lnkr national Environmental Health Criteria 60 Principles and Methods for the Assessment of Neurotoxicity Associated With Exposure to Chemicals Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization WORLD HEALTH ORGANIZATION GENEVA 1986 Other titles available in the ENVIRONMENTAL HEALTH CRITERIA series include: 1. Mercury 32. Methylene Chloride 2. Polychlorinated Biphenyls and 33. Epichlorohydrin Terphenyls 34. Chlordane 3. Lead 35. Extremely Low Frequency 4. Oxides of Nitrogen (ELF) Fields 5. Nitrates, Nitrites, and 36. Fluorides and Fluorine N-Nitroso Compounds 37. Aquatic (Marine and 6. Principles and Methods for Freshwater) Biotoxins Evaluating the Toxicity of 38. Heptachlor Chemicals, Part 1 39. Paraquat and Diquat 7. Photochemical Oxidants 40. Endosulfan 8. Sulfur Oxides and Suspended 41. Quintozene Particulate Matter 42. Tecnazene 9. DDT and its Derivatives 43. Chlordecone 10. Carbon Disulfide 44. Mirex 11. Mycotoxins 45. Camphechlor 12. Noise 46. Guidelines for the Study of 13. Carbon Monoxide Genetic Effects in Human 14. Ultraviolet Radiation Populations 15. Tin and Organotin Compounds 47. Summary Report on the 16. Radiofrequency and Microwaves Evaluation of Short-Term Tests 17. Manganese for Carcinogens (Collaborative 18. Arsenic Study on In Vitro Tests) 19. Hydrogen Sulfide 48. Dimethyl Sulfate 20. Selected Petroleum Products 49. Acrylamide 21. Chlorine and Hydrogen 50. Trichloroethylene Chloride 51. Guide to Short-Term Tests for 22. Ultrasound Detecting Mutagenic and 23. Lasers and Optical Radiation Carcinogenic Chemicals 24. Titanium 52. Toluene 25. Selected Radionuclides 53. Asbestos and Other Natural 26. Styrene Mineral Fibres 27. Guidelines on Studies in 54. Ammonia Environmental Epidemiology 55. Ethylene Oxide 28. Acrylonitrile 56. Propylene Oxide 29. 2,4-Dichlorophenoxyacetic 57. Principles of Toxicokinetic Acid (2,4-D) Studies 30. Principles for Evaluating 58. Selenium Health Risks to Progeny 59. Principles for Evaluating Health Associated with Exposure Risks from Chemicals During to Chemicals during Pregnancy Infancy and Early Childhood: 31. Tetrachloroethylene The Need for a Special Approach This report contains the collective views of an in­ ternational group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the Interna­ tional Labour Organisation, or the World Health Organization Environmental Health Criteria 60 PRINCIPLES AND METHODS FOR THE ASSESSMENT OF NEUROTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Organization Geneva, 1986 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisa­ tion, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by IPCS include the develop­ ment of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. ISBN 92 4 154260 8 ©World Health Organization 1986 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Conven­ tion. For rights of reproduction or translation of WHO publications, in part or in toto, application should be made to the Office of Publications, World Health Organization, Geneva, Switzerland. The World Health Organization welcomes such applications. The designations employed and the presentation of the material in this publica­ tion do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organiza­ tion in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. ISSN 0250-863X PRINTED IN FINLAND DHSS - V AMMALA - 5500 - 3 - CONTENTS PRINCIPLES AND METHODS FOR THE ASSESSMENT OF NEUROTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS PREFACE • • • • • 11 1. INTRODUCTION 13 1.1 The importance of studying the nervous system 13 1.1.1 Methylmercury . • • . • . • 14 1.1.2 Carbon disulfide (CS2) • • • • 16 1.2 Need to establish a comprehensive strategy for neurotoxicity testing 18 1.3 Scope of the book • . • • 19 1.4 Purpose of the publication 20 2. GENERAL PRINCIPLES FOR THE ASSESSMENT OF TOXIC EFFECTS OF CHEMICALS ON THE NERVOUS SYSTEM 22 2.1 Factors to be considered in the design of neurotoxicity studies 22 2.1.1 General considerations 22 2.1.2 Objectives 22 2.1.3 Choice of animals • • • 25 2.1.4 Dosing regimen 27 2.1.5 Functional reserve and adaptation 27 2.1.6 Other factors . • • • • 27 2.2 Statistical analysis • • • • • • . • . • 28 2.2.1 Type I and Type II errors • . 