PART I

    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization

    World Health Organization
    Geneva, 1978

    ISBN 92 4 154066 4

    (c) World Health Organization 1978

          Publications of the World Health Organization enjoy copyright
    protection in accordance with the provisions of Protocol 2 of the
    Universal Copyright Convention. All rights reserved. The designations
    employed and the presentation of the material in this publication do
    not imply the expression of any opinion whatsoever on the part of the
    Secretariat of the World Health Organization concerning the legal
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    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 Organization 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.





          1.1. Introduction
                 1.1.1. Defining toxicity, hazard, risk, and related terms
                 1.1.2. Laboratory testing
                 1.1.3. Toxicological field studies
                 1.1.4. Ecotoxicology
                 1.1.5. Priorities in the selection of chemicals for
                 1.1.6. The extent of toxicity testing required
          1.2. Dose-effect and dose-response relationship
                 1.2.1. Dose
                 1.2.2. Effect and response
                 1.2.3. Dose-effect and dose-response curves
                 1.2.4. Toxic effects due to a combination of chemicals
          1.3. Interpretation of laboratory data
                 1.3.1. Distinction between adverse and nonadverse effects
                 1.3.2. Threshold: practical and theoretical considerations
                 1.3.3. Extrapolation of animal data to man
                    Species differences and related factors
                    Safety factors
                    Low-dose extrapolation
                    Other methods of extrapolation
          1.4. Human data
                 1.4.1. Ethical considerations
                 1.4.2. Need for human investigations
          1.5. The use of toxicological data in establishing environmental
                 health standards
                 1.5.1. Environmental health standards
                 1.5.2. Assessment of health risk and evaluation of
                 1.5.3. An example of toxicological information used in
                          standard setting
          1.6. Limitations of safety evaluation

          2.1. Introduction
          2.2. Chemical and physical properties
                 2.2.1. General considerations
                 2.2.2. Physicochemical properties and the design of
                          toxicity studies
                 2.2.3. Impurities

          2.3. Probable routes of exposure
                 2.3.1. General considerations
                 2.3.2. Specific variables related to route of exposure
                    Rate of absorption
                    Site of action
                    Unintended route
                 2.3.3. Special tests related to route
          2.4. Selection and care of animals
                 2.4.1. General considerations
                 2.4.2. Animal variables
                    Selection of species
                    Animal models representing special
                                     populations at risk
                 2.4.3. Cyclic variations in function or response
                 2.4.4. Environmental variables
                    Diet and nutritional status
          2.5. Statistical considerations
          2.6. Nature of effects
                 2.6.1. Reversible and irreversible effects
                 2.6.2. Functional versus morphological changes
          2.7. Dynamic aspects of predictive toxicology
                 2.7.1. Traditional versus new techniques
                 2.7.2. Toxicity of chemical analogues
                 2.7.3. Relation between site of metabolism and site of
                 2.7.4. In vitro test systems

          3.1. Introduction
          3.2. General nature of test procedures
                 3.2.1. Housing, diet, and clinical examination of test
          3.3. Acute toxicity tests
                 3.3.1. Underlying principles
                 3.3.2. Experimental design
                    Selection of species
                    Selection of doses
                    Method of administration
                    Postmortem examination
                 3.3.3. Repeated high-dose studies

          3.4. Subacute and chronic toxicity tests
                 3.4.1. Underlying principles
                 3.4.2. Experimental design
                    Selection of species and duration of
                    Selection of doses
                    Method of administration
                    Biochemical organ function tests
                    Physiological measurements
                    Metabolic studies
                    Haematological information
                    Postmortem examination
                 3.4.3. Alternative approaches in chronic toxicity
                    Perinatal exposure
                    Use of nonrodent species
          3.5. Evaluation and interpretation of the results of toxicity

          4.1. Introduction
          4.2. Absorption
                 4.2.1. General principles
                 4.2.2. Absorption from the lungs
                 4.2.3. Absorption from the skin
                 4.2.4. Gastrointestinal absorption
          4.3. Distribution
          4.4. Binding
                 4.4.1. Plasma-protein binding
                 4.4.2. Tissue binding
          4.5. Excretion
                 4.5.1. Renal excretion
                 4.5.2. Biliary excretion
                 4.5.3. Enterohepatic circulation
                 4.5.4. Other routes of excretion
          4.6. Metabolic transformation
                 4.6.1. Mechanism of metabolic transformation
                    Microsomal, mixed-function oxidations
                    Conjugation reactions
                    Extramicrosomal metabolic transformations
                    Nonenzymatic reactions
                 4.6.2. Species variability
                 4.6.3. Enzyme induction and inhibition
                 4.6.4. Metabolic saturation
          4.7. Experimental design
          4.8. Chemobiokinetics
                 4.8.1. One-compartment open model
                 4.8.2. Two compartment/multicompartment open systems
                 4.8.3. Repeated administration or repeated exposure
                 4.8.4. Kinetics of nonlinear or saturable systems

          4.9. Linear and nonlinear one compartment open-model kinetics of
                 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T)
          4.10. Linear chemobiokinetics used to assess potential for
                 bioaccumulation of 2,3,6,7-tetrachlorodibenzo-p-dioxin

          5.1. Introduction
          5.2. General recommendations
          5.3. Gross observations
                 5.3.1. Autopsy techniques
                 5.3.2. Rat, mouse, guineapig, rabbit, monkey
                 5.3.3. Carnivores, swine
          5.4. Selection, preservation, preparation, and storage of
                 5.4.1. Selection of tissues
                 5.4.2. Oral toxicity tests
                 5.4.3. Inhalation toxicity studies
                 5.4.4. Dermal toxicity studies
                 5.4.5. Special studies
          5.5. Preservation of tissues
                 5.5.1. Immersion
                 5.5.2. Inflation
                 5.5.3. Perfusion
          5.6. Trimming
          5.7. Storage
          5.8. Histological techniques
          5.9. Special techniques
                 5.9.1. Enzyme histochemistry
                 5.9.2. Autoradiography
                 5.9.3. Immunofluorescence and immunoenzyme techniques
                 5.9.4. Electron microscopy
          5.10. Microscopic examination
                 5.10.1. Number of animals and number of organs and tissues
                          studied microscopically
                 5.10.2. Description of the lesions
          5.11. Presentation, evaluation, and interpretation of
                 pathological data

          6.1. Introduction
          6.2. Need for inhalation studies
          6.3. Fate of inhaled materials
                 6.3.1. Nature of aerosols
                 6.3.2. Deposition
                 6.3.3. Clearance
          6.4. Dose in inhalation studies

          6.5. Choice of species
                 6.5.1. Anatomical differences
                 6.5.2. Physiological considerations
                 6.5.3. Disease and susceptibility states
          6.6. Duration of exposure
                 6.6.1. Intermittent versus continuous exposure
          6.7. Inhalation systems
                 6.7.1. Facilities required
                 6.7.2. Static systems
                 6.7.3. Dynamic systems
                 6.7.4. Typical whole-body systems
                 6.7.5. Construction materials
                 6.7.6. Engineering requirements
                 6.7.7. Special systems
                    Isolation units
                    Head and nose exposures
                    Instantaneous exposure systems
                 6.7.8. Variables to monitor
                 6.7.9. Human exposure facilities
          6.8. Contaminant generation and characterization
                 6.8.1. Generation of vapours
                 6.8.2. Particle generators
                    Heterogeneous aerosols
                 6.8.3. Monitoring contaminant concentrations
                    Vapour sampling
                    Particulate sampling
          6.9. Other methods of respiratory tract exposure
                 6.9.1. In vivo exposures
                 6.9.2. In vitro exposures
          6.10. Biological end-points and interpretation of changes in
                 these end-points
                 6.10.1. Morphological changes
                 6.10.2. Functional changes
                   Measurement of respiratory frequency
                   Measurement of mechanics of respiration
                 6.10.3. Biochemical end-points
                 6.10.4. Other end-points in inhalation studies

          7.1. Introduction
          7.2. Carcinogenicity
                 7.2.1. Long-term bioassays
                    Species, strain, and sex selection, and
                                     size of groups
                    Route of administration
                    Inception and duration of tests
                    Dose-level and frequency of exposure

                    Combined treatment and cocarcinogenesis
                    Positive and untreated controls
                    Test material
                    Survey of animals, necropsy, and
                                     histological examination
                 7.2.2. Short-term tests (rapid screening tests)
                    Metabolic activation, reaction with DNA,
                                     and DNA repair
                    In vitro neoplastic transformation of
                                     mammalian cells
                    Mutagenicity tests
                    Submammalian assay systems
                    Mammalian somatic cells
                    Host and tissue-(microsome) mediated
                 7.2.3. Correlation between short- and long-term bioassays
                          for carcinogenicity
                 7.2.4. Significance of experimental testing for assessing
                          the possible carcinogenic risk of chemicals to man
          7.3. Heritable mutations
                 7.3.1. Whole-animal tests
                 7.3.2. Monitoring of human populations
                 7.3.3. Significance of tests for heritable mutations


          While every effort has been made to present information in the
    criteria documents as accurately as possible without unduly delaying
    their publication, mistakes might have occurred and are likely to
    occur in the future. In the interest of all users of the environmental
    health criteria documents, readers are kindly requested to communicate
    any errors found to the Division of Environmental Health, World Health
    Organization, Geneva, Switzerland, in order that they may be included
    in corrigenda which will appear in subsequent volumes.

          In addition, experts in any particular field dealt with in the
    criteria documents are kindly requested to make available to the WHO
    Secretariat any important published information that may have
    inadvertently been omitted and which may change the evaluation of
    health risks from exposure to the environmental agent under
    examination, so that the information may be considered in the event of
    updating and re-evaluation of the conclusions contained in the
    criteria documents.


          The use of chemicals in practically every aspect of life has
    grown very rapidly over the last few decades and international trade
    in bulk chemicals, specialty chemicals, and consumer products has
    increased proportionately, making imperative the need for continuous
    review and reappraisal of procedures for evaluating their safety.
    Concern about the possible health hazards that may arise from exposure
    to chemicals has increased throughout the world, especially in the
    industrialized countries. In many WHO Member States, this has resulted
    in new laws and regulations which, in turn, have created a need to
    assemble, analyse, and evaluate all available toxicological
    information with a view to assessing hazard. Toxicologists have
    responded by developing techniques for safety evaluation but these
    often differ from one country to another. The differences are
    sometimes slight, sometimes considerable; on occasion they have led to
    unfortunate misunderstandings, and often to needless duplication of

          Ever since the World Health Organization started programmes on
    food safety and drug evaluation, the need for some degree of
    uniformity and for generally accepted principles and requirements for
    toxicological testing and evaluation has been recognized. This has
    resulted, in the last 20 years, in a number of technical reports and
    guidelines on such topics as the general principles and methods for
    the testing and evaluation of intentional and unintentional food
    additives (WHO, 1957, 1958, 1967a, 1974a) and drugs (WHO, 1966, 1968,
    1975a), on the evaluation of teratogenicity (WHO, 1967b),
    mutagenicity, and carcinogenicity (WHO, 1961, 1969, 1971, 1974b,
    1976a) and, more recently, on environmental and health monitoring and
    the early detection of health impairment in occupational health (WHO,
    1973, 1975b), on chemical and biochemical methodology for assessing
    the hazards of pesticides to man (WHO, 1975c), and on the methods used
    in establishing permissible levels of occupational exposure to harmful
    agents (WHO, 1977). Several symposia have also been organized, to
    discuss, for example, the methods used in the USSR for establishing
    biologically safe levels of toxic substances (WHO, 1975d, 1975e), and
    screening tests in chemical carcinogenesis (IARC, 1976). All these
    publications remain a most useful source of information on selected
    aspects of toxicological evaluation.

          The need for more uniformity in methods of environmental health
    risk evaluation was again raised at the 1973 World Health Assembly, in
    resolution WHA26.58 on human health and the environment which  inter
     alia requested the Director-General to develop protocols for
    experimental and epidemiological studies, uniform terminology, and
    agreed definitions. Harmonization of toxicological and epidemiological
    methods is also one of the objectives of the WHO Environmental Health
    Criteria Programme (WHO, 1976b), initiated in 1973 in collaboration

    with Member States and the United Nations Environment Programme
    (UNEP), while a very recent (1977) World Health Assembly resolution
    WHA30.47 requested the Director-General "to examine the possible
    options for international cooperation with a view to accelerating and
    making more effective the evaluation of health risks from exposure to
    chemicals, and promoting the use of experimental and epidemiological
    methods that will produce internationally comparable results".

          Current concern about the health effects of chemicals is more
    intense in some countries than in others, with the consequent
    unevenness in political response reflected in variations in national
    safety regulations. This situation is likely to continue for some
    time. It is unrealistic and perhaps not really desirable at present,
    to seek international standardization in safety testing and evaluation
    as this might hinder the input of new ideas and the development of
    improved methods and might lead either to the application of needless
    tests or to failure to ask the essential questions. However, it is not
    too early for scientists and decision-makers to try to understand the
    similarities and differences in the safety evaluations made in
    different countries. The underlying objectives are the same
    everywhere, namely, to minimize harm and maximize safety and yet not
    impede the beneficial use of chemicals. Similarly, the basic
    scientific principles are globally accepted, so there is no reason why
    there should not be a gradual harmonization of methods and procedures
    for toxicological testing and evaluation.

          With these views in mind and taking into account past work of
    WHO, an attempt was made to set forth, comprehensively and on an
    international basis, the principles and procedures for the safety
    evaluation of all types of chemicals. More than 50 distinguished
    experts from some 11 countries collaborated with the Organization and,
    in a series of meetings and individual consultations, planned,
    drafted, and revised this compilation of toxicological procedures,
    providing at the same time an excellent example of international
    cooperation. In addition, there was valuable support for the project
    from the WHO collaborating centres at: the Institute of Hygiene and
    Occupational Health, Sofia, Bulgaria; the National Institute of Public
    Health, Bilthoven, Netherlands; the Department of Environmental
    Hygiene, The Karolinska Institute, Stockholm, Sweden; the National
    Institute of Environmental Health Sciences, Research Triangle Park,
    North Carolina, USA; and the Sysin Institute of General and Communal
    Hygiene, Moscow, USSR.

          The general approach in preparing this publication has been to
    present the underlying scientific principles, to evaluate the utility,
    strengths and weaknesses of various methods and procedures, to help
    the reader select the most suitable technique for a specific purpose
    (bearing in mind that circumstances will often dictate the most
    appropriate procedure) but not, as already mentioned, to prescribe

    standard tests. While aiming at agreement on purely scientific issues,
    it has not been sought on details of procedure, on the interpretation
    of results, or on methods for setting environmental health standards.
    Indeed, because there were often differences of opinion on these
    matters, the solution adopted has been to present the different
    viewpoints and interpretations. This explains a certain unevenness in
    the text, particularly in those chapters prepared jointly by many
    scientists from different countries.

          Although an effort has been made to avoid inconsistency in
    terminology, uniformity has not been possible; indeed, this is
    something beyond the scope of the present monograph. However, WHO and
    UNEP recently initiated another project that aims at internationally
    agreed definitions for those terms most frequently used in
    toxicological evaluation. Until this project is completed, it is
    important to understand that some terms may have various meanings and
    implications in different countries or in different scientific circles
    and that it may be highly misleading to employ them outside the
    national pattern of use or outside the context of a specialized field,
    without precise definition. The reader is therefore warned to be wary
    of the uncritical transfer of technical terms from one set of
    circumstances to another.

          Toxicology is a rapidly developing field, especially at this
    time; it is hoped, nevertheless, that this monograph provides a valid
    account of the present state of knowledge on the toxicity testing and
    evaluation of chemicals as practiced by some of the leading experts in
    the field. If it should also stimulate the exchange of knowledge and
    experience and so contribute to greater efficiency and reliability in
    toxicity testing and evaluation, it will have more than fulfilled its

          The work has been divided into two separate publications. The
    first part contains the broad principles and more general aspects of
    toxicity testing, the planning and evaluation of acute, subacute, and
    chronic toxicity tests, chemobiokinetics and metabolism, morphological
    tests, inhalation studies, and tests for carcinogenicity and
    mutagenicity. Part 2 systematically covers some more specialized
    procedures for safety evaluation, i.e. functional studies of organs
    and systems, effects on reproduction, neurological and behavioural
    studies, effects on the skin and the eye, cumulation and adaptation,
    and finally discusses factors that could modify the outcome of
    toxicity testing and evaluation.

          The main authors mutually reviewed the chapters of the treatise,
    which can therefore be considered to be a synthesis of various views
    and opinions, but this does not detract from the merit of their own
    contributions which are gratefully acknowledged. The WHO Secretariat
    at the Meeting of the Main Authors in Geneva (28 July to 1 August
    1975)a and at the Scientific Group in Lyons (1 to 5 December 1975)b
    comprised: Dr M. El Batawi, Chief, Occupational Healtha;
    Dr H. Bartsch, Unit of Chemical Carcinogenesis, IARC, Lyonsb:
    Dr J. F. Copplestone, Vector Biology and Controlb; Dr F. C. Lu,
    Chief, Food Additivesb; Dr R. Montesano, Unit of Chemical
    Carcinogenesis, IARC, Lyonsa,b; Dr H. Nakajima, Drug Evaluation and
    Monitoringa; Dr M. Vandekar, Vector Biology and Controla; and
    Dr G. Vettorazzi, Food Additivesa. Dr V. B. Vouk, Chief, Control of
    Environmental Pollution and Hazards was the Secretary of the Geneva
    meeting, while Dr L. Tomatis, Chief, Unit of Chemical Carcinogenesis,
    IARC, Lyons, and Dr Vouk were the Joint Secretaries of the Scientific
    Group at Lyons. Representatives of other organizations who were
    present at the meetings include: Dr M. Marcus (US Environmental
    Protection Agency)a; Dr W. J. Hunter (Commission of the European
    Communities)b; Mr C. Prior (Organization for Economic Cooperation
    and Development)b; Dr V. Smirnyagin (International Council of
    Scientific Unions)b. Miss S. Braman, Technical Assistant, Control of
    Environmental Pollution and Hazards, serviced the two meetings and
    helped throughout with the preparation of the manuscript.

          The final editing was carried out by a group headed by Professor
    N. Nelson who, indeed, presided over the whole project and to whom
    special thanks are due for, without his ideas, enthusiasm and, above
    all, profound knowledge of the subject, there would have been no


    a   Participated in the Meeting of Main Authors, Geneva, 28 July to
        1 August 1975.

    b   Participated in the Scientific Group on Methods of Toxicity
        Evaluation of Chemicals, Lyons, 1-5 December 1975.


    IARC (1976)  Screening tests in chemical carcinogenesis -- Proceedings
           of a Workshop organized by IARC and the CEC, Brussels 1975.
          IARC Sci. Publ. No. 12.

    WHO (1957) WHO Technical Report Series No. 129 (General principles
          governing the use of food additives: First report of the Joint
          FAO/WHO Expert Committee on Food Additives.) 22 pp.

    WHO (1958) WHO Technical Report Series No. 144 (Procedures for the
          testing of intentional food additives to establish their safety
          for use: Second report of the Joint FAO/WHO Expert Committee on
          Food Additives.) 19 pp.

    WHO (1961) WHO Technical Report Series No. 220 (Evaluation of the
          carcinogenic hazards of food additives: Fifth report of the Joint
          FAO/WHO Expert Committee on Food Additives.) 33 pp.

    WHO (1966) WHO Technical Report Series No. 341 (Principles for
          pre-clinical testing of drug safety: Report of a WHO Scientific
          Group.) 22 pp.

    WHO (1967a) WHO Technical Report Series No. 348 (Procedures for
          investigating intentional and unintentional food additives:
          Report of a WHO Scientific Group.) 25 pp.

    WHO (1967b) WHO Technical Report Series No. 364 (Principles for the
          testing of drugs for teratogenicity: Report of a WHO Scientific
          Group.) 18 pp.

    WHO (1968) WHO Technical Report Series No. 403 (Principles for the
          clinical evaluation of drugs: Report of a WHO Scientific Group.)
          32 pp.

    WHO (1969) WHO Technical Report Series No. 426 (Principles for the
          testing and evaluation of drugs for carcinogenicity: Report of a
          WHO Scientific Group.) 26 pp.

    WHO (1971) WHO Technical Report Series No. 482 (Evaluation and testing
          of drugs for mutagenicity: principles and problems -- Report of a
          WHO Scientific Group.) 18 pp.

    WHO (1973) WHO Technical Report Series No. 535 (Environmental and
          health monitoring in occupational health: Report of a WHO Expert
          Committee.) 48 pp.

    WHO (1974a) WHO Technical Report Series No. 539 (Toxicological
          evaluation of certain food additives with a review of general
          principles and of specifications: Seventeenth report of the Joint
          FAO/WHO Expert Committee on Food Additives.) 40 pp.

    WHO (1974b) WHO Technical Report Series No. 546 (Assessment of the
          carcinogenicity and mutagenicity of chemicals: Report of a WHO
          Scientific Group.) 19 pp.

    WHO (1975a) WHO Technical Report Series No. 563 (Guidelines for
          evaluation of drugs for use in man: Report of a WHO Scientific
          Group.) 59 pp.

    WHO (1975b) WHO Technical Report Series No. 571 (Early detection of
          health impairment in occupational exposure to health hazards:
          Report of a WHO Study Group.) 80 pp.

    WHO (1975c) WHO Technical Report Series No. 560 (Chemical and
          biochemical methodology for the assessment of hazards of
          pesticides for man.) 26 pp.

    WHO (1975d)  Methods used in the USSR for establishing biologically
           safe levels of toxic substances. Geneva, WHO, 171 pp.

    WHO (1975e)  Methods for studying biological effects of pollutants
           (A review of methods used in the USSR). Copenhagen, WHO
          Regional Office for Europe, 80 pp. (EURO publication 3109(4).)

    WHO (1976a) WHO Technical Report Series No. 586 (Health hazards from
          new environmental pollutants: Report of a WHO Study Group.)
          96 pp.

    WHO (1976b)  Background and purpose of the WHO Environmental Health
           Criteria Programme. (Reprint from  Environmental Health
           Criteria 1 Mercury.) Geneva, WHO, 9 pp.

    WHO (1977) WHO Technical Report Series No. 601 (Methods used in
          establishing permissible levels in occupational exposure to
          harmful agents: Report of a WHO Expert Committee with the
          participation of ILO.) 68 pp.


    Editorial Group

    Dr F. A. Fairweather, Department of Health & Social Security, London,

    Professor F. Kaloyanova, Institute of Hygiene & Occupational Health,
          Sofia, Bulgaria

    Dr G. N. Krasovskij, Laboratory of Water Toxicology, A. N. Sysin
          Institute of General & Communal Hygiene, Moscow, USSR

    Dr R. Kroes, Central Institute for Nutrition & Food Research, Zeist,

    Dr R. Montesano, Unit of Chemical Carcinogenesis, International Agency
          for Research on Cancer, Lyons, France

    Professor S. D. Murphy, Division of Toxicology, Department of
          Pharmacology, The University of Texas Health Sciences Center,
          Houston, TX, USA

    Professor N. Nelson, Institute of Environmental Medicine, New York
          University, NY, USA  (Chairman)

    Professor D. V. Parke, Department of Biochemistry, University of
          Surrey, Guildford, England

    Professor I. V. Sanockij, Department of Toxicology, Institute of
          Industrial Hygiene & Occupational Diseases, Moscow, USSR

    Dr I. P. Ulanova, Department of Toxicology, Institute of Industrial
          Hygiene & Occupational Diseases, Moscow, USSR

    Dr V. B. Vouk, Control of Environmental Pollution and Hazards,
          Division of Environmental Health, World Health Organization,
          Geneva, Switzerland  (Secretary)

    Professor J. G. Wilson, Department of Pediatrics, Children's Hospital
          Medical Center, University of Cincinnati, Cincinnati, OH, USA


    Contributors to Part 1

    b Dr H. Bartsch, Unit of Chemical Carcinogenesis, International
          Agency for Research on Cancer, Lyons, France (Chapter 7)

    Dr S. M. Charbonneau, Toxicology Research Division, Health Protection
          Branch, National Department of Health & Welfare, Ottawa, Canada
          (Chapter 3)

    a Dr R. T. Drew, Medical Department, Brookhaven National Laboratory,
          Upton, NY, USA (Chapter 6)

    a Dr H. L. Falk, National Institute of Environmental Health
          Sciences, Research Triangle Park, NC, USA (Chapter 2)

    Dr V. J. Feron, Central Institute for Food Research, Zeist,
          Netherlands (Chapter 5)

    a Dr P. Gehring, Toxicology Research Laboratory, Dow Chemical USA,
          Midland, MI, USA (Chapter 4)

    b Dr H. C. Grice, Toxicology Research Division, Health Protection
          Branch, Department of National Health & Welfare, Ottawa, Canada

    a,b Professor F. Kaloyanova, Institute of Hygiene & Occupational
          Health, Sofia, Bulgaria

    a Dr G. N. Krasovskij, Laboratory of Water Toxicology, A. N. Sysin
          Institute of General & Communal Hygiene, Moscow, USSR (Chapter 1)

    a,b Dr R. Kroes, Central Institute for Nutrition & Food Research,
          Zeist, Netherlands (Chapters 1 & 5)

    Dr J. E. LeBeau, Toxicology Research Laboratory, Dow Chemical USA,
          Midland, MI, USA (Chapter 4)

    a,b Dr S. Manyai, Biochemical Department, Institute of Occupational
          Health, Budapest, Hungary (Chapter 4)

    a,b Dr R. Montesano, Unit of Chemical Carcinogenesis, International
          Agency for Research on Cancer, Lyons, France (Chapters 1 & 7)

    a Dr I. C. Munro, Toxicology Research Division, Health Protection
          Branch, Department of National Health & Welfare, Ottawa, Canada
          (Chapters 1, 2 & 3)

    a Professor S. D. Murphy, Division of Toxicology, Department of
          Pharmacology, The University of Texas Health Sciences Center,
          Houston, TX, USA (Chapters 1 & 2)

    a,b Professor N. Nelson, Institute of Environmental Medicine, New
          York University, NY, USA (Chapters 1 & 7)

    b Professor G. Nordberg, Institute of Hygiene & Social Medicine,
          Odense University, Odense, Denmark

    a,b Professor D. V. Parke, Department of Biochemistry, University of
          Surrey, Guildford, England (Chapters 1 & 2)

    a,b Dr E. A. Pfitzer, Department of Toxicology, Research Division,
          Hoffman-La Roche Inc., Nutley, NJ, USA (Chapters 1 & 2)

    a Dr M. A. Pinigin, A. N. Sysin Institute of General & Communal
          Hygiene, Moscow, USSR (Chapter 1)

    Dr J. C. Ramsey, Toxicology Research Laboratory, Dow Chemical USA,
          Midland, MI, USA (Chapter 4)

    b Professor I. V. Sanockij, Department of Toxicology, Institute of
          Industrial Hygiene & Occupational Diseases, Moscow, USSR
          (Chapters 1 & 2)

    Dr K. K. Sidorov, Department of Toxicology, Institute of Industrial
          Hygiene & Occupational Diseases, Moscow, USSR (Chapter 6)

    b Dr L. Tomatis, Unit of Chemical Carcinogenesis, International
          Agency for Research on Cancer, Lyons, France (Chapter 7)

    b Professeur R. Truhaut, Laboratoire de Toxicologie et d'Hygine
          industrielles, Facult des Sciences pharmaceutiques et
          biologiques. Universit Ren Descartes, Paris, France

    a Dr I. P. Ulanova, Department of Toxicology, Institute of
          Industrial Hygiene & Occupational Diseases, Moscow, USSR
          (Chapter 6)

    a,b Dr V. B. Vouk, Control of Environmental Pollution and Hazards,
          Division of Environmental Health, WHO, Geneva, Switzerland
          (Chapter 1)

    Dr Z. Zawidski, Toxicology Research Division, Health Protection
          Branch, Department of National Health & Welfare, Ottawa, Canada
          (Chapter 3)


    a   Participated in the Meeting of Main Authors, Geneva, 28 July to
        1 August 1975.
    b   Participated in the Scientific Group on Methods of Toxicity
        Evaluation of Chemicals, Lyons, 1-5 December 1975.


    1.1  Introduction

          Toxicology is concerned both with the nature and mechanisms of
    toxic lesions and the quantitative evaluation of the spectrum of
    biological changes produced by exposure to chemicals. Every chemical
    is toxic under certain conditions of exposure. An important corollary
    is that for every chemical there should be some exposure condition
    that is safe as regards man's health (Lazarev, 1938; Pravdin, 1934;
    Smyth, 1963; Weil, 1972a) with the possible exception of chemical
    carcinogens and mutagens (WHO, 1974a).

          The quantitative evaluation of the biological changes caused by
    chemicals aims at the establishment of dose-effect and dose-response
    relationships that are of fundamental importance for health risk

    1.1.1  Defining toxicity, hazard, risk, and related terms

          In a general sense, the toxicity of a substance could be defined
    as the capacity to cause injury to a living organism (NAS/NRC, 1970;
    Sanockij, 1970). A highly toxic substance will damage an organism if
    administered in very small amounts; a substance of low toxicity will
    not produce an effect unless the amount is very large. Thus, toxicity
    cannot be defined without reference to the quantity of a substance
    administered or absorbed (dose), the way in which this quantity is
    administered (e.g. inhalation, ingestion, injection) and distributed
    in time (e.g. single dose, repeated doses), the type and severity of
    injury, and the time needed to produce that injury.

