4 Belgium Belgium regulates food contact materials under the Royal Arrete of 11 May 1992 on Materials Intended for Contact with Foodstuffs, as amended (the ‘1992 Decree’). This Decree governs the composition of food contact materials by means of ‘positive lists’ for various types of food contact materials, including the monomers, additives, and aids to polymerisation authorised for use in food contact plastics.

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14.3.5 Italy Food contact materials in Italy are regulated under the Decree of 21 March 1973 on Hygienic Requirements for Packaging, Containers and Utensils Intended to be Used in Direct Contact with Food and Substances for Personal Use (‘the 1973 Decree’), as amended. This decree establishes rules for the authorisation and control of objects intended to come into contact with food substances. Article 3, Title I of the 1973 Decree stipulates that food contact materials must be prepared exclusively from components specifically listed in Attachment II to the law for different categories of materials (such as plastic, rubber, regenerated cellulose, paper and cardboard, glass and stainless steel) and must otherwise comply with any conditions or limitations specified.

14.4 Council of Europe Work on Food Packaging 14.4.1 Introduction The Council of Europe (CoE) is a separate political grouping of European countries, which pre-dates the EU. There are currently 41 Member States, including all the EU countries. Hence there is close co-operation with the EU. The CoE plays an important role in harmonising various technical and safety measures and standards. The CoE started work on elaborating international standards for food contact materials in the early 1960s, and established the Partial Agreement in the Social and Public Health Field (PASPHF) in 1959. Current members of the Agreement are Austria, Belgium, Cyprus, Denmark, Finland, France, Germany, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. One of the stated aims of the Partial Agreement is: ‘to raise the level of health protection of consumers in its widest acceptation: constant contribution to harmonise – in the field of products having a direct or indirect impact on the human food chain as well as in the field of pesticides, pharmaceuticals and cosmetics – laws, regulations and practices governing, on one hand, quality, efficiency and safety controls for products; on the other hand, the safe use of toxic or noxious products.’ The Committee of Experts on Materials Coming into Contact with Food (CEMCCF) was founded in the late 1960s, under the aegis of the Partial Agreement. Around 13 countries are active participants on the CEMCCF, together with an EU representative, and representatives include toxicologists, analytical experts, food safety advisors, public health officials, and experts in food contact materials. The aim of the CEMCCF is to harmonise legislation on food contact materials, with emphasis on the safety of food contact materials from a public health perspective.

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Regulation of Food Packaging in the EU and US The CEMCCF has expanded its field of work markedly in the last few years and is now actively drafting resolutions and guidelines on many types of food contact materials; including paper and board. Other areas of interest include printing inks, surface coatings, cork, rubber, plastics, colourants, silicones and metals and alloys. The CEMCCF also has a watching brief on developments in active packaging materials. Although these resolutions and guidelines have no legal force, they are expert reference documents, which provide a benchmark for industry and can inform European Union legislation. This increase in scope of the CEMCCF’s work has meant that only limited time is available to progress individual topics at the twice-yearly meetings of the Committee. It has also been recognised by the Committee that certain topics required a greater input of technical expertise than could necessarily be provided for by the membership of the main Committee. Hence ad hoc committees have been established, each with a particular remit, to work on guidelines for recycled fibres, test conditions for paper and board, packaging inks, coatings, metals and alloys, cork and safety evaluation. The work of the CEMCCF has been given added impetus by the EU who have stated that they intend to use future CoE resolutions to ensure that food contact materials comply with the Framework Directive (89/109/EEC) [1] and that they will use future CoE resolutions as main reference documents in drafting specific EU directives. It should be noted that this does not mean that the European Commission necessarily agrees with all decisions taken by the CEMCCF, nor that all aspects of resolutions will form the basis of future directives. However, it is clear that the position of the Commission at the CEMCCF has changed from ‘observer’ status to a far more proactive role.

14.4.2 Completed Council of Europe Resolutions

14.4.2.1 Colourants in Plastic Materials Resolution AP(89)1 [22], which was adopted on 13 September 1989, defines colourants as substances which are intentionally added to plastics to impart colour, including dyes and organic and inorganic pigments. It requires that colourants do not pose a risk to health, or affect food quality, and are sufficiently integrated within the finished material so that there is no visible migration using a given analytical method, under normal conditions of use. It specifies that active ingredients should be tested and lays down purity criteria, compositional limits and analytical methods, for metals and metalloids, aromatic amines, sulphonated aromatic amines, carbon black, polychlorinated biphenyls (PCBs) and inorganic cadmium pigments.

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Practical Guide to Chemical Safety Testing It has been agreed to amend this resolution by adding an inventory list to it. It is intended that the list will distinguish between substances, which have been evaluated and those which have not. The basis of the list will be colourants already authorised in Japan, USA and France for which there is toxicological information. Colourants approved more recently in France on the basis of toxicological assessments will be classed together with colourants approved for use in food, and industry will be invited to add to the list. However, this work on updating the colourants resolution has been postponed because other projects are of higher priority.

14.4.2.2 Polymerisation Aids Resolution AP(92)2 [23], which was adopted on 19 October 1992, covers only substances which directly influence the formation of polymers and excludes substances which provide a medium for polymerisation. It requires that materials and articles intended to come into contact with food and which use aids to polymerisation in their manufacture, do not pose a risk to health, or affect food quality. Aids to polymerisation should be of good technical quality and should be monomers listed in the EU Plastics Directive 90/128/ EEC [7], subject to some additional limitations listed in the resolution, or substances listed in the resolution, with associated restrictions. Along with specific migration limits, the resolution also sets an overall migration limit, 60 mg/kg or 10 mg/dm2. These limits are to be tested according to the EU methods [3-6]. The inventory lists of starting substances, polymerisation aids and additives, in the Resolution specifies substances used in the manufacture of silicones which could be present in the finished material or article. The list is divided between substances which have been fully evaluated (lists 0-4), not fully evaluated (lists 6-8) and not evaluated (list 9) by an international body, e.g., the EU SCF.

14.4.2.3 Surface Coatings Resolution AP(96)5 [24], which was adopted on 2 October 1996, defines surface coatings as protective layers or functional barriers and describes their composition. It requires that such coatings do not pose a risk to health, or affect food quality. Surface coatings should be manufactured in accordance with good manufacturing practice using monomers, starting substances and additives listed in the resolution, as well as aids to polymerisation listed in Resolution AP(92)2 on polymerisation aids [23]. An overall migration of 60 mg/kg or 10 mg/dm2 applies, as measured by the EU method [3-6]. The inventory list of monomers and additives distinguishes between substances which have been fully evaluated (lists 0-4), not fully evaluated (lists 6-8) and not evaluated (list 9) by the EU SCF (see Table 14.1). Work is ongoing to update this resolution on surface coatings.

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14.4.2.4 Ion Exchange and Absorbent Resins Resolution AP(97)1 [25], which was adopted on 30 September 1997, defines ion exchange resins as synthetic organic macromolecular compounds which can be used in the processing of foodstuffs to bring about exchange of ions or absorption of food and excludes cellulosic ion exchangers. Such resins should be manufactured in accordance with a certified quality assurance system and under conditions specified in the resolution which requires that the resins do not release, or form, in foodstuffs, any substances at levels which pose a risk to health, or affect food quality. Test requirements are laid down including those for migration and residual limits. The inventory list of monomers, chemical modifiers and polymerisation aids, in the resolution distinguishes between substances which have been fully evaluated (lists 0-4), not fully evaluated (lists 6-8) and not evaluated (list 9) by the EU SCF (see Table 14.1).

14.4.2.5 Silicones Resolution AP(99)3 [26], which was adopted on 13 October 1999, defines silicones as a group of polymeric chemical substances and preparations, all containing polysiloxanes, and excludes silicones used as food additives as well as some specified starting substances. It requires silicones to be manufactured in accordance with a certified quality assurance system and under conditions specified in the resolution. Silicones should not pose a risk to health or affect food quality. An overall migration limit of 60 mg/kg or 10 mg/dm2 applies, as measured by the EU method [3-6].

14.4.3 Council of Europe Ongoing Work Various CoE recommendations and guidelines are in preparation, and are described briefly as follows. These draft documents are important in establishing that food packaging is safe, in the absence of other controls.

14.4.3.1 Paper and Board This resolution will apply to paper and board made from virgin as well as recycled fibres (but there will be separate guidelines for the latter) and, unless there is a functional barrier, also to paper in laminated materials. It will require that paper and board materials do not pose a risk to health or affect food quality and that they are manufactured in accordance with good manufacturing practice (GMP) (for which a guide will be drawn up), using substances listed in an inventory. Paper and board will have to be of suitable

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Practical Guide to Chemical Safety Testing microbiological quality for the end use and comply with the restrictions in the resolution, including for heavy metals. It is intended that the resolution will lay down, where appropriate, restrictions in the form of QMs (maximum quantity in the finished product) together with specific migration limits (SMLs). The resolution will also lay down test conditions for compliance with restrictions. The inventory list will distinguish between monomers, polymers with a molecular weight below 1000, non-polymeric additives and starches and natural polymers. Paper and board made from recycled fibres will be subject to specific requirements (in addition to those in the paper and board resolution). These requirements will be specified in guidelines being drawn up by an ad hoc Group. The approach taken in these guidelines is to integrate the nature of the recovered paper with the treatments in the mill and the proposed use of the end product. Therefore recovered paper is to be classified in a number of categories to reflect the potential presence of contaminants. The treatments in the paper mill are defined in terms of their function in removing potential contaminants. The foods intended to come into contact with the end product are differentiated to reflect the potential for chemical migration in use. Specific tests will be required to verify the inertness of the end product.

14.4.3.2 Packaging Inks This resolution covers preparations applied by a printing or varnishing process to the side of food contact materials which is not in contact with the food. It will require that such preparations do not pose a risk to health, or affect food quality and are manufactured in accordance with GMP. The guide for the latter will cover the manufacture, application and curing of inks, primers, lacquers and varnishes and the manufacture of pigments and dyes. Overall and specific migration limits will be laid down in the resolution together with analytical methods, and other specifications, like colourfastness for short duration applications. The inventory list will cover five classes of substances: plasticisers, dryers, solvents, pigments and dyes and additives used in organic pigments. Direct food contact inks, that is inks which are applied on the inner surface of food packaging, are excluded from the scope of the draft resolution on packaging inks, and are to be considered separately.

14.4.3.3 Rubber This draft resolution follows closely the provisions of the Netherlands legislation, as well as the recommendations of the German BgVV for food contact rubber. The resolution

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Regulation of Food Packaging in the EU and US introduces four factors (area, time, temperature and repeated use) which are combined to evaluate whether migration testing is required. In particular, the resolution will categorise products in three classes: products used to make materials which will come into contact with babies or baby food, products for which the evaluation of the four factors indicates that migration testing is necessary and products for which the evaluation of the four factors indicates that migration testing is not required.

14.4.3.4 Other Draft Resolutions and Guidelines and Future Developments Work is progressing both on a resolution and a GMP Guide for food contact cork. Guidelines are in preparation for food contact metals and alloys. The CoE is working on a project to deal with leaching of lead from glass tableware into food. The CoE is working on a compendium of national regulations on food packaging. Along with the proactive participation by the European Commission there has been a change in approach to the structure of future resolutions. In the past they have been standalone documents, laying down the requirements to be met for the particular materials involved. The CEMCCF is now moving towards a more integrated approach, with resolutions in a standardised format supported by technical documents. Inventory lists have been the subject of much debate amongst the CEMCCF over the past few years. They were compiled as state-of-the-art lists of the substances used in the manufacture of the particular food contact materials and to be included in the relevant resolution. There has been growing dissatisfaction with this approach for some time because it is not easy to have new substances added and in many cases there has been no risk assessment of the substance for the particular use. Hence the CoE has agreed a new policy on the listing of substances associated with resolutions and is to set up arrangements to evaluate toxicity and migration data before listing. Draft CoE guidelines concerning the safety evaluation of substances to be used in food contact materials and articles covered by CoE resolutions [27] are available. In essence, they apply the same criteria and data requirements as the EU SCF (see Section 14.2), with one significant difference. The CoE has agreed to accept only a very limited set of data for substances with a very low migration level (of below 0.5 µg/kg food), providing they do not contain structural alerts indicative of carcinogenicity. Note that this is the same value used in the US under the threshold of regulation policy (see Section 14.5). New substances will be approved by submitting the technical dossier, prepared in the EU format, to a new Safety Evaluation

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Practical Guide to Chemical Safety Testing Working Group, under the auspices of the CoE Committee of Experts. The list of substances for CoE recommendations will be subdivided into List 1 (substances approved by the CoE Committee of Experts) and List 2 (substances not yet approved). List 1 will include: •

Substances evaluated by the EU SCF, classified in list 0-4, and used in compliance with specific migration limits or other restrictions.

•

Substances approved in the CoE PASPHF member states or by the USA, based on an evaluation of a toxicological dossier which meets the present SCF criteria.

•

Substances authorised as direct food additives in compliance with specific migration limits or other restrictions.

•

Substances evaluated by the new CoE Working Group on Safety Evaluation and approved by the Committee of experts on materials coming into contact with food.

Note that List 2 will include all other substances which do not meet the criteria set for List 1 substances, as well as new substances notified by industry for use in the manufacture of food contact materials and articles.

14.5 Food Packaging in the USA

14.5.1 Introduction Chemicals used in food packaging materials have posed a number of problems for regulatory agencies in reaching safety determinations. Although the potential for components of food packaging to migrate to food in significant amounts is often quite small, the chemicals used in the manufacture of food packaging can often be relatively toxic compared to food ingredients. The gradual development of lower detection limits for analytical methods has shown that many substances previously not considered as indirect food additives do actually migrate into food. The US Food and Drug Administration’s (FDA) responsibility under the Federal Food Drug and Cosmetic Act (FFDCA) is to ensure that the products it regulates are wholesome, safe and effective. Pre-marketing approval by the FDA has been required for food packaging materials used in the US, unless they are generally recognised as safe (GRAS), ‘prior sanctioned’, or not reasonably expected to become a component of food. Through the food additive petition process, these so-called indirect food additives have had to be

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Regulation of Food Packaging in the EU and US shown to be safe for their intended use. Nevertheless, for some food packaging applications, the amount of migration of contaminants into food can be considered so small as to be negligible, and therefore present no public health or safety concern. In an effort to improve the speed and efficiency of the food additive petition process, in 1995 the FDA adopted a ‘Threshold of Regulation Policy’ to define this scenario of negligible exposure from migration. This procedure has proven to be reasonably effective at facilitating regulatory approval of food packaging, but it is not applicable in all cases. Hence the FDA, after full consultation with industry, has developed a Pre-marketing Notification system, referred to as food contact notification (FCN), to regulate most chemical substances used for food packaging. These chemical substances are ‘food contact substances’ (FCS). A FCS is a substance that is intended for use as a component of materials used in manufacturing, packing, packaging, transport, or holding food, if such use of the substance is not intended to have any technical effect in such food [28]. The FCN scheme began operating from 18 January 2000, and there will be important benefits to industry, and also to the FDA in reducing their workload without compromising public safety.