28 2.2.2 Selection of the appropriate statistical test(s) • • • . • • • • • . • 30 3. TEST MErHODS IN BEHAVIOURAL TOXICOLOGY 32 3.1 Introduction ••••• 32 3.2 Classes of behaviour • 33 3.2.1 Respondent behaviour 33 3.2.2 Operant behaviour .• 36 3.3 Test methods .•••••.• 38 3.3.1 General attributes of behavioural methods .••.•.••••.•• 38 3.3.1.1 Sensitivity and specificity 38 3.3.1.2 Validity ••• 38 3.3.1.3 Replicability 39 3.3.1.4 Costs 39 - 4 - 3.3.2 Primary tests . • • • • • • • • • • • • 39 3.3.2.1 Functional observation battery 39 3.3.2.2 Motor activity . • . • 40 3.3.3 Secondary tests • • • • • • • • 41 3.3.3.1 Intermittent schedules of reinforcement 41 3.3.3.2 Motor function • • 44 3.3.3.3 Sensory function • 45 3.3.3.4 Cognitive function 47 3.3.3.5 Eating and drinking behaviour 53 3.3.3.6 Social behaviour • • . • • 54 3.3.4 Strengths and weaknesses of various methods . • • • • • • • 55 3.4 Research needs • • • • • . • • • • 56 3.4.1 Compensatory mechanisms • • 56 3.4.2 Method development and refinement 57 4. NEUROPHYSIOLOGICAL METHODS IN NEUROTOXICOLOGY 59 4.1 Introduction • . • • • • • • • • • . • • 59 4.2 Methods for evaluation of the peripheral nervous system • • • . • • • • . • • • • 59 4.2.1 Conduction velocity • • • • • • • 59 4.2.2 Peripheral nerve terminal function 60 4.2.3 Electromyography (EMG) • • • • • 61 4.2.4 Spinal reflex excitability • • • • 61 4.3 Methods for evaluation of the autonomic nervous system • • • • • • • • • • 62 4.3.1 Electrocardiography (EKG) ••••• 62 4.3.2 Blood pressure 62 4.4 Methods for the evaluation of the central nervous system • • • • • • • • • • • . .• 63 4.4.1 Spontaneous activity- electroencephal­ ography (EEG) •••• 63 4.4.2 Sensory systems •...••• 64 4.4.3 General excitability •••. 65 4.4.3.1 Convulsive phenomena 65 4.4.3.2 Stimulation of the cerebral motor cortex • • . 66 4.4.3.3 Recovery functions 67 4.4.4 Cognitive function 67 4.4.5 Synaptic and membrane activity 67 4.5 Interpretation issues 67 4.6 Summary and conclusions 69 5. MORPHOLOGICAL METHODS • • • • 71 - 5 - 5.1 Introduction. • • • • • • • • • • • • • • 71 5.1.1 Role of morphology • • • • • • 71 5.1.2 Basis for morphological assessment 71 5.2 The nervous system and toxic injuries 73 5.2.1 The nervous system • • • • 73 5.2.2 Cellular structure of the nervous system • • • • • • • 74 5.2.3 Neurocellular reaction to LnJury 76 5.2.3.1 Biological principles 76 5.2.3.2 Neurons • • • • • 77 5.2.3.3 Myelinating cells 78 5.3 Experimental design and execution 80 5.3.1 General principles and procedure 80 5.3.2 Gross morphology • • • • • • • • 81 5.3.3 The role of histology • • • • • • 81 5.3.3.1 Biological principles dictating tissue response 81 5.3.4 Use of controls •• 82 5.3.5 Pattern of response • 83 5.3.6 Data acquired •• 83 5.4 Principles, limitations, and pitfalls of the morphological approach • 85 5.4.1 Tissue state •••• 85 5.4.2 Principles of fixation 85 5.4.3 Principles of tissue sampling 87 5.4.4 Preparation of tissue for examination 89 5.4.5 Recognition of artefact ••••• 91 5.4.6 Recognition of normal structural variations • • • • • • • • •• 91 5.4.7 Qualitative versus quantitative approaches 91 5.5 Specific procedures 92 5.5.1 Introduction 92 5.5.2 Primary methods 92 5.5.2.1 Formaldehyde/paraffin method 92 5.5.2.2 Glutaraldehyde/epoxy method 95 5.5.3 Special methods ••••• 96 5.5.3.1 Peripheral nerve microdissection 96 5. 5. 3. 2 Frozen sections 97 5.5.3.3 Histochemical methods 98 5. 5.3.4 Golgi method • • • •• 99 5.5.3.5 Transmission electron microscopy • • • 99 5.5.3.6 Other anatomical methods 99 5.6 Conclusions 100 - 6 - 6. BIOCHEMICAL AND NEUROENDOCRINOLOGICAL METHODS 101 6.1 Introduction • • • • • • 101 6.2 Fractionation methods 102 6.2.1 Brain dissection 102 6.2.2 Isolation of specific cell types 103 6.2.3 Subcellular fractionation • • • 104 6.3 DNA, RNA, and