          There is no generally agreed definition of "hazard" associated
    with a chemical, but the term is used to indicate the likelihood that
    a chemical will cause an adverse health effect (injury) under the
    conditions in which it is produced or used (Goldwater, 1968; NAS/NRC,
    1970, Pravdin, 1934).

          Risk is a statistical concept and has been defined by the
    Preparatory Committee of the United Nations Conference on the Human
    Environmenta, as the expected frequency of undesirable effects
    arising from exposure to a pollutant. Estimates of risk may be
    expressed in absolute terms or in relative terms. The absolute risk is
    the excess risk due to exposure. The relative risk is the ratio
    between the risk in the exposed population and the risk in the
    unexposed population (BEIR, 1972; ICRP, 1966).


    a   Preparatory Committee of the United Nations Conference on the
        Human Environment, Third Session, 13-24 September 1971
        (A/Conf. 4818, pp. 45 & 46).

          Safety is a term that has been used extensively but is difficult
    to define. One definition is that "safety" is the practical certainty
    that injury will not result from the substance when used in the
    quantity and in the manner proposed for its use (NAS/NRC, 1970). This
    definition is of little use unless "practical certainty" is defined in
    some way, for example, in terms of a numerically specified low risk.
    Another view is that "safety" should be judged in terms of socially
    "acceptable" risks. Such judgments are largely outside the scope of
    scientific evaluation but nevertheless require assessment both of the
    probabilitiesa of various adverse effects and of their severity in
    terms of human health or other concerns (NAS, 1975).

    1.1.2  Laboratory testing

          Human data on the toxicity of chemicals are obviously more
    relevant to safety evaluation than those obtained from the exposure of
    experimental animals (see section 1.4). However, controlled exposures
    of man to hazardous or potentially hazardous substances are limited by
    ethical considerations and information obtained by clinical or
    epidemiological methods must be relied on. Where such information is
    not available, as in the case of all new synthetic chemicals, data
    must be obtained from tests on experimental animals and other
    laboratory procedures. The degree of confidence with which human
    health risks can be estimated from laboratory data depends on the
    quality of the data, and the selection of appropriate laboratory
    testing procedures is the main subject of this monograph.

    1.1.3  Toxicological field studies

          In the laboratory, only a small number of animal species are
    available for testing. The testing of wild species, living in cages
    under field conditions, may be useful but sometimes presents a variety
    of problems. Successful trials require a large enough site (about
    8 ha; 20 acres) with adequate and varied populations of birds,
    mammals, fish, insects, and other species, and the area studied must
    be considerably greater than that treated (Brown & Papworth, 1974).
    Data obtained from field trials of chemicals are of considerable value
    in supplementing data obtained with laboratory animal species and in
    validating the projection of experimental results to the ecosystem,
    including man. Studies of random events in natural ecosystems can also
    provide useful data.


    a  i.e. the expected frequencies.

          Sensitive analytical techniques now make it relatively simple to
    conduct field studies in man by monitoring levels of a chemical or its
    metabolites in blood, urine, hair, or saliva; this biological
    monitoring together with environmental monitoring provides important
    information on the exposure of mana. Regular periodic determination
    of the profile of certain plasma enzymes and other biochemical
    variables in the subject provides another valuable method for
    monitoring health effects particularly under occupational exposure
    conditions (WHO, 1973, 1975a, 1975b); changes in these profiles may
    provide early warning of damage by toxic chemicals (Cuthbert, 1974).

    1.1.4  Ecotoxicology

          A new subdivision of toxicology, "ecotoxicology", has emerged
    following observations that some persistent chemicals can exert toxic
    effects at several points in an ecosystem. The appearance of a
    chemical or the manifestation of a toxic effect may occur far away
    from its initial point of introduction into the environment. Methods
    for assessing the extent and significance of the movement of
    pollutants and their degradation products through the environment to
    target systems are discussed in a recent publication (NAS, 1975).

    1.1.5  Priorities in the selection of chemicals for testing

          In principle, all new chemicals require safety evaluation before
    manufacture and sale, but, because of the large number of chemicals
    that represent a possible hazard to human health and limited
    resources, it is necessary to give priority to those that are directly
    consumed by man, such as drugs and food additives, and those that are
    widely used such as pesticides or household consumer products.
    Industrial chemicals that can escape into the working or general
    environment or can contaminate other products are another category of

          Compounds of suspected high acute, chronic, or delayed toxicity
    (such as carcinogenicity) or of high persistence in the environment,
    or compounds which contain chemical groups known to be associated with
    these properties, deserve the highest priority. This also applies to
    compounds known to inhibit metabolic deactivation of chemicals as they
    may represent a more insidious form of toxicity.


    a   Report of the Meeting of a Government Expert Group on Health
        Related Monitoring. Unpublished WHO document CEP/77.6.

          Chemicals resistant to metabolism, especially metabolism by
    microflora, will have a high environmental persistence. Many
    halogenated compounds come into this category, and should, therefore,
    have some degree of priority. Compounds that accumulate in food chains
    or are stored in the body, e.g. methylmercury and DDT, will be a
    matter of concern. Such compounds are often highly lipid-soluble or
    strongly bound to tissue proteins, or may undergo enterohepatic
    recirculation with consequent slow excretion resulting in accumulation
    in the organism.

          Physicochemical properties can be an important consideration in
    setting priorities for testing potential environmental pollutants. For
    example, biomagnification of stable, fat-soluble substances may lead
    to contamination of human food supplies as well as to adverse effects
    in wildlife at the higher levels of food chains, even though the
    intended use and sites of application of the substance would suggest
    that primary exposure of these species is unlikely (Edwards, 1970).
    Physicochemical properties such as vapour pressure, and particle size
    and density are important in predicting the atmospheric transport of
    chemicals (Fuchs, 1964; OECD, 1977). Adsorption of a chemical on soil
    particles may increase the likelihood that the material will become
    airborne or be transported by watercourses and subsequently deposited
    in areas remote from its site of application (Cohen & Pinkerton,
    1966), or it may retard the movement of a chemical through ground
    water and thus reduce the likelihood of contamination of ground water
    supplies near the site of application (Edwards, 1970; Hamaker et al.,

          Even though certain predictions and comparisons of environmental
    distribution and biomagnification of chemicals in the environment may
    be made theoretically on the basis of the physicochemical properties
    of the substances in question, more definitive information of this
    nature can be obtained experimentally by the use of model ecosystems
    such as those described by Metcalf et al. (1971) and Lu & Metcalf
    (1975). These model ecosystems may be oversimplified, and they should
    not replace experimental field studies or programmes for monitoring
    environmental contaminants. However, their use in an early phase of
    the overall evaluation of the toxicity of environmental chemicals may:
    ( a) help to determine order of priority of chemicals for study,
    ( b) identify the components of the environment (food, water, air)
    most likely to be a source of human exposure and ( c) suggest whether
    the chemicals are likely to accumulate in human tissues.
    Furthermore, the systematic application of such model systems to
    structure-distribution studies may help in determining with greater
    certainty those physicochemical properties of substances that are most
    useful in predicting the distribution and effects of chemicals in
    ecosystems (Lu & Metcalf, 1975).

          Information on production, use, and disposal are of great
    importance in determining the sources and quantities of a chemical
    released into the environment, in assessing the possible extent of
    human exposure, and in identifying human populations that are likely
    to be exposed.

          In conclusion, essential criteria for priority in the selection
    of chemicals for testing are: (a) indication or suspicion of hazard to
    human health and type and severity of potential health effects; (b)
    probable extent of production and use; (c) potential for persistence
    in the environment; (d) potential for accumulation in biota and in the
    environment, and (e) type and size of populations likely to be
    exposed. A chemical of first priority for testing would rate highly
    with respect to all or most of these criteria.

    1.1.6  The extent of toxicity testing required

          The extent of the toxicity testing required will depend on a
    variety of considerations, and generally valid procedures cannot be
    proposed. One scheme, proposed by Sanockij (1975a), for chemicals that
    are being developed, is shown in Table 1.1. As a first step, it may be
    useful to make an approximate estimation of toxicity based on the
    chemical structure and the physical and chemical properties of the
    substance, and on known correlations of these variables with
    biological activity (Andreyeshcheva, 1976; WHO, 1976a). These
    considerations may be of value for decisions on safety measures to be
    taken during initial laboratory work. Extrapolation and interpolation
    in homologous series may also be of value for decisions on safety
    measures to be taken during initial laboratory work (Ljublina &
    Miheev, 1974), but for some series of chemicals this is not

          A preliminary evaluation of toxicity should start when chemicals
    are synthesized in the laboratory stage of the development of an
    industrial process. The full evaluation of the chemicals involved,
    both in respect to occupational and general population exposure, and
    assessment of possible air, water, and food contamination, should be
    initiated later, when it has been decided to proceed with full-scale
    production of the chemical. Toxicity data obtained during the
    development stages of a technological process could provide
    information concerning the health hazards not only of the raw
    materials and products, but also of the various other substances used
    or produced as intermediates in the technological process, and of
    gaseous and other wastes. Toxicological evaluation may also help in
    the selection of an alternative technological process, less hazardous
    to health.

          Waste disposal by dispersion in air and water, the ease of
    environmental degradation of the chemical, and the toxicity of the
    degradation products, are other problems that need attention at an
    early stage in the toxicological evaluation of new chemicals. For
    example, resistance to degradation has to be taken into account when
    formulating health criteria regulating the application and disposal of
    pesticides (Medved & Spynu, 1970).

          This phasing of toxicological studies may be useful in
    coordinating testing at national and international levels.

          Environmental and health standards will need to be defined
    preferentially for those chemicals that show a significant degree of
    toxicity and represent a health hazard, and are likely to be used
    widely in industry, agriculture, or in consumer products.

          Changes and developments in industrial processes, the development
    of new chemicals, and changes in the use of existing chemicals, may
    lead to new or increased hazards. This calls for a continuous
    re-evaluation of priorities.

    1.2  Dose-Effect and Dose-Response Relationships

    1.2.1  Dose

          Most commonly, the term "dose" is used to specify the amount of
    chemical administered, usually expressed per unit body weight. If the
    dose is administered into the stomach, on the skin, or into the
    respiratory tract, transport across the membranes may be incomplete
    and the absorbed dose will not be identical with the dose
    administered. In environmental exposures, an estimate of the dose can
    be made from the measurement of environmental and food concentrations
    as a function of time, and involves the assessment of food intake,
    inhalation rate, and the appropriate deposition and retention factors.

          The doses in the organs and tissues of interest may be estimated

          (a)  administered dose or intake;

          (b)  measurement of the concentrations in tissues and organ

          (c)  measurement of concentrations in excreta or exhaled air.

        Table 1.1  The extent of toxicological evaluation required in relation to technological process development

    Stages of technological          Stages of toxicological       Toxicological studies
    development                      evaluation

    1. Theoretical concept           Preliminary toxicological     Analysis of literature data on toxicity and
       and process flow              assessment                    hazards of raw materials, reagents,
       diagram                                                     catalysers, semiproducts and additives

                                                                   Assessment of toxicological parameters on
                                                                   the basis of metabolic analogies, persistence,
                                                                   the relationship between chemical
                                                                   structure, chemical and physical properties.
                                                                   and biological activity. Interpolation and
                                                                   extrapolation in homologous series

    2. Laboratory development        Acute toxicity                Acute and subacute experiments on
       of the technological                                        animals. Toxicological evaluation of
       process                                                     technological unit processes

    3. Pilot plant stage             Subacute toxicity             Subacute toxicity experiments on animals.
                                                                   Studies of delayed effects. Medical
                                                                   examination of workers.

                                     Detailed toxicological        Chronic toxicity studies and, when indicated,
                                     evaluation                    effects on reproduction, carcinogenicity,
                                                                   mutagenicity. Formulation of medical and
                                                                   industrial hygiene requirements for
                                                                   full-scale production

    Table 1.1  (cont'd).

    Stages of technological          Stages of toxicological       Toxicological studies
    development                      evaluation

    4. Design of industrial          Additional studies            Studies of the mechanism of action, early
       scale process                                               and differential diagnosis, experimental

    5. Production and use            Field studies                 Assessment of working and environmental
       of chemicals                                                conditions and of health status of workers
                                                                   and general population

                                                                   Epidemiological studies

                                                                   Clinical evaluation of experimental
                                                                   prophylactic, diagnostic and therapeutic

                                                                   Adjustment and correction of requirements
                                                                   for health and environmental protection

    The use of these three types of information for the purposes of tissue
    and organ dose estimation requires the postulation of models to
    describe the absorption, distribution, retention, biotransformation,
    and excretion of the original chemical or its metabolites, as a
    function of time (see Chapter 4).

          When the site of toxic action is located at, or very near, the
    site of application, for example, the skin, then the tissue dose
    estimate may be very reliable. However, when the site of toxic action
    is remote, for example, a liver cell, then the estimates of
    toxicologically significant doses are much less reliable.

          The presence of a chemical in the blood indicates absorption;
    however, the blood concentration of a chemical is in a dynamic state,
    reaching higher levels with increasing absorption but decreasing as
    the distribution, tissue storage, metabolic transformation, and
    excretion increase. The blood concentration of a chemical is useful as
    an indicator of the dose only when it is related in a defined manner
    to the concentration at the site or sites of action (organs and
    tissues) (Task Group on Metal Toxicity, 1976).

    1.2.2  Effect and response

          "Effect" and "response" are often used interchangeably to denote
    a biological change, either in an individual or in a population,
    associated with an exposure or dose. Some toxicologists have, however,
    found it useful to differentiate between an effect and a response by
    applying the term "effect" to a biological change, and the term
    "response" to the proportion of a population that demonstrates a
    defined effect (Pfitzer, 1976; Task Group on Metal Toxicity, 1976).

          In this terminology, response means the incidence rate of an
    effect. For example, the LD50 value may be described as the dose
    expected to cause a 50% response in a population tested for the lethal
    effect of a chemical. This distinction will be made in the present
    monograph, although it should be recognized that this terminology is
    not generally accepted.

          An effect can usually be measured on a graded scale of intensity
    or severity and its magnitude related directly to the dose. Certain
    effects, however, permit no gradation and can be expressed only as
    "occurring" or "not occurring". Such effects are usually called
    "quantal" (see for example, Finney, 1971). Typical examples of quantal
    effects are death or occurrence of a tumoura.

          The toxic action of chemicals usually affects the whole organism
    but the primary damage may be localized in a specific target organ or
    organs in which the toxic injury may manifest itself in terms of
    dysfunction or overt disease (NIEHS, 1977). According to Sanockij
    (1975a), the specificity of acute toxic action can be expressed in
    terms of a "zone of specific action" (Zsp) which is the ratio
    between the thresholdb dose of an acute effect at the level of the
    total organism and the threshold dose for an acute effect at a
    specific organ or system. If Zsp > 1, the toxic action is specific;
    if Zsp < 1, it is non-specific.

          Acute effects are those that occur or develop rapidly after a
    single administration (Casarett, 1975) but acute effects may appear
    after repeated or prolonged exposure as well. Chronic effects may also
    result from a single exposure but more often they are a consequence of
    repeated or prolonged exposures. Chronic effects are characterized not
    only by their duration but also by certain pathological features. They
    may arise from the accumulation of a toxic substance or its
    metabolites in the body, or from a summation of acute effects. The
    latent period (or the "time-to-occurrence" of an observable effect)
    may sometimes be very long, particularly if the dose or exposure is
    low. Other aspects of the nature of toxic effects are discussed in
    section 2.6.


    a  A similar classification of effects is used in radiological
       protection where a distinction is made between "nonstochastic" and
       "stochastic" effects (ICRP, 1977). Nonstochastic effects are those
       for which the severity of effect varies with the dose. Stochastic
       effects are those for which the probability of occurrence, rather
       than their severity, is regarded as a function of dose. Hereditary
       effects and carcinogenesis induced by radiation are considered to
       be stochastic.

    b  The threshold concept is discussed in section 1.3.2.

          Not every effect is necessarily adverse or harmful. In some
    cases, a graded effect may be either within the so-called "normal"
    range of physiological variation, or an "adverse" effect, depending on
    its intensity. The distinction between a physiological change and a
    pathological effect (adverse effect) is sometimes very difficult to
    make and there is much disagreement on this subject which will be
    discussed in detail in section 1.3.1. The concept of biochemical
    lesion introduced by Peters and his collaborators (Gavrilescu &
    Peters, 1931; Peters, 1963, 1967), and based on the ideas of Claude
    Bernard (Bernard, 1898), is of fundamental importance in this respect.
    A biochemical lesion can be defined as the biochemical change or
    defect which directly precedes pathological change or dysfunction
    (Peters, 1967).

          The Task Group on Metal Accumulation (1973) and the Task Group on
    Metal Toxicity (1976) have defined the critical concentration for a
    cell as the concentration (of a metal) at which undesirable (adverse)
    functional changes, reversible or irreversible, occur in the cell.
    Critical organ concentration has been defined as the mean
    concentration in the organ at the time any of its cells reaches
    critical concentration and critical organ as that particular organ
    which first attains the critical concentration of a metal under
    specified circumstances of exposure and for a given population. This
    definition of "critical organ" differs from the generally accepted use
    of the term, i.e. that the critical organ is the organ whose damage
    (by radiation) results in the greatest injury to the individual (or
    his descendants) (ICRP, 1965). However, some toxicologists question
    the usefulness of the concept of a critical organ or tissue because it
    diverts attention from the role that the various regulatory systems of
    the body may have in relation to a toxic injury.

    1.2.3  Dose-effect and dose-response curves

          Dose-effect curves demonstrate the relation between dose and the
    magnitude of a graded effect, either in an individual or in a
    population. Such curves may have a variety of forms. Within a given
    dose range they may be linear but more often they are not. Finney
    (1952a) has discussed various transformations that can be used to make
    dose-effect curves linear.

          Dose-response curves demonstrate the relation between dose and
    the proportion of individuals responding with a quantal effect. In
    general, dose-response curves are S-shaped (increasing), and they have
    upper and lower asymptotes, usually but not always 100 and 0% (see for
    example Cornfield, 1954). One way of explaining the shape of
    dose-response curves is that each individual in a population has a
    unique "tolerance" and requires a certain dose before responding with
    an effect. There exists, in principle, a low dose to which none will
    respond and a high dose to which all will respond.

          For each effect there will usually be a different dose-response
    curve. Loewe (1959) and Hatch (1968) have discussed the relationship
    between dose, effect, and response and its graphical representation in
    a three-dimensional model.

          If the experiment or observation is well designed (Chapters 2 and
    3), the dose-response relationship will be based on data from many
    individuals over a range of doses from minimum to maximum response.
    Mathematical and statistical procedures are then used to establish the
    curvilinear relationship that provides the best fit to all of the
    data, expressed as mean values with their standard deviations at
    different doses. Mathematical expressions for dose-effect and
    dose-response relationships and the merits of applying normal,
    log-normal, and other types of distributions are discussed in the

          It should be pointed out that the shape of the dose-response
    curve for the same substance and the same animal species may vary with
    changes in experimental conditions, such as changes in the way in
    which the dose is distributed in time (Weil, 1972a).

          In evaluating human exposure to environmental chemicals, the dose
    will usually be estimated as a function of concentration and time. In
    some cases the concentration will be fairly constant and then the
    time-effect and time-response relationships will be similar to the
    dose-effect and dose-response relationships. However, in many cases
    the concentration will vary, as will the time of exposure to specific
    concentrations, and integrated relationships of dose-concentration-
    time must be considered as well as dose-effect and time-effect
    relationships (Druckrey, 1967; Golubev et al., 1973; Lazarev, 1963;
    Weil, 1972a).

          Haber's rule  (ct=k) states that the product of concentration
     (c) and time  (t) results in a constant intensity of effect  (k)
    for some gases. This formula was later changed to  ctb =  k (where
     b is constant) which fitted other biological data better (Lazarev &
    Brusilovskaja, 1934), although it also has its limitations. The
    extrapolation of concentration-time relationships has been used
    successfully to obtain predictions of response following long-term
    inhalation exposure to low concentrations (Pinigin, 1974).

          Concentration-time relationships, such as the variation of the
    fraction of the dose with time as in combinations of short-term peak
    concentrations and prolonged low-level concentrations in air
    pollution, and variable cycles of exposure, may influence the toxic
    effect. Few systematic attempts to evaluate these factors have been
    made, although Sidorenko & Pinigin (1975, 1976) have described some
    principles for setting air quality standards from this viewpoint, and
    Pinigin (1974) has dealt with the problems of intermittent inhalation
    exposure. This problem has also been discussed by Ulanova et al.
    (1973, 1976).

    1.2.4  Toxic effects due to a combination of chemicals

          When an organism is exposed to two or more chemicals, their joint
    action may be:

          (a)  independent -- when the chemicals produce different effects
               or have different modes of action;

          (b)  additive -- when the magnitude of an effect or response
               produced by two or more chemicals is numerically equal to
               the sum of the effects or responses that the chemicals would
               produce individually;

          (c)  more than additive -- often called potentiation or

          (d)  less than additive (antagonism, inhibition).

          More specific terminology may be used when the mechanisms of
    joint action are known or when definite assumptions are made about
    them (Finney, 1971; Hewlett & Pluckett, 1961). The time intervals and
    sequences between exposures to different chemicals are extremely
    important, and the quality as well as the degree of joint action may
    depend on these variables (Kagan, 1973; Kustov et al., 1974; Williams,
    1969). Furthermore, the joint action at lethal dose levels may be
    quite different from that at low dose levels, when the effects or
    responses are often only additive or independent (Smyth et al., 1969;
    Ulanova, 1969).

          Most statistical models for joint action have been developed for
    situations in which two or more chemicals are administered
    simultaneously or within a short (few minutes) time interval. A model
    proposed by Finney (1952b, 1971) is often used for predicting the
    acute joint toxicity of chemicals. The model is strictly applicable to
    mixtures of chemicals that act at the same site, producing the same
    type of acute toxic effect and having parallel regression lines of
    probits against log doses (see Appendix). For a mixture of, for
    example, three chemicals, the equation for the median effective dose
    (ED50) is

         1      contour integralA   contour integralB   contour integralC
              =                   +                   +                
    ED50 (A,B,C)      ED50 (A)             ED50 (B)             ED50 (C)

    where contour integralA, contour integralB and contour integralC are
    the fractions of substances  A, B, and  C in the mixture. When all
    the values on the right hand side of equation (1) are known, a predicted
    ED50 (assuming additive joint action) can be calculated and compared
    with the actual ED50 of the mixture determined experimentally. A
    smaller than predicted ED50 demonstrates a more than additive
    response (synergism), a greater than predicted ED50 indicates a less
    than additive response (antagonism). Smyth et al. (1969) demonstrated
    that this equation can give satisfactory results under conditions that
    are less restrictive than stated above, for example in identifying the
    type of acute joint action among randomly selected industrial
    chemicals. Ball (1959) applied the equation to the estimation of
    maximum allowable concentrations for occupational exposure to mixtures
    of substances that exercise a "similar joint action", e.g. benzene and
    toluene. Another model for estimating the results of joint action has
    been developed using the isoeffective concentrations instead of ED50
    in equation (1) (Pinigin, 1974).

          The possibility of predicting the type of joint action is
    enhanced if there is information on the metabolism and disposition of
    the chemicals (Murphy, 1969; Williams, 1969). Basic principles
    concerning the kinetics of reactions of chemicals with primary sites
    (tissue receptor sites) and with secondary sites are important in
    considering the joint action of chemicals (Gaddum, 1957; Schild et
    al., 1961; Veldstra, 1956; Williams, 1969). The relevant factors seem
    to be the relative affinities at the sites of action (e.g. target
    enzymes, neuroeffector sites, and other vital target sites), and at
    the sites of loss or sinks (e.g. detoxifying enzymes, nonvital tissue
    binding sites, pathways of excretion, and storage sites), and the
    intrinsic activity at the sites of actiona. Since there is a limited
    number of sites of action and sinks within any organism, there will be
    a limited dose range within which synergism or antagonism can be
    demonstrated. This, of course, is only one area where more information
    could help in predicting the effects of the joint action of chemicals.
    Other areas where knowledge is insufficient are the possible effects
    of low-level, prolonged exposures to mixtures of chemicals and the
    effects of multiple stresses including chemicals, physical factors
    such as heat and noise, and pre-existing disease (NIEHS, 1970).


    a  Relative affinity -- reciprocal of the dissociation constant for
       the chemical-receptor complex. Intrinsic activity -- the capacity
       of the chemical to produce an effect when it combines with a
       reactive tissue site. For precise definitions see for example
       Arins et al. (1957).

          Simultaneous exposures to the same chemical in different media
    (e.g. air, water, food) which is called "complex action" by some
    toxicologists (Korbakova et al., 1971; Kustov et al., 1974; Pinigin,
    1974; Spynu et al., 1972) is another aspect of multiple stresses which
    has considerable practical importance.

    1.3  Interpretation of Laboratory Data

          It is essential that all experiments to evaluate toxicity should
    be designed to be scientifically meaningful, and should not be
    conducted merely to comply with statutory regulations. Thus, the
    evaluation of each new chemical will not be an identical task and
    procedures will differ, to some extent, from one compound to another.
    The protocol for an experiment will evolve gradually during the
    experiment, in accordance with earlier findings. It is useful to have
    laboratory data validated by a study of the mechanisms involved in the
    development of the toxic lesion. Furthermore, numerous endogenous and
    environmental variables can modify the toxicity of chemicals, as
    discussed in subsequent chapters. In some instances, the influence of
    these variables is known, and can be controlled, but often this is not
    the case and this may cause serious difficulties in the interpretation
    of laboratory toxicity data.

          In the present context, we are mainly interested in the
    interpretation of laboratory data with a view to their application in
    the evaluation of the health risk to man. The discussion will
    therefore be limited to a few topics that are particularly relevant in
    this respect.

    1.3.1  Distinction between adverse and nonadverse effects

          An adverse, or "abnormal" effect has often been defined in terms
    of a measurement that is outside the "normal" range. The "normal"
    range, in turn, is usually defined on the basis of measured values
    observed in a group of presumably healthy individuals, and expressed
    in statistical terms of a range representing 95% confidence limits of
    the mean or, for individuals, in terms of 95% "tolerance" limitsa
    established with a derived degree of confidence (95% or 99%). An
    individual with a measured value outside this range may be either


    a  Tolerance limits are defined as  m   ks where  m is the sample
       mean,  s is the sample standard deviation and  k is a coefficient
       that depends both on the size of the sample  (N) and the required
       degree of confidence. If the "normal" mean has been determined on
       the basis of a very large sample, the 95% limits will be equal to
         1.96sigma where  and sigma are the "true" or population values
       of the mean and the standard deviation, respectively (see for
       example Owen, 1955).

    "abnormal" in fact, or one of that small group of "normal" individuals
    who have extreme values. According to Sanockij (1970), the distinction
    between "normal" and "abnormal" values based on statistical
    considerations may be used as a criterion for adverse effects, if the
    exposed population consists of adult, generally healthy individuals,
    subject to periodical medical examination, such as workers. Departures
    from "normal" values associated with a given exposure will then be
    considered as adverse effects, if the observed changes are:

          (a)  statistically significant ( P < 0.05) in comparison with a
               control group, and outside the limits  (m  2 s) of
               generally accepted "normal" values;

          (b)  statistically significant ( P < 0.05) in comparison with a
               control group, but within the range of generally accepted
               normal values, provided such changes persist for a
               considerable time after the cessation of exposure; and

          (c)  statistically significant ( P < 0.05) in comparison with a
               control group, but within the "normal" range, provided
               statistically significant departures from the generally
               accepted "normal" values become manifest under functional or
               biochemical stress.

          This statistical definition of adverse effects is less suitable
    for the general population which includes some groups that may be
    specially sensitive to environmental factors, particularly the very
    young, the very old, those affected with disease, and those exposed to
    other toxic materials or stresses. In this case, it is practically
    impossible to define "normal" values, and any observable biological
    change may be considered as an adverse effect under some
    circumstances. For this reason, attempts have been made to set
    criteria for adverse effects based on biological considerations and
    not only on statistically significant differences with respect to an
    unexposed population (control group). Although there is no general
    agreement on such criteria and the ultimate decision on what is an
    adverse effect will have to depend, in each case, on experience and
    expert judgment, it may nevertheless be useful to give examples of
    such criteria, which illustrate at the same time how different such
    criteria may be.

          A Committee for the Working Conference on Principles of Protocols
    for Evaluating Chemicals in the Environment (NAS, 1975) defined
    nonadverse effects as the absence of changes in morphology, growth,
    development, and life span. Furthermore, nonadverse effects do not
    result in impairment of the capacity to compensate for additional
    stress. They are reversible following cessation of exposure without
    detectable impairment of the ability of the organism to maintain
    homeostasis, and do not enhance susceptibility to the deleterious
    effects of other environmental influences.

          On the other hand, adverse effects may be deduced as changes

    "1.   occur with intermittent or continued exposure and that result in
          impairment of functional capacity (as determined by anatomical,
          physiological, and biochemical or behavioural parameters) or in a
          decrement of the ability to compensate additional stress;

    2.    are irreversible during exposure or following cessation of
          exposure if such changes cause detectable decrements in the
          ability of the organism to maintain homeostasis; and

    3.    enhance the susceptibility of the organism to the deleterious
          effects of other environmental influences."