14.5.2 History and Development of US Food Packaging Legislation There is a long history of regulation of food packaging in the USA. The 1938 Federal Food, Drug and Cosmetic Act was amended in 1958 by the Food Additives Amendment to require pre-marketing approval of chemicals which may become a component of food to ensure consumer safety. This broad definition of ‘food additive’ means that strictly all components of food packaging could be regulated as food additives, since in principle they could migrate from the packaging material and contaminate food. However, the legal principle of de minimus applies (often interpreted as ‘the law does not concern itself with trifles’), and the FDA have decided not to apply the strict interpretation of the FFDCA when there are clearly no public health or safety concerns. Since 1958, components of food contact materials (referred to as ‘indirect food additives’) have been regulated in virtually the same way as direct food additives which are intentionally added to food. This means that a supplier of a new chemical to be used in the manufacture of food packaging, and which therefore might contaminate food, generally has had to file a ‘food additive petition’ with the FDA. The petitioner has to establish that the indirect food additive is safe under the intended conditions of use. It is necessary to provide toxicity study data, which can be time consuming and expensive, information on manufacture and use of the chemical and its impurities, an estimation of potential human exposure from contaminated food and an assessment of the potential environmental impact. Needless to say, it takes some considerable time (generally perhaps

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Practical Guide to Chemical Safety Testing 2 to 4 years) for the packaging substance to be eventually authorised for use by publication of a formal Rule in the ‘Federal Register’. Any other company can then use the substance in the USA. The US regulatory system includes various alternative ways of clearing food contact substances without petitioning the FDA to obtain a food additive Regulation. A substance intended for use in packaging for food is not a ‘food additive’ if it is GRAS, under the conditions of its intended use, a concept unique to US food law. Alternatively, the substance may have been ‘prior sanctioned’ (i.e., approved) by the FDA before the Act was adopted in 1958. Also, a substance used in food packaging may not be regulated as an indirect food additive because it is not reasonably expected to become a ‘component’ of food based on a ‘no-migration determination’ or because it can be demonstrated that the substance is separated from the food by a barrier that does not allow its migration to food (the ‘functional barrier concept’). In addition to these explicit exemptions, the FDA had traditionally not regulated ‘housewares’ as food additives, i.e., articles like cookware, dishes and cutlery used exclusively in the home for food preparation. Finally, there is the ‘basic polymer doctrine’, not specifically stated in US legislation but elaborated in numerous papers and by FDA policy, which subjects catalysts, chain transfer agents, reaction-control agents and other substances used in small quantities during the polymerisation stage of food packaging, to control by due diligence and GMP. From the 1950s, even before the 1958 Act, the FDA offered informal advisory opinion letters on food contact substances. In particular, they issued letters for those food packaging materials with very little migration potential, setting the scene for the formal ‘Threshold of Regulation’ policy nearly 40 years later. The number of letters issued varied from hundreds per year in the early 1960s to only a few annually in the early 1980s. In the 1970s the FDA attempted to issue a Regulation to revoke all these opinion letters, in order to rationalise the extraordinarily complicated regulatory scheme that had evolved. In practice, however, the letters were never revoked, because the FDA was advised that this action could be an illegal usurpation of Congressional power which could be subject to legal challenge. Nevertheless, from the mid 1980s the number of informal opinion letters issued by the FDA was greatly reduced. There was also serious concern about the high cost of FDA resources to regulate the virtually nonexistent risk to public health from indirect food additives present in food in extremely small quantities from food packaging. After much debate and public consultation, the FDA decided to introduce formally a ‘Threshold of Regulation’ approach. The final Regulation, which came into effect on 16 August 1995, formalised this policy which the FDA had in effect been using on a case-by-case basis to make pragmatic decisions since the 1958 Act.

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14.5.3 The FDA Petition A formal Petition can be made to the FDA for approval of an indirect food additive, i.e., a substance which can migrate into food from packaging. The FDA have provided detailed guidelines, in the so-called ‘Red Book’ first issued in 1982 and now in an updated 1993 draft form [29], for direct food additives, and in practice these are also used for indirect additives. Part of the long-awaited ‘Red Book 2000’ is now available on the FDA web site (www.cfsan.fda.gov/~redbook/red-toca.html). The web version of Red Book 2000 provides guidance for the safety assessment of food ingredients, including direct food additives, colour additives used in food, GRAS substances, food contact substances and constituents or impurities of any of the above. For the Petition, it is necessary to provide toxicological data on the substance depending upon a number of factors, the most important of which are the degree to which the substance migrates into food and the structure and properties of the substance itself. In general the best way to proceed is to have a ‘pre-petition meeting’ with the FDA, to present the available information and obtain FDA’s comments and guidance on what is required for the Petition. One of the advantages to the applicant in holding a pre-petition meeting is to enable the cost of the Petition to be estimated, including the migration, toxicological and other studies. It also ensures that the most appropriate regulatory approach (i.e., petition, FCN, to Threshold of Regulation (TOR), etc.) is taken. It should be noted that the FDA methods for the migration studies differ to those used for EU approval, as shown in Table 14.2 [30]. Also, depending upon the indirect food additive’s use and the concentration in which it will be present in the food contact material, the FDA may also require an environmental assessment. The FDA operates a tiered system for safety assessment, in which the amount of toxicological data initially required is related to the potential risk from the substance, represented by a ‘Concern Level’. In the absence of any toxicological data, the initial Concern Level is based on the estimated human exposure and a prediction of toxicity from the chemical structure (see Table 14.3). Hence a substance is assigned to a category of high, medium or low potential toxicity based on its functional groups. For each structure category there are three human exposure threshold levels which are then used to create the three distinct ‘Concern Levels’, with exposure weighed more heavily than structure. The cost of safety testing can range from a few thousand pounds to several hundred thousand, or even higher. Hence in order to make business plans for the commercial viability of a new ingredient for food packaging, it is essential to know early on in the development programme the likely cost of such testing. A key factor is the time taken to conduct these studies, which could easily be over 2 years, and there is an even longer subsequent period, normally 2 to 4 years, needed for FDA approval and publication of the final Regulation. Another factor to take into account is that once the Regulation is in

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Table 14.2 US Food and Drug Administration Guidance for Migration Studies for Food Packaging with Single-Use Applications [30] A. High temperature, heat sterilised or retorted above 100 °C 10% Ethanol 121 °C for 2 hours 50% Ethanol 71 °C for 2 hours Food oil (e.g., corn oil) or HB307 or Miglyol 812 121 °C for 2 hours 50% or 95% Ethanol 121 °C for 2 hours After 2 hours at elevated temperature, the tests are continued at 40 °C to a total of 10 days. Test solutions are analysed at 2, 24, 96 and 240 hours. B. Boiling water sterilised Protocol as for condition of use A, except that the highest test temperature is 100 °C C. Hot filled or pasteurised above 66 °C Either 100 °C (or the hot fill temperature) for 30 minutes, allowed to cool to 40 °C, then at 40 °C for 10 days or 66 °C for 2 hours followed by 238 hours at 40 °C. Analysis carried out at the same intervals as A. D. Hot filled or pasteurised below 66 °C Protocol as for condition of use C, except that the initial conditions are 66 °C for 30 minutes, before cooling to 40 °C. E. Room temperature filled and stored (no thermal treatment in the container) 10 days at 40 °C. Analysis should be performed at 24, 48, 120 and 240 hours. F. Refrigerated storage (no thermal treatment in the container) Protocol as for E, but 10 days at 20 °C. G. Frozen storage (no thermal treatment in the container) Protocol as for F, but 5 days at 20 °C. H. Frozen or refrigerated storage; ready-prepared foods intended to be reheated in container at time of use: 10% Ethanol 100 °C for 2 hours Food oil (e.g., corn oil) or HB307 or Miglyol 812 100 °C for 2 hours 50% or 95% Ethanol 100 °C for 2 hours I. Other use conditions Special conditions for the migration study apply to model heating and cooking of food above 121 °C and microwave cooking and heating. Note: Migration to fatty food is evaluated using liquid fat or aqueous ethanol, migration to aqueous, acidic and low alcohol foods is evaluated using 10% ethanol and migration to high alcohol foods is tested using 50% ethanol.

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Table 14.3 Toxicity studies recommended by the US Food and Drug Administration for a food contact petition Study

Concern level I

II

III

+

+

+

+

+

Subchronic toxicity study, 90 days, rat (note 1)

+

+

Subchronic toxicity study, 90 days, dog (note 1)

+

Reproduction study, 2 generation, with teratology phase

+

Short-term tests for genetic toxicity Metabolism and pharmacokinetic studies Short-term toxicity study, 14 to 28 days, rat (note 1)

+

+

Chronic toxicity study, one year, dog

+

Carcinogenicity studies, rat and mouse (note 2)

+

Chronic toxicity and carcinogenicity combined study, rat (notes 2 and 3)

+

Notes: These studies are recommended in the FDA Red Book [28]: 1. Including immunotoxicity and neurotoxicity screens. 2. An in utero evaluation is recommended for one of the two carcinogenicity studies with rodents, preferably the rat study. 3. The combined study can be performed as two separate studies if preferred

force, any company can use the new substance for food packaging, and the original applicant who has made considerable investment in their new product can be undercut by competitors who have incurred no such costs. The problem is that the FDA Regulations are not comprehensive, ‘transparent’ or ‘userfriendly’. In effect they are a partial listing of substances that can be used in food contact applications, a partial statement of the permitted uses of such substances, and a set of customised specifications. Each Regulation was uniquely designed for the petitioner and FDA at the time of submission, and hence there can be poor consistency in treatment and there may be overlap between Regulations or even regulatory duplication. For example, there are Regulations for specific polymers or copolymers based on starting materials, which give specifications for particular starting substances used to manufacture these

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Practical Guide to Chemical Safety Testing plastics and also for other plastic additives (referred to as ‘adjuvants’). There are also separate Regulations for such additives and ‘omnibus Regulations’ that contain lists of substances that can be mixed and matched, and reacted to form can enamels, paper coatings, adhesives, and rubber articles for repeated use. Some of the Regulations for food contact polymeric resins or groups of resins contain limits on overall migration, but these so-called ‘end tests’, mainly for non-volatile extractives, require various test methods and have different specifications. The US Regulations also contain some ‘specific migration limits’, generally to control migration from plastic into food of residual monomers and impurities which are of toxicological concern. The Regulations for adjuvants used in food contact polymers frequently contain both composition limits, to specify the level permitted in the polymer, and limitations on the polymer type. The newer Regulations especially also rely on the use conditions to ensure safety, restricting the permitted use temperature of the packaging and/or the types of food which can be packaged with it. Note that the US specific migration tests, which estimate the degree of migration of components from food packaging into food simulants, differ from the European methods, so for worldwide marketing, a supplier incurs the cost of two sets of testing.

14.5.4 Threshold of Regulation Process In 1995 the FDA set up the TOR procedure [31] to deal with the backlog of pending Petitions. Substances shown to have a potential human exposure below the threshold of toxicological concern do not require formal FDA approval. Instead the applicant notifies the FDA with a reduced technical dossier containing information on the identity and use of the substance (temperature, type of food with which it will be in contact, contact duration and whether application will be for single or repeated use), migration studies with food simulants and the available existing toxicity data. If there is no objection from the FDA within 90 days the substance can be marketed. Again, as with the petition process it is possible to hold a pre-notice meeting with the FDA to ensure that the TOR will apply to the food contact substance (FCS). This is particularly important if the FDA have any internal concerns regarding carcinogenicity of the FCS type, which may not be well documented. Furthermore, as the review time is not codified in the regulations, FDA are not obliged to respond to TOR applications within a set time frame. However, 90 days is the normal target review time. FCSs are published by the FDA in a publicly available list, and hence again there is no protection against competitor companies marketing the product. Where exposure in the human diet exceeds 0.5 ppb, or for an existing regulated direct food additive where the new food packaging use results in dietary exposure that exceeds 1% of the ADI, a standard food additive Petition or notification must be submitted. Materials that are known to be carcinogenic are also excluded from this simplified procedure.

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Regulation of Food Packaging in the EU and US Data submitted under a TOR application must be sufficient to allow FDA (and the applicant) to calculate daily dietary concentration resulting from the proposed uses. The daily dietary concentration is calculated based on either the measured concentration of migrant in the food simulant or on the theoretical concentration assuming 100% of the substance migrates. For the former method of determining migration, fully validated analytical methods are required to be submitted including evidence that the technique used is sufficiently sensitive to detect levels below the ‘cut-off’ of the TOR. Additional data on the substance and its impurities such as literature toxicological data, e.g., carcinogenicity, is also provided to justify the view that the FCS is below the TOR. If an impurity is known to be carcinogenic its TD50 value must not be below 6.25 mg/kg of bodyweight. Note that for the purposes of this criterion the TD50 value is the concentration, based on chronic feeding studies, that causes cancer in 50% of the test animals when corrected for tumours found in control animals.

14.5.5 The Pre-Marketing Notification Scheme As already described, as from 18 January 2000, the FDA have introduced a pre-marketing notification scheme referred to as Food Contact Notification (FCN) [32] as their primary method to regulate indirect food additives. Guidance documents are available [28, 30, 33]. Any still-pending indirect food additive Petitions which qualify for evaluation as a FCN can be converted to a FCN. Since most new indirect food additives will not now require a Petition with the subsequent Regulation, other suppliers of the substance will have to make a separate FCN. Nevertheless, the notification details (i.e., chemical name, notifier and FCN number) will be published on the FDA web site to enable users to confirm the regulatory status of the substance. The FDA has 120 days to evaluate a FCN after up to an initial 30 days review time, and unless they object within this time, the notifier can market the substance. The initial review time allows the FDA to check that the applicant has used the correct regulatory procedure and that all administrative requirements have been fulfilled, after which a letter of acknowledgement is issued. This letter of receipt will indicate to the applicant: (i) That the FCN has been received by FDA and is considered to meet administrative requirements (ii) The start of the 120-day review period. A FCN is not acceptable in certain circumstances, as summarised below, and in these cases a traditional Petition is needed with FDA approval before marketing, unless the applicant can justify why this is unnecessary: •

The estimated cumulative dietary concentration (CDC) from food use is above 1 ppm (or 200 ppb for biocides).

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There are new carcinogenicity studies not already evaluated by the FDA which are not clearly negative.

To ensure the correct regulatory approach is being taken it is recommended to contact FDA prior to submission of the proposed FCN. Any concerns with regard to ingredients in the food contact packaging, e.g., instability, analytical methodology, etc., can be discussed in advance and any additional FDA requirements built into the testing programme. This is particularly important where products intended for high temperature/ microwave applications are being considered, as a migration testing plan is not prescribed in the FDA chemistry guidelines [30]. Furthermore, it may be possible to treat the whole food packaging product as a FCS, as opposed to notifying individual ingredients, and hence reduce the testing burden. However, again this requires ‘clearance’ by FDA prior to starting testing. Notifications for food contact substances must contain sufficient scientific information to demonstrate that the substance that is the subject of the notification is safe for the intended use. The safety criteria used in evaluating indirect food additives are the same irrespective of the administrative procedure used, and hence the same information is required for a FCN as for a petition. The FCN contains a safety narrative (SN), summarising the information justifying the substance as safe when used as intended. There is also a comprehensive toxicological profile (CTP), which includes an evaluation of the ADI. Standardised notification forms are used, which contain information on chemical structure, safety data, intended use and estimated daily intake (EDI), including cumulative estimated daily intake (CEDI) to account for all sources of the substance. The FDA has proposed recommended toxicology testing to assess the safety of indirect food additives for a FCN based on the CDC (which can be used to derive a CEDI), on the principle that the potential risk is likely to increase with exposure. However, the notifier may be able to justify omitting studies on a case-by-case basis as unnecessary for risk assessment. Conversely, extra studies may be needed if there are structural alerts. Ideally GLP-compliant studies on standard technical grade substance, or appropriate component or degradant, conducted to FDA ‘Red Book’ methods [29], OECD guidelines [13] or International Conference on Harmonisation (ICH) guidelines [34] are used for the safety assessment, but non-standard studies may be adequate if they contain adequate information to establish the ADI. The safety studies are based on the CDC (see Table 14.4). This new FCN process offers many advantages to industry and the FDA without compromising public safety. The FDA can continue to conduct an adequate evaluation while industry can obtain more timely clearance for new food contact substances. The fixed 120-day review period enables businesses to plan their marketing and supply strategies. The FDA clearance of the FCN is proprietary to the notifier, with competitors having to make separate notifications. Hence the original notifier has a business advantage

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Table 14.4 Minimum safety studies for US pre-marketing notification for indirect food additives CDC

Test requirements

CDC < 0.5 ppb

no testing (but a discussion of potential carcinogenicity)

CDC > 0.5 ppb but < 50 ppb

Ames test and preferably an in vitro mouse lymphoma assay (or alternatively an in vitro chromosome aberration test), with the discussion of potential carcinogenicity

CDC > 50 ppb but < 1 ppm

as for the CDC < 50 ppb plus an in vivo chromosome aberration test and subchronic oral toxicity studies in rodents and non-rodents to decide if further long-term or specialised studies are needed

and protection of their expensive and time consuming test data. The clearance of the FCN extends to the notifier’s customers, who only have to have a ‘paper trail’ to record that they have used a notified substance to manufacture the food packaging. The whole process becomes much quicker and more efficient, with considerably less burden on the FDA. The pre-submission meeting with FDA officials to plan the FCN does become even more important however, to make sure adequate chemistry and toxicology information will be included to enable the review to be completed within the 120 days. Industry will have to learn the new FCN procedures, making use of the new FCN form and guidelines to prepare high standard ‘reviewer friendly’ notifications. The exact wording of the FCN acknowledgement and clearance letters will have to be considered carefully, and negotiated with the FDA, since these define the notifier’s proprietary rights and will probably be used to inform customers. Finally, there will be a need to educate customers about the new FCN scheme, because they have been used to relying on Regulations published in 21 CFR to establish FDA compliance. They will now need to verify a substance has been notified by using the FDA web site or from the FDA final letter to the notifier. They will also need to understand that there may be multiple notifiers for the same substance, perhaps with differing clearances granted to the various suppliers. The new FCN scheme to approve most starting substances and additives for use in food packaging materials in the USA will be of great benefit to industry in greatly speeding up FDA approval and offering protection against competitors. The FDA will have a considerably reduced administrative burden, to enable work to be focused on areas of greater potential concern. Above all, however, public health will not be compromised.

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References 1.

Council Directive 89/109/EEC of 21 December 1988, Official Journal of the European Communities, 11:2:89, L40, 38.

2.

Commission Directive 80/590/EEC of 9 June 1990, Official Journal of the European Communities, 19:6:80, L151, 21.

3.

Council Directive 82/711/EEC of 18 October 1982, Official Journal of the European Communities, 23:10:82, L297, 26.

4.

Commission Directive 93/8/EEC of 15 March 1993, Official Journal of the European Communities, 14:4:93, L90, 22.

5.

Commission Directive 97/48/EC of 29 July 1997, Official Journal of the European Communities, 12:8:97, L222, 10.

6.

Council Directive 85/572/EEC of 19 December 1985, Official Journal of the European Communities, 13:12:85, L372, 140.

7.

Corrigendum to Commission Directive 2002/72/EC of 6 August 2002, relating to plastic materials and articles intended to come into contact with foodstuffs (OJL 220 of 15/8/2002) Official Journal of the European Communities, 31:02:03, L39, 1 which consolidates and supercedes Commission Directive 90/128/EEC, as amended.

8.

Synoptic Document Provisional List of Monomers and Additives notified to European Commission as Substances which may be used in the manufacture of Plastics intended to come into contact with Foodstuffs, European Commission, Brussels, Belgium, latest version.

9.

Food Contact Materials Practical Guide, European Commission, Brussels, Belgium, latest version.

10. Food Contact Materials Note for Guidance, European Commission, Brussels, Belgium, latest version. 11. Guidelines of the Scientific Committee on Food for the presentation of an application for safety assessment of a substance to be used in food contact materials prior to its authorisation, European Commission Scientific Committee on Food, Brussels, Belgium, 8 December 2000, reference SCF/CS/PLEN/GEN/90 Final.

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Regulation of Food Packaging in the EU and US 12. Annex V of Council Directive 67/548/EEC of 27 June 1967 as amended and adapted to technical progress. 13. OECD Guidelines for the Testing of Chemicals, OECD, Paris, France, 1993, as updated. 14. Commission Directive 93/10/EEC of 15 March 1993, Official Journal of the European Communities, 17:4:93, L93, 27. 15. Council Directive 84/500/EEC of 15 October 1984, Official Journal of the European Communities, 20:10:84, L277, 12. 16. Council Directive 78/142/EEC of 30 January 1978, Official Journal of the European Communities, 15.02.78, L44, 15. 17. Commission Directive 80/766/EEC of 8 July 1980, Official Journal of the European Communities, 16.08.80, L213, 42. Commission Directive 81/432/EEC of 29 April 1981, Official Journal of the European Communities, 24.6.81, L167,6. 18. Commission Directive 93/11/EEC of 15 March 1993, Official Journal of the European Communities, 17.04.93, L93, 37. 19. Commission Directive 2002/16/EC of 20 February 2002, Official Journal of the European Communities 22:02:02, L51, 27. 20. References of the European and National Legislation, European Commission, Brussels, Belgium, latest version. 21. R. Ashby, I. Cooper, S. Harvey, P. Tice, Food Packaging Migration and Legislation, Pira International, Leatherhead, UK, 1997. 22. Resolution AP(89)1 on colourants in food contact plastics, Council of Europe, Strasbourg, France, 1989. 23. Resolution AP(92)2 on aids to polymerisation for food contact plastics, Council of Europe, Strasbourg, France, 1992. 24. Resolution AP(96)5 on surface coatings, Council of Europe, Strasbourg, France, 1996. 25. Resolution AP(97)1 on ion exchange and absorbent resins used in food processing, Council of Europe, Strasbourg, France, 1997.

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Practical Guide to Chemical Safety Testing 26. Resolution AP(99)3 on Silicones, Council of Europe, Strasbourg, France, 1999. 27. Guidelines concerning the safety evaluation of substances to be used in food contact materials and articles covered by Council of Europe Resolutions, Council of Europe, Strasbourg, France, latest version. 28. Guidance for Industry Preparation of Food Contact Notifications: Administrative, US Food and Drug Administration, Washington DC, USA, May 2002. 29. Red Book II (Draft): US Food and Drug Administration Center for Food Safety and Applied Nutrition, 1993 (www.cfsan.fda.gov/~redbook/red-toca.html). 30. Guidance for Industry Preparation of Food Contact Notifications and Food Additive Petitions for Food Contact Substances: Chemistry Recommendations, US Food and Drug Administration, Washington DC, USA, April 2002. 31. FDA, HSS, Threshold of regulation for substances used in food contact articles, Federal Register, 21 CFR 170.39, July 17, 1995. 32. FDA, Federal Food, Drug and Cosmetic Act, US Code Title 21 – Food and Drugs, 9(IV) Section 348, 1938, as amended. 33. Guidance for Industry Preparation of Food Contact Notifications for Food Contact Substances: Toxicology Recommendations, US Food and Drug Administration, Washington DC, USA, April 2002. 34. Harmonised Tripartite Guidelines, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.

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15

Regulation of Biocides Derek J. Knight and A. Mel Cooke

15.1 Introduction Biocidal products are preparations, containing one or more active substances, that control harmful organisms by chemical or biological means. They protect health, improve product performance and prevent spoilage, and are increasingly important to modern life in ensuring safe, long-lasting and effective products. Biocides encompass a wide range of applications including disinfection, preservation and pest control. The worldwide biocides market has an annual sales value of up to £3 billion, with a growth rate currently of ca. 4% per annum. The major user is North America, followed by Europe then Japan. The biocides industry involves mainly small and medium-sized businesses, delivering its products to the end user by a complex supply chain. Biocidal active substances may also be used in plant protection products, so active ingredient manufacturers can be involved in both types of product. Biocides are intended to be toxic, but only to the target organism. Such biologically active chemicals potentially pose a risk to humans and the environment. Hence biocides are one of the most tightly regulated chemical products. The risk from a biocide is determined from its hazardous properties, and the likely exposures of humans and the environment throughout its life-cycle. Such an in-depth science-based risk assessment gives a realistic estimation of the potential impact of a biocide. However, this is a time consuming process that can delay regulatory approval, which can be costly to industry. Different regulatory jurisdictions (e.g., the EU and the USA) have different definitions for biocides and varying requirements for their registration (the process which a product must undergo to allow it to be marketed). For example, some countries consider disinfectants and sanitisers as biocides, but in others they are regulated as medicines or general chemicals. As a result, the regulatory pattern is complex, with many countries having different regulatory bodies for different biocidal product types. The European market is fragmented, with different regulatory requirements in different EU Member States. In the EU, the Biocidal Products Directive (BPD) will gradually

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Practical Guide to Chemical Safety Testing supersede the various current national schemes and harmonise the requirements for the registration of biocidal active ingredients and their formulations, reducing barriers to trade, and improving levels of protection of humans and the environment. In the USA, biocidal products are covered by the same regulations as agricultural pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). With the passage of the Food Quality Protection Act (FQPA) in 1996, registration of antimicrobial pesticides in the USA has become more complex. The act stipulates that no pesticide will cause unreasonable adverse effect on man or the environment, and that it is safe for sensitive subpopulations, such as children. Furthermore, the risk assessment also takes into account the effect of active substances with a comparable mode of action. The US FQPA, as with the European comparative assessment, may mean that an application is assessed against competitive products during the authorisation procedure. These various aspects of worldwide biocide regulation, including a description of some national schemes, and well-established chemical notification schemes that are used for biocide control, are covered in detail in a recent book [1]. Here we concentrate chiefly on those jurisdictions with specific biocide legislation of major commercial importance, namely the EU and the USA.

15.2 Control of Biocides in the EU

15.2.1 Introduction The outlook for the biocides industry transformed dramatically with the implementation by 14 May 2000 of the Biocidal Products Directive (BPD) [2]. The two main intentions of the BPD are to harmonise the EU internal market for biocides and to ensure a high level of protection for humans and the environment. The BPD impacts manufacturers, distributors and users of biocides. It is more demanding than any prior legislation, and may cost the industry over £350 million. Active substance suppliers have to co-operate to register biocidal products. The industry is concerned that the cost of testing of active substances will stifle innovation and lead to the withdrawal of many existing products. The BPD fills a gap in EU legislation. In some EU Member States, most biocides were covered only by EU general chemical regulation, notably the Dangerous Substances Directive [3] for substances, and the Dangerous Preparations Directive [4] for formulated products, which meant that new substances for biocide use had to be notified according to the ‘Seventh Amendment’ scheme [5]. Some EU countries, in particular Belgium, 67/548/EEC 1999/45/EC 402

Regulation of Biocides Denmark, Finland, the Netherlands, Sweden and the UK, did have systematic and comprehensive national controls of some types of biocidal products. The BPD replaces these national schemes and harmonises the authorisation of biocidal products within each Member State, while the active substances are evaluated at EU level. Many of the existing national or other EU provisions will continue to apply for several years for existing active substances (those on the market before 14 May 2000) and biocidal products containing them, until the various aspects of the Directive come into force. The timetable for this transition will depend on the progress made with the evaluation of existing active substances. Note that new active substances will have to be approved under the BPD scheme before being marketed.

15.2.2 Main Features of the Directive The main provisions of the BPD are as follows: •

Establishment of a positive list of biocide active substances approved for use in the EU, with any restrictions, as Annex I of the Directive. Active substances for low risk biocidal products will be in Annex IA and basic substances (i.e., commodity substances with minor biocide use) will be in Annex IB.

•

National authorisation of biocidal products containing active substances on the positive list. Low risk biocidal products can be registered with a simplified technical dossier and basic substances can be marketed without application.

•

Automatic acceptance (mutual recognition) by Member States of biocidal products authorised or registered in other Member States, subject to certain safeguards.

•

New products within a frame formulation (i.e., with only minor changes to an approved product) can be authorised within 60 days.

•

There will be a 10-year review programme to evaluate existing active substances.

15.2.3 System of Approval Biocidal products containing new active substances will require two regulatory submissions, the first on the active substance, and the second on the formulated biocidal product. If the dossiers show that the product can be used safely, the active ingredient will then be approved for use in one or more of the 23 product types (Table 15.1).