          Soviet toxicologists emphasize that criteria for differentiating
    between adverse and nonadverse effects should not be based on overt
    pathology (e.g. inflammation, necrosis, hyperplasia), and have
    proposed,  inter alia, a number of criteria based on metabolic and
    biochemical changes. Such changes are considered to be adverse if:

          (a)  the metabolism of a substance becomes less efficient or the
               elimination of a substance (expressed in terms of biological
               half-time, T) slows down with increasing doses of the
               substance (Sanockij, 1956);

          (b)  enzymes that have a key significance in metabolism are
               inhibited (Kustov & Tiunov, 1970);

          (c)  the inhibition of a certain enzyme results in an increase in
               the concentration of the corresponding natural substrate in
               the body and/or in a decreased capacity to metabolize the
               specific substrates in a loading test (Kustov & Tiunov,

          (d)  the relative activities of different enzyme systems are
               changed (e.g. the ratio of the activities of asparagine and
               alanine transaminases (Kustov & Tiunov, 1970)).

          Pokrovskij (1973) also attaches great importance to the changes
    in the pattern of isoenzymes in the blood, and to the changes in the
    subcellular membranes (e.g. lysosomal membranes) resulting from the
    action of toxic substances.

          Differentiation between "nonadverse" and "adverse" effects
    requires considerable knowledge of the importance of reversible
    changes and subtle departures from "normal" physiology and morphology
    in terms of the organism's overall economy of life, ability to adapt
    to other stresses, and their possible effects on life span. Newer and

    improved methods of research have increasingly provided more sensitive
    tests for subtle biological deviations such as induction of enzymes of
    the smooth endoplasmic reticulum of the liver, or reversible
    hypertrophy of the liver. These types of changes are produced by
    relatively low doses of many chemicals and they are considered by some
    authors to be adaptive and generally useful to health, and by others
    to be indicative of injury (Hermann, 1974; Kustov & Tiunov, 1970;
    Parke, 1975). One of the most challenging areas for basic research in
    toxicology today is the acquisition of data that can be used to
    estimate whether, or under what conditions, subtle changes in enzyme
    activities, nerve action potentials, altered behavioural reaction etc.
    indicate impairment of physiological function or predict impending
    development of more serious irreversible injury, should exposure to
    the chemical continue.

          In addition to all these considerations, the possibility must be
    kept in mind that an effect may not be seen because the number of
    animals studied was inadequate, the observation time was too short, or
    for other reasons.

    1.3.2  Threshold: practical and theoretical considerations

          The concept of "threshold" is complex and the term has to be
    carefully defined, so that statements concerning this concept in
    relation to the protection of human health are not confused by
    semantic differences. A distinction should be made between the
    threshold for individuals and thresholds for limited groups of
    individuals or general populations.

          The dependence of effect or response on the dose of a chemical
    has already been discussed (section 1.2). As a rule, the intensity of
    the effect or response decreases with reduction in dose, and a
    biological reaction often reaches zero before the dose becomes equal
    to zero. Below a certain limiting exposure level, or dose, i.e. below
    the threshold, a chemical substance may not elicit a toxic effect. The
    threshold for an adverse effect of a chemical is defined by some
    toxicologists as the minimum exposure level or dose that gives rise to
    biological changes beyond the limits of homeostatic adaptation. True
    homeostatic adaptation should be carefully distinguished from
    pathological processes (Sanockij, 1975a).

          The existence of a threshold for all adverse effects is, however,
    still a matter for discussion. Sanockij (1975b) has provided data
    which show that small quantities of environmental chemicals may not
    reach their receptor because the rate of elimination or metabolic
    degradation is relatively more effective with smaller doses. It has
    also been suggested that where effective repair processes are present,
    even if a substance interacts with the receptor, it need not
    necessarily produce an adverse effect.

          For some toxic effects, such as neoplastic disease or mutations
    of genetic material, it has been assumed by some authors that a single
    molecule of a chemical is sufficient to initiate a process that may
    progressively lead to an observed, harmful effect. For this reason, it
    may not be possible to demonstrate that a threshold dose for a
    carcinogen or a mutagen exists (Saffiotti, 1973).

          Other scientists view carcinogenic or mutagenic chemicals as
    toxic entities that may have special properties with regard to the
    nature and characteristics of their adverse effects, but are subject
    to the same physicochemical and biological interactions that are
    considered to result in a threshold dose for other chemicals (Dinman,
    1972; Sanockij, 1970; Stokinger, 1972; Weil, 1972a).

          The question of the existence of a threshold for carcinogens and
    mutagens was recently discussed by a WHO Scientific Group (WHO,
    1974a), which concluded that "the existence of a threshold may be
    envisaged. Nevertheless, the difficulties of determining a threshold
    for a population are great. Therefore, mathematically derived
    conclusions that it is impossible to demonstrate no-effect levels
    experimentally cannot be ignored". A "no-effect" level for a group of
    animals may occur because the dose is really below the theoretical
    no-effect level (i.e. below the threshold) or because the number of
    animals is too small. For example, in an experiment with 20 animals,
    it is possible that none of the animals will show an effect whereas in
    an experiment with 100 animals some response might be seen. However,
    an upper limit for the probable response can be estimated
    statistically. For instance, if in an experiment with 100 animals, no
    response has been observed, it can be shown that there is a 95%
    probability that, under the conditions of the experiment, the upper
    limit of response is 3%, and that there is a 99% probability that the
    response will not exceed 4.5%. Even in an experiment with 1000 animals
    showing no response, the upper 95% confidence limit of response is 3
    animals showing an effect per 1000 treated animals (Food & Drug
    Administration, 1970).

          Another reason for not having seen a response in an experiment
    may be that the time of observation was too short. This may be the
    case, for example, when the quantal effect considered is a cancer,
    with a long latent period between exposure and appearance of tumours.

          For these reasons the "no-effect level" has no real meaning and a
    better term is "no-observed-effect level" (NAS, 1975).

    1.3.3  Extrapolation of animal data to man

          In many cases, studies with laboratory animals make it possible
    to predict the toxic effects of chemicals in man. However, it is
    important to realize that experimental animal models have their
    limitations, and that the accuracy and reliability of a quantitative
    prediction of toxicity in man depend on a number of conditions, such
    as choice of animal species, design of the experiments, and methods of
    extrapolation of animal data to man.

          Hoel et al. (1975) considered the criteria for the adequacy of an
    experiment to be used for the extrapolation of animal data to man.
    They include: test animal species and strain (the animal should be
    susceptible to induction of the effects under consideration); the
    number of animals; the route of administration (which should include
    the routes of human exposure); and the physical state and chemical
    form of the agent. The side effects of the chemical and its organ
    specificity should also be taken into account in the design of the
    experiment. In interpreting the results, attention should be paid to
    adequate survival of the animals, to possible intercurrent disease,
    the quality and extent of pathological data, the quality and extent of
    relevant data collection during the experiment, and the availability
    of data at the time of interpretation.  Species differences and related factors

          The most difficult problem in the extrapolation of animal data to
    man is the conversion from one species to another. For most
    substances, the pathogenesis of poisoning is the same in man and other
    mammals, and for this reason the signs of intoxication are also
    analogous. Thus, quantitative rather than qualitative differences in
    toxic response are most common. Man may be more sensitive than certain
    laboratory animals but there are also many cases where some animal
    species are more sensitive than man. For example, the mouse is most
    sensitive to atropine, the cat is less sensitive, while the dog and
    the rabbit tolerate atropine in doses 100 times higher than the lethal
    dose for man. However, the dog is more sensitive to hydrocyanic acid
    than man (Elizarova, 1962).

          Species differences in sensitivity can often be explained by
    differences in metabolism, in particular by quantitative and
    qualitative differences in the ability of an enzyme to detoxify
    chemicals, and also by differences in the rates of absorption,
    transport, distribution, and elimination of chemicals (Curry, 1970;
    Ecobichon & Cormeau, 1973; Flynn et al., 1972; Hucker, 1970; Portman
    et al., 1970; Sato & Moroi, 1971). Rall (1970) discussed various
    factors to be considered in the selection of animal models for
    pharmacotherapeutic studies in relation to the steps that intervene

    between administration of the drug (or chemical) and the arrival of
    the compound at the ultimate sites of action. After oral
    administration, absorption in standard laboratory animals is generally
    considered to be very similar to man, although there are quantitative
    differences for some compounds. For example, species differences in
    the absorption and action of some compounds are related to differences
    in the bacterial flora of the gastrointestinal tract (Williams, 1972).
    Rall further concluded that the distribution and storage of drugs are
    reasonably consistent in mammalian species, including man, although
    plasma binding tends to be more extensive in man than in small
    mammalian species. Urinary excretion in different animal species
    depends to some extent, on their different diets, since diet
    influences urinary pH and thus the extent of ionization of compounds.
    Biliary excretion is quite variable from species to species and
    apparently is more extensive in mice and rabbits than in rats or man.
    Species differences in response to chemicals appear to be mainly
    related to rates of biotransformation which are generally more rapid
    in small laboratory animals than in man.

          One of the most potent bladder carcinogens, 2-naphthalenamine
    (2-naphthylamine), produces bladder cancer in the dog, hamster, and
    man, but not in the rat, rabbit, or guineapig. Species differences in
    the carcinogenicity of 2-fluorenylacetamide (2-acetaminofluorene) have
    been attributed to the different extents of metabolism to the
    proximate carcinogen, the  N-hydroxy derivative (Miller et al.,
    1964). Similarly, strain differences in metabolism may also affect
    toxicity (Mazze et al., 1973).

          If metabolic information is available, differences in absorption,
    distribution, biotransformation, and elimination of toxic substances
    in man and animals should be taken into account when selecting
    experimental animals.

          Species differences in toxicity may also be due to differences in
    cellular transport. Aflatoxin, which is more toxic to rats than to
    mice, both as an acute poison and as a carcinogen, is transported more
    slowly into the liver cells and is metabolized more rapidly in the
    mouse than in the rat (Portman et al., 1970).

          In determining the required duration of an animal experiment, it
    is often useful to compare the life span of the animal with that of
    man. Using the "body weight rule", the average life span for 70
    species of mammals showed a linear correlation with body weight, but
    the average life span of man was found to be an exception (Krasovskij,
    1975). The regression equation obtained from a study of many mammals
    showed that the average life span for a mammalian representative,
    having the same body weight as man (70 kg) was equal to 15 years.

    Thus, if this assumption is accepted the average life span of a rat
    (about 2.5 years) corresponds to only 15-17 years of a man's life.
    This inconsistency in the life spans of man and experimental animals
    should be taken into account in the design and interpretation of
    animal experiments for the evaluation of toxicity to man.

          There are other problems in the evaluation of toxicity to man
    from experiments on animals, such as where an effect is difficult to
    measure or where similar conditions are difficult to obtain in animal
    models, for example, intelligence and the more esoteric behavioural
    changes. Furthermore, in animal experiments, the effects of social
    factors, so important to man, cannot be evaluated.

          For these reasons, when extrapolating from animals to man it is
    prudent to apply a species conversion factor which should be
    determined on the basis of biological considerations and the available
    information on the test species (Hoel et al., 1975). There is no
    definite rule for the species conversion factor. If the extrapolation
    of data is based on the most sensitive species tested, some
    toxicologists use a factor of 1 (Sabad et al., 1973), but others
    recommend a factor as large as 10 (Weil, 1972a).

          The unit of dose to be used has also to be considered in the
    extrapolation of data to man and it has recently been recommended that
    the dose per unit surface area approximately equivalent to the weight
    raised to the power 2/3 should be used. If the dose is given in terms
    of dietary concentration, there seems to be no need to make the
    surface area adjustment (Hoel et al., 1975; Mantel & Schneiderman,

          A separate problem, to which there appears to be no satisfactory
    answer at present, is the conversion from an inbred animal strain to a
    genetically highly heterogeneous human population (Hoel et al., 1975).  Safety factors

          In almost all instances, laboratory data on the toxicity of
    chemicals are drawn from experiments in which the adverse effect
    occurs at a considerably higher incidence rate than would be
    acceptable in man. For this reason alone, and apart from the
    biological differences between laboratory species and man, an
    extrapolation from a known dose-response range to an unknown range is
    necessary. Indeed, essentially the same problem arises when a human
    accident or epidemiological data are used as the starting point.

          Traditionally, a safety factor has been introduced to provide for
    uncertainties in extrapolation from animals to man, and from a small
    group of individuals to a large population. Such safety factors have
    ranged from 1 to as much as 5000. Because of the current uncertainty
    regarding the mathematical and biological reliability of methods for
    extrapolating from high doses to low doses, primary dependence on
    somewhat arbitrary safety factors continues. However, means of
    extrapolating from high to low doses are being intensively studied at
    the present time, especially with respect to carcinogenicity.

          Most regulatory authorities rely on the use of safety factors but
    there are no precise guidelines for deciding the appropriate size of
    such a factor. Sanockij (1962) and Sanockij & Sidorov (1975) have
    discussed the rationale for different safety factors. In general, the
    size of the safety factor will depend on (a) the nature of the toxic
    effects, (b) the size and type of population to be protected, and (c)
    the quality of toxicological information available. A factor of 2 to 5
    or less may be considered as sufficient if the effect against which
    individuals or a population are to be protected is not regarded as
    very severe, if only a small number of workers are likely to be
    exposed, and if the toxicological information is derived from human
    data. On the other hand, a safety factor as large as 1000 or more may
    be required if the possible effect is very serious, if the general
    population is to be protected, and if the toxicological data are
    derived from limited experiments on laboratory animals. In some cases,
    the safety factor may be a value that has been used with reasonable
    success and is, therefore, perpetuated.

          For most food additives that are not considered to be
    carcinogenic, it has been the accepted practice to divide the
    no-adverse-effect dose (i.e., the maximum ineffective dose) in animals
    by 100, to arrive at an acceptable daily intake (ADI) for man
    (Vettorazzi, 1977; WHO, 1958). For pesticides and certain
    environmental chemicals, safety factors ranging from less than 100 to
    several thousand have been used (Vettorazzi, 1975). For some
    occupational exposures, and for certain air pollutants (WHO, 1977)
    much smaller safety factor have been proposed in the range of 2-5.
    Safety factors have also been proposed for carcinogenic chemicals
    ranging from 100 (Druckrey, 1967; Janyseva, 1972) to about 5000 (Weil,
    1972a) but they have not been generally accepted.  Low-dose extrapolation

          Low-dose extrapolation is based on mathematical models that are
    used to predict the response at a given low dose or to predict that
    dose which gives a predetermined low response. Such models may relate
    the incidence of a quantal effect to dose, or they may consider the
    distribution of the "time to occurrence" of a condition and its
    relation to dose. In both cases, the results of extrapolation are

    strongly dependent on the choice of the model. For example, the
    Advisory Committee on Food Additives (FDA, 1970) noted that
    dose-response data may fit several models equally well in the 2% to 5%
    range, but the doses extrapolated to very low responses would differ
    very strikingly: the ratio of ED1 to ED0.000001 would be either
    100, 100 000, or 1 000 000 for the probit, logistic, or one-hit
    curves, respectively.

          Several extrapolation procedures have been proposed which will
    give an upper limit to the dose corresponding to a low response. In
    other words, the result of extrapolation will not be the best estimate
    of the unknown dose required to give the desired response but a dose
    that is most likely to be below the dose required to give this
    response. Two procedures based on this approach have received
    particular attention: one is based on the one-hit model, the other on
    the probit model.

          The one-hit model assumes that an effect can be induced after a
    single susceptible target has been reached by a single biologically
    effective unit of dose (see for example Cornfield, 1954). At low
    doses, this model is numerically equivalent to the linear
    dose-response model which is compatible with animal data for some
    carcinogens (Druckrey, 1967) and with some human data such as the
    incidence of lung cancer in relation to the number of cigarettes
    smoked per day (Doll, 1967). In their simplest form (i.e. when the
    true response at zero dose is assumed to be zero), the currently used
    extrapolation procedures based on this model (Gross et al., 1970; Hoel
    et al., 1975; Schneiderman, 1971) operate as follows: (1) the upper
    99% confidence limit (UCL) is estimated for the observed response at a
    dose  d; (2) a desired limit is set for a low response ( R) e.g. 1
    in 1 000 000; and (3) the dose ( de) that would produce a response
    which is, with a 99% probability, lower than  R is calculated from
    the equation  de =  d*R/(UCL). Such procedures are more
    conservative than the procedures based on any other currently used
    dose-response model (probit, logit, or extreme-value models). In
    addition, the one-hit model seems to have a reasonable biological
    basis for carcinogenesis at low doses (Hoel et al., 1975).

          Mantel & Bryan (1961) proposed the use of probits (see Annex to
    this Chapter) and a log-normal distribution to describe the
    variability of the sensitivities (tolerances) of individuals in a
    population. The probit model gives a dose-response curve that is
    concave at low-dose levels, and is less conservative than the linear
    model based on the one-hit hypothesis. The Mantel-Bryan procedure (see
    for example Schneiderman & Mantel, 1973) involves (1) the choice of a
    desired limit of response  (R) (e.g. 1 in 1 000 000); (2) the
    estimation of the upper 99% confidence limit (UCL) for the observed
    response at dose  d, and (3) imposing a probit-log dose straight line

    through UCL, with a slope () equal to 1 (i.e. one probit per 10-fold
    dose-range). The choice of the slope () is critical in this
    procedure.  = 1 has been proposed because a slope greater than 1 is
    usually (but not always) observed in carcinogenesis experiments. The
    Mantel & Bryan procedure has been modified to take into account
    response levels in control groups (Mantel et al., 1975).

          A second category of models is based on the observation that the
    median "time to occurrence" (latent period) of an effect such as
    cancer may increase as the dose decreases but not proportionally. A
    thousand-fold change in the dose usually causes an approximately
    ten-fold change in the median time to tumour appearance and, with
    decreasing dose levels, a dose may be reached which would predict
    tumour occurrence beyond the life expectancy of the exposed
    individuals. This would still be consistent with the hypothesis that
    molecular changes in the cells, occurring in proportion to the
    concentration of carcinogens, are the initiating event (for a recent
    review see Jones & Grendon, 1975). One model (Altschuler, 1973; Blum,
    1959; Druckrey, 1967) considers a log-normal distribution of the
    time-to-occurrence with median time depending on dose but with
    standard deviation independent of the dose. The dose  d is related to
    the median time  (t) by d= c/tn, where  n is assumed to be greater than
    one, and  c is a constant. Peto et al. (1972) compared this model
    with another model in which the time to occurrence is considered to
    have a Weibull distribution (Day, 1967; Peto & Lee, 1973). They found
    that the Weibull distribution agreed with experimental data better
    than the log-normal, but this no doubt depends on the type of cancer
    involved. The low-dose extrapolation using these models would also be
    strongly dependent on the choice of the model (Chand & Hoel, 1974).
    Dose-"time-to-occurrence"-response relationships in cancer risk
    assessment have also been considered by Janyseva & Antomonov (1976).

          The application of all these procedures presents practical
    difficulties (Hoel et al., 1975). Low-dose extrapolation is thus a
    very difficult problem that cannot be solved by statistical methods
    alone. Great caution should be exercised in using the existing methods
    and their inherent limitations should always be kept in mind. Good
    experimental data, combined with human data if available, and an
    understanding of the mechanisms of toxic action are essential if the
    task of low-dose extrapolation is to be accomplished satisfactorily.  Other methods of extrapolation

          A method for extrapolation from one species to another based on
    an established relationship between the indices of toxicity and body
    weight for different animal species has also been suggested
    (Krasovskij, 1976a). In mammals, the weights of internal organs, and
    many physiological variables (pulse and respiration rates, consumption

    of oxygen, food, and water, liver microsomal enzyme activity) show a
    log-log linear relationship with body size of the animals (allometric
    ratios). This regularity appears to be valid for more than 100
    different variables including the period of gestation, litter size,
    erythrocyte life span, and latent period of tumour development but
    there are also other variables to which it does not apply. This "body
    weight rule" (Krasovskij, 1975) may be expressed as follows: the
    logarithms of biological variables of mammals show a linear regression
    to the logarithms of the body weight.

          Krasovskij (1976b) showed that values for the lethal dose for
    dogs of several chemicals obtained from regression analysis of
    toxicity in four other species of small mammals compared well with
    predictions made from direct extrapolation from albino rats or from
    the most sensitive animal species, or from the relationship of body
    surface area, and also with predictions obtained by the method of
    Ulanova (1969) and Van Noordwijk (1964). For the calculation of
    extrapolation coefficients from regression equations, see Krasovskij

    1.4  Human Data

    1.4.1  Ethical considerations

          In research involving human subjects, a number of elements, such
    as the assessment of risk, potential benefit, and quality of consent,
    have to be evaluated to ascertain whether ethical considerations are
    satisfied. The essential provisions for protecting human subjects in
    experimentation and research have been expounded by many international
    and national organizations. Key factors include the right to informed
    consent and freedom from coercion. The international instruments in
    dealing with this matter are the Declaration of Helsinki (as revised
    in Tokyo in 1975) and Article 7 of the International Covenant on Civil
    and Political Rights, adopted by the United Nations General Assembly,
    December 1966. Article 7 provides that "no-one shall be subjected
    without his free consent to medical or scientific experimentation"
    (Cranston, 1973; WHO, 1976b). Some countries possess specific codes of
    ethics relating to human experimentation, and special problems of
    experimentation that involve the use of fetuses, children, the
    mentally ill, and prisoners require special consideration.

          It is essential that human experimentation should only be
    undertaken when there is adequate evidence from animal and other
    studies that both the chemical and the circumstances of administration
    are safe. Every experiment with human volunteers should be subject to
    prior review and approval by a local ethical committee in order to
    ensure that the intended study complies with the ethical principles
    embodied in the Declaration of Helsinki and with other requirements of
    national and local bodies.

          Ideal conditions of truly informed consent may not always be
    achieved in practice, consequently the burden of responsibility rests
    mainly with the investigator and, to a lesser extent, with the peer
    review body. Because of these difficulties, the guidelines and
    procedures for the protection of human subjects should be constantly
    reviewed and updated (WHO, 1976b).

          In any case, collection of data from human subjects must be
    accomplished with due respect for human rights and dignity. The use of
    ethics committees with broad representation to review and approve all
    such experimentation is recommended to protect the rights of human
    subjects and to ensure responsible investigation.

    1.4.2  Need for human investigations

          Although there is general repugnance at the idea of using human
    subjects to assess the safety of environmental chemicals, the question
    is not whether or not human subjects should be used in toxicity
    experiments but rather whether such chemicals, deemed from animal
    toxicity studies to be relatively safe, should be released first to
    controlled, carefully monitored groups of human subjects, instead of
    being released indiscriminately to large populations with no
    monitoring and with little or no opportunity to observe adverse
    effects (Paget, 1970).

          The prediction and prevention of possible toxic hazards that may
    arise from the introduction of chemicals into the environment can be
    made more valid if data from studies of the chemical in human subjects
    are available. Three particular aspects of human toxicology have need
    of such information, namely: (a) the selection, through comparative
    consideration of metabolism, of the most appropriate animal species
    for studies to predict the human response; (b) investigation of a
    specific, reversible effect of the compound in the most sensitive
    animal species, to determine whether there is a correlation with a
    similar effect in man; and (c) study of effects specific to man.

          Certain types of information about the effects of chemicals can
    only be obtained by direct observations on man. Often, carefully
    controlled experiments can provide significant information at doses
    well below those anticipated to be "safe"; measurement of subtle
    changes of reaction time, behavioural functions, and sensory responses
    may be examples. In other cases, useful information may be obtained by
    careful studies on human cells or tissue maintained by culture

          Human toxicological data include both the data obtained from
    epidemiological surveys of populations exposed to a toxic chemical
    under normal conditions of use, in cases of acute accidental poisoning
    and in occupational exposure, and the data from experiments in
    volunteers. Although an experiment is defined as observations under
    controlled conditions of exposure, there is, at times, only a grey
    area that distinguishes an experiment with human subjects from
    observations on human subjects under natural conditions. For example,
    some segments of human populations are at higher risk and should be
    particularly closely monitored, e.g., those exposed to chemicals at
    work or those receiving continuous treatment with medicines. The
    periodic clinical evaluation of workers is normally the responsibility
    of the employer and careful records of these examinations coupled with
    measurement of exposure conditions often exist. If accidental
    excessive exposure of an individual or a population should occur, it
    is both ethical and pertinent to learn as much as possible,
    recognizing always the right of the patient. Because of the wide
    individual variation in the toxicity of chemicals to man, the final
    evaluation should be based on information obtained from as widely
    varied a human population as is compatible with the various ethical
    principles involved.

    1.5  The Use of Toxicological Data in Establishing Environmental
         Health Standards

    1.5.1  Environmental health standards

          The aim of environmental health standards is to protect
    individuals, human populations, and their progeny from the adverse
    effects of hazardous environmental factors, including chemicals. A
    sound principle of health protection is to keep all exposures as low
    as reasonably achievable, subject to the condition that the
    appropriate exposure limits, defined by the standard, are not

          Environmental health standards for chemicals may be formulated
    either in terms of concentrations in environmental components (e.g.,
    air, water, food, consumer products) or in terms of amounts of
    substances that may be taken into the body. These concentrations and
    amounts should be sufficiently low that the threshold dose (if it
    exists and can be determined) will not be reached, or that the
    population of concern will not be subject to "unacceptable" risk, even
    following life-time or working life-time exposure. In some cases, as
    for irritant air pollutants, the distribution of exposure
    concentrations in time should also be considered. Standards may also
    prescribe the quantity of a substance to be used at any time and the
    manner of its use.

          Social, cultural, and economic considerations should be taken
    into account in setting standards, but never to the detriment of
    health protection which should be of primary concern.

          It is obvious that a standard setting process will necessarily
    involve many considerations besides toxicology. This process is often
    very different in different countries and different types of society.
    In general, however, it involves appraisal of toxicological data,
    particularly of dose-response relationships, including the effects on
    non-human targets (plants, animals, materials); social and economic
    analysis, policy analysis and review of experience elsewhere, leading
    eventually to an administrative or policy decision concerning the
    standard. Other relevant questions include the technological
    feasibility of achieving a standard, the cost and benefit of
    implementing it, means of enforcement, other public health priorities
    etc. Many of these topics are outside the scope of the present

    1.5.2  Assessment of health risk and evaluation of benefits

          Assessment of health risk from a given exposure to an
    environmental factor is an essential step in any procedure for setting
    environmental health standards. Assessment of health risk involves
    more than routine application of "safety factors", or low dose
    extrapolation which provides estimates of response that are, strictly
    speaking, applicable only to the conditions of the experiment. The
    application of a "species conversion" factor has been discussed and
    the difficulties pointed out (section Questions such as the
    incidence of effects in various age groups and the degree of life
    shortening in affected individuals are all relevant to standard
    setting. For this reason the study of "time-to-occurrence" models in
    the extrapolation of data should be encouraged (Albert & Altschuler,
    1976; Hoel et al., 1975). In addition, the seriousness of adverse
    effects will have to be evaluated from the public health and social
    viewpoint. Attention should also be paid to the heterogeneity of human
    populations, and, at present, it is not clear how the existence of
    susceptible groups, and the influence of nutrition and pre-existing
    disease in human populations should be taken into account. The
    existing methods of extrapolation from animal data to man deal with
    exposures to single substances whereas the actual human environment
    contains a large number of hazardous chemicals and other factors that
    can interact and considerably modify the effects, for example, in
    cancer induction (Bingham & Falk, 1959; Montesano et al., 1974). From
    this viewpoint, the importance of epidemiology and systematic
    surveillance of high risk groups cannot be overemphasized.

          The acceptability of a given risk should also be considered in
    standard setting. This, as well as the judgment on safety (which
    involves decision on the acceptability of risk) exceeds the expertise
    of toxicologists. This is a domain where society at large has a role
    to play. Political decisions are also required on various social,
    economic, and ecological concerns. The same applies to the evaluation
    of benefits. As pointed out by a WHO Expert Committee (WHO, 1974a)

    "the expertise needed for the evaluation of risk is different from
    that needed for benefit evaluation. On the risk side, concern is
    focused on adverse health effects on man, damage to the environment,
    and misuse of natural resources. On the benefit side, the emphasis is
    on value to the consumer and the country". The interaction of all
    these factors is often described by the term "risk and benefit
    analysis" (see for example Falk, 1975), which is only partly within
    the area of toxicological expertise. The final judgment as to whether
    the benefit does or does not justify a risk is for society to make.

    1.5.3  An example of toxicological information used in standard

          Although standard setting procedures will differ from country to
    country, and the requirements for toxicological information will vary
    to a considerable extent, it may be useful, nevertheless, to describe,
    as an example, the procedure used in the USSR for setting standards
    for chemical pollutants in surface waters (Sysin, 1941; WHO, 1975a).

          Information is first obtained on the likely concentrations of the
    chemical in industrial waste waters and the physical and chemical
    properties of the chemical. The stability of the substance under
    environmental conditions is then evaluated by standard analytical
    methods and the influence of the chemical on the self-purification
    processes of natural waters is studied.