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Table 15.1 Product types according to the EU Biocidal Products Directive Main Group 1: Disinfectants and general biocides Product types: 1. Human hygiene products 2. Private and public health area disinfectants 3. Veterinary hygiene biocides 4. Food and feed area disinfectants 5. Drinking water disinfectants

Main Group 3: Pest control Product types: 14. Rodenticides 15. Avicides 16. Molluscicides 17. Piscicides 18. Insecticides, acaricides and products to control other arthropods 19. Repellents and attractants

Main Group 2: Preservatives Product types: 6. In-can preservatives 7. Film preservatives 8. Wood preservatives 9. Fibre, leather and polymerised materials preservatives 10. Masonry preservatives 11. Preservatives for liquid cooling systems and processing 12. Slimicides 13. Metal-working fluid preservatives

Main Group 4: Other biocides Product types: 20. Preservatives for food or feedstocks 21. Antifouling products 22. Embalming and taxidermist fluids 23. Control of vertebrates

The biocidal product is assessed for adverse effects on human or animal health, efficacy, humaneness on target organisms, and environmental impact, especially considering its fate and distribution and effect on groundwater. Some criteria for the assessment of biocidal products are given in the Common Principles for the Evaluation of Dossiers in Annex VI of the BPD. The overall assessment includes a risk assessment for the intended use(s) and a reasonable worst-case scenario. A technical note for guidance (TNG) detailing the risk assessment and decision-making processes for approval of biocidal products is available to supplement the framework of Annex VI [6].

15.2.4 Assessment for the Inclusion of Active Substances in Annex I of the Biocidal Products Directive The Scientific Committee on Biocidal Products will assess the scientific content of the dossier on behalf of the Commission. An approved active substance will be listed in

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Regulation of Biocides Annex I for up to 10 years, with renewals being granted for up to 10 years. The inclusion of an active substance in Annex I will be restricted to those product use types (Table 15.1) for which adequate data have been submitted. Low risk active substances, defined as those that are not carcinogenic, mutagenic, toxic for reproduction, sensitising, or both bioaccumulative and not readily biodegradable, will be listed in Annex IA. There is a TNG on evaluation of active substances for Annex I listing [7]. A new active substance for use in biocidal products can only be placed on the EU market if an application for Annex I listing has been made. Furthermore, the substance has to be labelled as being for biocide use.

15.2.5 Authorisation of Biocidal Products Authorisation of the formulated biocidal products is the responsibility of the competent authorities of individual Member States. Full authorisation of the product may be granted only if its active substance is listed in Annex I, IA or IB. Biocidal products containing active substances in Annex I of the BPD require authorisation, while those containing low risk active substances from Annex IA can undergo faster registration with much reduced information. Biocidal products containing a new active substance for which a decision for Annex I listing is pending may be provisionally authorised for up to three years. New products containing existing active substances can be authorised under existing national schemes for up to ten years during the review program. The previous national schemes continue to apply to biocidal products containing existing active substances until the Commission makes a decision on whether to include that active substance into Annex I. If the listing in Annex I is not approved, the biocidal products containing them will be subject to a phase-out period. If the active substance is approved the existing biocidal products will have to be re-authorised by the Member States. Under the BPD, applicants may adopt a frame formulation to facilitate authorisation of a group of similar biocidal products. The biocidal products in the frame formulation must have the same use pattern, technical grade of active substance, and only minor differences in composition, such as in the colouring and perfume ingredients. New biocidal products within a frame formulation can be authorised within 60 days. Once one Member State approves a biocidal product, other Member States must approve the product, subject to certain safeguards, according to the principle of mutual recognition. Thus a Member State receiving an application for a biocidal product that has been authorised or registered in another Member State must approve that product within 405

67/548/EEC 1999/45/EC Practical Guide to Chemical Safety Testing 120 or 60 days, respectively. The Member State can refuse the application only if the target species does not exist in that country, there is proven unacceptable resistance to the active substance, or if the circumstances (e.g., climate or breeding period) differ significantly from the lead country. The Standing Committee on Biocidal Products will resolve any dispute between Member States. Member States may opt out of the mutual recognition procedure for avicide, piscicide and vermin-control biocidal products.

15.2.6 Hazard Communication The classification, packaging and labelling of biocides will be the same as for general industrial chemicals, except for insecticide, acaricide, rodenticide, avicide and molluscicide products which are currently covered under a separate scheme [8]. Neat active substances are classified, packaged and labelled according to the Dangerous Substances Directive [3], while biocidal formulations are covered by the Dangerous Preparations Directive (modified for biocidal products from 30 July 2004 [4]). Safety data sheets for biocide active substances and products are also required as for chemicals. Biocidal products classified as very toxic, toxic, or as category 1 or 2 carcinogens, mutagens or reproductive toxicants cannot be made available to the public.

15.2.7 The Review Programme for Existing Active Substances Annex I is currently empty. It will be added to by a few new active substances, but mainly through the evaluation of active substances that were on the market before 14 May 2000 (existing active substances). The European Chemicals Bureau (ECB) produced an unofficial and indicative list of around 1500 existing active substances [9], when corrected for duplicate entries. Existing substances and products containing them will benefit from the transitional arrangements of the BPD, being allowed to be marketed during the evaluation period. Conversely, any substance not on the final list of existing active substances will be considered new and, together with biocidal products containing it, will have to meet the various provisions of the BPD before it can be marketed. Hence it is commercially advantageous for industry to ensure their active ingredients qualify as existing. The process for producing the definitive list of existing active substances is underway. The first Review Regulation [10] came into force on 28 September 2000, and establishes the procedures to nominate existing active substances, determine whether they will be supported for full review for Annex I listing, and provides a framework for phasing out unsupported active substances and the biocidal products containing them. It also schedules the full review of the active substances for wood preservatives and rodenticides (phase

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Regulation of Biocides 1). A second Review Regulation will give the definitive list of identified and notified existing active substances, and will list the substances with the rapporteur Member States for the first and second stages of the review. The deadlines for submission of full dossiers are 28 March 2004, 30 April 2006, 31 July 2007 and 31 October 2008 for the four phases. The first Review Regulation describes a two-tier process for nominating active substances as existing. The lesser ‘identification’ consists of commercial, technical and administrative data which had to be submitted to the ECB by 28 March 2002. Around 2700 identifications were made covering 759 active substances. The draft list is on the ECB web site (http://ecb.ei.jrc.it/biocides), and the definitive list will be in the second Review Regulation. Identified, but non-notified, active substances will be phased out before 1 September 2006. Substances that are neither notified nor identified cannot be used unless approved as a new substance. By proceeding with the more involved ‘notification’ process, the applicant indicates their intention to support the active ingredient through full review for Annex 1 listing. The information required for notification, comprises the identification data plus summaries of the physico-chemical, toxicological and ecotoxicological studies corresponding approximately to the EU ‘Base Set’ for notification of a new chemical substance [5]. The notification should have been sent to the ECB, again by 28 March 2002, using the electronic format called the International Uniform Chemicals Information Database (IUCLID). A Prolongation Regulation 1687/2002 [11] allows for notification of existing active substances which were only identified and further product types of notified substances to be added by 31 January 2003. The exhaustive list of existing active substances will specify notified active substances by product types. The notified existing active substances can continue to be marketed in products until they are reviewed, but only for the listed product types. The notifier is committed, unless exceptional circumstances apply, to submit all the information needed for full review. As the first phase of the review programme, successful notifiers for active substances for wood preservatives and rodenticides have to submit a full dossier to the designated rapporteur Member State by 28 March 2004.

15.2.8 Technical Guidance Four Expert Working Groups (EWGs) will develop TNGs to cover toxicology, ecotoxicology, environmental fate and behaviour and general issues. Two other EWGs will examine data waiving and scope issues. The scope EWG aims to define which products are covered by the BPD, and has drafted documents on the potential overlap of the BPD with plant protection products, human and veterinary medicinal products, and cosmetics.

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Practical Guide to Chemical Safety Testing However, further work is needed to deal with cosmetic products, food additives, and treated articles and to cover the definition of basic substances. Successfully notified active substances will be evaluated in a phased timetable. The remainder of the BPD Annex IIA common core data will be required, perhaps along with additional data from Annex IIIA. The applicant also has to include a dossier for at least one biocidal product containing the active substance, which contains the necessary common ‘core’ data of Annex IIB and appropriate additional data from Annex IIIB. Criteria for their selection are covered in a Technical Note for Guidance (TNG) on data requirements [12]. Studies should be GLP-compliant, and conducted to the standard EU methods [13], or OECD Guidelines [14], where such guidelines are available. Any studies which are not necessary or unfeasible can be omitted, but the applicant has to supply justification. The acceptability of these ‘data waivers’ may vary between national competent authorities, and causes uncertainty in the evaluation process. General decisions on successful data waiving may be published by the ECB in a non-confidential ‘Manual of Decisions’. The key TNGs on data requirements [12], the inclusion of active substances in Annexes I [7], and the supporting document for the ‘Common Principles’ of Annex VI for the authorisation of biocidal products [6] have been issued in draft form with final adoption after a period of practical use. Practical guidelines for the structure, presentation and formatting of complete dossiers have been developed [15]. The so-called ‘all in one’ approach harmonises the format of the summary dossier and the competent authority report. The structure of the dossier includes study summaries, summary tables, and completeness checks to make the assessment process easier. Dossiers will probably be in English, although Competent Authorities can ask for them in their own language. A revised technical guidance document for risk assessment to include biocide active substances has been developed from the current guidance for chemical substances [16]. Exposure is a key area of risk assessment, and the EUBEES (EU biocide emission scenarios) project is underway to address this. There is a corresponding project on emission scenarios and predictive exposure models for human health. Finally, the Danish and Finnish Competent Authorities have validated the evaluation procedure in a pilot project on the full review dossiers for the active substances tebuconazole (as a wood preservative) and glutaraldehyde (as a paper slimicide and preservative for cooling water systems) [17].

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15.3 Control of Biocides in the USA

15.3.1 Introduction The basic national pesticide law in the USA is the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) [18], administered by the Environmental Protection Agency (EPA). The EPA make a statutory risk/benefit analysis on the product before authorising its sale. The regulations implementing FIFRA, including the data requirements, are contained in Parts 150 to 189 of Title 40 of the Code of Federal Regulations (CFR) [19]. Other information is available in the form of advisory documents issued by the EPA. FIFRA was enacted in 1947 and last amended in October 1988. The amended Act contains a major section on the re-registration of pesticides. All pesticides containing an active ingredient first registered prior to 1 November 1984 will be re-registered in 5 phases over a 9 year period. Pesticides are to be re-registered every 15 years. In addition, new regulations were included on storage, disposal, transportation and recall of pesticides. FIFRA also covers pesticide labelling, post-regulation obligations (such as reporting requirements and generation of new data to support continued registration) and pesticide export and import. The other statute regulating pesticides, including biocides, in the USA is the Federal Food, Drug and Committee Act (FFDCA) [18], which sets forth the criteria for legal limits for residues of pesticides used on food crops, and also regulates food additive and food contact substances. The Act is administered jointly by the EPA and the Food and Drug Administration (FDA). The two most recent amendments of FIFRA are the Food Quality Protection Act (FQPA) [18] of 1996 and the Antimicrobial Reform Technical Corrections Act (ARTCA) [20] of 1998. The FQPA amends the risk assessment of pesticide residues which may be present on food, and has also impacted the assessment of food contact biocides. Before an EPA registered pesticide can be sold, state approval is also required. Most states issue regulations but do not review data, with notable exceptions such as California, New York, Florida and Massachusetts, which require submissions comparable to FIFRA, and make regulatory decisions independent of the EPA.

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15.3.2 Data Requirements for Registration Part 158 of 40 CFR [19] includes a matrix relating data requirements to the nine general use patterns of the pesticide. Within the matrix, tests are specified as being required, conditionally required or not required. The regulations indicate whether the testing should be conducted on the manufacturing use product (MP), the technical grade of the active ingredients (TGAI) or the end-use product (EP). For most products a minimum set of data is required irrespective of the general use pattern. Pesticide Assessment Guidelines [21] give guidance in the design and validation of suitable protocols for testing. Standard Evaluation Procedures [22] give guidance on the scientific procedures to be used during the evaluation of the data by the authorities. Studies are normally conducted to the Office of Prevention, Pesticides and Toxic Solution’s Guidelines [23], although OECD guidelines [14] are acceptable providing that the requirements of CFR Part 158 are satisfied where these are more restrictive. The studies normally have to be GLP compliant. Information for a new active ingredient typically includes data on product chemistry, acute, subchronic and chronic toxicity, human exposure, environmental fate, ecotoxicity, and residues, when registered for a food use. The toxicology data typically required for biocides, including antimicrobials (both food and non-food contact) that have a potential for high exposure (e.g., used in swimming pools, drinking water, and outdoor aquatic applications), is referred to as a full ‘CORT’ (chronic, oncogenicity, reproductive and teratogenicity) database. As a starting point, the data tables in 40 CFR Part 158 [19] indicate specific data requirements, but these 1984 tables are, in practice, out of date, so a ‘pre-registration’ meeting with EPA is highly desirable to finalise the data package. Data requirements for some antimicrobial pesticides differ from other biocides and agricultural pesticides. Since 1987 the EPA has used a tiered approach for toxicology data requirements for these products, dependent on the potential exposure: those with high exposures have the same data requirements as other pesticides, whereas those with low-exposures require minimum Tier 1 data. For low exposure, food contact uses, e.g., surface sanitizers and slimicides used in manufacturing food contact paper products, the Tier 1 toxicology data set includes an acute toxicity battery (acute oral, dermal and inhalation toxicity; eye and dermal irritation and dermal sensitisation), two subchronic (90-day) oral toxicity studies, teratogenicity (one species), a mutagenicity battery and a two-generation reproductive toxicity study. When the residue concentrations from indirect food contact use exceed 200 ppb in food, the full CORT toxicity database is required. Fewer toxicity data are needed for low exposure, non-food contact uses of antimicrobials, e.g., non-food contact material preservatives. The Tier 1 database includes an acute

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Regulation of Biocides toxicity battery, one 90-day study (usually by the most common route of exposure), teratogenicity (one species) and a mutagenicity battery. In general, follow-on registrations of an active ingredient require the same data as the original registration. The new registrant may generate an additional, complete data package or offer compensation to the original data submitter (see Section 15.3.4). Product specific data necessary to support each new end-use formulation include product chemistry data and acute toxicity data. The EPA does not normally consider efficacy of pesticides but they can elect to review this information on giving 15 days notice. It is the registrant’s responsibility to ensure that the product performs in accordance with its labelling claims. Efficacy data however, are routinely required for products with labels that make public health claims, such as antimicrobial and vertebrate biocides and some mammalian repellents. Insecticides and other biocides that make public health claims may also be required to submit efficacy data, although EPA has not consistently required efficacy data for these products. Even when EPA does not require submission of efficacy data, such data must be generated by the registrant, maintained in its files and submitted to EPA upon request.