          The toxicological investigations required include LD50 studies
    for mice, rats, guineapigs and rabbits (Krasovskij, 1965) and subacute
    experiments lasting 1-2 months, to provide data on functional
    disturbances of organs and systems and on any cumulative properties of
    the chemical (Krasovskij, 1970). These tests are followed by chronic
    toxicity experiments lasting 6-8 months. Study of specific effects of
    chemical water pollutants (e.g., mutagenicity, teratogenicity, and
    effects on reproductive function) is also a necessary component of
    toxicological investigations.

          The subthreshold (maximum ineffective) concentration determined
    by chronic experiments is then compared with the threshold
    concentrations established for the other two indices of water quality
    (i.e. effects on the self-purification of water and its organoleptic
    properties), and the smallest concentration is assumed to be the
    "hygienic" standard.

          The total number of hygienic standards for hazardous substances
    in water, developed in the USSR, has reached 500; of these, about 60%
    have been established according to organoleptic criteria and 30%
    according to the toxicological hazard index. These standards have been
    incorporated in the water legislation of the country and serve as the
    basis for practical control measures in protecting water bodies from
    chemical pollution.

    1.6  Limitations of Safety Evaluation

          Experimental toxicology is a highly complex, multidisciplinary
    science. The extrapolation of animal data to man requires
    well-informed contributions from several scientific disciplines.
    Absolute proof of safety for man of a chemical substance cannot be
    obtained from the results of toxicological tests (Coon, 1973).
    However, toxicological tests do provide guidance on the relative
    toxicity of a compound and help in identifying likely modes of action
    in man.

          Acute toxicity studies in animals are of value in predicting
    potential toxic effects of a chemical in human beings exposed to near
    fatal doses. From the results of such studies, the nature of acute
    responses in man may be anticipated with a view to initiating
    life-supporting measures or first-aid or therapeutic procedures.

          Short-term and subacute studies are particularly valuable in
    determining the more subtle toxic effects of a chemical, whether or
    not it has potential for cumulative toxicity and whether or not the
    toxic effects are reversible upon cessation of exposure. These tests
    are of value in estimating the potential hazard to man following
    exposure of intermediate duration, usually 2-7 years.

          Of greatest concern to toxicologists, regulatory officials, and
    the general public are the possible chronic toxic effects of
    chemicals. Chronic toxicity tests assist in establishing the degree of
    risk to man that may be expected from low-level long-term exposure to
    a chemical substance. Chemicals that tend to persist or concentrate in
    the biosphere and as a result have the potential to affect large
    segments of the population are of particular concern.

          In extrapolating animal data to man, several factors must be
    taken into consideration. These include the "no-effect level" derived
    from animal experiments, the nature of the dose-response curve and the
    nature of the toxic effects produced (Friedman, 1969). The known or
    anticipated level of exposure in man and the potential number of
    exposed individuals must also be considered (NAS, 1975). It is worth
    pointing out that the so-called "no-effect level" is a statistically
    derived value usually estimated within a 95% confidence interval and
    that a 5% probability exists that the value is in error. It has been
    noted, for example, that if a toxic effect occurs in only 1% of the
    test animals, the effect will be entirely missed 37% of the time if
    only 100 animals are used in each test (Friedman, 1969). In addition,
    if the same effect occurs spontaneously in control animals, the
    chances of detecting that response in treated animals becomes even
    more remote.

          Predictions of toxicity from laboratory animal studies are
    dependent on the relevance of these studies to man, to wild-life, and
    to environmental ecosystems. They are also dependent on the genetics,
    nutrition, general health, and environmental circumstances of the
    individuals exposed.

          There may be a hereditary disposition in man to an increased
    susceptibility to toxic chemicals, such as an increased tendency to
    malignant tumours (Kellerman et al., 1973). Similarly, persons under
    stress or treatment with immunosuppressive drugs may also be at
    greater risk to chemical toxicity and chemical carcinogenesis. These
    individuals will constitute abnormal populations for which the degree
    of risk may not be predictable from animal studies or from human
    studies carried out on healthy subjects, but these abnormal
    populations may be of sufficient magnitude to merit special
    consideration. Furthermore, genetic variations in laboratory animals
    are paralleled by variations in the toxic response to chemicals, and
    this puts additional limitations on predictions of possible human
    toxicity from such animal data.

          Similarly, the nutritional status of individuals may also result
    in wide variation in susceptibility to toxic chemicals because
    malnutrition may lead to reduction of natural protection afforded by
    detoxication mechanisms.

          Safety evaluation of chemicals is too frequently empirical and
    there is often a tendency to mistake quantity of data for quality.
    Regulatory agencies and toxicologists must be flexible and keep
    abreast of new experimental techniques and methods and of fundamental
    developments in the understanding of the mechanisms of toxicity. The
    application of new methods could be more relevant and informative than
    the routine use of old traditional ones, but these new methods should
    not necessarily be expected to replace traditional procedures, nor
    should they be applied routinely until adequately evaluated for
    significance and reliability.

          Whether new or old procedures are employed, it is very important
    that the specific conditions under which the experiments are conducted
    should be accessible to other scientists, so that the results from
    different laboratories may be compared. Where it is not possible to
    set forth such details in publications of toxicological
    investigations, a central listing of detailed experimental procedures
    and conditions would be desirable.



          Dose-effect and dose-response relationships may be plotted and an
    empirical "best fit" of a curvilinear correlation may be expressed as
    a mathematical equation. Alternatively, a visual inspection of the
    graph may suggest a mathematical equation, such as linear,
    exponential, or power function, and then the best-fit of the data
    points to the equation may be calculated. A single set of data could
    fit several mathematical equations equally well when the range of data
    is limited. Therefore, care must be taken not to assume that
    biological events follow a specific mathematical model unless the data
    have been collected over a wide range of values.

          Whenever possible, it is useful to develop a hypothesis for a
    mechanism of toxic action on biological grounds, to derive the general
    mathematical expression for the mechanism, and then to fit the data to
    the equation to obtain the values for the constants in the equation
    that will be specific for the conditions of the experiment. For
    example, one mechanism of action may indicate that the law of mass
    action (or chemical equilibrium) applies to the dose-effect
    relationship. If one assumes 1) that one molecule of the chemical
    binds reversibly with one receptor site; 2) that effect  (E) is
    directly proportional to the fraction of the total receptors bound by
    the chemical; and 3) that the amount of bound chemical is very small
    compared to the total concentration or dose  (D), then the
    application of the law of mass action leads to a relationship:
     E =  K1 D/( K2 +  K1 D) where  K1 and  K2 are constants specific to the
    experiment. Clark (1933) noted that this mathematical equation, which
    gives an equilibrium curve asymptotically approaching the maximum
    effect, has a very similar shape, over certain dose ranges, to a
    logarithmic curve, such as  E =  K1 log( K2 D + 1), or a power function
    curve, such as  E =  K1 DK2.

          The sigmoid or S-shaped curve is a commonly observed curvilinear
    expression for some dose-effect and most dose-response relationships.
    The biological basis for this relationship may be partially understood
    by the nature of the frequency distribution of individual
    susceptibilities or resistances in a population. Most of the
    individuals in a population will respond close to a central dose
    level, and a few will respond only at very low or at very high dose
    levels. This leads to a frequency distribution for the individual
    responders as a function of dose. A frequency distribution, however,
    does not describe a biological mechanism for susceptibility or
    resistance, but the random occurrence of individuals with different

          Fig. 1.1 shows a normal frequency distribution; the curve is
    symmetrical around a central point. Its cumulative frequency
    distribution provides the often observed sigmoid curve. A
    dose-response relationship will be observed as a cumulative frequency
    distribution because an individual who responds at a low dose will, of
    course, also respond at higher doses. Thus the frequency of responders
    at a given high dose includes all those that respond at that and all
    lower doses.

          Fig. 1.2 presents a distribution which is skewed towards the
    high-dose levels. This distribution is often described as log-normal
    because a logarithmic transformation of dose values results in a
    normal frequency distribution (Fig. 1.3). In nature, many frequency
    distributions are log normal in shape. This shape is also observed in
    distributions where the central point is near zero; since the dose
    level cannot be less than zero, there is only a narrow range in which
    the more susceptible responders will cluster. The logarithmic scale
    expands the zero point towards negative infinity, thus producing a
    more symmetrical distribution around the central point. In addition to
    normal and log-normal distributions, there are various other types of
    skewed distributions.

          The mathematical equation for the S-shaped curve is difficult to
    handle and it is therefore often transformed into a straight line for
    the presentation and evaluation of data. It is a mathematical
    characteristic of the normal distribution that the points of
    inflection of the curve on either side of the peak (or mean value) are
    at values equal to plus and minus one standard deviation (S.D.) from
    the mean  (m). The integration of the normal distribution function
    shows that the area under the curve from  m  - 1 S.D. to  m + 1 S.D.
    includes 68.3% of all members of the population. Thus 15.9% of the
    population will be responders at doses equal to or less than the mean
    minus 1 S.D., and 84.1% will be responders at doses equal to or less
    than the mean plus 1 S.D. It may also be calculated that approximately
    95.4% of the population will respond within a dose range given by the
    mean  2 S.D., and approximately 99.9% will respond between the mean 
    3 S.D.

    FIGURE 1

    FIGURE 2

    FIGURE 3

          Since  m - 3 S.D.,  m - 2 S.D.,  m - 1 S.D.,  m, m + 1 S.D.,
     m + 2 S.D., and  m + 3 S.D. indicate equal dose intervals, the
    corresponding percentage of responders, i.e. 0.1, 2.3, 15.9, 50, 84.1,
    97.7, and 99.9 respectively, will give a straight line when these
    percentages are plotted at equidistant intervals. Fig. 1.4 illustrates
    this transformation; both the percent scale and the commonly used
    "probit" scale are presented. Finney (1971) has presented the history
    of the development and the utility of probit transformationa. Many
    toxicologists use log-probability graph paper to express dose-response
    relationships as a linear function for log-normal distributions of an

          Fig. 1.5 shows two dose-response curves on the log-probability
    graph paper. The ED50 (50% effective dose) value for chemical A is
    10 dose units, while that for chemical B is 0.01 dose units. ED50
    data are sometimes presented in the literature as a single dose value,
    without providing confidence limits or the slope of the dose response
    curve. It is clear in Fig. 1.5 that for chemicals A and B, not only
    are the ED50 values three orders of magnitude apart but the test
    systems respond in a very different manner.

          As an example of the necessity for taking into account the
    slopes, the practice of some toxicologists to study the effects of
    repeated doses at 1/10 of the single dose ED50 can be considered.
    For chemical A an effect would already be seen in 16% of the
    population (ED16) after the first of the repeated doses, while for
    chemical B it is quite probable that an effect will never be seen even
    after many repeated doses (Fig. 1.5). This is predictable, if the
    slopes of the dose-response curves are known.

          Flat slopes, as for chemical A, are often indicative of such
    factors as poor absorption, rapid excretion or detoxication, or of
    toxic effects that become manifest some time after administration.
    Steep slopes, as for chemical B, most frequently indicate rapid
    absorption and rapid onset of toxic effects, as for example with
    hydrogen cyanide or irritant gases. While slope is not an absolutely
    reliable indicator of physiological or toxicological mechanisms, it is
    useful to the experienced toxicologist, and should always be reported
    along with its confidence limits.


    a  "Probit" = probability unit. Probit is the "standard deviate" or
       the "normal equivalent deviate" (NED) increased by 5. The NED is
       defined as the abscissa corresponding to a probability  P in a
       normal distribution with mean 0 and standard deviation 1.

    FIGURE 4

    FIGURE 5

          Fig. 1.6 illustrates the importance of parallelism of
    dose-response curves for making any general statements about relative
    effects. Chemicals C and D have identical ED50 values. However, any
    statement about the relative equality of effect would only be true at
    that particular dose. In fact, at higher doses, chemical C would be
    more effective than D, and at lower doses, chemical D would be more
    effective. Chemicals E and F, on the other hand, show relative
    equi-effects in the ratio of 1 to 10 dose units over the entire dose
    range. This parallelism of dose-response curves is essential for the
    validity of general statements about relative toxicities. Special note
    should be made, however, that the curves for chemicals E and F apply
    only to one specific effect and one set of experimental conditions.
    Observations of response for a different toxic effect, or
    administration by a different route or to a different species may not
    produce parallel dose-response curves for the same chemicals.

    FIGURE 6


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    2.1  Introduction

          The choice and sequence of toxicity tests will depend on the
    questions or hypotheses that are developed. The nature and sequence of
    tests used to satisfy requirements of regulatory agencies may differ
    markedly from those used in an investigation of the basic mechanisms
    of toxic action. Differences in approach will also depend on whether
    the investigation is initiated to evaluate the toxicity of a chemical
    prior to its introduction into use, i.e. prospective toxicology, or to
    confirm in laboratory animals an epidemiological association that
    suggests chemical-induced disease in man, i.e. retrospective
    toxicology. Under ideal conditions, prospective toxicology will
    eliminate the need for retrospective toxicity evaluation.

          National or international regulatory or advisory bodies have
    developed fairly specific guidelines or test protocols which are
    expected to be applied to the toxicity evaluation of certain groups of
    chemicals, introduced deliberately into our environment, e.g. food
    additives and pesticides (Council of Europe, 1973; FDA, 1959; WHO,
    1967). The development of guidelines for the systematic evaluation of
    the toxicity of chemicals to which man is exposed, through his
    occupation or through incidental contamination of the ambient
    environment, is less common. Where specific guidelines have been
    formulated, they usually require: test information on acute toxicity
    in several species of experimental animals; some knowledge of the
    biochemical disposition of the compounds; various short-term toxicity
    tests; tests of the effects of the chemical on reproductive function;
    chronic toxicity tests in one or more species; special tests on organ
    function, clinical biochemistry and haematology, and other specific
    tests as determined by the particular type and intended uses of the
    chemical under consideration. Some commercial firms have developed
    their own guidelines for toxicity tests and their sequence in the
    premarket toxicological evaluation of products. Often the sequence of
    tests will have certain checkpoints at which decisions will be made as
    to whether continued development of the product (and more extensive
    toxicity testing) is warranted.

          In this chapter, various topics will be discussed from the
    standpoint of the usefulness of certain types of information and the
    influence that various factors may have in designing protocols for the
    interpretation of data obtained in toxicity evaluation programmes.

    2.2  Chemical and Physical Properties

    2.2.1  General considerations

          The late Horace Gerarde stated in an address that "toxicity is
    the capacity of a substance to cause injury. It is an inherent,
    unalterable molecular property which is dependent upon chemical
    structure. There is nothing we can do about the toxicity of a chemical
    except to know it"a.

          The nature or quality of the toxic action inherent in a chemical
    will depend to a large extent upon the functional group or groups
    present in the molecule. Knowledge of the reactions that these
    functional groups may undergo with reactive groups in critical
    endogenous biochemical constituents provides a means of predicting the
    nature of the toxic effects that may be expected. Smyth (1959) used
    the permanence of threshold limit values over a period of years as a
    criterion for evaluating various types of information used in setting
    safety limits for industrial chemicals. For a limited number of
    compounds, occupational threshold limit values (TLVs) had been
    established on the basis of analogy with better known substances and
    the permanence of these TLVs appeared to equal that of TLVs based upon
    data from experimental toxicity studies and from human experience.
    However, evaluation of toxicity by analogy with chemically-related
    substances contains considerable potential for error, and requires a
    great deal of toxicological information on very closely related
    chemical substances. Very minor changes in structure may be
    accompanied by profound changes in toxicity. The relationships between
    physicochemical characteristics and toxicity, including the biological
    activities of homologous series, have been reviewed by Ljublina &
    Filov (1975).

    2.2.2  Physicochemical properties and the design of toxicity studies

          Zbinden (1973) included fourteen chemical and physical variables
    in a check list of types of information useful in the toxicity
    evaluation of new drugs. Although some of these variables may be
    determined in the course of a toxicological evaluation, all of them
    apply equally as well to environmental chemicals as to therapeutic


    a  Presented at the Flavor Manufacturers' Association of the United
       States. Fall Symposium, 16 November 1972, Washington, DC.

          Knowledge of the chemical structure is essential for the
    preliminary prediction of the nature and site of toxic action,
    assuming, of course, that some prior knowledge of the toxicity of
    chemically-related compounds is available. It is also essential for
    developing extraction and assay procedures for the determination of
    tissue concentrations and allows for logical estimates of the nature
    of metabolites that may be found. Indeed, without such knowledge,
    logical design of an experiment is impossible.

          The stability of the chemical at various pH values and the
    photochemical properties are variables that must be considered as soon
    as a substance arrives for testing in the toxicology laboratory, as
    they may determine the manner in which the chemical should be stored
    prior to administration to test animals or indicate the stability of
    residues in tissue extracts. Many organic chemicals undergo
    photochemical reactions that lead to either more or less toxic
    products (Crosby, 1972) and organic esters are often readily
    hydrolysed under conditions encountered during their laboratory
    investigation (Eto, 1974). It may be necessary to exercise special
    precautions to avoid chemical reactions during the preparation and
    storage of test solutions or diets and during the analysis of tissues
    and metabolic reaction mixtures. Furthermore, if a chemical is likely
    to become activated by photochemical reactions, special tests for
    phototoxicity may be required.

          The organic solvent/water partition coefficient and pK are
    physical properties of particular importance in the determination of
    the absorption and distribution of a compound in living organisms as
    well as in the development of appropriate extraction and assay
    procedures for the chemical. Hansch & Dunn (1972) reviewed numerous
    studies which suggest that characterization of the lipophilic nature
    of compounds may allow systematic predictions of their relative
    biological activities. Dillingham et al. (1973) applied these
    principles when they compared the toxicity of substituted alcohols in
    a tissue culture with their acute toxicity in mice and concluded that
    tissue culture test systems may be useful in determining predictive
    correlations between  in vivo toxicity and the physicochemical
    properties of compounds.

          The extent of ionization of an organic compound will influence
    its passage through lipoidal membranes (La Du et al., 1971). In
    general, the unionized lipid-soluble form of an organic compound will
    most readily pass through biological membranes. Although most of the
    physicochemical principles of absorption and distribution have been
    developed through systematic studies on medicinal chemicals, these
    principles also apply to organic chemicals in the environment and to
    the design of toxicity experiments (Loomis, 1974; also Chapter 4).
    Patty (1958) discussed the influence of oil and water solubility and
    of the coefficient of distribution of a vapour between blood and
    alveolar air on the rates of equilibrium saturation and desaturation
    of the body during inhalation experiments.

          Particle size, shape, and density are of obvious importance in
    studying the inhalation toxicities of aerosols, as they are important
    factors in the determination of the site of deposition and the rates
    and mechanisms of clearance from the respiratory tract (Hatch & Gross,
    1964; also Chapter 6). Furthermore, the particle size of substances
    given orally as suspensions can also markedly influence their toxicity
    (Boyd, 1971). If critical judgements based on the relative toxicities
    of the same or different substances, administered orally in
    suspension, are to be made, it is necessary to ensure uniformity of
    particle size.

          Vapour pressure of a chemical substance is important in the
    practical consideration of the likelihood of exposure of man through
    inhalation, and the design of experimental inhalation toxicity studies
    will be influenced by the ease with which a solid or liquid vaporizes
    under controlled conditions. However, a high vapour pressure may
    produce technical problems if the objective of the test is to
    determine toxicity by the oral route of administration. Studies by
    Jones et al. (1971) showed that for a large series of food flavouring
    agents mixed in laboratory animal diets, the amount of loss from the
    diet was inversely related to the boiling points of the flavouring
    agents. Frequent chemical analyses of the diet, as well as frequent
    preparation of fresh diets and/or restricted feeding periods to limit
    the time for loss by vaporization are necessary to provide accurate
    estimates of intake in feeding studies on substances with low boiling

          Knowledge of reactivity with, or binding to, macromolecules may
    allow specific design of mechanism experiments, when these
    macromolecules are essential tissue and cell constituents. Knowledge
    of the chemical reactivity of a substance may also be of considerable
    importance in the early planning of feeding studies, if the chemical
    under test is likely to react with the macromolecules present in the
    laboratory diet. On the one hand, chemical binding or adsorption on
    macromolecules in the diet may markedly alter the rate and extent of
    absorption of the test compound from the gastrointestinal tract; in
    some cases, biologically reactive groups on the test chemical may be
    neutralized by dietary constituents. On the other hand, reaction of
    the test chemical with essential dietary constituents may contribute
    to nutritional deficiency states, or new and more toxic compounds may
    be formed. Several examples of these types of reactions have been
    summarized by Golberg (1967).

    2.2.3  Impurities

          In the design of toxicity experiments, it is extremely important
    to consider the chemical purity of the sample to be tested. In certain
    cases, e.g. food additives and pesticides, the regulations will often
    provide specifications of purity for the compound in actual use and
    recommended test protocols may specify that toxicity evaluations are

    to be conducted with samples that meet these specifications (WHO,
    1967). However, there is always the possibility that, when testing the
    technical product, the biological effects observed may be due to, or
    modified by, trace contaminants. If the contaminants are unknown or
    their biological activity unsuspected, toxicity tests may lead to
    erroneous conclusions concerning the primary chemical in question. In
    contrast, tests on highly purified samples may not detect the toxic
    action of contaminants present in samples used commercially.
    Furthermore, for many chemical substances used in manufacturing or
    incidentally released into the environment, specifications of purity
    may not be standardized. Therefore, one of the earliest and most
    difficult decisions that must be made in the design of a toxicity
    evaluation programme is the selection of the sample to be studied
    (technical grade, highly purified, etc.)

          Requirements for the purity of the compounds selected for
    toxicological testing depend on the purpose of the testing as
    discussed in section 2.1. During the development of a new
    technological process, it may even be useful to test mixtures of
    unknown composition. The information so obtained may alert organic
    chemists in the research laboratory or pilot plant operators to the
    possible hazards of these unknown mixtures which may vary as
    procedures develop and improve. However, data obtained from such
    studies will have a limited value. The determination of health
    standards requires a compound of a high degree of purity or of highly
    standardized composition combined with a precise knowledge of various
    impurities. Only in this way will health standards have a universal
    value. However, for practical purposes, and for extrapolation to human
    exposure conditions it may be prudent, when a technical grade product
    standardized by specifications is used in commerce, to select this
    grade for toxicity testing and carefully characterize it with respect
    to the nature and amounts of any impurities present. Scheduled
    analytical spot checks during the course of the experiment to provide
    assurance of chemical constancy is also desirable.

          When a chemical substance used in commerce is not standardized
    with respect to specifications for purity, experimental toxicity
    evaluation on a test sample of high purity would, indeed, seem to be
    the most rational selection. Data derived from this compound could
    then be used to characterize, toxicologically, the action of the
    primary chemical under consideration. When certain quantifiable
    indices of toxicity have been identified, selected tests with typical
    samples of commercial products could be conducted and the results
    compared with the purified product to detect possible differences. Of
    course, if the impurities represent a significant portion of the
    product, or if their chemical properties or their chemical analogy to
    other known substances suggest they may have serious toxic properties,
    the impurities must be evaluated separately.

          An alternative approach is to select a sample of a chemical that
    most nearly represents the impure product used commercially, subject
    this to comprehensive toxicity evaluation and then make selected,
    critical toxicity comparison with purified samples of the primary
    chemical as well as with the impurities.

          Either approach contains uncertainties, and little would be
    gained by short-term spot checks if the toxic action of the impurity
    were only detectable after long-term exposure or a latent period. It
    is in problem situations such as these that some of the
    chemicophysical principles discussed earlier must be applied at
    several levels of decision making: the sample to choose for testing;
    the design of the evaluation protocol; or the decision (or regulation)
    to produce a purer substance for routine use in commerce.

          A recent, most controversial problem arising from the
    contamination of a primary commercial product involves the apparent
    teratogenic action of the herbicide (2,4,5-trichlorophenoxy)acetic
    acid (2,4,5-T) (Panel on Herbicides 1971). It is now known that the
    first studies to reveal this action were conducted with a sample of
    the herbicide that contained a rather high concentration (about
    30 mg/kg) of the contaminant 2,3,7,8-tetrachloro-dibenzo-4-dioxin
    which is formed during the synthesis of the trichlorophenol precursor.
    Tetrachlorodioxin is extremely toxic. For guineapigs, the ratio of the
    LD50 for 2,4,5-T to the LD50 of the dioxin is about 630 000. In
    female rats, the acute oral LD50 for dioxin is about 1/10 000 of the
    oral LD50 for 2,4,5-T. The daily dose of dioxin in pregnant rats
    that produced fetal toxicity was only about one four-hundredth of the
    maternal LD50 of dioxin or about one four-millionth of the
    single-close oral LD50 of 2,4,5-T for female rats. Thus, even if the
    concentration of dioxin as a contaminant of 2,4,5-T is kept below
    0.5 mg/kg, the major concern for the toxicity of 2,4,5-T should
    apparently still be directed towards the contaminant rather than
    towards the herbicide itself (at least insofar as effects on
    reproduction are concerned).

          This kind of problem is not new. Twenty-five years ago, marked
    differences in the toxicity of different samples of the insecticide
    parathion were traced to contamination with small quantities of the
    oxygen analogue and the phosphorothiolate isomer, which are much more
    potent anticholinesterases and more acutely toxic than the parent
    insecticide (Diggle & Gage, 1951).

    2.3  Probable Routes of Exposure

    2.3.1  General considerations

          Many chemicals will become distributed in various environmental
    media or will be used for different purposes, and substantially
    different populations may be at risk. Thus, it may be necessary to
    obtain extensive test data by different routes of exposure. The choice

    of route for practical purposes is generally dictated by: (a) the
    likely route by which man will be exposed; and (b) whether the
    chemical will produce local injury at the site of exposure. The second
    question will often be resolved by acute or short-term studies on
    animals dosed by oral, inhalation, dermal, and possibly ocular routes.
    Details of test procedures are included in subsequent chapters.
    Although it is usually wise to conduct experiments using the route
    through which man will be exposed, other more convenient routes may be
    chosen for many of the tests if it is determined that the major toxic
    effects of a chemical are systemic, occurring only after absorption
    and distribution in the body. Data on blood and tissue levels should
    be obtained by several routes of exposure, including those that are
    considered primarily experimental; with this information, it may be
    possible to relate toxic effects to blood or tissue concentrations of
    the test chemical and its metabolites. Such information greatly
    facilitates comparison of experiments using different routes and may
    either confirm or deny the validity of extrapolating data, obtained
    experimentally by one route, to the evaluation of potential toxicity
    by another perhaps more realistic route of exposure.

    2.3.2  Specific variables related to route of exposure  Rate of absorption

          As a general rule, one can predict that for the usual routes by
    which man may be exposed, absorption of chemicals will be most rapid
    when given by inhalation, less rapid when given by gavage, and slowest
    with dermal application. This order may, however, be modified
    depending upon various physicochemical properties of the substance
    under test in relation to the microenvironment of the absorbing
    surface (Klassen, 1975; Loomis, 1974). The rate of absorption will be
    one determinant of the rate of onset of signs of acute poisoning. If
    the rates of detoxification and excretion or of injury repair exceed
    the limiting rate at which a chemical is absorbed, it is possible that
    toxic signs observed by one route of administration will not be
    detectable by another route for which absorption is slower (Casarett,
    1975; Murphy, 1975). Comparative absorption-distribution kinetics by
    different routes would determine such a possibility.  Site of action

          Specific tests should be conducted to evaluate effects related to
    local reactions with specific receptors present in the organ of
    absorption. These may be morphological tests to detect evidence of
    irritation, inflammation, or oedema, or they may be functional to
    detect biochemical or reflex action or bronchoconstriction. In
    addition, the route of exposure may determine the organ or
    physiological system in which effects will be first observed or
    detected at lowest doses. For example, pesticides, which are direct
    inhibitors of acetylcholinesterase, when given in sufficient doses by

    any route, will produce a characteristic toxic syndrome involving
    essentially all organs or structures innervated by cholinergic nerves.
    However, at low doses, only specific organs may be involved. If such
    compounds are applied to the skin, local sweating and fasciculations
    may occur in the absence of signs of systemic poisoning. Exposure by
    inhalation may result in bronchoconstriction, exposure by ingestion
    may cause gastrointestinal upset before, or at lower doses than,
    generalized systemic effects (Henderson & Haggard, 1943; Holmstedt,
    1959).  Biotransformation

          The route of exposure may determine the likelihood and type of
    biotransformation before the chemical contacts the specific sites of
    action. Thus, when chemicals are administered by the oral (or
    intraperitoneal) routes, they will be absorbed and transported first
    through the portal circulation to the liver (Lukas et al., 1971). For
    example, if, with low oral or dietary doses, the capacity of the liver
    to detoxify the compounds exceeds the rate of absorption, an effective
    injurious concentration may never reach critical sites of action in
    other tissues. Absorption of the same quantity through the lung or
    skin, which generally have less detoxifying capacities, may result in
    toxic action. It is now known that the lung, skin, and intestinal
    mucosa, although generally less active than the liver, also have the
    capacity for biotransformation of foreign organic chemicals (Alvares
    et al., 1973; Fouts, 1972; Lake et al., 1973; Wattenberg, 1972).
    Although knowledge is incomplete with regard to the tissue
    distribution of both activating and detoxifying enzyme systems, it is
    likely that their relative distribution will determine, to some
    extent, the specific tissues that will be most affected by low doses
    of some compounds, when given by different routes of exposure.  Species

          The relative susceptibility of different species to the action of
    chemicals may differ depending upon the route of exposure. When
    administering compounds by the oral route, such factors as vomiting
    reflex (absent in rats) and/or differences in type and distribution of
    microflora that may detoxify (or activate) the test compound can
    influence the interpretation of the results (Williams, 1972). The
    rates of penetration of compounds through the skin and the acute
    dermal toxicities of various compounds differ markedly among species
    and not always in a predictable manner (McCreesh, 1965). Many of the
    problems encountered in dermal toxicity testing and suggestions for
    further research have been discussed by Barnes (1968). Roe (1968)
    discussed various problems encountered in the design and
    interpretation of inhalation toxicity studies related to species
    differences in the anatomy of the respiratory tree. Enzootic lung
    infections are an additional problem in the use of some species of
    animals for long-term inhalation studies.  Unintended route

          Interpretation of results and measurement of actual dose-response
    relationships can be made difficult, because appreciable oral
    ingestion may occur with inhalation or dermal exposures. Animals
    exposed by either of these routes may ingest the material as a result
    of preening, unless dermal applications are covered or made
    inaccessible to licking or unless special exposure chambers (e.g. head
    only) are used for inhalation exposures (see Chapter 6). In addition,
    in particle inhalation experiments, the physiological protective
    mechanisms of clearance by mucous transport of the particles out of
    the respiratory tree (Hatch & Gross, 1964) with subsequent swallowing
    may result in gastrointestinal exposure. Some degree of lung exposure
    to volatile compounds administered in the diet or by dermal
    application is also likely.

    2.3.3  Special tests related to route

          When exposure to a compound is most likely to occur by
    inhalation, it is useful to know the effect of variations in
    ventilation rates, since this will be a common variable among an
    exposed human population under different conditions of activity. This
    may be accomplished by the use of exercise wheels or treadmills.

          When dermal exposure is the likely route, it will be useful to
    conduct some tests to determine the effect of different solvents on
    penetration of the test compound through the skin. Studies of the
    influence of factors such as sweating, abrasions, or the presence of
    detergents on dermal absorption and toxicity will also aid in
    estimating toxicities under conditions likely to be experienced by man
    (see Chapter 11, Part 2).

          Interpretation and implications of toxicity data obtained with
    oral exposures can be enhanced by examination of the influence of
    fasting, dietary variations, and, particularly, administration by
    gavage versus inclusion in the diet or drinking water. These factors
    are discussed in more detail in subsequent chapters, but it should be
    stressed that quite different results and interpretations may ensue if
    the same daily dose is given rapidly by gavage or gradually in the
    diet. Interpretation of such experiments is greatly aided, if the
    design includes comparative absorption and distribution kinetics.

    2.4  Selection and Care of Animals

    2.4.1  General considerations

          The selection and care of laboratory animals to be used in
    toxicity tests is especially important in determining the success of
    the experiment itself, the extrapolation of the data to man, and the
    cost of the evaluation programme. In order to provide data on a

    sufficient number of animals for valid statistical analyses, it has
    become common practice to use small laboratory rodents for most
    large-scale toxicity test programmes. Dogs or nonhuman primates are
    frequently included in some of the studies, and studies on at least
    one nonrodent species are often required by the test protocols
    recommended by regulatory and advisory agencies. Recommendations (with
    appropriate references for detailed information) concerning the
    selection and care of laboratory animals to be used in the usual broad
    scale toxicity evaluation studies are included in Chapter 3. The
    selection of animals to be used in various special test procedures is
    discussed in subsequent chapters.

          Animals and animal care practices should be selected to provide a
    scientifically sound and reproducible experiment; however, some of the
    variables that contribute to nonuniformity may actually be exploited,
    in special studies, to obtain data that may be useful in extrapolation
    to nonuniform human populations. For example, if the inherent toxicity
    of an air pollutant is to be characterized, the occurrence of chronic
    lung infections should be avoided. On the other hand, specially
    designed experiments to test the influence of air pollutants on the
    susceptibility of animals to lung infections have provided a sensitive
    procedure for measuring the adverse effects of air pollutants on the
    physiological protective mechanisms that confer resistance to
    respiratory infections (Ehrlich, 1966). Epidemiologists could
    certainly use such information in the design of studies on human
    populations exposed to air pollutants and to infectious microorganisms
    present in the environment.

          The choice of animals and the environment in which they are used
    in toxicity studies will ultimately be determined (as for any other
    decisions relating to experimental design) by the nature of the
    questions asked or the hypotheses formulated. Controlled introduction
    of additional variables may be desired for special studies. The
    important principle is that appropriate control conditions should be
    included in such studies to allow comparisons with results obtained
    under more conventional procedures.

    2.4.2  Animal variables

          The objective of most experimental toxicity studies is to predict
    the adverse effects of chemicals in man. Therefore, in addition to
    uniformity of response, the guiding principle for the selection of
    appropriate test species is that the test animals should resemble man
    as closely as possible with respect to absorption, distribution,
    metabolic transformation, excretion, and effect at site(s) of action
    of chemicals. Both male and female animals should be tested and the
    test protocol should encompass exposures of animals at both ends of
    the age spectrum (see Chapter 3).

          It is generally recommended that random-bred rather than highly
    inbred strains of animals be used in broad-scale toxicity testing, at
    least until the action of the chemical is well characterized (Food &
    Drug Administration, 1970). In more specialized toxicity tests, it may
    be desirable to use inbred strains, for example, when animal models
    are needed that represent a genetic variation in human population, or
    when hypotheses on the mechanism of action are tested.  Selection of species

          The Food Protection Committee (1970) indicated that sensitivity,
    convenience, and similarity in metabolism to man are the prime factors
    to be considered in the selection of animal species for toxicity
    testing. In the absence of information to the contrary, it is
    generally recommended that data obtained from the most sensitive
    species should be used as the basis for the extrapolation of test
    information to man.

          There is now ample evidence of wide quantitative variations among
    species in their rates of biotransformation of foreign compounds
    (Committee on Problems of Drug Safety, 1969; Parke & Williams, 1969;
    Williams, 1967). Since many organic chemicals are subject to
    biotransformation at several reactive groups in the molecule, it is
    important to identify and quantify the biotransformation and
    distribution pathways of a chemical in man and in several laboratory
    animal species as early as possible in toxicity evaluation studies. It
    seems axiomatic that for costly chronic studies on experimental
    animals, the species that is most representative of man with respect
    to the metabolism of the test chemical should be chosen. Often, the
    only information concerning the metabolism and distribution of the
    test compound in man may be derived from limited studies on
    individuals accidentally or occupationally exposed to uncontrolled or
    unknown doses.

           In vitro studies of metabolism using animal tissues and human
    tissues obtained at autopsy or biopsy could help in comparisons of
    similarities or differences in metabolism between man and laboratory
    animals. Although this approach cannot, in itself, provide information
    that may be obtained in studies on intact animals, it can, coupled
    with knowledge of the physicochemical properties of the compound and
    the kinetics of enzymatic biotransformation reactions in tissues of
    various species, provide a logical basis for selection of species for
    long-term toxicity tests. Decisions based on comparative human and
    experimental animal metabolism data should take into account
    information concerning several pathways of metabolism. This will help
    to ensure the inclusion of data on quantitatively minor pathways of
    metabolism that may result in products of major toxicological
    importance. These considerations of variation in biotransformation can
    also be applied to intraspecies variations related to age, sex, and
    strain (Benke & Murphy, 1973; Jori et al., 1971a; MacLeod et al.,
    1972; Parke & Williams, 1969).

          Anatomical and morphological variations can also determine the
    selection of species. This source of variation is likely to be of
    particular importance in inhalation toxicity studies (Roe, 1968).
    Tyler & Gillespie (1969) compared anatomical characteristics of the
    lungs of human beings with several laboratory and domestic animal
    species when considering appropriate animal models for human
    emphysema. They grouped anatomically similar species into four
    classes: (a) cattle, sheep, and swine; (b) dogs, cats, and rhesus
    monkeys; (c) rabbits, rats, and guineapigs; and (d) horses and man.
    From their studies on horses, they concluded that the pathophysiology
    and the morphological characteristics of emphysema in horses closely
    resembled the disease in man and that the horse could be a
    particularly suitable laboratory animal for studies of this disease.
    Obviously, in the usual toxicity studies on air pollutants, the costs
    of using horses would be prohibitive. The reactivity of a chemical at
    primary target sites must also be considered as a potential variable
    contributing to species differences in toxicity. The acute toxicity of
    certain organophosphorous insecticides in representative mammalian,
    avian, and fish species appeared to be more readily related to species
    differences in the reactivity of the target enzyme
    (acetylcholinesterase ( than to differences in hepatic
    biotransformation rates (Murphy et al., 1968).

          In the absence of specific knowledge of comparative metabolism
    and sites of action, it is appropriate to apply the principle that
    quantitative and qualitative similarity of response in several
    mammalian laboratory species enhances the confidence that man will
    respond similarly. Tests on several species seem equally as useful for
    predicting effects in a heterogenous human population as the selection
    of test species based on the results of limited studies of metabolism
    in a very few individual human subjects, who may or may not be
    representative of a broad cross-section of the human population at
    risk. Of course, any quantitative information on the disposition and
    action of chemicals in man is useful, as it adds to the accumulation
    of knowledge from which more specific guidelines for species selection
    may be derived in the future.  Animal models representing special populations at risk

          Because many chemicals in the environment are widely dispersed,
    all segments of the human population may sustain some exposure. For
    this reason, it may be useful to design special experiments to
    evaluate toxicity in animal models that represent potentially
    hypersusceptible segments of the human population. The very young and
    the aged represent such segments generally, because in the very young,
    natural protective mechanisms such as metabolic detoxification systems
    may be incompletely developed and in the aged, cell repair processes
    may be less active than in younger individuals. Evaluation of the

    toxicity of chemicals in animal models of commonly occurring human
    diseases may be of value. Thus, for example, epidemiological studies
    suggest that individuals suffering from coronary artery disease may be
    particularly susceptible to carbon monoxide, the severity of signs and
    symptoms in patients suffering from cardiorespiratory disease appears
    to be aggravated by air pollution, and asthmatic patients appear to
    have a higher frequency of attacks during periods of high oxidant air
    pollution (Heimann, 1967). Few attempts have been made to evaluate
    experimentally the interactions between exposure to toxic chemicals
    and model disease conditions. Taylor & Drew (1975) reported that an
    inbred strain of cardiomyopathic hamsters was more susceptible to
    acute toxicity and cardiac arrhythmias produced by inhaled
    trichlorofluoromethane than were random-bred hamsters that were not
    cardiomyopathic. Easton & Murphy (1967) suggested that their
    observation of greater mortality and respiratory distress in
    ozone-preexposed than in air-exposed guineapigs given histamine
    injections or inhalation exposures might be analogous to the apparent
    increase in frequency and severity of asthmatic attacks reported for
    peak periods of photochemical air pollution.

          Problems of standardization of disease conditions add another
    dimension to toxicity studies. However, it seems that animal disease
    models should be given more consideration in toxicity evaluations that
    are intended to provide the basis for the safe use of chemicals to
    which large human populations are exposed. Jones (1969) summarized
    reference sources for animal models of a large number of specific
    human diseases. Several papers in a series of symposia proceedings
    published by the US National Academy of Sciences provide discussion
    and references to (among other topics) animal models for commonly
    occurring disease states of the lungs (Tyler & Gillespie, 1969), the
    cerebrovascular system (Luginbuhl & Detweiler, 1968), the heart (Jobe,
    1968), the kidney (Lerner & Dixon, 1968), atherosclerosis (Clarkson et
    al., 1970), diabetes (Hackel et al., 1968), and chronic degenerative
    diseases (Abinanti, 1971). Since gut microflora are changed in certain
    gastrointestinal diseases in man, modifications of the quality and
    distribution of microflora in experimental animals might be a useful
    model for special tests (Williams, 1972). There are at least three
    possible applications of these disease models to toxicity studies: (a)
    evaluation of the susceptibility of diseased tissues to chemicals
    known to exert their action on that tissue; (b) influence of the
    disease state on the metabolism and distribution of chemicals that may
    act on the diseased tissue or at other sites; and (c) research on the
    mechanism of action of toxic chemicals using specific modification of
    receptor function or biochemistry.

    2.4.3  Cyclic variations in function or response

          Many physiological variables undergo cyclic peaks and troughs of
    activity (Altman & Dittmer, 1966) some of which are diurnal (24-h) and
    others of longer duration. These rhythms may be completely under
    intrinsic control or they may be partly or largely regulated by
    environmental variables such as light and temperature. Boyd (1972)
    considers most diurnal variations in susceptibility to drug toxicity
    to be mainly related to eating and sleeping habits. Since rats are
    nocturnal feeders, the greater quantity of food in the stomach early
    in the morning compared with the afternoon may alter the acute
    toxicity of chemicals given intragastrically. Attempts to standardize
    this variable have led to recommendations that acute toxicity tests by
    intragastric administration should be conducted on animals that have
    fasted overnight (Food & Drug Administration, 1959). Intragastric
    LD50 values are generally lower in rats fasted overnight compared
    with those fed  ad libitum; however, the differences are usually only
    of the order of two- to three-fold (Boyd, 1972; Loomis, 1974). The
    influence of fasting may, in some cases, be related to rates of
    absorption from the gut in the presence or absence of food but this
    cannot account for all such variations. A striking example has been
    reported by Jaeger et al. (1975) in which acute toxicity and liver
    injury in rats exposed through inhalation to several halogenated
    olefins were enhanced 10- to 20-fold by overnight fasting. A diurnal
    cycle of susceptibility of rats to the toxicity of inhaled vinylidene
    chloride appeared to be related to the diurnal cycle of liver
    glutathione concentrations (Jaeger et al., 1973) which may be
    secondary to a diurnal cycle in feeding activities. The duration of
    pentobarbital anaesthesia in mice under usual laboratory housing
    conditions exhibited a diurnal cycle with the longest duration at
    14h00 and the shortest (40-60% of that at 14h00) duration at 02h00
    (Davis, 1962). The amplitude of the cycle was considerably reduced,
    when animals were caged individually as opposed to group caging, and
    constant light abolished the cycle. That circadian variation in the
    action of certain organic chemicals may be related to circadian
    variation in their biotransformation is suggested by the work of Jori
    et al. (1971b).

          Beuthin & Bousquet (1970) reported seasonal or circannual rhythms
    for drug action and biotransformation rates in rats. The induction of
    increased drug metabolism by phenobarbital also exhibited a seasonal
    variation. Basal levels of hexobarbital metabolism were highest during
    the winter months and lowest in summer, whereas the opposite cycle for
    induction of hexobarbital oxidase by phenobarbital was observed. It
    should be noted that studies of seasonal variations in the metabolism
    or toxic action of chemicals must be carefully controlled with respect
    to environmental variables that might produce similar variations in
    response (see 2.4.4). Boyd (1972) suggests that seasonal variations in
    susceptibility may be related to the hibernation reaction or to
    weather conditions in the geographical area concerned.

          Circadian variation in adrenocortical activity in rats was
    investigated by Szot & Murphy (1971) in animals exposed acutely or
    subacutely to the pesticides parathion and DDT. Although the degree of
    stimulation of corticosterone secretion after single doses of
    parathion varied depending upon the phase of the cycle at the time of
    administration, feeding parathion or DDT in the diet at rather high
    concentrations did not change the phase or the amplitude of the
    natural adrenocortical rhythm or alter the stimulation of
    corticosterone secretion produced by irritant stress.

          In rodents, locomotor activity is greatest at night and Boyd
    (1972) suggests that depression of activity is best demonstrated at
    night or in rats starved for 3 days when their daytime activity is as
    great as at night time. However, a more convenient approach may be to
    reverse the lighting schedule, a procedure used successfully for
    measuring the effects of various inhaled air pollutants on locomotor
    activity in mice (Murphy, 1964).

          The time of day at which biochemical or other tests are conducted
    in control and experimental animals may influence the reproducibility
    of the test data, if the biological variables under test display
    rhythmic variation. An investigator may exploit these rhythmic
    variations to advantage in special studies of factors that influence
    susceptibility to chemical injury. However, if the aim is a broad
    scale characterization of the toxicity of a chemical, the choice may
    be to carefully standardize times of administration of chemicals and
    of animal sampling to minimize both known and unrecognized circadian
    variations as much as possible.

    2.4.4  Environmental variables

          There are numerous possible variations in the environment in
    which experimental animals are housed or tested that can influence
    their response to toxic chemicals. General considerations of these
    variables are discussed by several authors (Boyd, 1972; Doull, 1972,
    1975; Hurni, 1970; Morrison, 1968). Unless the purpose of the
    experiment is to use these variables to predict possible alterations
    in effects in man exposed to chemicals under similar environmental
    variations, it is generally possible to minimize their influence on
    the toxicity of chemicals by adopting good principles of animal care.
    Reference sources are available to provide guidelines for proper
    housing, diets, cage size requirements, etc. (DHEW, 1972; Universities
    Federation for Animal Welfare, 1972).

          Only brief comments will be made on some of the major
    environmental variables that affect toxicity experiments or that can
    be used for predicting mechanisms or possible implications to man.  Temperature

          Major variations from the recommended environmental temperatures
    and relative humidities can contribute not only to the impairment of
    general health and increased susceptibility to infection of the
    animals, but also to variation in their response to toxic chemicals.

          The mechanisms of interactions between environmental and body
    temperatures and drugs or toxic agents have been reviewed by Doull
    (1972) and by Cremer & Bligh (1969). Since absorption, distribution,
    metabolic transformation, excretion, and reactivity with receptor
    sites depend on various temperature-dependent chemical reactions, it
    might be expected that the toxicity of chemicals would be readily
    influenced by temperature. However, since toxicity studies are usually
    conducted with homotherms, only minor changes in core body temperature
    occur with moderate changes in environmental temperatures. By the same
    token, changes in environmental temperature will elicit homeostatic
    changes in various physiological or biochemical systems. These may
    then alter some of the physiological variables (e.g. ventilation,
    circulation, body water, intermediary metabolism) that are
    rate-limiting determinants of the absorption, deposition, and action
    of toxic chemicals. Furthermore, toxic chemicals may exert their
    action by disruption of the thermoregulatory mechanism as suggested
    for cholinesterase inhibitors (Meeter & Wolthuis, 1968). Exposure to
    toxic chemicals can also mimic the actions of extremes in
    environmental temperature or other physical stressors (Murphy, 1969;
    Szot & Murphy, 1970). Thus, fluctuations in environmental temperatures
    can lead to functional changes that might be mistakenly attributed to
    the action of the chemical or they may actually alter the toxicity. If
    interference with physiological thermoregulatory mechanisms is a
    likely action of the chemical, careful control of environmental
    temperatures is necessary to ensure reproducibility of measurements of
    this action.  Caging

          The type of cage, grouping, bedding, and other factors related to
    caging can markedly influence the toxicity of some chemicals and drugs
    (Boyd, 1972; Doull, 1972; Hurni, 1970). The acute toxicity of
    (isoproterenol) was markedly greater in rats caged singly for more
    than three weeks than in rats caged in groups (Hatch et al., 1965).
    Winter & Flataker (1962) reported that grouped rats held in "closed"
    (sheet metal on four sides and bottom) cages were more resistant to
    the acute toxicity of morphine and 1-[2-(4-amino-phenyl)ethyl]-
    4-phenyl-4-piperidine carboxylic acid ethyl ester (anileridine) than

    rats held in "open" (wire mesh) cages. These differences were
    attributed to mechanical factors that prevented depressed rats from
    maintaining an open airway in the wire mesh cages. Altered toxicity of
    chemicals related to caging effects are generally purely experimental
    variables and can be controlled by good practices of laboratory animal
    housing.  Diet and nutritional status

          Dietary variables can influence the toxicity of chemicals in
    several ways. The toxicities of several pesticides were enhanced to
    different degrees in rats given low protein diets (Boyd, 1969).
    Protein-deficient diets protected rats against the acute
    hepatotoxicity of carbon tetrachloride and  N-methyl-
     N-nitrosomethanamine (dimethylnitrosamine) (although the number of
    kidney tumours after a single dose of the latter increased), while the
    acute toxicity of chloroform was unchanged and the acute toxicity of
    aflatoxins was markedly enhanced (McLean & McLean, 1969). These
    effects could be explained, at least in part, by the reduction of
    activity of hepatic mixed-function oxidases that generally results
    from feeding low-protein diets. Whether or not a compound's toxicity
    is increased or decreased in such circumstances will depend upon
    whether microsomal biotransformation leads to the formation of more or
    less toxic metabolites. Numerous other examples of macro and
    micronutrient deficiencies, which alter the activity of the
    drug-metabolizing enzyme systems and the toxicity of chemicals, have
    been reviewed by Campbell & Hayes (1974). Intestinal and pulmonary
    aryl hydrocarbon hydroxylase activity is modified by diet. Of
    particular interest is the observation that changing rats from a
    commercial, natural diet to a balanced, purified diet resulted in an
    almost total loss of activity of this enzyme system in these tissues
    (Wattenberg, 1972). Flavonoid compounds, present as natural
    constituents of alfalfa meal (and other plants), may account for the
    apparent induction of aryl hydrocarbon hydroxylase by natural diets.
    The trace mineral content of diets can also influence the metabolism,
    distribution, and action of toxic chemicals (Moffitt & Murphy, 1974).

          The results of toxicity experiments can be markedly influenced if
    care is not taken to ensure constancy of diets free from residues of
    contaminating chemicals. However, since nutritional imbalances are
    widespread in the human populations, controlled variation of
    experimental diets to simulate major human deficiency states (e.g.
    kwashiorkor resulting from protein deficiency) is an important area
    for research in toxicology and should, perhaps, be included in
    standard toxicity evaluations of select groups of chemicals.

    2.5  Statistical Considerations

          Although various protocols for toxicity testing recommend
    specific numbers of animals to be used for various acute and chronic
    tests (see Chapter 3), a useful guiding principle is that sufficient
    animals should be used to allow statistically valid conclusions
    concerning differences in the response of test animals compared to
    controls and to provide a base for statistical extrapolations to
    larger population samples. Statistical procedures allow the
    experimenter to make ( a) descriptions of sets of data or population
    characteristics, and ( b) statements of probability of events.
    Various procedures provide for both enumerative data, or yes-no type
    characteristics, and measurement data, or graded effects or
    characteristics. Some procedures (t-test, F-test) are restricted to
    observations that have specific frequency distributions, while others
    (signed rank test, rank run test) are free of any assumptions about
    distribution (i.e. nonparametric). Standard texts should be consulted
    for the application of biostatistics to the design and analyses of
    experiments. In practice, it is highly advisable to involve a
    statistician in the experimental design as well as in the analysis.

          The number of animals required to make statistically valid
    conclusions regarding the differences between experimental and control
    animals will depend upon the degree of confidence desired and the
    magnitude of the possible sources of variation in the experiment. The
    second consideration will depend upon the uniformity of the test
    animals with respect to the biological system or systems under test.
    This, in turn, will depend upon both genetic and environmental
    factors. The reproducibility of the bioassay and chemical procedures
    used in the tests will be another source of variation. A further
    source of variation in toxicity testing is related to the constancy
    and stability of the test chemical. Finally, there are the variables
    introduced by the investigators (often the most difficult to control),
    beginning with the care and attention given to accurate dosing
    throughout the various steps of the experiment. Attempts must be made
    to minimize all these sources of variation as far as possible, without
    sacrificing any important aspect of the experiment.

          When testing whether or not two sets of data may both be valid
    samples from the same population with normal frequency distribution
    (i.e. null hypothesis) or whether the control group is not
    significantly different from the treatment group, statisticians
    describe the appropriate sample size in terms of the desired "power"
    of the test. Two types of decision errors exist. One is that a
    significant difference between groups is stated to exist when, in
    fact, there is no difference. This is called the type I error and the
    experimenter must state what probabilities (alpha) for this error he
    will accept; most commonly a probability of 0.05 is used, but an
    experimenter may sometimes require a probability as low as 0.01, or

    any other he chooses. The second type of decision error is that no
    significant difference between groups is stated to exist when, in
    fact, the groups are different. This is called the type II error, and
    1- (the probability that one will not make this error) is the power
    of the test. The power is directly related to the sample size and the
    ratio of "differences between true means of the samples" to
    "differences between experimental error of the means of the samples".
    Once this ratio is fixed, the power increases solely as a function of
    sample size. The experimental error (pooled variance) can be estimated
    from previous experiments or a pilot study. The acceptable magnitude
    of the differences between true means of the samples is at the
    discretion of the experimenter; he must use expert judgment and should
    have a reasonable rationale; it will usually be the smallest value
    that is considered to be of practical importance.

          Another important statistical consideration is related to the
    selection of the valid number of sampling units. This may be
    particularly true in considering quantal (all-none, yes-no) effects
    that may be multiple occurrences within a single test animal, as, for
    example, in reproduction and carcinogenesis studies where,
    respectively, there may be a number of affected offspring or a number
    of tumours resulting from the treatment of a single animal. The
    selection of the appropriate unit, either the number of animals
    exposed or the number of occurrences of an effect, can determine the
    statistical significance of an observed effect. Weil (1970) suggests
    that in reproduction studies the number of maternal animals (or
    litters) and not the number of affected fetuses or offspring is the
    valid sampling unit, and that in carcinogenesis studies the number of
    tumour-bearing animals should be the sampling unit and not the total
    number of tumours. Furthermore, in carcinogenesis studies, animals
    risk death from factors other than the tumours; in some cases, animals
    may have died before they had time to develop a tumour; in other
    cases, information from some animals may be lost from the study
    because of unexpected death and autolysis of tissues preventing tumour
    identification. An adjusted tumour incidence may be estimated by the
    life-table techniques in such experiments (McKinney et al., 1968).

          Another problem may be associated with gross or histopathological
    examination where, because of cost and time considerations, only
    tissues of a fraction of the total number of animals exposed are
    subjected to complete examination. This will reduce the likelihood
    that statistically valid conclusions can be drawn from the data on
    occurrence of lesions. In practice, reasonable compromises are usually
    necessary. Irrespective of the statistical methods of analysis used in
    both the design and interpretation of results of toxicity tests on
    chemicals, they cannot replace careful experimentation and
    comprehensive knowledge of the underlying biological mechanisms of the
    various steps between exposure to a chemical and injury.

    2.6  Nature of Effects

    2.6.1  Reversible and irreversible effects

          Reversible effects are characterized by the fact that the
    deviation from normal structure or function induced by a chemical will
    return to within normal limits (controls) following cessation of
    exposure. With irreversible effects, the deviation persists or may
    progress, even after exposure ceases. This might be further qualified
    by time limits, that is, the time required for return to normality
    after exposure should be a reasonable fraction of the remaining
    lifetime of a young animal for it to be considered reversible.
    Reversibility may also be qualified by the normal lifetime of a
    specific cell or macromolecule that serves as the end-point for the
    effect. For example, cholinesterase-inhibiting insecticides are
    generally considered irreversible inhibitors if the rate of reversal
    of inhibition corresponds approximately to the time required for
    synthesis and replacement of the enzyme, a process with different
    rates in different tissues. Certain effects of toxic chemicals are
    unmistakably irreversible, including the production of terata, or
    malignant tumours, production of mutations in offspring of exposed
    animals, certain chronic neurological diseases, production of true
    cirrhosis, or emphysema. These are rather gross manifestations of
    certain specific chemical-cell interactions, and, either at the level
    of the first affected molecule or at intervening points leading to
    these manifestations, there are probably reversible effects.
    Understanding these effects and determining the critical dose that
    produces them will make it possible to predict truly adverse effects
    more rapidly.

          The rate of reversibility of an effect will depend upon the rate
    of cellular injury and the rate at which this injury is repaired
    (Casarett, 1975). The rate of injury will depend upon the
    concentration and duration or frequency with which a test chemical
    contacts responsive tissue constituents. It is, thus, dose and
    dose-rate dependent. The rate of repair is determined intrinsically
    and may involve several cell processes. It may vary between different
    tissues and probably between different species and strains. From a
    practical standpoint, it is generally impossible to measure the
    specific processes involved in injury and repair in a standard
    toxicity evaluation study. However, it is important to make
    measurements of the reversibility of effects in early, acute and
    subacute studies. Thus, the time required for a process to return to
    normal after single doses (which produce various degrees of injury)
    will provide a guideline for the selection of doses to be used in
    subsequent acute or chronic studies. The predictive value of such
    information will depend upon the persistence of the chemical in the
    test organism. If the chemical produces an effect and then is rapidly
    detoxified or excreted, it may be possible to predict, with reasonable

    accuracy, doses or exposure schedules that would not produce
    cumulative effects. The manner of exposure and possible actions other
    than the one being measured would, of course, be important in drawing
    such conclusions. For example, rapid reversibility after a single dose
    might not be indicative of the rate of reversal with a repeated dosing
    if the first dose, in addition to the measured effect, also altered
    either the repair processes or the processes responsible for
    detoxification of the chemical. An example of an apparent
    self-inhibition of detoxification is the insecticide malathion which
    is rapidly hydrolysed by carboxylesterases. These are, in turn,
    inhibited by metabolites or contaminants of malathion (Murphy, 1967).
    Repeated exposure studies are necessary to evaluate such
    possibilities; thus, the design of short-term feeding or inhalation
    studies should include extra groups of animals that can be removed
    from exposure either at the end of the experiment or, preferably, at
    selected intervals for measurements of rate of reversal of any
    observed effect.