15.3.3 Registration Applications The EPA separates pesticides into two general categories: conventional chemical pesticides and biochemical and microbial pesticides. The EPA further categorises pesticide applications as new chemical, new use or ‘me-too’ applications. The application for registration of a conventional chemical pesticide should include the information specified in Table 15.2 as applicable. It should be noted that the source of active ingredient used must be registered, otherwise the applicant must provide as a minimum, product chemistry data on the TGAI as well as the formulated product. Pesticide registrations are granted for 5 years. If additional data are required to support an existing registration, a list is sent to relevant parties who then have to provide evidence within 90-days that they are taking appropriate steps to secure the additional data. The registration can be suspended if they fail to comply. Applicants are encouraged to share data and any costs involved. A notice of application is published in the Federal Register for registration of any pesticide if it contains a new active ingredient or would entail a change in use pattern. The notice provides for a period of 30 days in which any Federal Agency or interested person may

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Table 15.2 Information needed for US registration of a pesticide • Application of Registration form • For non-US applicants, a US agent to act on their behalf • Confidential Statement of Formula form • Product Label • Registration Data Matrix form • Certification with Respect to Citation of Data form • Certification relating to child-resistant packaging • Restricted use classification (for use of the pesticide by Certified Applicators) • Registration standards • Petition for a tolerance (where applicable)

comment. If the EPA refuse registration, the applicant has 30 days to correct the application. The reason that registration has not been granted will be published in the Federal Register. Each end-use pesticide formulation also has to be registered prior to distribution or sale. Usually the formulated product comprises one or more registered active ingredient and one or more excipients. The application package consists of administrative forms, labelling and product specific data, together with Application for Registration and Confidential Statement of Formula forms, which are the same as used for an active ingredient registration (see Table 15.2). When a formulator purchases a registered active ingredient for use in an end-use formulated product, they can obtain a registration without offering to compensate the original data submitter by the so-called Formulator’s Exemption. If the formulator of the end-use product is also the registrant of the active ingredient, EPA does not permit use of the Formulator’s Exemption. The applicant must instead complete Certification with Respect to Citation of Data and Data Matrix forms, which must be consistent with those submitted for the active ingredient registration. Alternatively, a registrant using an active ingredient from an unregistered source in the product formulation is responsible for satisfying all generic data requirements for the active ingredient, either by providing new studies or by offering to compensate the original data submitter (see Section 15.3.4).

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Regulation of Biocides The most common end-use registration is known as a ‘me-too’ registration. The formulation must be substantially similar to another registered pesticide product in composition, use patterns and use directions. The EPA evaluate substantial similarity for composition by comparing the Confidential Statements of Formula submitted by the new and previous applicants. They must then notify an applicant within 45 days on the completeness of the ‘me-too’ application, and 90 days thereafter whether a complete application has been approved. Applications for a technical or manufacturing use product do not qualify for me-too status, because, at a minimum, the EPA must assess manufacturing process data, batch analyses and physical and chemical characteristics, which thus precludes expedited processing. After registration, a registrant can amend the product’s formulation or the labelling, by applying for an amendment of the registration using a Confidential Statement of Formula or draft revised labelling. Extra test data may be required, depending on the nature of the amendment. Alternatively, some limited labelling, formulation or other changes can be made by notifying the EPA. Such changes can be implemented immediately, except for any change in the labelling of an antimicrobial pesticide, which is subject to a maximum 60-day waiting period. Finally, minor corrections to the label can be made without even notifying the EPA.

15.3.4 Data Compensation Although data submitted under FIFRA may be disclosed to the public after 30 days, there are limitations on another applicant’s right to cite these data. In 1978, an amendment to FIFRA granted ‘exclusive use’ rights, for a 10-year period, to the original data submitter for certain data that were submitted to support the first registration of a product containing a new active ingredient. Where the first registration was granted after 30 September 1978, these data are termed ‘exclusive use’ and the EPA may not use these data for a period of 10 years after the date of initial registration, unless the second applicant has written authorisation from the original data submitter. After the 10 year exclusive use period, the second applicant can cite the original data without authorisation, but must offer to compensate the data submitter for a period of 15 years from the original submission.

15.3.5 Re-Registration of Existing Pesticides Congress amended FIFRA to require EPA to re-register (re-evaluate) all pesticide products containing any active ingredient registered prior to 1984. The re-registration process

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Practical Guide to Chemical Safety Testing was to be completed by fiscal year 1997, but has now been postponed until 2006. EPA was directed to ensure the study data supporting pesticide registrations were brought up to the current standards, i.e., the now out-of-date 1984 40 CFR 158 data requirements [19] ‘Data call-ins’ were issued for the active ingredients that did not meet these 1984 requirements. In order to improve the efficiency of the registration and re-registration processes, the EPA issued registration standards (a statement on all formulated pesticide products containing the same active ingredient). To obtain a new registration or re-registration, the product must comply with the relevant standard. When a product does not comply with the standard, the applicant must either change the product so that it does comply, or request an amendment to the standard to bring the product into compliance. Each standard consists of two parts: one covering manufacturing use products, and the other covering formulated end use products. Each part of the standard contains the following elements: •

EPA’s regulatory position based on a review of available data on an active ingredient and its formulations.

•

An explanation of the regulatory position.

•

Identification of the data reviewed and the EPA’s conclusions on their significance.

•

Tolerances for residues on food and feed (when appropriate).

•

A bibliography of all the data considered by the Agency in developing the standard.

When insufficient data were available on a group of products, an interim standard was issued, and a schedule for the additional data required was included in the regulatory position. Once submitted, the interim standard was amended, and re-issued as a completed standard. Also, data call-in notices indicated the data gaps, especially long-term toxicology studies, which must be filled by registrants before a registration standard could be prepared. It was apparent that the EPA would not complete its re-registration work on time, and FIFRA was amended in 1988 to facilitate re-registration. Under FIFRA 1988, the 600 cases undergoing re-registration were divided into four lists: the first were those which had already had registration standards issued, and the other three were ranked according to human exposure. FIFRA 1988 included a timetable to complete the processes of the reregistration, e.g., declaration of intent, identify studies, data call-in, and summarise studies, which culminate in the publication of a Re-registration Eligibility Decision (RED) stating the completeness of the database and the eligibility of the pesticide for re-registration. Those pesticides that already had a registration standard went straight to the RED process,

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Regulation of Biocides since they had already been substantially reviewed by the EPA. The RED therefore effectively replaced the registration standard. The EPA evaluates the risks posed by each active ingredient and its use to make a reregistration decision. Active ingredients may not qualify for re-registration, or in other instances some uses of a particular active ingredient may not be registered. Frequently, risk mitigation measures are imposed during re-registration. The EPA review of the use, chemistry, toxicology, and environmental impacts, and the resultant risk assessments are published in a RED document. Thereafter, each end-use formulation containing the active ingredient also must be re-registered, which involves updating product-specific chemistry, toxicity and where required, efficacy data, and revising the label to be consistent with the decisions and limitations in the RED. The FQPA requirement for procedures for the periodic 15 year review of existing registrations are under development.

15.3.6 Petition for a Pesticide Tolerance A tolerance is a legal maximum residue concentration of a pesticide chemical allowed in food or animal feed. Tolerances are set under the authority of Section 408 of the FFDCA [18], and have been changed by the FQPA [18]. Before a pesticide can be registered for food use, a tolerance or the exemption from the requirement for a tolerance must be established. A pesticide tolerance petition has to contain the information specified in Table 15.3. Usually a petition request is accompanied by an application for registration, although an import tolerance generally would not require one. The FQPA requires the EPA to re-assess all existing tolerances and exemptions from tolerances by 2006. Re-registration (see Section 15.3.5) is therefore being conducted in conjunction with the tolerance re-assessment activities. As a result, all re-registration decisions involving pesticides regulated under both FIFRA and FFDCA now must take into consideration the new risk assessment criteria established by the FQPA, namely application of an uncertainty factor of ten (which may be altered); aggregation for a single active ingredient from all sources (e.g., diet, drinking water); and cumulative risk assessments when a group of active ingredients has a common mechanism of toxicity. The FQPA also requires the evaluation of the pesticide for endocrine disrupter effects.

15.3.7 Regulation of Food Contact Biocides Section 409 of the FFDCA [18], which is administered by the FDA, regulates ‘food additives’, i.e., substances reasonably expected to be a component of food as a result of 415

Practical Guide to Chemical Safety Testing their intended use. A food additive petition may be required for a biocide proposed for a food contact use, or alternatively a food contact pre-market notification may be suitable. The FQPA [18], owing to an inadvertent drafting error, transferred jurisdiction for antimicrobials, historically regulated as food additives by the FDA, to the EPA as ‘pesticide chemical residues’ subject to regulation under Section 408 of the FFDCA. In 1998 ARTCA [20] was enacted, and partially restored jurisdiction for food contact antimicrobial pesticides to the FDA, while also continuing EPA’s regulatory authority for these substances. The result is a complex scheme of both separate and shared responsibility for antimicrobial food contact uses between EPA and FDA (see Table 15.4).

Table 15.3 Information required for a US pesticide tolerance petition • Identity and composition of the pesticide, manufacturing process, analysis of the active ingredient, certified limits for the ingredients and analytical methods. • Amount, frequency and time of application. • Full reports on safety of the pesticide (according to 40 CFR Part 158.340 [19], which identifies the required toxicity data). • Results of residue tests. • Methods for removing residues that exceed any proposed tolerance. • Proposed tolerances. An exemption can be granted when the total quantity of the pesticide in all raw agricultural commodities will involve no hazard to human health. • Reasonable grounds in support of the petition.

Table 15.4 Examples of antimicrobials regulated in the US by EPA and FDA Antimicrobial applied to water that contacts food in food processing

Dually regulated by FDA as a food additive under FFDCA Section 409 and by EPA as a pesticide under FIFRA

Antimicrobial preservative in an article with food contact (e.g., a plastics preservative incorporated into equipment used in food processing)

Dually regulated by FDA as a food additive under FFDCA Section 409 and by EPA as a pesticide under FIFRA

Dually regulated by FDA as a food additive Antimicrobial used in food packaging material (e.g., slimicides used in paper mills under FFDCA Section 409 and by EPA as a pesticide under FIFRA and preservatives in food contact paper coatings)

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15.4 Regulation of Biocides in Other Countries The regulation of biocides is very varied in those countries with developed chemical control schemes. Many countries do not recognise biocides as a separately identifiable group of chemicals, and often there is no legal definition. Biocides, as recognised in the EU, therefore often fall under a mix of legislative schemes intended for industrial chemicals, cosmetics, agricultural pesticides, veterinary products and pharmaceuticals. Each scheme has its own detailed requirements and is administered by different authorities. Often, there are difficult issues of scope between these different schemes, so that an insect repellent used on skin may be viewed as a cosmetic, a biocide, an industrial chemical or as a medicine. These details, while important, are extremely complex, and the information is difficult to ascertain for most countries. Some major markets such as Japan have no specific scheme for biocide regulation, and they are treated as ordinary industrial chemicals (see Chapter 10). Other countries have rigorous schemes, but applied only to certain biocide use categories such as wood preservatives, public use disinfectants or rodenticides, either through special schemes, or through legislation aimed at other chemical categories such as pesticides. The EU BPD has led towards harmonisation within the EU but there has been a knock-on effect in other countries, such as Switzerland and Central Europe (Macedonia, Slovenia, Poland, Hungary and Czech Republic), which have taken steps to harmonise their existing chemicals regulations to the BPD. Some of these central European countries are preparing for future expansion of the EU, while Switzerland aims to reduce trade barriers with the EU. The BPD is seen as the most rigorous of the regulatory regimes, so that a supplier of biocides in compliance with the BPD will have few extra data requirements in other jurisdictions. However, the OECD have published a useful review of biocide regulation in OECD countries [24]. The OECD strives to harmonise requirements for biocide registration amongst its membership of 30 industrialised nations and beyond, through the initiation of a biocides programme, which takes many of the BPD requirements and integrates them with the US biocides regulations. The OECD programme will attempt to bring the requirements for biocides more in line with those for pesticides and pharmaceuticals. Although the OECD is composed of so-called developed countries, developing nations are involved to ensure that OECD policies do not have a negative impact upon non-member states. Therefore, there is a tendency for non-member states to abide by OECD decisions.

Acknowledgements The authors are grateful for information on control of biocides in the USA from Chapter 4 of reference [1] by Sue Crescenzi and for comments on Section 15.2 on the BPD from Dr Sara Kirkham of Safepharm Laboratories Ltd. 417

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References 1.

D.J. Knight and M. Cooke, The Biocides Business: Regulation, Safety and Applications, Wiley-VCH, Weinheim, Germany, 2002.

2.

Council Directive 98/8/EC of 16 February 1998, Official Journal of the European Communities, 24:4:98, L123, 1.

3.

Classification, packaging and labelling of dangerous substances in the European Union, European Commission, Luxembourg, 1997, as updated.

4.

Council Directive 88/379/EEC of 7 June 1988, Official Journal of the European Communities, 16:7:88, L187, 14, as replaced by Directive 1999/45/EC of 31 May 1999, Official Journal of the European Communities, 30:7:99, L200, 1.

5.

Council Directive 92/32/EEC of 30 April 1992, Official Journal of the European Communities, 5:6:92, L154, 1.

6.

Technical Notes for Guidance in Support of Annex VI of Directive 98/8/EC of the European Parliament and the Council concerning the placing of Biocidal Products on the Market Common Principles and Practical Procedures for the Authorisation and Registration of Products, European Commission, Brussels, Belgium, final draft version, July 2002.

7.

Technical Notes for Guidance Document in Support of the Directive 98/8/EC concerning the placing of Biocidal Products on the Market Principles and Practical Procedures for the inclusion of active substances in Annex I, IA and IB, European Commission, Brussels, Belgium, final draft version, April 2002.

8.

Council Directive 78/631/EEC of 26 June 1978, Official Journal of the European Communities, 29:7:78, L206, 13.

9.

Biocidal Products Directive: The Provisional List of Existing Active Substances (98/8/EC). European Chemicals Bureau, Ispra, Italy, 2000.

10. Commission Regulation No 1896/2000 of 7 September 2000, Official Journal of the European Communities, 8:9:00, L228, 6. 11. Commission Regulation No. 1687/2002 of 25 September 2002, Official Journal of the European Communities, 26:9:02, L258, 15-16.