          If the chemical persists or accumulates in the organism,
    measurements and interpretation of rates of reversal of effects are
    more complicated. For this reason, it is useful to have kinetic data
    on absorption and disposition to correspond with data on rates of
    production and reversal of effects. Further discussion of these and
    related principles is provided by Hayes (1972) in relation to his
    proposal that determination of "chronicity factors" (1-dose LD50
    (mg/kg)  90-dose LD50 (mg/kg/day) in diet i.e. the ratio of single
    dose LD50 to the daily dose given in diet for 90 days which results
    in 50% mortality at that time) is useful in predicting candidate
    chemicals requiring long-term studies. The use of such predictive
    methods must also take into consideration potential for other effects
    that could never be detected in a subacute study (e.g. tumorigenesis).

    2.6.2  Functional versus morphological changes

          Toxic effects are often classed as functional or morphological in
    nature. There has been a traditional attitude that changes in gross or
    microscopic structure are more serious than functional changes.
    Indeed, altered structure often seems to have taken on the implication
    of irreversibility while altered function is often considered a
    reversible effect. This conclusion, of course, depends on the level of
    understanding of mechanisms of injury, rates and mechanisms of repair,
    and causal associations between related functional and morphological
    changes. Furthermore, whether the change is regarded as functional or
    morphological may depend on the manner of detection. For example,
    accumulation of fat in a cell observed through a microscope will most
    often be considered a structural change, but, if the same cells or
    tissues were assayed for triglyceride content, the increased
    triglyceride would probably be classified as a functional or

    biochemical change. The introduction of enzyme histochemistry and
    electron microscopy into toxicity evaluation studies makes the
    distinction between morphological and functional effects even less
    clear. It may therefore be inappropriate to attempt to make these

          Rowe et al. (1959) reviewed data from studies on a large number
    of chemicals repeatedly administered to animals over periods ranging
    from one month to two years, and summarized the frequency with which a
    certain effect was found and the frequency with which it was the only
    effect found. The effects were considered on: mortality; food intake;
    body weight; organ weights; the histopathology of virtually every
    major organ; haematology; blood urea nitrogen; clinical urinalyses;
    central nervous system (most probably gross behaviour); gross
    pathology; and cholinesterase activity. The authors found that if
    growth, liver weight, kidney weight, liver pathology, and kidney
    pathology had been studied, the lowest dose level that caused any
    effect would have been detected in 96% of the studies. Changes in food
    intake, central nervous system depression, excessive mortality,
    increased lung weight, testicular injury, haematological changes, and
    cholinesterase depression were the most sensitive effects in one or
    more of the remaining 4% of cases. The reader should consult the
    original reference for details but it is important to note that of the
    commonly used criteria of effects, liver and kidney micropathology
    were quite sensitive indices.

          There are, of course, well-known examples where functional
    changes are the only manifestations of toxicity. Many of the
    organophosphate and carbamate insecticides inhibit cholinesterase
    ( activity and produce signs and symptoms (even death) that
    can be characterized as purely functional without the production of
    morphological lesions, detectable by conventional techniques.
    Similarly, irritant air pollutants can often cause bronchoconstriction
    and respiratory distress, without any accompanying morphological
    changes. Both the functional cholinergic signs and symptoms produced
    by the anticholinesterase insecticides and the bronchoconstriction
    produced by irritants provide means of early detection at low levels
    of exposure. Although these effects are reversible, they are no less
    important during the period of exposure than certain kinds of
    morphological effects. On the other hand, certain kinds of
    "functional" changes, e.g. increased level of plasma transaminase
    activity, usually reflect some type of structural change in cells that
    allow these enzymes to "leak" into plasma (Cornish, 1971).

          It is not possible, with present knowledge, to conclude that
    either functional or morphological changes represent the most
    sensitive, the earliest, or the most serious effects of toxic
    chemicals. Since maintenance of both integrated function and
    integrated structure ultimately depends on chemical reactions among
    cell constituents, it is logical to conclude that specific biochemical
    changes are the first and most sensitive effects. Unfortunately, with
    relatively few exceptions, the specific biochemical receptors for
    toxic chemicals are unknown. The more information that can be obtained
    with respect to time- and dose-relationships for functional and
    morphological effects, the more predictive the tests will become. This
    requires an approach to toxicity studies in which proof of a mechanism
    will require an integrated biochemical, physiological, and
    morphological approach. Dawkins & Rees (1959) provide a useful short
    treatise on an integrated biochemical-pathological approach to studies
    of several toxic chemicals. Although advances in both biochemistry and
    pathology now allow even more precise studies than those outlined by
    these authors, the general principles which they develop are still

    2.7  Dynamic Aspects of Predictive Toxicology

    2.7.1  Traditional versus new techniques

          The objective of any toxicity test programme is prediction:
    prediction of biological disposition from physicochemical constants,
    prediction of altered cell or organ system function from reaction with
    macromolecules, prediction of irreversible consequences of reversible
    changes, prediction of implications of selected measurable variables
    to overall health and survival of the test organisms, prediction of
    effects in individuals of one species from tests conducted in another,
    and finally predictions of incidence in large populations from tests
    on small samples. All of these predictions must be related
    quantitatively to a dose and dose-rate or schedule that can ultimately
    be related to probable amounts used, the manner of use or the
    occurrence of the chemicals in the environment.

          Generally, traditional approaches to toxicity evaluation have not
    attempted to make predictions far removed from the final application
    or interpretation of the data. Thus, as outlined in Chapter 3, test
    organisms are exposed to a range of doses and their health status is
    examined by biochemical, physiological, or pathological procedures
    analogous to those used in clinical medicine. When this approach has
    been comprehensive, judicious application of the data usually appears
    to have been successful in preventing chemically-induced disease.
    Abandoning this approach in favour of new, different, or short-cut
    methods cannot be advocated without thorough verification of their
    validity. On the other hand, serious consideration must be given to
    the application of some short-term ways of predicting toxicity in

    order to provide a practical means of evaluating the many chemicals
    already in the environment and those new compounds that are
    continuously being added to the environment and have not been
    subjected to traditional tests. Preceding sections have discussed some
    possibilities, the following sections contain brief comments and
    examples for consideration in selecting tests.

    2.7.2  Toxicity of chemical analogues

          Although it may be possible to predict the toxicity of individual
    compounds in a homologous series from detailed knowledge of some
    members of the series, some special exceptions should be noted. A
    classical example involves the series of fluorine-substituted
    aliphatic alcohols and acids, in which high acute toxicity alternates
    with odd and even numbers of carbon atoms, the latter being the most
    toxic (Pattison, 1959). The odd number of total carbon atoms confers
    high toxicity in a homologous series of fluoronitriles, however. This
    demonstrates the possibility that detailed information concerning only
    a few members of a homologous series might fail to predict the
    toxicity of another member of the series.

          Recently, Johnstone et al. (1974) examined a number of
    biochemical effects in a series of isomerically pure compounds for
    their potency as liver enzyme inducers. Potency for this effect
    increased with increasing chlorination that was related to differences
    in biotransformation and excretion rates; however, there were also
    striking differences in the potency of positional isomers in the lower
    chlorinated biphenyls.

          The mechanism of the toxic action of organophosphorus
    insecticides was known to be the inhibition of acetylcholinesterase
    even before they were introduced into use 30 years ago. However,
    quantitative prediction of their acute toxicity from  in vitro tests
    of their relative potency as anticholinesterases is still inadequate
    because of incomplete knowledge of the dynamic relationships between
    several pathways of metabolism which yield both more and less potent
    metabolites (Murphy, 1975). Nevertheless, these compounds have been
    subjected to a great deal of research on both their physicochemical
    and biological properties which should be applicable to predictions of
    their relative environmental persistence, interaction with other
    compounds, and, ultimately, to the design of safe molecules (Eto,

          Using model ecosystems, Lu & Metcalf (1975) studied
    bioaccumulation, biodegradability, and comparative detoxification
    mechanisms in several benzene derivatives with widely-varying
    physicochemical constants and biological activities. They concluded
    that biological disposition and action could be predicted by the basic
    molecular properties of water solubility, the partition coefficient
    for lipid/water, and reactivity as determined by electron density.

          Johnson (1975) recently reviewed the problems encountered in the
    pursuit of the mechanism of delayed peripheral neuropathy produced by
    some organophosphorus esters. The structure-activity relationships
    identified in this research may be considered as a model of thorough
    investigation that began as a problem in retrospective toxicology and
    led to promising developments applicable to prospective toxicology.
    Some interesting aspects of this problem are: the particular
    usefulness of a non-mammalian species, the hen, as a predictor of a
    toxic action that occurs in man; the concept of primary metabolic
    effects on central neurons as a precursor to pathological change
    detected in peripheral nerve fibres; and the difficulties of detecting
    a specific critical esterase inhibition that represented only a small
    percentage of the total esterase activity. Production of peripheral
    neuropathy appears to be characteristic of organophosphorus esters
    which may not only phosphorylate the specific "neurotoxic esterase"
    but are also capable of dealkylation (or aging) following
    phosphorylation. Although the steps between this primary
    phosphorylating-aging process and the eventual manifestation of
    peripheral neuropathy are still unclear, it appears that it may be
    possible to predict probable occurrence of a delayed, chronic disease
    from studies of the primary chemical-macromolecular interaction of
    neurotoxic esterase inhibition that occurs immediately following

    2.7.3  Relation between site of metabolism and site of injury

          Although for many years it was thought that the biotransformation
    of organic chemicals represented detoxification mechanisms, it is now
    apparent that numerous compounds are enzymatically converted to
    intrinsically more active compounds  in vivo (Fouts, 1972; Murphy,
    1975; Parke, 1968). The liver is generally the most active tissue in
    catalysing these "activation" reactions, but it is not always the most
    susceptible target tissue as, for example, in the case of activation
    of phosphorothioate insecticides to phosphate insecticides. This may
    be explained, in part, by the presence of detoxifying enzymes or
    reactive but noncritical binding sites in the liver that may prevent
    the phosphates from escaping to inhibit cholinesterase in nerve target
    tissues. The brain tissue has only a small fraction of the liver's
    capacity to activate phosphorothioates, but because the activation
    occurs in the same tissue as the critical target site, activation in
    the brain may be the most important in determining toxicity.

          Recently, the characteristic hepatotoxicity of several chemicals
    has been related to their enzymatic conversion to highly reactive
    derivatives that covalently bind to essential liver cell constituents
    at, or near, the site of activation (Brodie et al., 1971). A similar
    possibility may explain the bronchiolar neurosis produced in rats and
    mice by bromobenzene and other aromatic hydrocarbons (Reid et al.,

          The detailed study of the metabolism, storage, or binding and
    distribution of foreign chemicals in the lung is a relatively recent
    activity and has focused largely on therapeutic chemicals (Bend et
    al., 1973; Brown, 1974; Orton et al., 1973). Because inhalation is a
    common route of exposure to a wide variety of air contaminants in
    industrial and community environments, future toxicity studies would
    benefit by the inclusion of metabolic studies concerning rates of
    absorption from, and local actions in the lung. Witschi (1975) has
    reviewed biochemical approaches that may be used in the evaluation of
    toxic injury to the lung.

          Since the intensity and duration of the toxic action of a
    chemical depends on the concentration of the active form at critical
    receptor sites of action, kinetic aspects of absorption, distribution,
    and excretion (as well as biotransformation) will influence the
    specific sites of action. This topic is discussed in detail in Chapter
    4 but it is worthy of note that Dedrick (1973) developed several
    useful kinetic models that might be applied in predicting species
    differences or similarities in response.

    2.7.4  In vitro test systems

          Where appropriate, studies in experimental animals should be
    supplemented by isolated perfused organ, tissue slice or extract, and
    tissue culture techniques. Where possible, attempts should be made to
    compare the tissues of human subjects available from autopsy or
    therapeutic biopsies, with those of other species in their response to
    toxic chemicals (Worden, 1974). When the mechanism(s) of toxicity have
    been elucidated and the target organ(s) identified, specific species
    comparisons and dose-response relationships can be studied by these
     in vitro techniques.

          Knowledge of a specific enzyme or biological macromolecule that
    serves as a target for reaction with toxic chemicals may provide a
    means for screening and predicting relative potencies or specific
    actions of chemicals in intact organisms. However, as pointed out
    previously for the organophosphorus insecticides, biotransformations
    and membrane barriers to the distribution of chemicals in intact
    animals will often invalidate conclusions drawn from  in vitro
    assays. This problem may be partly overcome by incorporating enzymic
    biotransformation systems with the target macromolecule (or organism)
    in the  in vitro test system. Such an approach is used for screening
    for potential mutagens in microorganism test systems (Malling &
    Frantz, 1973) and has been applied to studies of the biochemical
    actions of pesticides (Chow & Murphy, 1975; Cohen & Murphy, 1974). A
    major problem in the use of  in vitro test systems for predicting
    toxicity is the difficulty of quantitatively relating concentrations
    in the simplified  in vitro systems to action in complex intact

    organisms. With adequate correlative data in both  in vitro and
     in vivo systems this may become possible, but such information is
    generally lacking at present. For the most part, therefore,  in vitro
    model test systems are qualitative predictors rather than
    quantitative. This need not decrease their usefulness, however, as
    long as this limitation is recognized in the interpretation of

          In general,  in vitro test systems have been useful in
    qualitatively predicting acute actions. However, as discussed earlier,
    neurotoxic esterase inhibition provides promise for predicting delayed
    chronic neuropathy produced by certain organophosphate compounds.
    Major research efforts are now being devoted to  in vitro test
    systems for the prediction of mutagenesis and carcinogenesis. These
    are discussed in detail in Chapter 7. There is general recognition of
    the value of these test systems (Council of Environmental Mutagen
    Society, 1975; Food & Drug Administration, 1970; Food Protection
    Committee, 1970) as screening procedures, but much less agreement as
    to the priority that they should have in the conduct and
    interpretation of toxicity evaluations.

          When it is possible to obtain comparisons between exposures of
    organs or tissues of experimental animals and humans to toxic
    chemicals, such comparisons will provide useful baseline data for the
    future extrapolation of data from intact animal studies to man.
    Culture systems of human cells may also be useful as comparative
    systems. The difficulties of maintaining some human cell lines are
    well documented, but primary cultures of differentiated mammary and
    liver epithelia have been established and maintained (Buehring, 1972;
    Lasfargues & Moore, 1971; Potter, 1972). Human lymphocytes have also
    been used  in vitro (Kellermann et al., 1973).

          It may be possible to use these isolated systems to determine the
    susceptibility to toxic chemicals of different cell types in different
    organs and to determine the reversibility of adverse effects in these
    cell lines and organs. Of particular usefulness would be the
    determination of dose-response curves for many tissues and their
    interspecies comparison. Such information could be used to predict
    target cells and organs with a high degree of susceptibility or
    resistance. However, as mentioned earlier, the usefulness of the data
    may be limited if the cells, tissues, or organs are incapable of
    metabolizing the chemical to a toxic form in the intact animal. Such a
    biotransformation may even occur in a different tissue or organ from
    the one under test  in vitro. To overcome this problem,
    biotransformation systems from animal or human tissues (e.g.
    microsomal activating systems) are often added with the chemical to
    the isolated culture systems.

          One problem with these methods is the uncertainty that all the
    steps of metabolism are equally duplicated, that is, in addition to
    activation of a chemical there should also be an opportunity for the
    chemical to be detoxified or conjugated and eliminated. Some of these
    detoxification steps require different coenzymes or metabolites, and
    the enzyme systems may not be limited to microsome fractions or liver
    tissue. However, as long as it is realized that these  in vitro test
    systems may exaggerate the situation that occurs  in vivo they can
    prove useful, especially for tests where only very small quantities of
    material are available, as might be the case with some impurities or

          In summary, short-term,  in vitro tests both for carcinogenicity
    and other forms of toxicity show great promise (Golberg, 1974), and
    although no single test is likely to be reliable, appropriate
    combinations may provide valuable information concerning the
    fundamental toxicity of environmental chemicals. This would currently
    provide a useful adjunct to long-term studies in animal populations,
    and may develop further in the future to provide the more reliable
    method of assessment. Such tests will require much development and
    will take years to validate and perhaps even longer to win public
    confidence with regard to their reliability.


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    3.1  Introduction

          The primary objective of toxicological testing is to determine
    the effects of chemicals on biological systems and to obtain data on
    the dose-response characteristics of the chemical. These data may
    provide information on the degree of hazard to man and the environment
    associated with a potential exposure related to a specific use of this
    chemical. Elucidation of the metabolic behaviour of the chemical in
    test animals increases confidence in defining the hazard (see Chapter
    4). The degree of confidence with which hazard may be estimated
    depends on the quality of the toxicological data. Selection of the
    most appropriate test procedures coupled with strict adherence to
    accepted experimental practices and astute observation are of
    paramount importance in experimental toxicology.

    3.2  General Nature of Test Procedures

          Several types of toxicity testing procedures have been developed.
    These include acute, subacute, and chronic studies. The major
    difference between these tests is the dose employed and the length of
    exposure to the chemical agent, but other differences in intent and
    nature do exist and will be discussed. All of the tests share some
    common characteristics. Each requires that groups of healthy animals,
    housed under suitable conditions, be exposed to graded doses of the
    test chemical. Rats, mice, guineapigs, rabbits, and hamsters are
    commonly used for this purpose, but in some cases it may be necessary
    to use dogs, swine, nonhuman primates, or other species. As a rule, a
    control group is given the dosing vehicle or is sham treated.
    Following treatment, the animals are closely observed for signs of
    toxicity. Laboratory procedures designed to measure biological effects
    are carried out on the treated and control animals. Detailed records
    are maintained on each animal. Following completion of the test, all
    animals, including controls, are subjected to a pathological
    examination. Data should be analysed by appropriate statistical

    3.2.1  Housing, diet, and clinical examination of test animals

          Animals should be healthy, genetically stable, and adequately
    identified as to colony source. The controls and treated animals
    should be of the same strain and species, age, and weight range, and
    be supplied from the same source. Before starting the experiment, the
    health status of all animals should be determined and monitored for
    some time. During this time, a small randomly selected number of
    animals from each shipment should be sacrificed and examined for
    disease, parasites, and other specific pathogens. During the

    quarantine period, animals may be caged together according to the
    weight-space specifications. Acceptable standards for the housing and
    care of experimental animals have been published (DHEW, 1972; Canadian
    Council on Animal Care, 1973; Sontag et al., 1975).

          During toxicity studies, rodents should be housed singly or in
    pairs in stainless steel or plastic shoe-box cages while nonrodents
    should be housed in suitable runs. The animals should be randomly
    allotted to the cages and treatment regimes should be randomly applied
    (Cox, 1958). Rodents should be allowed free access to food and water.
    Nonrodents should be fed meal and given water  ad libitum. The diet
    fed to the animals should meet all of their nutritional requirements
    (National Academy of Sciences, 1975) and should be free of toxic
    chemical impurities that might influence the outcome of the test.
    Periodic analysis of the diet to ensure its nutrient composition
    should be undertaken since nutritional status may affect the nature of
    toxic responses (Arcos, 1968). Although commercially available diets
    of recognized quality are suitable for most subacute studies,
    semipurified diets may be preferred because the nutrient and
    nonnutrient components of the diet may be altered readily, where
    necessary (Munro et al., 1974; Newberne, 1968).

          Careful clinical observation of test animals is the most
    neglected area in experimental toxicology. Few investigators are aware
    or recognize that the skills required of a good medical diagnostician
    are also required in assessing or diagnosing the toxic state or
    condition of an animal. In toxicity studies, many animals may be lost
    for evaluation because of death from intercurrent disease and
    subsequent autolysis. With concerned, reliable staff, these losses can
    be greatly reduced if a conscientious effort is made to recognize
    early clinical signs of disease in the test animal. Ideally, each
    animal on test should be looked upon as an individual patient. In this
    way, there is an awareness of the idiosyncrasies of the animal and
    departures from the normal will be more easily recognized. Once a
    routine of careful clinical assessment has been established, it is
    possible either to treat diseased test animals or, if necessary,
    sacrifice them. In the latter case, the tissues are available for
    histological examination. Otherwise, chronic disease effects might
    render the tissues useless for the assessment of effects due to test

          Detailed clinical examinations should be conducted weekly on the
    test animals by competent, laboratory animal technicians under the
    supervision of a veterinarian skilled in laboratory animal medicine
    (Health and Welfare, Canada, 1973). These should include general
    observation of the animals for overt signs of toxicity, quality of
    hair, coat, general condition of the eyes, mouth, teeth, nose, and
    ears (Leclair & Willard, 1970; Loomis, 1968). Assessment of cardiac
    and respiratory function should be conducted by auscultation. If

    neurological effects are anticipated, a detailed neurological
    examination should be conducted. In larger species, this can be done
    by skilled personnel using the methods of Charbonneau (1974), McGrath
    (1960), and Mowbray & Cadell (1962). Examination of the eyes using
    opthalmoscopic and slit-lamp techniques (Marzulli, 1968) may assist in
    detecting ocular toxicity. The external and internal structures should
    be carefully palpated and any tissue masses should be noted. Detailed
    records of clinical evaluation should be maintained and should be
    accessible to the attendant pathologist.

    3.3  Acute Toxicity Tests

    3.3.1  Underlying principles

          Acute toxicity has been defined as the adverse effects occurring
    within a short time of administration of single dose or multiple doses
    given within 24 h (Hagan, 1959). When data are unavailable concerning
    the toxicity of the test agent, acute toxicity studies are indicated
    to identify the relative toxicity of the compound, to investigate its
    mode of action and its specific toxic effect, and to determine the
    existence of species differences.

          The most frequently used acute toxicity test involves
    determination of the median lethal dose (LD50) of the compound. The
    LD50 has been defined as "a statistically derived expression of a
    single dose of a material that can be expected to kill 50% of the
    animals" (Gehring, 1973). The basic protocol for the determination of
    the LD50 is well established and consists of treating groups of
    animals with a mathematically-related series of doses in order to
    determine the dose that kills 50% of the group and the dose-response
    function. The LD50, being a calculated value, is always accompanied
    by some estimation of the error of the value, such as the confidence
    limits. The most commonly used methods for calculation of the LD50
    are the graphic method of Litchfield & Wilcoxon (1947), the
    logarithmic probit graph paper method of Miller & Tainter (1944), and
    the method of moving averages of Thompson (1947) and Weil (1952). A
    comparative review of these and other methods was published by
    Armitage & Allen (1950). Death which occurs after the first 24 h is
    more likely to be due to delayed toxic effects, which may be direct or
    indirect. Signs occurring after the first 24-h period may give some
    indication of the effect that the chemical may have at lower levels,
    when administered for longer time periods.

    3.3.2  Experimental design  Selection of species

          The extent of species variation in toxicity testing has been well
    documented in the reviews of Brodie (1964) and Rumke (1964). The
    usefulness of determining species variability in order to assess the
    applicability of toxicity data to man has been discussed by Hagan

    (1959). Litchfield (1962) has postulated that if the toxicity of a
    compound is the same in several species, there would appear to be an
    increased likelihood that man would react in a similar manner.

          The mouse, rat and dog are the most commonly used species for
    acute toxicity testing. Both the rat and mouse should be used, as
    marked differences in the LD50 between these two species are not
    uncommon (Morrison et al., 1968).

          The LD50 determination should be conducted in both male and
    female animals, as differences in the LD50 between sexes have been
    well documented (Hurst, 1958; Rumke, 1964) and are probably related,
    in part, to differences in hepatic metabolism (Conney et al., 1965).

          Acute toxicity may vary substantially with the age of the test
    animal (Dieke & Richter, 1945; Lu et al., 1965; Scott et al., 1965;
    Yeary & Benish, 1965), and animals of various ages should be used in
    LD50 determinations. The effect of the age of the animal on the
    LD50 is well documented and may be related to different levels of
    drug metabolizing enzymes, absence of sex hormonal influences, or an
    altered sensitivity of the central nervous system (Fouts & Hart, 1965;
    Jondorf et al., 1959; Setnika & Magistretti, 1964).

          The animals should be derived from previously untreated healthy
    females. Weinberg et al. (1966) have demonstrated an effect of
    treatment of dams during gestation with various compounds on the acute
    oral toxicity in the newborn.

          Furthermore, the animals should not have been previously used for
    other studies, nor should there be a history of recent exposure to
    anthelminthics or any other drug treatment.

          The number of animals used should be sufficient for statistical
    analysis and will depend on the method used for the calculation of the
    LD50. Usually 8-10 rodents (4-6 animals of each sex) are used per
    dose group (Leclair & Willard, 1970). Diechmann & LeBlanc (1943)
    described a method using a total of 6 animals, while other methods
    involved the use of 4-5 animals per dose group (Horn, 1956; Litchfield
    & Wilcoxon, 1947; Thompson, 1947).  Selection of doses

          The doses are selected to provide data for estimating the LD50
    and to obtain information on the slope of the dose-response curve. At
    least four doses, selected in logarithmic progression, should be used
    (Weil, 1952).

          In general, however, the doses can be arrived at only by
    experimentation. The initial dose may be such that no effect is
    manifested in the animals. In subsequent groups of animals, the dose
    should be increased by a constant multiple until the dose of the
    compound administered is sufficiently high that all of the animals in
    the group die. Under these conditions, data can be obtained that can
    be plotted to give a dose-response curve and from which the LD50
    value may be calculated.  Method of administration

          Generally, the chemical should be administered by the route by
    which man would be exposed. If the route is oral, the compound should
    be administered by gavage rather than mixed in the diet. In some
    cases, the administration of the chemical along with the diet has been
    shown to increase its toxicity compared with gavage dosing (Bein,
    1963; Worden & Harper, 1963), but, in general, the oral toxicity of a
    compound is greatest when it is administered by gavage to animals that
    have fasted (Griffith, 1964). Griffith (1964) has demonstrated the
    effect of the type and concentration of the vehicle on the LD50
    value. The amount of the liquid or carrier administered should be
    appropriate and the carrier should not, in itself, be toxic to the

          In certain cases, even though the route of human exposure would
    be oral, acute dermal, eye, and inhalation studies may be indicated to
    assess the hazard to personnel handling the compound in the
    laboratory.  Postmortem examination

          In general, all animals dying during the observation period and
    all surviving animals should be autopsied by a qualified pathologist
    (Leclair & Willard, 1970). The autopsy should include gross and
    histopathological examination of all organs.

          If death is almost instantaneous and due to a pharmacological or
    physical effect, i.e. massive gastrointestinal haemorrhage or acute
    respiratory collapse, detailed histopathological examination of all
    organs may not be indicated.

    3.3.3  Repeated high-dose studies

          Because of the inherent limitations of the LD50 in predicting
    long-term toxicity, a short but intensive study or a series of such
    studies may be indicated before commencing subacute tests. The purpose
    of such studies is to define more precisely the doses to be used in
    subacute tests and to elucidate more fully the organ systems affected.
    The design of these repeated high-dose studies may vary but consists,
    essentially, of repeated daily administration of a mathematically-
    related series of doses to groups of animals for 5-21 days.

          One type of repeated-dose study (Sontag et al., 1975) consists of
    treating groups of five young adult animals of each sex at each of
    five dose levels, the upper level being the one that is estimated to
    produce no more than 10% lethality following a single dose, the
    remaining doses being fractions of this dose.

          A seven-day feeding study described by Weil et al. (1969)
    consisted of treating five rodents of each sex at each of three or
    four dose levels for seven days. Criteria of effects were mortality,
    body weight gain, relative liver and kidney weights, and feed
    consumption. This study showed that the results of the seven-day
    feeding test were of significantly greater value in predicting dose
    levels for the 90-day toxicity test than the LD50 values.

          Daily observations, as described in section 3.1.3, should be
    conducted and weekly body weight and food consumption (if the animals
    are caged individually) monitored. For some test agents, especially
    those with delayed toxicity or cumulative effects, other measurements,
    such as organ function, body burden, absorption, and excretion of the
    compound may be indicated. Animals should be necropsied and the
    tissues should be examined for gross pathological changes and studied
    histopathologically, if indicated.

    3.4  Subacute and Chronic Toxicity Tests

    3.4.1  Underlying principles

          The subacute toxicity test generally involves daily or frequent
    exposure to the compound over a period up to about 90 days. It
    provides information on the major toxic effects of the test compound
    and the target organs affected (Barnes, 1960). The latency of
    development of the effect as related to dose, the relationship of the
    blood and tissue levels of the compound to the development of lesions,
    and the reversibility of the effects may also be studied. Data derived
    from these studies are used for designing chronic toxicity tests in
    which animals are exposed to the chemical for longer periods of time.

          Man may be exposed for the greater part of his life-time to low
    levels of a wide variety of environmental chemicals. Usually, the
    degree of exposure is insufficient to produce overt signs of toxicity;
    thus, cause-effect relationships cannot be easily established.
    Epidemiological studies may assist in this respect, but, because man
    is exposed simultaneously to several chemicals, it is difficult to
    establish unequivocally the degree of hazard associated with any one
    chemical. Acute and subacute toxicity tests are of limited value in
    predicting chronic toxic effects because: (a) chemicals may produce
    different toxic responses when administered repeatedly over a period;
    and (b) during the aging process, factors such as altered tissue

    sensitivity, changing metabolic and physiological capability, and
    spontaneous disease may influence the degree and nature of toxic
    responses. In addition, several important diseases such as heart
    disease, chronic renal failure, and neoplasia are associated with
    advancing age. These are multicausal in nature and thought to be due,
    in part, to the presence of chemical substances, both natural and
    synthetic, in the environment (WHO, 1972). Chronic toxicity tests, in
    which animals are exposed for their entire lifetime to environmental
    chemicals, have provided useful means of identifying those substances
    of greatest public health concern. The tests are usually conducted
    with the aim of establishing "no-observed-adverse-effect levels" that
    may be used in setting acceptable daily intakes (ADIs), tolerance
    limits for chemicals in food or water, or threshold limit values in
    the case of occupational exposure. Since chronic toxicity testing is
    expensive and requires specialized facilities and personnel, great
    care must be taken in the design, execution, and interpretation of the
    results of such studies.

    3.4.2  Experimental design  Selection of species and duration of studies

          In the subacute studies, if the compound has produced evidence of
    toxicity in man and if sufficient toxicological and metabolic
    information is available, it is often possible to select an
    appropriate species on the basis of these data. For compounds about to
    be put on the market about which little is known toxicologically, the
    recommendations of the World Health Organization (WHO, 1958) and
    competent national agencies (Friedman, 1969; Leclair & Willard 1970;
    National Academy of Sciences, 1975) should be followed in selecting
    appropriate test species. As a minimum recommendation, subacute
    studies should be undertaken in two species, one rodent and one
    nonrodent. Traditionally, the rat and dog are selected for subacute
    toxicity testing because of their availability and the large amount of
    background information available on them. When rats are used, the test
    should be initiated just after weaning so that observations may be
    made during the period of most rapid growth. A conventional strain
    should be selected, so that the results in control and treated animals
    can be compared with known literature values, and both sexes should be
    tested to ascertain the influence of the sex hormones on the toxic
    response. At least 10 animals of each sex should be included in each
    dose group and the experiment should continue for 10% of the animals'
    lifetime or about 3 months. If it is desired to study the pathogenesis
    and reversibility of induced lesions or biochemokinetics, it is
    recommended that observations be made at 3-week intervals during
    exposure and last up to 3 months following termination of exposure.

          In chronic toxicity testing, it is usual to expose the animals to
    the chemical for the greater part of the life span. A wide variety of
    animal species have been used in this type of work, although in most
    cases rodents are the animals of choice, since large numbers can be
    used to aid in the statistical interpretation of the results. Larger
    animals should also be used (e.g. dog and monkey) for such species
    have the advantage that larger samples of blood can be obtained on a
    routine basis.

          If the objective of the test is to study the carcinogenic
    potential of a compound, the rat, mouse, or hamster is usually chosen
    because of its shorter lifetime and the fact that large numbers may be
    used to increase the sensitivity of the test.

          When data on the metabolic fate of the test chemical in man is
    not available, the species showing the greatest sensitivity in
    subacute studies should be selected as the test species, provided the
    species does not react atypically to the compound due to metabolic

          Sufficient numbers of animals should be included in the test to
    ensure that a statistically valid design is achieved. Based on the
    incidence of effects observed in subacute studies and the anticipated
    incidence of chronic effects, the number of animals that should be
    used can be calculated (Snedecor & Cochran, 1967).

          Since it is usually the intention in chronic toxicity studies to
    expose animals over the major portion of their life span, it is
    essential to commence exposure early in life.  Selection of doses

          Guidance on the selection of doses for subacute studies may be
    obtained from the results of acute and repeated high-dose studies. For
    compounds having a tendency to bioaccumulation, selection of doses is
    particularly difficult. Kinetic studies may assist in establishing
    acceptable dose levels since the half-time ( t) for elimination
    (Chapter 4) may provide guidance on the degree of bioaccumulation that
    could be anticipated. To establish the nature of the toxic reaction,
    the highest dose should provide a distinct toxic effect while the
    lowest dose should not produce any detectable toxic reaction (Leclair
    & Willard, 1970). To obtain maximum information on the dose-response
    characteristics of the compound, at least two intermediate doses
    should be included.

          Information from subacute toxicity tests is of value in the
    selection of appropriate dose levels, when commencing chronic toxicity
    studies. In general, however, it is highly desirable to establish the
    chemobiokinetic behaviour (Chapter 4) of the test compound and if
    possible its major metabolites in the test species prior to
    undertaking a chronic toxicity test. Particular attention should be

    given to evidence for dose-dependent detoxification. Studies of this
    nature will provide information on the degree to which the chemical
    may be expected to accumulate in various body compartments and
    unexpectedly produce evidence of toxicity. Since it is the object of
    chronic toxicity tests to establish dose-response patterns and
    "no-observed-adverse-effect levels", a minimum of three dose levels
    should be used. The upper dose level should produce some slight
    evidence of toxicity, but should be compatible with normal
    physiological function (Leclair & Willard, 1970). The lowest dose
    level would not be expected to produce evidence of toxicity (Health &
    Welfare, Canada, 1973).  Method of administration

          The route of administration in subacute and chronic studies
    should be that through which man is likely to be exposed. For gases
    and volatile industrial solvents, inhalation studies are recommended
    (Magill et al., 1956) (Chapter 6), while for food additives,
    pesticides, and other chemicals likely to come into contact with food
    or water, the oral route is recommended (Leclair & Willard, 1970;
    National Academy of Sciences, 1975). Incorporation of the test
    chemical into the diet or drinking water is an appropriate means of
    administration; however, care must be taken to ensure the stability of
    the chemical in the dosing medium. The concentration of the test
    chemical in the diet should be determined periodically to ensure
    uniform dispersion and to aid in the quantification of achieved doses.
    In some cases, the chemical may be unpalatable and administration by
    gavage, or, in the case of dogs, by capsules may be necessary.

          The diet is the preferred vehicle of administration, but it is
    absolutely essential that the chemical be present in the diet in an
    unaltered form; toxicity may be altered by interaction with dietary
    constituents (Kello & Kostial, 1973). In rodent studies, the compound
    may be administered in the diet as a fraction of the total diet, or a
    sufficient quantity of the chemical may be added to the diet to
    achieve predetermined dose levels (in mg per kg body weight per day).
    In the latter case, it is necessary to adjust the dietary
    concentration weekly or biweekly to maintain a constant dose level,
    since food consumption per unit of body weight decreases as the animal
    gets older. If, in rodent tests, the concentration of the test
    compound in the diet is kept constant from weaning to maturity, the
    actual dose received will be reduced by approximately 2.5 times over
    the dosing period. This may have profound effects on the severity of
    the toxic response and may be mistaken for tolerance. In chronic
    toxicity tests, the chemical should be administered daily over the
    entire treatment period. As an aid to interpretation of the test, only
    one lot chemical should be used for the entire test unless the purity
    of the chemical is definitely assured.  Biochemical organ function tests

          In subacute studies, the use of a species such as the dog instead
    of a rodent species permits the application of a wider range of
    biochemical organ function tests because larger samples of blood can
    be collected on a routine basis. Organ function studies should be
    undertaken prior to initiation of the test, 3 and 10 days after the
    start of dosing, at 30-day intervals thereafter throughout the test,
    and terminally. The tests described for the chronic studies are also
    applicable in subacute studies.

          In the course of chronic toxicity tests, studies should be
    undertaken to evaluate the functional integrity of various organ
    systems. Assessment of the urinary system should commence with an
    examination of the urine. Freshly-voided urine samples should be
    obtained every one to three months from the test animals and examined
    for the presence of occult blood, glucose, protein, and bilirubin
    using simple diagnostic procedures. If positive effects are noted,
    quantitative methods should be applied as outlined by Bergmeyer
    (1965). Samples of freshly-voided urine should also be filtered
    through Millipore filters and the filters stained according to the
    Papanicolaou method (Frost, 1969) to detect the presence of renal
    tubular cells or other cell types derived from the urinary system.
    Urinary calculi and parasite eggs (Chapman, 1969) may be detected by
    this method. Blood urea-nitrogen levels and other standard tests of
    kidney function may be applied but they usually lack sufficient
    sensitivity to detect subtle changes in kidney function.

          Several test procedures are available for the assessment of liver
    function. Most of these methods involve an examination of the serum
    levels of hepatic enzymes that may be released in the serum following
    liver injury (Czok, 1965; Henley et al., 1966; Zimmerman, 1974).
    Korsrud et al. (1972) compared the sensitivity of various liver
    function tests in the rat and noted that serum sorbitol dehydrogenase
    ( activity (an enzyme specific to the liver) correlated well
    with the degree of histological alteration produced by hepatotoxic
    agents such as carbon tetrachloride, 2,2'-iminobisethanol
    (diethanolamine), and ethanethioamide (thioacetamide). However, Grice
    et al. (1971) noted that pathological changes induced by these
    compounds must be reasonably advanced before elevations are noted in
    serum glutamic-oxaloacetic transaminase (, lactate
    dehydrogenase (, or lactate dehydrogenase isoenzymes,
    suggesting that changes in serum enzyme activity may not be as
    sensitive an indicator of toxicity as pathomorphological examination.
    Tests of liver function such as serum enzyme activities and various
    clearance tests were reviewed recently by Balazs (1975). A complete
    review of the principles and applications of these tests is given by

    Cornish (1971) and further discussion of these methods is found in
    Part II, Chapter 8. Suffice it to say, that a transient increase in
    the activity of serum enzymes or other organ-derived constituents may
    result from a transient change in organ homeostasis that produces no
    lasting toxic effect.

          For routine screening of organ function in large animals,
    Charbonneau et al. (1974) used clinical procedures that can measure
    the concentration of several serum enzymes and inorganic and other
    constituents by automated methods. In general, these methods are not
    sufficiently standardized or reproducible to detect minor alterations
    in organ function but they do serve as a useful guide to general
    clinical status.  Physiological measurements

          In subacute studies, it is often possible to detect ensuing
    pathological events through application of physiological function

          In all studies, food consumption and body weight should be
    recorded weekly in all animals. Weight gain per unit of food consumed
    should be calculated (Munro et al., 1969). This gives a measure of the
    efficiency of food use. The daily dose of chemical should be
    calculated from data on food intake and body weight. Similar
    measurements of food intake and body weight must be carried out in
    chronic toxicity tests. If the test chemical is incorporated into the
    drinking water, water intake must be measured. These measurements
    should be conducted on a weekly basis during the entire test. The data
    can be used to estimate the dose of chemical received and are
    necessary in the establishment of dose-response relationships. Body
    weight changes serve as a sensitive indication of the general health
    status of test animals. Any rapid loss in body weight may signal the
    onset of intoxication or disease. Computerized methods for recording
    and analysing this type of data are available (Munro et al., 1972).

          Under special circumstances, when the target organs of toxicity
    have been identified during subacute studies, it is appropriate to
    conduct measurements of the physiological function of organ systems.
    Procedures such as electrocardiography (Grice et al., 1971),
    electroencephalography (Flodmark & Steinwall, 1963; Harada et al.,
    1967; Mann, 1970), electromyography (Chaffin, 1969), nerve conduction
    studies and measurement of evoked potentials (Barnet et al., 1971;
    Hrbek et al., 1972) may greatly assist in defining the functional
    effects of chemicals (Chapter 8). Such tests are expensive to perform
    and require highly specialized equipment and personnel. They have
    limited application in routine testing but may be used to define
    mechanisms of action. It is imperative that the results of such
    studies be correlated with clinical and pathological findings (Grice,
    1972; Osborne & Dent, 1973).  Metabolic studies

          Subacute studies provide an excellent opportunity to undertake
    metabolic investigations under conditions of repeated exposure that
    may alter the nature of the metabolites and the rate of metabolic
    transformation of the test compound. Urine and faeces can be collected
    and examined for the presence of metabolites and, by undertaking
    serial sacrifices at three-week intervals, the kinetics of
    accumulation of the compound in various body compartments can be

          To gain an understanding of the metabolic fate of a chemical that
    may have a long biological half-time, such as hexachlorobenzene (Grant
    et al., 1975), three extra groups of animals need to be studied for
    tissue distribution to provide information that is necessary for
    estimating the potential hazard to man. The principles of these
    methods are reviewed in Chapter 4. Often it is desirable, in subacute
    studies, to study the kinetics of the test compound and its
    metabolites following completion of the dosing period. If extra groups
    of animals are initially included for this purpose, much valuable
    additional information on the compound may be obtained.  Haematological information

          In subacute studies involving rodents, haematological studies
    should be undertaken on randomly selected subgroups of animals prior
    to initiation of the test, at 30-day intervals, and on all animals
    terminally. Bone marrow should be examined terminally. Nonrodent test
    animals should be examined at similar intervals.

          In chronic toxicity studies involving rodents, haematological
    studies of circulating blood cells should be undertaken on randomly
    selected subgroups of animals prior to initiation of the test, at 3 to
    6-month intervals and, on selected animals, terminally. Bone marrow
    should be examined terminally and, if indicated, at interim times by

          To assess the clinical state of nonrodents, haematological
    variables should be examined frequently.

          A set of test procedures is necessary for routine haematological
    screening and the tests must be of sufficient sensitivity and accuracy
    to be of practical value for use in large numbers of laboratory
    animals (Cartwright, 1969; Schalm, 1967; Sirridge, 1967).
    Quantification of blood cells and thorough study of cellular
    morphology by a haematologist, experienced in small animal medicine,
    is necessary in the study of haematological disorders. Haematological
    evaluation of experimental animals is facilitated by the fact that
    repeated sampling is relatively easy and small amounts of blood are

    required, and that single-cell systems can be studied to obtain
    information on cell production, destruction, defects, and dysfunction.
    For erythroid evaluation, the numbers of circulating erythrocytes must
    be counted and the haematocrit and haemoglobin concentration measured.
    As an index of erythropoietic activity in the bone marrow, a
    reticulocyte count should be carried out. Morphological assessment of
    erythrocytes is mandatory. The number of circulating leucocytes should
    be quantified and a differential count and morphological assessment
    should be made. To evaluate the functional capacity and malignant
    changes in the blood-forming organs, bone marrow should be examined
    terminally. From bone marrow smears a differential count and
    morphological assessment can be carried out. Imprints of lymph nodes
    or spleen permit a detailed cytological study of normal and abnormal
    cells present that may be of diagnostic significance.

          To assess haemostatic function, it will be necessary to evaluate
    platelets, coagulation systems, and fibrinolysis. Screening tests
    include platelet count, clot retraction, one stage prothrombin time,
    and activated partial thromboplastin time. More specific evaluation
    may require factor assays, thrombin time, fibrinogen determination,
    euglobulin clot lysis time, prothrombin consumption time, platelet
    aggregation, and adhesiveness.  Postmortem examination

          In every toxicity evaluation, all animals should be given a
    thorough gross autopsy and detailed records kept on each animal.
    Samples of all organs and supporting structures should be saved for
    histopathological examination. Detailed autopsy methods are outlined
    in Chapter 5.

          In chronic toxicity testing it is often useful to incorporate
    interim autopsy dates so that the progression of lesions may be
    studied. At interim sacrifices and terminally (if sufficient animals
    are in a healthy state), the major organs should be weighed. Organ
    weights may serve as a useful index of toxicity; however, care must be
    taken in the interpretation of the data. Decreased absolute organ
    weights in treated animals may be merely a reflection of lower body
    weight and calculation of organ to body weight ratios may increase the
    usefulness of the data (Feron, 1973).  Controls

          In the evaluation of both subacute and chronic toxicity, special
    attention must be given to the control animals. The quality of data
    obtained from the control animals has an important bearing on the
    interpretation of results from the treated animals. Suitable numbers
    of control animals of the same age and body weight as the treated
    animals must be included in the experimental design in a statistically
    randomized fashion.

          Except for treatment with the test chemical, these animals should
    be handled identically to the test subjects and all measurements
    conducted on the treated animals must be carried out on the controls
    with the same precision and frequency. In studies in which the
    chemical is administered by gavage, the control animals should receive
    the suspending vehicle in an amount equivalent to the treated animals.
    The incidence of spontaneous lesions or of other changes in control
    animals must be carefully noted and the interpretation of data
    obtained from treated animals must include an appreciation of the role
    that spontaneous disease processes may play in the manifestation of
    chemical toxicity. It is particularly important, in studies with
    rodents, to have detailed information on the incidence of neoplastic
    diseases, since some species and strains (Sher, 1972) may have a high
    background incidence of certain tumours which tends to reduce
    longevity and decrease the chance of observing chronic toxic effects.
    In addition, the chemical under test may alter the incidence of
    spontaneous tumours and other diseases or may induce new tumours, and
    this possibility must be taken into consideration in the evaluation of
    the chronic toxicity of chemicals. In all cases, responses
    attributable to the test compound must be compared with background
    observations in controls. For this reason, the quality of the
    toxicological data rests heavily on the adequacy of the control values
    (Weil & Carpenter, 1969).

    3.4.3  Alternative approaches in chronic toxicity  Perinatal exposure

          The majority of chemicals to which man may be exposed are present
    in air, food, or water for his entire lifetime. Recently, there has
    been an attempt to duplicate the human situation in the chronic
    toxicity test by exposing the test animals during the neonatal period
    as well as throughout life (Friedman, 1969). In this approach, groups
    of weaning animals (usually rodents) are exposed to the test chemical
    until they reach sexual maturity. They are then mated, within dose
    groups, and the treatment is continued during pregnancy and lactation.
    Following weaning, the offspring are transferred to their parents'
    diet and exposed for the balance of their lifetime to the test
    chemical. The details of this test procedure have been outlined in a
    recent Canadian Government publication (Health & Welfare, Canada,
    1973) and by Epstein (1969). It is not known yet whether this
    technique increases the sensitivity of the chronic toxicity test, but
    it is known that exposure to carcinogens in the perinatal period will
    often increase the incidence and decrease the latent period of
    carcinogenesis (Tomatis & Mohr, ed., 1973).

          Further study of this method is required to evaluate its
    usefulness fully. It should be pointed out, however, that this
    procedure adds considerably to the cost and length of the chronic
    toxicity test.  Use of nonrodent species

          In chronic toxicity studies with nonrodent species such as
    nonhuman primates, dogs, or cats, it is often not feasible to expose
    the animals to the test compound for their entire lifespan, even
    though they may be the species of choice. Under such conditions,
    careful examination of the kinetic and metabolic behaviour of the test
    compound in these species may substitute, to some extent, for the
    decreased treatment period (provided the anticipated endpoint is not
    carcinogenesis). Carefully conducted kinetic studies will assist in
    establishing when steady-state tissue concentrations of the test
    chemical and its metabolites have been achieved. If treatment is
    continued for a substantial period after the establishment of
    steady-state kinetics without any increase in the degree of toxic
    effects observed clinically, or during interim sacrifice, this may
    partially substitute for a lifetime study and provide increased
    assurance for those having to make regulatory decisions. If this
    approach is not feasible, it may be possible to test human metabolites
    in rodent species (Health & Welfare, Canada, 1973).

    3.5  Evaluation and Interpretation of the Results of Toxicity Tests

          The evaluation and interpretation of toxicity studies starts with
    a clear definition of experimental objectives. The design of the
    experiment should be such that the objectives can be reasonably
    achieved. Well designed and carefully executed experiments add greatly
    to the ease with which results can be evaluated and interpreted and
    also to confidence in the experimental data.

          The primary usefulness of the LD50 determination is to obtain
    some idea of the magnitude of the acute toxic dose (Frazer & Sharratt,
    1969) and information concerning the type of toxic effects of the
    chemical. Such information includes whether death is immediate or
    delayed, whether recovery from a near lethal dose is rapid or complete
    or both, or whether the cause of death is narcosis with respiratory
    failure, lung oedema, or liver necrosis.

          However, the LD50 provides little information for the
    assessment of the hazard from compounds to which the human population
    is exposed for extended periods of time. Although it has been
    suggested that compounds that do not show adverse effects when given
    in doses of 3-5 g per kg body weight are essentially non-toxic
    (National Academy of Sciences, 1975), there are numerous examples in
    the literature of compounds with LD50 values greater than 5 g per kg
    which produce toxic effects, when given in low doses for extended
    periods of time (Frazer & Sharratt, 1968). If the main object of an
    acute toxicity test is not to establish a value for the LD50 with

    precision, but to learn something about the way in which the chemical
    acts as a poison, as suggested by Paget & Barnes (1964), this can best
    be accomplished by tests involving repeated daily administration to a
    few animals for a period of 5-21 days. The information provided by the
    LD50 regarding the effects of acute exposure to toxic compounds may
    be useful as a guide for selecting doses for such studies.

          The primary objective of subacute and chronic toxicity studies is
    to determine the nature and severity of toxic effects and the
    "no-observed-adverse-effect" dose level. These data may then be used
    in the establishment of acceptable levels of exposure for man.

          Data on group weight gain or body weight change should be plotted
    against time, and differences between groups should be evaluated
    statistically. Changes in body weight with time are best evaluated
    statistically using trend analysis procedures (Armitage, 1955). Food
    (and water) consumption data should be handled in a similar fashion.
    Reduced body weight or weight gain in otherwise healthy treated
    animals may be due to reduced food intake owing to its unpalatability
    or to a specific toxic effect of the chemical resulting in reduced
    efficiency of food use. Using data on the dietary concentration of the
    test chemical, food consumption, and body weight, the mean daily dose
    of chemical received (in mg/kg body weight/day or similar units)
    should be calculated. Automated data processing procedures to
    accomplish this are available (Munro et al., 1972).

          Data on organ weights should be evaluated and interpreted with
    great care. Increased relative (to body or brain weight) organ weights
    may also result from adaptation to stress phenomena or from metabolic
    overloading of biochemical pathways or physiological processes.
    Increased liver weight, for example, may result from a stimulation of
     de novo protein synthesis in the smooth endoplasmic reticulum (SER).
    This results in a morphologically detectable increase in SER. The
    biochemical counterpart of this increase is an increased ability of
    the liver to metabolize certain foreign substances, sometimes
    including the test compound and endogenous substrates, due to a
    stimulation in the activity of hepatic mixed function oxidases
    (Staubli et al., 1969). These adaptative changes may manifest
    themselves clinically as tolerance. Often these changes are reversible
    upon cessation of dosing and do not produce lasting toxicological
    effects but the implications of chronically elevated levels of these
    enzymes is not known. Certain enzyme inducers may cause impairment of
    liver function and produce pathological and biochemical changes (Feuer
    et al., 1965).

          Data on biochemical and haematological effects should be
    tabulated and compared with control values using statistical
    procedures (Johnson, 1950). Any observed effects should be correlated
    with clinical and pathological findings. A biochemical or
    haematological change such as reduction in liver glycogen or an
    alteration in white cell count may not be indicative of a toxic
    effect, but an adaptation to a stress situation (National Academy of
    Sciences, 1975). In general, changes in homeostasis must be carefully
    evaluated since reversible shifts do not necessarily imply a toxic
    effect in the absence of other toxic manifestations.

          Changes in the functional state of physiological or neurological
    processes, such as an alteration in the electrocardiogram or abnormal
    behaviour, may result from pharmacological or pathological effects of
    the test compound. Changes in functional state must be closely
    correlated with their morphological counterpart in order to evaluate
    their toxicological importance properly (Grice, 1972).

          The cornerstone of experimental toxicology is the pathological
    examination. Usually, decisions regarding the safety of a compound are
    based on this evidence. All pathological findings in test animals
    should be graded carefully and their incidence tabulated (see Chapter
    5). Spontaneous lesions in control animals should also be noted and
    compared to the observations in control animals in previous
    experiments or in the literature (Peck, 1974) to ensure that the
    incidence and nature of the lesions is representative of the strain.
    Pathological data should be analysed rigorously using appropriate
    statistical methods (Fleiss, 1973) and spurious observations
    apparently unrelated to treatment should be identified. Lesions that
    are dose-related should be studied in detail and correlated with gross
    pathological findings, clinical observations, and other variables
    (Grice, 1972).

          It is not uncommon in chronic toxicity testing to find
    pathological or other changes that occur in low incidence and that are
    not dose-related but occur only in treated animals. Such reactions may
    be idiosyncratic in nature or may be due to the hypersensitivity of
    certain animals. Nevertheless, they deserve special attention since
    they may be indicative of a hitherto unsuspected toxic effect. The
    clinical history and other data from such animals should be reviewed
    with great care and an attempt should be made to determine the reason
    for the observed effects. Toxic effects that occur in extremely low
    incidence present special problems in interpretation. There is no
    substitute for experience in this respect and the prudent investigator
    will consult the knowledgeable experts in this field (Zbinden, 1973).


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    4.1  Introduction

          The objective of chemobiokinetic studies is to obtain data that
    allow reliable assessment of the hazard of environmental chemicals to
    man. Since effects are related to the amounts or concentrations of a
    chemical in tissues and cells, it is imperative to elucidate the
    dynamics of the toxicant at the target site. It should be emphasized
    that the toxicant may be either the parent chemical or a metabolite or
    degradation product formed from it. Thus, the qualitative
    identification of the degradation products of a chemical together with
    a quantitative characterization of their fate, as well as the fate of
    the parent chemical, as a function of time, are inextricably
    associated in a proper chemobiokinetic evaluation. In the context of
    this chapter, the word "chemobiokinetics" has been used in place of
    "pharmacokinetics" because too often the latter implies restriction of
    this scientific discipline to drugs. The term chemobiokinetics is
    proposed to emphasize its importance in evaluating the biological
    effects of all chemicals.

    4.2  Absorption

    4.2.1  General principles

          Absorption of a chemical into the body can take place,
    potentially, by all routes of exposure. In assessing the toxicity and,
    ultimately, the hazard of a chemical, the oral, dermal, and inhalation
    routes of exposure are of primary importance. Following absorption,
    the chemical is distributed by the blood to the various tissues.
    Therefore, the rate of absorption is frequently estimated by
    determining the concentration of the chemical in the plasma as a
    function of time following exposure.

          The route of administration can greatly influence the rate at
    which a foreign chemical enters the body. Upon ingestion, the gastric
    contents and pH of the stomach can influence the rate of absorption of
    the chemical. In the small intestine, food may either enhance or delay
    absorption. Indeed, the environment of the gastrointestinal tract (pH,
    food, bacteria) may change the parent chemical into another chemical.
    The inhalation route allows a chemical to pass rapidly into the blood
    without encountering drastic changes in pH, food, or microflora. The
    skin effectively retards the absorption of many chemicals; however, it
    should not be considered as an absolute barrier. Some chemicals
    readily penetrate intact skin and a minor abrasion of the skin may
    greatly enhance the absorption of many chemicals.

          In order that a chemical may be absorbed into the bloodstream, it
    must cross one or more semipermeable membranes, such as the
    gastrointestinal epithelium, the lining of the respiratory tract, or
    the epidermis of the skin. Membranes are essentially lipoproteins with
    aqueous pores through which water-soluble molecules can pass. The pore
    size varies from 4  (intestinal epithelium and mast cells) to 30 
    (capillaries), allowing the passage of molecules with molecular
    weights less than 100-200 to approximately 60 000, respectively. Most
    membranes have an electrical potential that may effectively preclude
    the ready penetration of charged chemical species. Thus it is obvious
    that the absorption of a chemical depends on its physicochemical
    properties, molecular size, shape, degree of ionization, and lipid
    solubility. For a more thorough discussion, the reader is referred to
    Davson & Danielli (1952) and Schanker (1962a).

          Three mechanisms have been proposed to explain how a chemical
    passes across a cell membrane: (a) passive diffusion through the
    membrane, (b) filtration through membranous pores, and (c) specialized
    transport systems that carry water-soluble and large molecules across
    the membrane by means of a "carrier".

          Passive diffusion is considered to be the principal mechanism by
    which chemicals can cross cell membranes. The rate of passive
    diffusion of a molecule is proportional to the concentration gradient
    across the membrane, the membrane thickness, the area available for
    diffusion, and the diffusion constant, in accordance with Fick's Law
    (La Du et al., 1972). The rate of passage is related directly to the
    lipid solubility (Brodie, 1964). However, since absorption requires
    passage through an aqueous- as well as a lipo-phase, the absorption of
    a chemical with an extremely low solubility in water may be impeded in
    spite of a high lipid-to-water partition coefficient. The passive
    diffusion also depends on the extent of the ionization and the lipid
    solubility of the ionized and nonionized species (Brodie, 1964).

          Filtration is a process by which a chemical passes through the
    aqueous pores in the membrane, and is governed by the size and shape
    of the molecule. The bulk flow of water across the membrane produced
    by an osmotic gradient or hydrostatic pressure can act as a carrier
    for chemicals.

          Specialized transport processes are needed to explain the
    transport and kinetic behaviour of large, lipid insoluble molecules
    and ions. Two types of carrier-mediated transport systems have been
    recognized: active transport and facilitated diffusion. The carrier in
    both systems is some component of the membrane that combines with the
    chemical and assists its passage across the membrane. It has a limited
    capacity and when it is saturated, the rate of transfer is no longer
    dependent on the concentration of the chemical and assumes zero order
    kinetics. Structure, conformation, size, and charge are important in
    determining the affinity of a molecule for a carrier site, and
    competition for carrier site will occur.

          Active transport is a carrier-mediated transport system which
    moves a molecule across a membrane against a concentration gradient,
    or, if the molecule is an ion, against an electrochemical gradient. It
    requires the expenditure of metabolic energy and can be inhibited by
    poisons that interfere with cell metabolism. Active transport plays an
    important role in the renal and biliary excretion of chemicals.