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Regulation of Biocides 12. Technical Guidance Document in Support of the Directive 98/8/EC concerning the placing of Biocidal Products on the Market Guidance on Data Requirements for Active Substances and Biocidal Products, European Community, Brussels, Belgium, final draft version, October 2000. 13. Annex V of Council Directive 67/548/EEC of 27 June 1967 as amended and adapted to technical progress (available at http://erb.jrc.it/testing-methods/). 14. OECD Guidelines for the Testing of Chemicals, OECD, Paris, France, 1993, as updated. 15. Technical Notes for Guidance on Dossier Preparation including Preparation and Evaluation of Study Summaries under Directive 98/8/EC Concerning the Placing of Biocidal Products on the Market, European Commission, Brussels, Belgium, latest version. 16. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No. 1488/94 on Risk Assessment for Existing Substances, European Commission, Brussels, Belgium, 1996 and the 2003 partial update and revision. 17. Final Report of the Projects: Pilot Evaluations on Existing Biocidal Active Substance, Finnish Environmental Institute Chemicals Division, Helsinki, Finland, 2001 (available at http://www.vyh.fi/eng/environ/risk/biocid/pilot.htm). 18. Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and Federal Food, Drug, and Cosmetic Act (FFDCA) as amended by the Food Quality Protection Act (FQPA) of August 3 1996, US Environmental Protection Agency Office of Pesticide Programmes, Washington DC, USA, March 1997, reference 730L97001. 19. Code of Federal Regulations Title 40 Parts 150 to 189, US Government Printing Office, Washington DC, USA, latest edition. 20. Antimicrobial Regulation Technical Corrections Act of 1998 (ARCTA), Public Law L105-324, US Government Printing Office, Washington DC, USA, 1998. 21. Pesticide Assessment Guidelines, National Technical Information Service, Springfield, USA, latest version. 22. Standard Evaluation Procedures, National Technical Information Service, Springfield, USA, latest version.

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Practical Guide to Chemical Safety Testing 23. Office of Prevention, Pesticides and Toxic Substances Test Guidelines, US Environmental Protection Agency, Washington DC, USA, latest version, (available at http://www.epa.gov/opptsfrs/home/guidelin.htm). 24. Report on the Survey of OECD Member Countries’ Approaches to the Regulation of Biocides, OECD Environmental Health and Safety Publications, Series on Pesticides No. 9, OECD, Paris, France, 1999.

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Abbreviations and Acronyms

Abbreviations and Acronyms

ADG Code

Australian Dangerous Goods Code

ADI

acceptable daily intake

ADME

Absorption, Distribution, Metabolism and Excretion

ADR

road transport agreement

AICS

Australian Inventory of Chemical Substances

ANSI

American National Standards Institute

ARTCA

Antimicrobial Reform Technical Corrections Act

ASTM

American Standards for Testing and Materials

ASV

air saturation value

ATLA

Alternatives to Laboratory Animals

ATP

Adaptation to Technical Progress of an EU Directive

BADGE

2,2-bis (4–hydroxyphenyl) propane bis (2,3-epoxypropyl)ether

BAPEDAL

Indonesian Environmental Impact Management Agency

BCF

bioconcentration factor

BCOP

bovine cornea opacity and permeability

BFDGE

bis (hydroxyphenyl) methane bis (2,3-epoxypropyl) ethers

BgVV

German Federal Institute for Health Protection of Consumers and Veterinary Medicine, i.e., Bundesinstit für Gesundheitlichen Verbraucherschutz und Veterinarmedizin

BMD

benchmark dose

BNS

Binational Toxics Strategy

BOD

biological oxygen demand

BPD

EU Biocidal Products Directive

BS

British Standards

CA

Competent Authority

421

Practical Guide to Chemical Safety Testing CAAT

Center for Alternatives to Animal Testing, John Hopkins

CAM-TB

Chorioallantoic membrane – trypan blue staining

CAS

Chemical Abstracts Service

CBER

US Center for Biologics Evaluation and Research

CBI

Confidential Business Information

CChemRTK

US Chemical Right-to-Know initiative

CCO

Philippine Chemical Control Order

CDC

Cumulative Dietary Concentration

CDRH

US Center for Devices and Radiological Health

CE

capillary electrophoresis

CEC

Commercial Evaluation Chemicals

CEDI

cumulative estimated daily intake

CEMCCF

Council of Europe’s Committee of Experts on Materials Coming into Contact with Food

CEN

European Committee for Standardisation

CEPA

Canadian Environmental Protection Act

CFR

US Code of Federal Regulations

CHARM

Chemical Hazard Assessment and Risk Management

ChemRTK

chemical right-to-know

CHL

Chinese hamster lung

CHO

Chinese hamster ovary

Clocaleff

concentration of the chemical substance in the sewage treatment plant effluent

COD

chemical oxygen demand

CoE

Council of Europe

COLIPA

European Cosmetic, Toiletry and Perfumer’s Association

COMET

single cell gel-electrophoresis

CORT

chronic, oncogenicity, reproductive and teratogenicity

CSCL

Japanese Chemical Substances Control Law

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Abbreviations and Acronyms CSTEE

EU Scientific Committee for Toxicity, Ecotoxicity and the Environment

CTP

comprehensive toxicological profile

DENR

Philippine Department of Environment and Natural Resources

DEPTHsoil

mixing depth of soil

DEPtotalann

annual average total deposition flux

DEREK

Deductive Estimation of Risk from Existing Knowledge

DES

Data Entry Screens

DIN

Deutsches Institut Für Normung

DMF

dimethyl formamide

DNA

deoxyribonucleic acid

DOC

dissolved organic carbon

DOL

Department of Labour

DOSH

Malaysian Department of Occupational Safety and Health

DOT

US Department of Transport

DPD

EU Dangerous Preparations Directive

DSC

differential scanning calorimetry

DSD

EU Dangerous Substances Directive

DSL

Canadian Domestic Substances List

DSS

decision support system

DTA

differential thermal analysis

DTI

UK Department of Trade and Industry

EAR

Canadian Environmental Assessment Regulations

EASE

estimation and assessment of substance exposure

EC50

an effective concentration that causes a 50% response in the population

ECB

European Chemicals Bureau

ECETOC

European Centre for Ecotoxicology and Toxicology of Chemicals

ECI

Existing Chemicals Inventory

423

Practical Guide to Chemical Safety Testing ECR

EU Existing Chemicals Regulation

ECVAM

European Centre for Validation of Alternative Methods

EDI

estimated daily intake

EEA

European Economic Area

EFTA

European Free Trade Association

EINECS

European Inventory of Existing Commercial Substances

ELINCS

European List of Notified Chemical Substances

EMB

Philippine Environmental Management Bureau

EMFIC

Chinese Regulations for Environmental Management on the First Import of Chemicals and the Import and Export of Toxic Chemicals

ENCS

Existing and New Chemical Substances

EP

end-use product

EPA

US Environmental Protection Agency

EPCA

Singapore Environmental Pollution Control Act

ERMA

New Zealand Environmental Risk Management Authority

ESAC

ECVAM Scientific Advisory Committee

EST

embryonic stem cell test

ETAD

Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers

EU

European Union

EUBEES

EU Biocide Emission Scenarios

EUSES

European Union Systems for the Evaluation of Substances

EWG

Expert Working Group

F&DA

US Food and Drugs Act

FAO

Food and Agriculture Organization

FCF

fat consumption factor

FCN

US Food Contact Notification

FCS

food contact substance

FDA

US Food and Drug Administration

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Abbreviations and Acronyms FFDCA

US Federal Food, Drug and Cosmetic Act

FIFRA

US Federal Insecticide, Fungicide and Rodenticide Act

FOPH

US Federal Office of Public Health

FQPA

US Food Quality Protection Act

GCMS

gas chromatography mass spectrometry

GESAMP

Joint Group of Experts on the Scientific Aspects of Marine Pollution

GHS

globally harmonised system

GHTF

Global Harmonisation Task Force

GLP

Good Laboratory Practice

GMP

good manufacturing practice

GPC

gel permeation chromatography

GPRA

US Government Performance and Results Act

GRAS

generally recognised as safe

HCS

Hazard Communication Standard

HEDSET

harmonized electronic data set

HENRY

Henry’s law constant

HET-CAM

hen’s egg test – chorioallantoic membrane

HOCNF

Harmonised Offshore Chemical Notification Format

HPLC

high performance liquid chromatography

HPRT

hypoxanthine-guanine phosphoribosyl transferase

HPV

high production volume chemical

HSE

UK Health and Safety Executive

HSNO

New Zealand Hazardous Substances and New Organisms Act

IARC

International Agency for Research on Cancer

IATA

International Air Transport Association

ICAO

International Civil Aviation Organization

ICC

Philippine Import Clearance Certificate

ICCA

International Council of Chemical Associations

425

Practical Guide to Chemical Safety Testing ICCVAM

US Interagency Co-ordinating Committee on the Validation of Alternative Methods

ICH

International Conference on Harmonisation

IDE

US Investigational Device Exemption

IDLH

immediately dangerous to life or health

ILO

International Labour Organization

IMCO

International Consultative Organization

IMDG

International Maritime Dangerous Goods

IMO

International Maritime Organization

IPCS

International Program on Chemical Safety

IPS

Informal Working Group on Priority Setting

ISHL

Korean Industrial Safety and Health Law

ISO

International Standards Organization

ISP

Philippine Interim Status Permit

IUCLID

International Uniform Chemicals Information Database

IUPAC

International Union of Pure and Applied Chemistry

IUR

US Inventory Update Rule

IVC

in vitro chromosome aberration test

IVD

in vitro devices

IVTIP

Industrial Platform on In Vitro Testing

JECFA

Joint Expert Committee on Food Additives

JIS

Japanese Industrial Standards

JRC

EU Joint Research Centre

K

first order rate constant for removal from top soil

Ka

dissociation constant

Kair-water

Air:water partitioning coefficient

KBwS

German Commission for Classification of Substances Hazardous to Water, i.e., Geschaftsstelle der Kommission Bewertung Wassergefahrdender Stoffe

426

Abbreviations and Acronyms KCMA

Korean Chemical Management Association

KECI

Korean Existing Chemicals Inventory

Koc

adsorption coefficient

Kow

n-octanol:water partition coefficient, also known as Pow

Kpsusp

concentration of suspended matter in the receiving water

Ksoil-water

soil:water equilibrium partitioning coefficient

L(E)C20

lethal effect concentration required to kill 20% of the population

L(E)C50

lethal effect concentration required to kill 50% of the population

LC50

median lethal concentration

LCMS

liquid chromatography mass spectrometry

LD50

median lethal dose

LEL

lower explosion limit

LLNA

local lymph node assay

LMBG

German Foodstuffs and Commodities Law, i.e., Lebensmittel und Bedarfsgegenständegesetz

LOAEL

lowest observable adverse effect level

LOEC

lowest observed effect concentration

LoREX

US low release and exposure

LVE

low volume exemption

M and K

Magnusson and Kligman guinea pig maximisation test

MAFF

Japanese Ministry of Agriculture, Forestry and Fisheries

MARPOL

International Convention for the Prevention of Pollution from Ships

MDCK

Madin-Darby canine kidney

MDR

Medical Device Reporting

METI

Japanese Ministry of Economy, Trade and Industry

MHLW

Japanese Ministry of Health, Labour and Welfare (formerly MHW and MoL)

MHW

Japanese Ministry of Health and Welfare (now MHLW)

MI

mitotic index

427

Practical Guide to Chemical Safety Testing MLA

mouse lymphoma assay

MM

micromass

MMAD

mean mass aerodynamic diameter

Mn

number-average molecular weight

MoE

Ministry of the Environment

MoH

Ministry of Health

MoL

Ministry of Labour (Note: Japanese Ministry of Labour is now MHLW)

MP

manufacturing use product

MPD

OECD Minimum Pre-Marketing Data Set

MRA

Mutual Recognition Agreement

MSDS

material safety data sheet

MTD

maximum tolerated dose level

MTP

modified test procedure

MW

molecular weight

NADPH

nicotinamide adenine dinucleotide phosphate

NAFTA

North American Free Trade Association

NAMW

number average molecular weight

NCE

normochromatic erythrocyte

NDSL

Canadian Non-Domestic Substances List

NEC

no-effect concentration

NEDB

UK National Exposure Database

NEPA

Chinese National Environmental Protection Agency (now SEPA)

NICE

Thai National Institute for the Improvement of Working Conditions and Environment

NICNAS

Australian National Industrial Chemicals Notification and Assessment Scheme

NIER

Korean National Institute of Environmental Research

NIHS

Japanese National Institute of Health Sciences

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Abbreviations and Acronyms NOAEL

no observed adverse effect level

NOC

US Notice of Commencement

NOEC

no observed effect concentration

NOEL

no observed effect level

NOEQ

Canadian Notice of Excess Quantity

NOGE

novolac glycidyl ethers

NRCC

Chinese National Registration Centre for Chemicals

NRUPT

Neutral Red Update Phototoxicity Test

NSFR

Canadian New Substances Fee Regulations

NSNR

Canadian New Substances Notification Regulations

NTP

US National Toxicology Program

OCNS

Offshore Chemical Notification Scheme

OECD

Organisation for Economic Co-operation and Development

OEHS

Swiss Ordinance on Environmentally Hazardous Substances

OPPTS

US Office of Prevention, Pesticides and Toxic Substances

OSHA

US Occupational Safety and Health Act

OSPAR

Oslo and Paris Commissions

OSPARCOM

Oslo Paris Convention, Harmonized Off-Shore Chemical Notification

OTS

Swiss Order relating to Toxic Substances

PASPHF

Council of Europe Partial Agreement in the Social and Public Health Field

PB-PK

physiologically-based pharmacokinetic model

PBT

persistent, bioaccumulative and toxic

PCB

polychlorinated biphenyl

PCD

Singapore Pollution Control Department

PCE

polychromatic erythrocyte

PCL

Priority Chemicals List

PCT

polychlorinated terphenyl

429

Practical Guide to Chemical Safety Testing PEC

predicted environmental concentration

PEC

Priority Existing Chemical

PEClocalwater

local concentration in surface water

PFS

Japanese Polymer flow scheme

PHA

phytohaemagglutinin

PIC

prior-informed consent

PICCS

Philippine Inventory of Chemicals and Chemical Substances

pKa

dissociation constant

PLC

polymer of low concern

PLONOR

pose little or no risk to the environment

PMA

pre-market approval

PMD

pre-marketing data set

PMN

US Pre-Manufacturing Notice

PMPIN

Philippine Pre-Manufacture Pre-Importation Notification

PNEC

predicted no effect concentration

PNECaqua

predicted no effect concentration in water

PNECsoil

predicted no effect concentration in soil

POEM

predictive occupational exposure model

POP

persistent organic pollutant

PORD

Process-Orientated Research and Development

Pow

n-octanol:water partition coefficient, also known as Kow

PPP

plant protection product

PRC

People’s Republic of China

PRTR

Pollutants Release and Transfer Register

QA

quality assurance

QM

quantitative maximum of residual substance in food packaging

QMA

quantitative maximum of residual substance per unit area of food packaging

QSAR

quantitative structure-activity relationships

430

Abbreviations and Acronyms R

gas constant

R&D

research and development

RCR

risk characterisation ratio

REACH

EU Registration, Evaluation and Authorisation of Chemicals

RED

US Re-registration Eligibility Decision

REET

rabbit enucleated eye test

RfD

reference dose

RHOsoil

bulk density of wet soil

RID

regulations concerning the carriage of dangerous goods by rail

RNA

ribonucleic acid

RTP

EU Reduced Test Package for Polymers

SAEFL

Swiss Agency for Environment, Forestry and Landscape

SAR

structure-activity relationship

SCAS

semi-continuous activated sludge test

SCC

EU Scientific Committee for Cosmetology (now SCCNFP)

SCCNFP

Scientific Committee on Cosmetic Products and Non-Food Products intended for consumers

SCF

EU Scientific Committee for Food

SDS

safety data sheet

SEPA

Chinese State Environmental Protection Administration (formerly NEPA)

SETC

Chinese State Economic Trade Commission

SHE

Syrian hamster embryo

SIAM

SIDS initial assessment meeting

SIAR

SIDS initial assessment report

SIDS

OECD screening information data set

SLIM

EU Simpler Legislation for the Internal Market

SML

specific migration limit

SN

US Safety Narrative

431

Practical Guide to Chemical Safety Testing SNAc

Canadian Significant New Activity

SNIF

EU Summary Notification Interchange Format

SNUN

US Significant New Use Notice

SNUR

US Significant New Use Rule

SOL

water solubility

SPR

structure-property relationship

SS

suspended solids

STP

sewage treatment plant

STP

EU Standard Test Package for Polymers

TCCL

Korean Toxic Chemicals Control Law

TD50

the concentration of a substance that causes cancer in 50% of test animals in chronic feeding studies

TDI

tolerable daily intake

TEMP

temperature at the air-water interface

TER

transcutaneous electrical resistance assay

TFT

trifluorothymidine

TGAI

technical grade active ingredient

TGD

technical guidance document

THF

tetrahydrofuran

ThOD

theoretical oxygen demand

TK

thymidine kinase

TME

test market exemption

TNG

technical note for guidance

TOC

total organic carbon

TOR

threshold of regulation

TRI

US Toxics Release Inventory

TSA

Toxic Substances Act

TSCA

US Toxic Substances Control Act

TWA

time-weighted average

432

Abbreviations and Acronyms UDS

unscheduled DNA synthesis

UEL

upper explosion limit

UN

United Nations

UNCED

United Nations Conference on Environmental Development

US TS

US Tracking System

USG

United States Government

UV

ultraviolet

VCCEP

US Voluntary Children’s Chemical Evaluation Program

VIS

visible

VP

vapour pressure

VPVB

very persistent and very bioaccumulative

VwVwS

German Administrative Regulation on the Classification of Substances Hazardous to Waters, i.e., Verwaltungsvorschrift wassergefahrdende stoffe

WAF

water accommodated fraction

WEC

whole embryo culture

WGK

German Water Endangering Classes, i.e., Wassergefahrdungsklassen

WHMIS

Canadian Workplace Hazardous Materials Information System

WHO

World Health Organization

433

Index

Index terms

Links

A Acartia tonsa

75

Acceptable daily intake

152

Active Implantable Medical Devices Directive

348

Acute oral toxicity alternative methods Acute toxic class method Acute toxicity

351

12 15 16 9

Adsorption coefficient batch equilibrium method

97 98

high performance liquid chromatography

98

Adsorption onto aerosols

169

Adsorption onto soil

169

Adverse events

356

Aerosol studies

15

Air concentration

176

Air quality standards

142

Air:water partition coefficient

170

Algal growth tests

361

362

365

59 320

123

130

73

American National Standards Institute

266

Ames test

47 284

Anaerobic biodegradation testing

71

Aneuploidy

47

Antifoulant

404

Antimicrobials

416

243

50

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435

436

Index terms

Links

Aquatic ecosystem

180

Aquatic plant tests

79

Aquatic toxicity testing

72

Aquatic toxicology analytical measurements

77

chronic tests

79

difficult compounds

78

Arrhenius relationship

101

Asbestos

191

Australia

280

polymer notification

320

Australian Dangerous Goods Code

282

Australian Inventory of Chemical Substances

281

320

Auto-ignition temperature test method

112

Auxoploses

111

Azo dyes

48

B Bacterial mutation test

47

Bacterial toxicity test

65

Base Set

192

Basic Data Record

221

Benthic compartment

180

Binational Toxics Strategy

268

Bioaccumulation

22 206 236

95 209 242

Biocidal Products Directive

401

402

Annex I

207

105 219

404

Biocidal Products Directive identification procedure Biocides

407 225

active ingredient/substance

403

405

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406

152 221

169 235

437

Index terms

Links

Biocides (Continued) authorization

405

data requirements

410

efficacy

411

food contact

415

frame formulation

405

low risk

403

market

401

me-too registration

411

product types

192

regulations

401

review programme

403

risk assessment

404

US regulations

409

Bioconcentration factor

405

404 406

81

97

Biodegradability

235

236

Biodegradable polymers

316

Biodegradation

92

Biodegradation tests

65

Biological oxygen demand

68

Biological risk assessment

353

Biological safety

361

medical devices

352

Biotransformation

22

Block polymer

315

Blue Book Memo

361

Blue Card

244

Bluegill sunfish

75

Boiling point

90

Bovine cornea opacity and permeability assay Brine shrimp By-product

172 219

365

129 80 265

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171

242

207

209

438

Index terms

Links

C Cadmium

191

Canada

270

New Substances Notification Regulations

271

polymer notification

311

Canadian Environmental Protection Act

270

Capillary method

89

Carcinogenicity

30

tests

311 33

43

157

351

352

358

238

256

310

123

Carcinogens

191

Carp

81

CE mark

348

Cell cycle

50

Cell transformation assay

123

Center for Biologics Evaluation and Research

359

Center for Devices and Radiological Health

359

Centromere

55

Chemical Abstracts Service

220

Chemical Hazard Assessment and Risk Management

226

Chemical oxygen demand

219

Chemical Right-to-Know (ChemRTK) Initiative

266

Chemical Substances Control Law

236

Children’s Health Test Rule

267

315

China polymer notification

331

Regulation on the Manufacturing of Packaging and Containers for Dangerous Chemicals

290

Regulations on Licensing for the Business and Sale of Dangerous Chemicals

290

Regulations on the Registration of Dangerous Chemicals

290

China’s State Economic Trade Commission

290

Chironomus

81

Chorioallantoic membrane-trypan blue staining

129

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439

Index terms Chromosome aberration test

Links 243

247

284

72

87

Chromosome damage

50

Chromosomes

44

Classification

10

biocides

406

dangerous

203

EU

154

explosivity

112

Japan

250

medical devices

349

351

UN

110

217

Clastogen

26

Clastogenicity

50

Clausius-Clapeyron equation

91

Clean Air Act

255

Clean Water Act

255

Clinical chemistry

212

364

92

30

33

12

23

145

Clinical trials

353

361

365

COLIPA

224

Colours permitted in cosmetics

223

125 229

130 348

Clinical data

23

320

145

355

Clinical observations

COMET assay

60

Commercial Evaluation Chemicals

282

Commission for Classification of Substances Hazard

221

Competent Authority

122 200

German

132

Compressed gases

216

Conformity assessment

351

Consumer exposure assessment

151

CORROSITEX™

126

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193 352

194 357

440

Index terms

Links

Corrosivity skin tests

223

tests

125

CORT

410

Cosmetics

126

128

192

402

406

223

239

407 toxicity tests

224

Council Directive 67/548/EC

192

Council Directive 73/404/EEC

224

Council Directive 76/768/EEC

223

Council Directive 76/769/EEC

191

Council Directive 92/32/EEC

191

Council Directive 93/35/EEC

223

Council Directive 1999/45/EC

402

Council Regulation Number 793/93

207

406

Cytotoxicity testing mammalian cells

59

medical device

35

D Dangerous preparations

214

Dangerous Preparations Directive

214

Dangerous substances

212

Dangerous Substances Directive

10 406

Dangerous to the environment

72

Daphnia

74

reproduction test

413

Data gaps

211

Data sharing

193

Decision support system

132

Degradation

340

402

406

125

192

227

402

194

207

79

Data compensation under FIFRA

Decomposition

227

91 168

172

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441

Index terms

Links

Delayed hypersensitivity

19

Density

99

gas comparison pycnometer method

100

pycnometer method

100

Deposition

169

Dermal exposure

150

Dermal irritation

410

Dermal toxicity

11

Detergents

13

21

28

29

91

111

225

Developmental toxicity

26

tests

123

Diatom

73

Differential scanning calorimetry

89

Differential thermal analysis

91

Diploid

46

Direct contact method, testing of medical devices

38

Disinfectants

404

Dissociation constant

103

conductometric method

103

spectrophotometric method

103

titration method

103

Dissolved organic carbon

66

Distillation

91

417

Domestic Substances List

271

311

Dose-response

144

164

Dosing

12

Draize

13

Duckweed

79

E Earthworms

82

EC Method A11

110

EC Method A12

110

EC Method A13

110

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202

159

442

Index terms

Links

EC Method A14

111

EC Method A15

112

EC Method A16

112

EC Method A17

113

EC Method A9

109

EC50

72

ECB information ECETOC

74

205

209 66

skin sensitisation

19

Ecosystems

163

179

Ecotoxicity

63

92

ECB information

209

full notification

196

hydrolysis

102

QSAR

130

reduced notification

197

tests

214

241

17

121

122

278

331

ECVAM

125

133 EINECS

192

Eisenia foetida

82

ELINCS

193

331

Emissions

164

166

167

Emulsion

105 30

229

Endocrine disruptors tests

28 124

Environment risk assessment

204

Environmental Assessment Regulations

272

Environmental behaviour

164

Environmental classification

214

Environmental concentrations

164

Environmental degradants

241

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176

131

443

Index terms Environmental distribution

Links 169

advection

169

dispersion

169

Environmental exposure assessment

164

Environmental fate

103

104

Environmental fate modeling

93

95

Environmental Impact Report

279

115

Environmental impact biocides

415

Environmental legislation

191

Environmental Pollution Control Act

296

Environmental Protection Agency

256

inhalation studies Environmental risk assessment

308

15 65

163

Environmental Risk Management Authority

292

324

Environmentally Hazardous Substances

277

EpiDerm™

126

EpiOcular™

129

EPISKIN™

125

Equilibrium partitioning coefficient

169

Essential Requirements for medical devices

348

Estimation methods

409

128

129

227

406

88

See also Prediction methods EU

191

biocide regulations

402

medical device regulation

345

polymer notification

331

EU Annex V

125

EU Chemicals Policy

340

EU Method A21

114

EU Method B41

126

EU Method B6

127

EU risk categories European Chemicals Bureau

11 199

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168

209

444

Index terms

Links

European Cosmetic Toiletry and Perfumer’s Association

224

European Economic Area

191

European Free Trade Association

191

European Standard ISO 14155

356

EUSES

207

Exclusive use of pesticides data in the US

413

Exemptions

263

Existing and New Chemical Substances

315

Existing chemicals

208

OECD review

298

Existing Chemicals Inventory

325

Existing Chemicals Regulation

207

Existing Substances Inventory

331

Existing Substances Regulation

227

Exothermic

90

Explosivity

90

test method

111

Explosophores

111

Export-only

208

Exposure assessment

139

345

111

164

Exposure prediction EASE

151

Exposure scenarios

137

Exposure-triggered testing

228

148

Extraction tests flask method

108

polymer test method

108

testing of medical devices Extractivity

35

36

332

Extrapolation statistical

182

Eye irritation test method

18

128

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202

204

445

Index terms

Links

F Family approach

336

Fat solubility

106

test method

106

Fathead minnow

75

Federal Chemicals Law

279

Federal Food, Drug and Committee Act

409

Federal Food, Drug, and Cosmetic Act

359

Federal Insecticide, Fungicide and Rodenticide Act

409

Federal Office of Public Health

341

Fertility

26

Film preservatives

404

Fire hazard

109

Fire Service Law

249

Fish

169

Fish tests

80

159

75

bioaccumulation

81

early life stage

80

embryo and sac-fry stage

77

flow-through

76

growth tests

80

semi-static

76

Fixed dose procedure

16

Flammability

90

test method

110

tests

114

Flash point

91

closed cup method Fluorescein leakage assay Foetal toxicity

109

109

109 129 29

Food and Drug Administration

360

Food contact biocides Food packaging

410

415

239 This page has been reformatted by Knovel to provide easier navigation.

446

Index terms

Links

Food, pesticide residue in

415

Food Quality Protection Act

409

Formulator’s Exemption

412

Frame formulation

403

Freezing temperature

89

Freshwater invertebrate

74

Freshwater species Pseudokirchneriella subcapitata subspicatus

73

Scenedesmus subspicatus

73

Freund’s complete adjuvant

19

Functional Observation Battery

23

G Gas chromatography mass spectrometry

99

Gavage

12

Gel permeation chromatography

107

Gene expression

46

Gene mutation assays

53

General Law of Ecological Equilibrium and Environmental Protection

295

Genotoxic carcinogens

34

Genotoxicity

31

Germany

220

GESAMP

218

Gestation

26

Global harmonization

10

Good Laboratory Practice

88

Government Performance and Results Act

269

Graft polymer

315

Groundwater

166

43

123

157

88

364

365

178

Guinea Pig sensitization tests Guppy

19 75

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447

Index terms

Links

H Haematology

23

30

33

145

140 204

158

164

178

252

255

266

Half-life environment

173

Haploid

46

Harm

138

Harmful

10

Harmonized Electronic Data Set

208

Harmonized Mandatory Control System

225

Harmonized offshore chemical notification format

225

Harmonized European standards

350

Hazard

138

Hazard communication

212

Australia

282

biocides

406

EU

212

Japan

247

Korea

286

New Zealand

295

People’s Republic of China

292

Hazard identification

139 202

Hazard quotient

226

Hazardous Substance and New Organism Bill

324

Hazardous Substances and New Organisms Act

293

Head-only exposure Health and Safety Executive

14 131

212

93

95

Henry’s law

170

175

Hen’s egg test-chorioallantoic membrane

129

Henry’s Constant

Hepatocytes

58

High production volume (HPV) chemicals

208 298

High-Pressure Gases Control Law

250

210

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448

Index terms Histopathology

Links 12

HPLC

23

24

242

HPRT assay

54

Hydrolysis

97

172

101

114

242

121

126

127

test methods

I ICCVAM Immunotoxicology

33

Implantation

28

medical device testing

32

Impurity

265

In Vitro Diagnostic Medical Device Directive

348

In vitro tests

354

351

59

Indirect contact test testing of medical devices

37

Indonesia

297

Industrial Safety and Health Law

246

Industrial Safety and Hygiene Law

315

Inhalable

101

Inhalation

93

calculating exposure

148

intake-uptake relation

148

Inhalation toxicity Inherent biodegradation

11

14

174

modified Zahn-Wellens/EMPA test

70

semi-continuous activated sludge test

70

Intermediate

285

265

International Agency for Research on Cancer

43

International Air Transport Association

218

International Civil Aviation Organization

218

International Convention for the Prevention of Pollution from Ships (MARPOL) International Council of Chemical Associations

218 252

299

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21

410

449

Index terms

Links

International Labour Organization

247

International Maritime Consultative Organization

218

International Maritime Organization

218

International Programme on Chemical Safety

299

International Regulations Concerning the Carriage of Dangerous Goods by Rail

218

219

International standards medical devices

345

Interspecies variations

179

Intraspecies variations

179

Inventory Update Rule

268

Investigational Device Exemption

361

Irritation tests

17

ISO standards

350

ISO 10993

35

354

ISO 10993-1

361

365

Isocyanate polymers

319

IUCLID

210

IUPAC

240

IVTIP

121

407

J Japan

235

biocide regulation

417

medical device regulation

363

polymer notification

312

Japan Chemical Daily Handbook

238

Japanese Product Liability Law No.85

249

K Killifish

75

Korea

283

Chemical Control Order

288

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450

Index terms

Links

Korea (Continued) polymer notification

325

Priority Chemicals List

288

Korean Chemical Management Association

284

Korean Existing Chemicals Inventory

283

L Labelling

10

biocides

406

Japan

247

Labelling Working Group Lactation

72

87

154

149

219

212 26

Law concerning the Protection of the Ozone Layer

250

LC50

10

64

LD50

10

149

Leaching food packaging

152

groundwater

166

leachate

108

medical devices

353

Lemna

169

79

Level 1 testing

194

Level 2 testing

198

Lewalle

140

Life-cycle

165

Liquid chromatography mass spectrometry

99

List of Existing Chemical Substances

238

Lists of permitted ingredients in cosmetics

223

Local lymph node assay

229

19

LoREX exemption

263

Low Environmental Release and Low Human Exposure

308

Low Volume Exemption

245

Japan

240

Korea

284

127

247

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178

451

Index terms

Links

Low Volume Exemption (Continued) USA

262

Lowest observable adverse effect level Lumbriculus variegatus

155 81

M Malaysia

297

Malformations

29

Mammalian cell assays

59

Mammalian toxicity

9

Manual of Decisions

408

Marine Pollution

218

Marine species

75

Skeletonema costatum Marketing and Use Directive

73 191

Mating

26

Maximisation test

19

Me-too

212

227

127

411

biocide registration

413

device

360

Medical Device Reporting Medical device testing Medical devices

362 34 239

345

357

361

Medical Devices Directive

345

348

Medicinal Products Directive

349

custom made devices

Melting temperature

89

Mesocosm

83

Metabolic activation

47

Mexico

295

Microcosm

83

Micronucleus

55

59

247

284

Micronucleus test

This page has been reformatted by Knovel to provide easier navigation.

351

452

Index terms

Links

Migration food stuffs

106

Mineralisation

242

Minimum pre-marketing data set

87

256

Ministry of Economy, Trade and Industry

236

315

Ministry of Health and Welfare

236

Ministry of Health, Labour and Welfare

315

Ministry of the Environment

236

Mitosis

51

Mitotic index

52

Modified Test Procedure

340

Molecular weight

308

Monomers

307

Mouse lymphoma assay

331

53

Mutagen

157

Mutations

43

Mutual recognition

403

Mutual Recognition Agreement

352

405

N National Environmental Protection Agency

291

National Exposure Database

151

National Industrial Chemicals Notification and Assessment Scheme

280

National Institute of Environmental Research

283

National Institute of Health Sciences

363

National Toxicology Program

43

Necropsy

12

Neurotoxicity

23

developmental

326

24

29

Neutral red release assay

129

New Chemical Substance Card

244

New Substances Fee Regulations

274

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298

334

340

341

453

Index terms New Zealand

Links 292

polymer notification

324

N-Nitroso compounds

48

No longer polymers

332

No observed adverse effect level

20

145

148

155

No observed effect level

20

72

73

205

Non-Domestic Substances List

271

311

Non-hazardous

208

North American Free Trade Association

295

Nose-only exposure

14

Notice of Commencement

274

Notice of Commencement of Manufacture or Import

261

Notice of Excess Quantity

274

Notification

192

Australia

280

Canada

273

China

290

confidential

201

exemptions

200

Japan CSCL

237

Korea

283

Philippines

288

procedure

407

Prolongation Regulation

407

Switzerland

278

Notified Body

352

Nucleotide bases Number average molecular weight test method

309

44 107

244

107

O Occupational exposure standards

142

Occupational Safety and Health Act

265

n-Octanol:water partition coefficient

219

242

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308

243

454

Index terms OECD

Links 298

biocide registration

417

OECD Guideline 102

89

OECD Guideline 103

90

OECD Guideline 104

91

OECD Guideline 106

97

OECD Guideline 107

95

OECD Guideline 109

99

OECD Guideline 110

100

OECD Guideline 111

101

OECD Guideline 112

103

OECD Guideline 115

104

OECD Guideline 116

106

OECD Guideline 117

95

OECD Guideline 120

108

OECD Guideline 121

97

OECD Guideline 209

65

OECD Guideline 301A

68

OECD Guideline 301B

68

OECD Guideline 301C

68

OECD Guideline 301D

68

OECD Guideline 301E

69

OECD Guideline 301F

69

OECD Guideline 302A

70

OECD Guideline 302B

70

OECD Guideline 302C

70

OECD Guideline 303

71

OECD Guideline 303A

173

174

174

OECD Guideline 306

69

OECD Guideline 307

174

OECD Guideline 401

15

OECD Guideline 407

23

OECD Guideline 408

23

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455

Index terms

Links

OECD Guideline 420

16

OECD Guideline 425

16

OECD Guideline 429

20

OECD Guideline 471

48

OECD Guideline 474

55

OECD Guideline 476

53

OECD Minimum Premarketing Data-Set

87

OECD polymer definition

307

OECD SIDS Initial Assessment Report

267

OECD test guidelines

87

Oestrous cycles

26

Offshore Chemical Notification Scheme

225

Oligomer content

244

Oligomers

309

One generation study

104

Oocyte evaluation

28

316

262

Opthalmoscopy

24

Oral toxicity

11

Order relating to Toxic Substances

276

Ordinance on Environmentally Hazardous Substances

340

Organization for Economic Co-operation and Development OSHA

265

225

Oslo Paris Convention, Harmonized Offshore Chemical Notification

69

Osmolarity

52

effect on cells

150

7 43

Oslo and Paris Commissions

52

OSPAR

225

Oxidizing properties test method

298

26

Onsager equation OPPTS

256

113

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21

456

Index terms

Links

P Particle size distribution

100

aerodynamic method

100

sieving method

100

Partition coefficient

95

calculation method

96

HPLC

95

shake-flask method

95

People’s Republic of China

290

Peroxides

113

Peroxisome proliferators

169

331

33

Persistent, bioaccumulative and toxic

230

267

Persistent Organic Pollutants

230

245

Pesticide

404

413

re-registration

413

tolerance

415

Petroleum oils

90

Pharmaceuticals

239

Pharmacopoeia

350

355

43

54

Philippine Inventory of Chemicals and Chemical Substances

287

328

Philippines

286

Phenotype

polymer notification

328

Philippines Republic Act polymer definition

328

Photodegradation

172

Photographic articles

265

Phototoxicity

126

neutral red uptake test Phototoxicity test

125

127

223

Physico-chemical properties ECB information

209

full notification

195

risk assessment

204

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268

457

Index terms

Links

Physico-chemical properties notification reduced notification Plant growth tests

197 83

Plants

169

PLONOR list

225

Poisonous and Deleterious Materials Control Law

249

Pollutants Release and Transfer Register

249

Pollution Control Department

296

Polychlorinated biphenyls

191

Polychlorinated terphenyls

191

Polyester

309

Polymer

200

crosslinking

319

crystallinity

319

flow scheme

244

light stability

318

notification, Japan

244

notification, Korea

285

physico-chemical tests

89

salts

265

solubility

318

water solubility

236

106

94

Polymer exemption Japan

247

Polymer Exemption Guidance Manual

308

Polymer Guidance Document for the Implementation of Annex VIID

332

Polymers of Low Concern

275

Polymer Specific Data

326

Polymer Working Group

340

Polyploidy

47

Porous pot test

71

Post-market surveillance Pour point

356

319

311

321

358

363

89 This page has been reformatted by Knovel to provide easier navigation.

458

Index terms

Links

Pre-consultation system

235

Pre-manufacture Notice

308

Pre-manufacture Pre-importation Notification

328

Pre-manufacturing and pre-importation notification

287

Pre-manufacturing notice

261

Pre-market approval

360

Pre-market notification

360

Precautionary principle

153

Predators

171

Predicted environmental concentration

175

Predicted no effect concentration

205

environment

206

226

178

Prediction methods

88

114

See also Estimation methods Predictive Occupational Exposure Model

151

Preservatives

223

Priority existing chemicals

282

Priority setting

208

Priority substances

211

Process Orientated R&D

201

336

3 211

88

124

80

81

404

Q Quantitative structure-activity relationships KOC

171

R R&D exemption Korea

289

Rabbit enucleated eye test

129

Radiolabelled

58

Rainbow trout

75

Rat

10

Re-registration Eligibility Decision

414

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129

204

459

Index terms Read-across

Links 100

228

See also: QSAR Ready biodegradability

221

DOC die-away

68

seawater

69

tests

66

Red Book

139

Reduced notification

194

Reduced test package polymers

332

Reference dose

155

172

241

284

157

354

410

101

334 130

Registration dossier EU

228

Registration, Evaluation and Authorization of Chemicals

227

Releases

167

Repeated dose toxicity studies

20

Replacement, reduction and refinement

30

Reproductive organs

354

Reproductive toxicity

25

tests

123

Research and Development and Process Development Respirable

336 15

Risk

138

Risk assessment

5 202

93 208

404

415

biocides carcinogenicity

33

testing strategy

154

Risk benefit analysis medical devices

352

Risk characterization

140

environment

164

198

184

Risk characterization ratios

184

Risk evaluation

137

140

155

Risk management

137

138

159

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137

139

460

Index terms Risk phrases

Links 21

Risk reduction

208

Risk:benefit ratio

355

Road Transport Agreement

218

Rodenticides

406

Royal Society Study Group

138

Russell and Burch

119

213

417

S S9

48

Safe Drinking Water Act

255

Safety data sheet

247

biocides

406

China

292

European format

215

Korea

286

Mexico

296

New Zealand

295

Safety phrases

213

SCCNFP

224

Scientific Committee for Cosmetology

224

Scientific Committee on Cosmetic Products and Non-Food Products

224

Screening Information Data Set

299

Sediment

176

Sediment toxicity

322

180

81

Sediment:water partitioning coefficient

181

Sensitization

410

tests

282

18

Sewage treatment plant

172

Sexual maturation

28

Sheepshead minnow

75

Shonin

364

SIDS Initial Assessment Report

300

174

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175

461

Index terms

Links

Significant New Activity

273

Significant new use rule

262

Simple Legislation for the Internal Market

227

Singapore

296

Site limited polymers

321

Site-limited intermediates

208

239

Site-limited substances Canada

275

‘Siwoloboff’ method

91

Skin irritation tests

18

Skin reactions

128

13

Skin sensitisation tests Buehler method

127

Magnusson and Kligman

127

SkinEthic™

129

SLIM

227

Soil

166

Soil ecosystem

182

Sole-representative facility

200

Solids:water partition coefficient

170

Spermatogenesis computer assisted sperm analysis

177

26 28

Spontaneous combustion

110

Standard test package polymers

332

113

Standards for medical devices horizontal standards

350

product standards

350

semi-horizontal standards

350

State Environmental Protection Administration

291

Stockinger-Woodward approach

149

Summary Notification Interchange Format

199

Surface tension

94

ring test method

96

105

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104

462

Index terms

Links

Surface water

174

Surfactants

224

Swiss Agency for Environment, Forestry and Landscape

278

340

Switzerland

192

276

biocide regulation

417

polymer notification

340

175

Systemic toxicity tests

124

Taiwan

297

Tanker Safety and Pollution Prevention

218

Target compartments

164

Technical Guidance

407

TER assay

125

Test battery

122

Test market exemption

263

T

Test methods ASTM

87

BS

87

DIN

87

EU ‘Annex V’

87

JIS

87

OPPTS

87

Testing programmes

256

Thailand

297

Thermal hazards

113

Three Rs

119

Thymidine

58

Thymidine kinase

54

Tolerance

415

Total organic carbon

94

Toxic

10

Toxic Chemical Business

285

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180

463

Index terms Toxic Chemicals Control Law

Links 283

Toxic effects man

204

Toxic Substances Act

292

Toxic Substances and Hazardous and Nuclear Wastes

286

Toxic Substances Control Act

256

324 308

Toxic Substances List Switzerland

276

Toxicant

144

Toxicity testing programme

243

Toxicokinetics

103

130

Toxicological studies full notification

196

reduced notification

197

Toxicology

144

Toxics Release Inventory

269

Transport regulations

215

TSCA inventory

256

Tumour promoters

34

Turbot

75

Two generation study

26

U Ultraviolet filters

223

UN Recommendations

216

UN Transport scheme

109

UN Transport Tests

110

88

UN Transportation Classification danger classes

217

environmentally hazardous

217

Uncertainty factors

178

Unscheduled DNA synthesis

57

Up-and-down procedure

16

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243

464

Index terms USA

Links 255

biocidal regulations

402

HPV Chemical Tracking System

267

medical device regulation

359

polymer notification

308

Uterotrophic assay

409

30

V Vapour pressure

90

balance method

92

calculation method

92

isoteniscope method

92

static method

92

Very persistent and very bioaccumulative substance Very Toxic

91

230 10

Vigilance requirements

357

Volatilisation

169

Voluntary Children’s Chemical Evaluation Program

269

VwVwS

220

W Wassergefahrdungsklassen Water accommodated fraction Water Hazard Classification Water solubility

220 78 220 93

column elution method

93

flask method

93

tests

114

visual estimate

93

Water:biota partition coefficient

171

WGK

220

White Paper

227

Wick-effect

113

Wood preservatives

406

417

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194

465

Index terms Workplace

Links 93

Workplace Hazardous Materials Information System

276

Worst-case scenario

404

Z Zebra fish

75

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