          Facilitated diffusion is a carrier transport mechanism by which a
    water-soluble molecule (i.e. glucose) is transported through a
    membrane down a concentration gradient. No apparent energy is required
    and metabolic poisons will not inhibit this process. The difference
    between facilitated diffusion and active transport is that the latter
    moves molecules against a concentration gradient, whereas the former
    does not. For more complete discussion of membrane transport, refer to
    La Du et al. (1972) and Goldstein et al. (1974).

          Another active process, pinocytosis, has been implicated as a
    mechanism for transferring large molecules and particles into cells.
    In this process, the membrane engulfs the material and pinches off an
    envelope containing the material within the cell.

    4.2.2  Absorption from the lungs

          The pulmonary epithelial lining is very thin, possesses a large
    surface area, and is highly vascular. Thus, absorption of foreign
    chemicals can take place at a very rapid rate. Most rapidly absorbed
    are gases and aerosols with small particle size and a high
    lipid-to-water partition coefficient. In most inhalation studies,
    absorption may occur by routes other than the lungs and the
    investigator should be aware of this in the interpretation of data.

          A more complete discussion of the inhalation of chemicals is
    presented in Chapter 6.

    4.2.3  Absorption from the skin

          The structure of the skin enables rapid penetration of
    lipid-soluble compounds through the epidermis, a lipoprotein barrier,
    whereas the highly porous dermis is permeable to both lipid- and
    water-soluble substances (Katz & Poulsen, 1971). Factors which govern
    penetration through the skin are hydration, pH, temperature, blood
    supply, and metabolism as well as vehicle-skin interactions. Abrasion
    of the skin may enhance absorption greatly. For a more complete
    discussion of the principles for absorption through the skin and
    experimental methods, refer to Part II, Chapter 11.

    4.2.4  Gastrointestinal absorption

          The gastrointestinal tract is one of the most important routes of
    absorption of foreign compounds (Schanker, 1971). Chemicals can be
    absorbed along any section of the gastrointestinal tract, but because
    of the large surface area and rich blood supply, absorption is
    favoured from the small intestines. In most parts, the movement of a
    chemical across the epithelial lining of the gastrointestinal tract is
    by diffusion and carrier transport mechanisms are involved to a lesser

          Although therapeutic amounts of drugs may be absorbed from the
    buccal mucosa (Beckett & Hossie, 1971), absorption of environmental
    chemicals from the mouth is minimal compared with that from the
    stomach and intestine. Chemicals absorbed from the mouth are not
    exposed to the gastrointestinal digestive juices and drug-metabolizing
    enzymes. Furthermore, since they are not transported by the hepatic
    portal system directly to the liver, their normally rapid metabolism
    may be precluded, thus prolonging their effect.

          The stomach is a significant site of absorption by passive
    diffusion of many acid, and neutral, foreign compounds (Schanker et
    al., 1957). Due to the acidity of the stomach, weak acids will exist
    in the diffusible, nonionized, lipid-soluble form, whereas weak bases
    will be highly ionized and therefore not generally absorbable.

          Absorption from the small intestine is similar in principle to
    that from the stomach (passive diffusion), except that the pH of the
    intestinal contents (pH 6.6) may alter the fraction of the chemical in
    the nonionized form favouring the absorption of both weakly-acid and
    weakly-alkaline chemicals. The aqueous pore size, 4 , limits
    absorption by filtration to molecules having a molecular weight of
    less than 100-200. Rarely, an environmental chemical may be absorbed
    from the intestinal tract by an active transport system that is
    normally involved in the absorption of nutrients, e.g. sugars and
    amino acids (Schanker, 1963).

          Many factors can affect the absorption of foreign compounds from
    the gastrointestinal tract (Brodie, 1964; Levine, 1970; Place &
    Benson, 1971; Prescott, 1975): ( a) increased gastric emptying can
    decrease gastric absorption and increase intestinal absorption; ( b)
    increased intestinal peristalsis generally inhibits intestinal
    absorption; ( c) gastric acid, intestinal digestive juices, and gut
    microflora can all degrade chemicals to other absorbable or
    nonabsorbable chemical species; ( d) food in the gastrointestinal tract
    can impair absorption by producing a nonabsorbable complex, by
    decreasing gastric emptying (especially fats), and by reducing,
    mixing, or altering pH; ( e) normal digestion produces increased

    gastrointestinal blood flow which will enhance absorption; ( f)
    absorption of a solid will be impaired if dissolution in the
    gastrointestinal tract does not take place. The practice of
    administering chemicals admixed with the diet must take these factors
    into account, especially the possible reaction of the chemical with
    dietary constituents.

    4.3  Distribution

          Once absorbed, the distribution of a chemical is determined by
    the relative plasma concentration, the rate of blood flow through
    various organs and tissues, the rate by which the chemical penetrates
    cell membranes, and the binding sites that are immediately available
    in the plasma and tissues. After the initial distribution phase, the
    rate by which a chemical penetrates cell membranes and the available
    sites for binding are the predominating factors influencing the final
    distribution of a chemical in the body.

          When the plasma concentration of a chemical is high and the cell
    membranes do not provide significant barriers to diffusion,
    distribution is mainly to organs with high blood flow, e.g. brain,
    liver, and kidney. A classic example of distribution and
    redistribution is thiopental, a highly lipid-soluble chemical that,
    after administration, is first distributed to the brain and
    subsequently to muscle and body fat which have poor blood flow (Price
    et al., 1960). Lipid-soluble, foreign compounds tend to be distributed
    and localized in adipose tissue (Mark, 1971), in accordance with their
    lipid-to-water partition coefficients, e.g. the chlorinated
    hydrocarbon pesticides, dieldrin, DDT, and DDE (Rodomski et al., 1968)
    and polychlorinated biphenyls (PCBs) (Allen et al., 1974).
    Distribution of chemicals into organs and tissues is influenced by
    membraneous barriers in the same way as absorption (see 4.2). For a
    more detailed treatment, see Quastel (1965). The capillary membrane,
    unlike other body or cell membranes, is freely permeable to foreign
    compounds of a molecular weight of 60 000 or less, whether
    lipid-soluble or not (Pappenheimer, 1953; Renkin, 1964); generally
    chemicals pass these membranes readily, except in the brain,
    testicles, and the eye (Gehring & Buerge, 1969).

          The movement of foreign chemicals to the brain represents a
    unique example that cannot be explained by the physicochemical
    properties of the chemical and the tissue distribution. Many chemicals
    fail to penetrate into the brain tissue or cerebrospinal fluid as
    readily as into other tissues (Brodie & Hogben, 1957). The boundary
    between blood and brain consists of several membranes; those of the
    blood capillary wall, the glial cells closely surrounding the
    capillary, and the membrane of the neurons or nerve cells. The
    so-called "blood-brain barrier" is located at the capillary wall-glial
    cell region. The capillary walls in the brain tend to be more like
    cell membranes than capillary membranes. Therefore, ionized substances

    and large water-soluble molecules such as proteins are almost entirely
    excluded from passage (Rall, 1971). The chief mode of exit of both
    lipid-soluble and polar compounds is by filtration across the
    arachnoid villi. The method for studying the movement of chemicals
    into and from the brain has been discussed by Rall (1971).

          The red blood cell has unusual permeability in that organic
    unions penetrate much more readily than cations. This may be explained
    by the presence of positively charged membrane pores that will accept
    anions but repel cations (Schanker et al., 1957).

    4.4  Binding

          A major factor, that can affect the distribution of a chemical,
    is its affinity to bind to proteins and other macromolecules of the
    body. Foreign chemicals have been shown to bind reversibly to such
    substrates as albumen, globulins, haemoglobin, mucopolysaccharides,
    nucleoproteins, and phospholipids (Shore et al., 1957). For a survey
    of the biological implications of the protein binding of chemicals,
    the reader is referred to Gillette (1973a).

          Once a chemical is bound to a body constituent, it is temporarily
    localized. This localization modifies the initial pattern of
    distribution and affects the rates of absorption, metabolism, and
    elimination of the chemical from the body.

    4.4.1  Plasma-protein binding

          Most chemicals show some degree of binding to plasma proteins,
    the most important fraction of which is albumen. Albumen at pH = 7.3
    contains a net negative charge; however, cationic groups must be
    accessible because albumen has been shown to bind anions as well as
    cations. Although the plasma proteins show an appreciable capacity for
    binding many chemicals, this is limited, making it important to
    understand such binding as a function of the concentration of the

          Since plasma proteins possess a limited number of binding sites
    and the sites are somewhat nonspecific, two chemicals with an affinity
    for the same binding site will compete with one another for binding.
    The plasma proteins of various laboratory animals and man show
    differences in the degree and nature of binding. This is due to
    differences in the total concentrations and relative proportions of
    the various plasma proteins as well as the composition and
    conformation of albumens (Gillette, 1973b).

    4.4.2  Tissue binding

          The binding of chemicals to tissue constituents also contributes
    to the localization of a chemical. Certain chemicals show a much
    greater affinity for tissue than for plasma proteins, and in some
    instances the affinity for tissue is quite specific. For example,
    polycyclic aromatic compounds have been shown to have particular
    affinity for the melanin in the eye (Potts, 1964).

          Some metals and several chemicals and organic anions are bound to
    proteins (Y and Z proteins or ligandins) in the liver (Levi et al.,
    1969). These proteins may play a key role in the transfer of organic
    anions from plasma to liver (Levi et al., 1969; Reyes et al., 1971),
    and they also bind corticosteroids and azo-dye carcinogens (Litwack et
    al., 1971). For further details concerning the nature and effects of
    binding of chemicals by proteins and for methods of study, see
    Chignell (1971), Gillette (1975), Keen (1971) and Settle et al.

          Many inorganic ions, particularly metals, as well as
    tetracycline, are concentrated in various tissues and organs,
    particularly in bones and teeth (Foreman, 1971). A convenient method
    for studying the accumulation of chemicals in organs and tissue is
    autoradiography (Roth, 1971). Valuable measurements may also be
    obtained with classical chemical and radio-chemical techniques which
    have the added advantage of being quantitative.

    4.5  Excretion

          Chemicals are excreted as the parent chemical, as metabolites, or
    as conjugates of the parent chemical or its metabolites. The principal
    routes of excretion are the urine and bile, and to a lesser degree
    expired air, sweat, saliva, milk, and secretions of the
    gastrointestinal tract.

    4.5.1  Renal excretion

          The kidneys are the most important route of excretion of foreign
    compounds (Weiner, 1971). The three mechanisms of renal excretion are:
    glomerular filtration, active tubular transport, and passive tubular
    transport. Only compounds of high molecular weight or those bound
    tightly to plasma proteins escape glomerular filtration and the
    resulting filtrate contains approximately the same concentration of
    foreign compounds as that found in the plasma in an unbound state.

          Water and endogenous substrates are reabsorbed from the
    glomerular filtrate as it passes down the tubule. In the tubule,
    lipid-soluble, unionized chemicals pass in either direction by passive
    diffusion. Thus, lipid-soluble chemicals may be reabsorbed by the
    tubule, prolonging their retention in the body. Ionic chemicals, such
    as conjugates and other metabolites, are poorly reabsorbed and pass
    directly out of the body in the urine.

          Active transport takes place in the proximal tubule of the
    kidney. There are two distinct active transport processes. One process
    is specific for organic anions and the other specific for organic
    cations. Chemicals transported by the same transport process compete
    with each other, and the excretion rate of one compound can be reduced
    by the administration of the other. The active transport process can
    be saturated as the concentration of the chemical in the plasma is
    increased. When the active tubular secretion is saturated, that is,
    when an increase in the concentration of the chemical in the plasma is
    no longer accompanied by a proportional increase in the concentration
    of the chemical in the urine, the concentration in the plasma is
    referred to as the renal-plasma threshold.

          The anionic secretory process is responsible for the excretion of
    metabolites formed through conjugation of the parent chemical or its
    degradation products with various endogenous substrates such as
    glycine, sulfate, or glucuronic acid. These relatively polar,
    lipid-insoluble metabolites are poorly reabsorbed from the tubules and
    more readily excreted.

    4.5.2  Biliary excretion

          Biliary excretion is a major route for the excretion of foreign
    chemicals (Smith, 1971a, 1973). It is has been demonstrated (Brauer,
    1959; Schanker, 1962b; Sperber, 1963; Williams, 1965) that compounds
    with high polarity, anionic and cationic conjugates of compounds bound
    to plasma proteins, and compounds with molecular weights greater than
    300 are actively transported against a concentration gradient into the
    bile. It has also been shown that, once these compounds are in the
    bile, they are not reabsorbed into the blood and are excreted into the
    gastrointestinal tract (Schanker, 1965). Factors that influence the
    biliary excretion of foreign chemicals and metabolites are considered
    to be of two types: (a) physicochemical, relating to molecular size,
    structural features, and polarity; and (b) biological, relating to
    protein binding, renal excretion, metabolism, species, and sex. For a
    comprehensive and detailed discussion of these subjects, the reader is
    referred to Smith (1971a, 1973), and Stowe & Plaa (1968).

    4.5.3  Enterohepatic circulation

          Enterohepatic circulation is the phenomenon that occurs when a
    compound is excreted via the bile into the gastrointestinal tract,
    reabsorbed from the gastrointestinal tract and carried via the portal
    system back to the liver, where it is again excreted via the bile and
    recycled. Physiologically, enterohepatic circulation is important
    because it permits reuse of endogenous biliary excretion products.
    However, when a foreign compound is involved in enterohepatic
    circulation, it must make its way either to the faeces or to the
    peripheral blood to be excreted from the body. Thus, enterohepatic
    circulation of a foreign compound serves to enhance its retention in
    the body. There are examples in the literature (Gibson & Becker, 1967;
    Keberle et al., 1962) which demonstrate that the half-life of a
    compound involved in enterohepatic circulation can be decreased after
    surgically interrupting the enterohepatic cycle. Administration of a
    sequestering agent that binds the compound in the gastrointestinal
    tract would serve the same purpose.

          Smith (1973) has described the following factors that can affect
    the enterohepatic circulation of a compound: ( a) the extent and rate
    of excretion of the compound in the bile; ( b) the activity of the gall
    bladder; ( c) the fate of the substance in the small intestine; and ( d)
    the fate of the compound after reabsorption from the gut. Since many
    foreign chemicals are excreted in the bile as unabsorbable conjugates,
    the hydrolysis of these conjugates in the intestine may play a key
    role in enterohepatic circulation. For a thorough discussion of
    enterohepatic circulation, the reader is referred to Plaa (1975).

    4.5.4  Other routes of excretion

          In addition to excretion in bile and urine, other routes for the
    excretion of foreign chemicals and their metabolites should not be
    overlooked. In accordance with the pH partition theory, organic bases
    highly ionized at the pH value of gastric juice may be secreted into
    the stomach (Shore et al., 1957). Similarly, weak acids ionized at
    neutral pH may be transferred from the plasma to the lumen of the
    intestine. These chemicals are sequestered by the intestinal contents,
    augmenting their excretion in the faeces.

          Many volatile organic chemicals are excreted readily via exhaled
    air (see Chapter 6). This route of excretion is common for carbon
    dioxide, an ultimate end-product of an extensively metabolized organic
    chemical. For this reason, the quantification of expired radiolabelled
    carbon dioxide (14CO2) is very important in chemobiokinetic
    studies using carbon-14-labelled compounds.

          Many foreign compounds are excreted, to different degrees, in
    milk, in either the aqueous or lipid phase (Rasmussen, 1971). Although
    this route may be of minor importance for the elimination of a
    chemical from the body, it should be given particular attention in
    evaluating the hazard of chemicals to man. First, consumption of cow's
    milk may constitute an important vehicle of exposure. Secondly, the
    consumption of mother's milk by the newborn may provide very high
    doses of a chemical that is concentrated in the milk. It should also
    be noted that the volume of milk consumed by the newborn per unit body
    weight may, in itself, magnify the dose received by this segment of
    the population.

          Chemicals are also excreted in sweat and saliva. The presence of
    a chemical in sweat may lead to dermatitis. Although saliva is usually
    swallowed and thus does not lead to elimination of the agent from the
    body, recent work has shown that analysis of saliva for the presence
    of a chemical may preclude the necessity for venepuncture to obtain
    plasma for analysis.

    4.6  Metabolic Transformation

          Metabolic transformation or biotransformation are terms that have
    been used to describe the process which converts a foreign chemical to
    another derivative (metabolite) in the body. Metabolic transformation
    has been the subject of several excellent reviews (Conney, 1967;
    Conney & Burns, 1962; Dahm, 1971; Daly, 1971; Dutton, 1971; Garattini
    et al., 1975; Gillette, 1971a,b & 1974a,b; Gillette et al., 1974;
    Kuntzman, 1969; McClean, 1971; Smuckler, 1971; Weisburger &
    Weisburger, 1971). It usually results in the formation of more polar
    and water-soluble derivatives of a foreign chemical which can be more
    readily excreted from the body. Generally, such metabolic
    transformation of a foreign chemical also results in the formation of
    a less toxic chemical. However, there are many cases where the
    metabolites are more toxic than the parent chemicals (McLean, 1971;
    Miller & Miller, 1971a).

          A few compounds resist metabolic transformation. Most strong
    acids and bases are excreted unchanged. Also the resistance of
    long-acting nonpolar compounds (barbital, halogenated benzene, etc.),
    to metabolic transformation might explain their slow elimination from
    the body.

          A metabolic activation is suggested, if a compound is more toxic
    when given orally than intravenously, if there is a long delay between
    the administration of a chemical and the onset of its biological
    effect, or, if there is an increased effect following pretreatment
    with compounds that induce metabolic transformation (Garattini et al.,

    4.6.1  Mechanisms of metabolic transformation

          Usually, the metabolic transformation of chemicals takes place to
    the greatest extent in the liver and is catalysed by enzymes found in
    the soluble, mitochondrial, and microsomal fractions of the cell.
    Enzymes metabolizing foreign chemicals are also found, to a lesser
    degree, in the cells of the gastrointestinal tract, kidney, lung,
    placenta, and blood (Aitio, 1973; Gillette, 1963; Gram, 1973;
    Hietanen, 1974; Hietanen & Valinio, 1973; Wattenberg & Leong, 1971;
    Wattenberg et al., 1962; Witschi, 1975). It must be emphasized that
    for a particular chemical, or a particular route of administration,
    other organs may play a more important role in the metabolic
    transformation of the chemical than the liver. The role of enzymatic
    reactions carried out by the intestinal flora may be very important
    and should not be overlooked (Scheline, 1968; Smith, 1971a).
    Enzyme-catalysed, biochemical transformations can be classified into
    four main types: (a) oxidations, (b) reductions, (c) hydrolyses and
    (d) synthetic reactions (see Table 4.1 in Annex to this Chapter).

          The metabolic transformation of a chemical can occur via various
    pathways which can consist of a single reaction or multiple reactions.
    If the metabolic pathway consists of one reaction it is usually
    oxidation, reduction, or hydrolysis which tends to increase the
    polarity of the compound. Multiple-reaction metabolic pathways can
    consist of a series or any combination of oxidation, reduction, or
    hydrolysis. The final reaction in a multiple-reaction pathway is
    usually a conjugation reaction involving the addition of polar
    endogenous functional groups (D-glucuronic acid, glycine etc.) which
    usually render the molecule more polar, less lipid-soluble, and
    therefore more readily excretable. The predominant sequence of
    reactions or metabolic pathways is determined by many factors such as
    the dose of the chemical, species, strain, age, sex, and certain
    environmental variables.  Microsomal, mixed-function oxidations

          The metabolism of a large variety of foreign compounds involves
    oxidative processes. Microsomal oxidation refers to reactions
    catalysed by the enzymes found in the microsomes of the endoplasmic
    reticulum. These enzymes are sometimes referred to as microsomal,
    mixed-function oxygenases (mono-oxygenases) (Mason, 1957). The
    reactions require molecular oxygen and nicotinamide adenine
    dinucleotide phosphate, reduced form (NADPH). The reduction
    equivalents from NADPH are used to reduce molecular oxygen so that it
    can be carried by a cytochrome called P-450 to the compound to be
    oxygenated. The oxygen is then fixed into the compounds, usually as a
    hydroxyl group (Estabrook, 1971; Estabrook et al., 1971).

          The apparent sequence of events in the course of a mixed function
    oxidation has been described (Boyd & Smellie, 1972; Estabrook et al.,
    1972; Gillette, 1971c). The compound (substrate) forms a complex with
    the oxidized cytochrome P-450; this is reduced either directly by
    NADPH-cytochrome- c-reductase ( or indirectly via an
    unidentified electron carrier. The reduced cytochrome P-450-substrate
    complex then reacts with oxygen to form an "active oxygen" complex,
    which decomposes with the formation of the oxidized substrate and
    oxidized cytochrome P-450. Substantial progress has been made in
    elucidating this mechanism by the development of a method involving
    the resolution and reconstitution of the components of the liver
    microsomal hydroxylating system (Lu & Levin, 1974).

          Measurement of mixed-function oxidase activities of liver
    microsomes  in vitro has become an important aspect in evaluating the
    toxicity of chemicals. The mixed-function oxidase system may be either
    a biotransformation system or a site of action of chemicals.
    Measurements of the activity of this system can be performed using
    either a 9000  g supernatant fraction (Henderson & Kersten, 1970;
    Klinger, 1974) of a liver homogenate prepared in buffered KCl
    solution, or a microsome fraction sedimented by centrifugation at
    about 105 000  g (Flynn et al., 1972; Hewick & Fouts, 1970a,b; Liu et
    al., 1975).

          The reaction mixtures consisting of the particle-bound enzymes
    have to be supplemented with an NADPH-generating system. This may be
    fulfilled by the addition of NADPH and glucose-6-phosphate, if the
    9000  g fraction is used, but if washed microsomes are used,
    glucose-6-phosphate dehydrogenase ( must also be added.

          Isolated tissue cells, tissue cultures, or slices of organs, as
    well as perfused organs can also be used for metabolic studies.

          Because cytochrome P-450 is intimately associated with the
    metabolism of many foreign chemicals, the following methods and
    variables have been developed for ascertaining its activity in the
    tissues of animals used in toxicological investigations.

          The method of Omura & Sato (1964a,b) has been used to measure the
    change in the microsomal content of cytochrome P-450 and cytochrome
    b5. This method relies on a spectral shift of the pigment upon
    exposure to carbon monoxide. An increase in the cytochrome P-450
    content can be explained as a consequence of enzyme induction, whereas
    the decrease of the haem pigment content may be the result of enhanced
    permeability of microsomal membranes due to the damaging effects of
    the chemical (Bond & De Matteis, 1969). The concentration of
    cytochrome P-450 in the liver, however, is not always directly
    proportional to the activity of the mixed-function oxidases.

          Spectral changes of cytochrome P-450, determined in the presence
    of various substrates, provide information about the binding between
    the pigment and substrate (Hewick & Fouts, 1970a; Remmer et al.,
    1966). Compounds may be classified into type I or type II according to
    their spectral reactions with cytochrome P-450. When type I compounds
    bind to cytochrome P-450, the characteristic spectral shift, spectral
    difference, gives a peak at 385-390 nm and a trough at 418-427 nm,
    whereas with type II compounds, the peak occurs at 425-435 nm and the
    trough at 390-405 nm. Originally, it was thought that the magnitudes
    of these spectral shifts, especially type I spectra, could be
    correlated with microsomal biotransformations (Schenkman et al.,
    1967). This correlation, however, is not universally applicable
    (Davies et al., 1969; Gigon et al., 1969; Holtzman et al., 1968).
    Thus, differences in the magnitude of these spectral changes are
    difficult to interpret when they are detected in animals treated with
    a chemical (Gillette et al., 1972). The same is true for the
    ethylisocyanide difference spectra of cytochrome P-450 which are
    characterized by two peaks at about 455 nm and 430 nm (Omura & Sato,

          Determination of the NADPH-cytochrome P-450 reductase activity,
    the assumed rate limiting step in microsomal oxidations, has proved
    useful in evaluating the effectiveness of the cytochrome P-450 system
    prior to the oxygenation step (Fouts & Pohl, 1971; Gigon et al., 1969;
    Hewick & Fouts, 1970b; Holtzman et al., 1968; Zannoni et al., 1972).
    Measurement of the NADPH-cytochrome-c-reductase activity may give
    information about the rate of flow of reducing equivalents from NADPH
    to cytochrome P-450.

          Determination of the rate of enzymatic conversion of a substrate
    is a most valuable tool in elucidating the metabolic process. For this
    purpose, however, it is essential to know the pathway for the
    transformation of the chemical, and analytical methods are essential
    to quantify the parent chemical and its reaction products. Selected
    methods for monitoring some compounds and enzymatic reactions are
    listed in Table 4.2 (see Annex to this Chapter).

          There are large variations in the metabolism of foreign chemicals
    as well as in susceptibility to metabolic inducers depending on the
    species (Hucker, 1970), strain, age, and sex of animals.

          Many variables must be considered as important factors in species
    differences in the metabolism of foreign chemicals. Among these are
    differences in binding, either to tissues or to plasma components,
    such as albumen. Considerable variations in binding have been reported
    for the same chemical in different species (Borg et al., 1968; Kurz &
    Friemel, 1967; Scholtan, 1963; Sturman & Smith, 1967; Witiak &
    Whitehouse, 1969). More obvious are the concentrations and types of
    foreign chemical-metabolizing enzymes in each species (Flynn et al.,
    1972).  Conjugation reactions

          The major conjugation mechanisms are: glucuronide synthesis,
    "ethereal" sulfate synthesis, glutathione conjugation, glycine
    conjugation, methylation, acetylation, and thiocyanate synthesis.
    Glutamine conjugation has also been shown to occur in man and monkey.
    The conjugates formed by these mechanisms are usually nontoxic,
    therefore conjugation has also been referred to as a detoxification

          These conjugations are biosynthetic reactions in which foreign
    compounds or their metabolites containing suitable groups (hydroxyl,
    amino, carbonyl, or epoxide) combine with some endogenous substrates
    to form conjugates (Parke, 1968; Williams, 1967a, 1971). These
    reactions require ATP as source of energy, coenzymes, and transferases
    which are usually specific for the formation of conjugates of foreign
    compounds. The conjugations usually proceed in at least two steps:
    first, the extramicrosomal synthesis of acylcoenzyme and next the
    transfer of the acyl moiety to the aglycone, which, in some but not
    all cases, is localized in the microsomes. Thus, these reactions
    cannot be considered as transformations, characteristic of microsomes.

          In accordance with the coenzymes participating in these
    reactions, they include:

          formation of glucuronides (via uridine diphosphate glucuronic
          acid, UDPGA);
          formation of sulfate esters (via 3-phosphadenosine-5-
          phosphosulfate, PAPS);
           O-, N-, and  S-methylation via 5'-[(3-amino-3-carboxypropyl)
          methylsulfonio]-5'-dioxyadenosine( S-adenosylmethionine);
          acetylations (via acetyl coenzyme A);
          formation of peptide conjugates (via different acylcoenzyme A
          formation of glutathione conjugates and mercapturic acids
          (conjugations with glutathione).

          Formation of glucuronides is probably the most important
    microsomal conjugation mechanism (Dutton, 1971). It occurs in the
    liver and to a lesser extent in the kidney, gastrointestinal tract,
    and the skin. Biosynthesis of glucuronides can be measured in intact
    animals by determining D-glucaric acid (Marsh, 1963) and
    D-glucuronolactone dehydrogenase ( (Marselos & Hanninen,
    1974), by enhancement of D-glucuronolactone and aldehyde dehydrogenase
    ( by inducers of microsomal metabolism (Marselos & Hanninen,
    1974), glucuronides (Gregory, 1960; Yuki & Fishman, 1963) and
    L-ascorbic acid in urine. Elevation in urinary excretion of these
    compounds may be an indicator of an adaptive acceleration of hepatic

    glucuronide formation (Notten & Henderson, 1975). It should be
    emphasized that increased excretion of D-glucaric acid can result from
    enzyme induction; therefore it cannot be assumed that this occurrence
    is indicative only of an increased glucuronide formation. Methods for
    the measurement of glucuronide synthesis in whole organs and tissue
    cultures, as well as in tissue slices, have been summarized by Dutton
    (1966). In assays with homogenates and cell fractions the reaction
    mixtures have to be supplemented with added UDPGA.

          UDP-glucuronosyl transferase ( activity can be
    determined using 2-aminophenol (Burchell et al., 1972; Dutton &
    Storey, 1962), 4-nitrophenol (Isselbacher 1956; Zakim & Vessey, 1973),
    bilirubin (Heirwegh et al., 1972), 7-hydroxy-4-methyl-2H-I-
    benzopyran-2-one (4-methylumbelliferone) (Aitio, 1973; Arias, 1962) or
    morphine (Strickland et al., 1974).

          In contrast to glucuronide synthesis, the formation of sulfate
    esters is most probably an extramicrosomal process and is catalysed
    generally by sulfate-conjugating enzymes in the presence of
    3-phosphoadenosine-5-phosphosulfate as a co-enzyme (Roy, 1971). Among
    the compounds of toxicological interest, phenols are converted by
    sulfation to esters and excreted in the urine. Aminophenols yield
    sulfamates. There are specific assays for the determination of
    sulfotransferase (2.8.2) activity using 4-nitrophenol (Gregory &
    Lipmann, 1957), or 3-(2-aminoethyl)-1H-indol-5-ol (serotonin) (Hidaka
    et al., 1967) as acceptors.

          The methyltransferases (2.1.1) catalyse  O-, N- and
     S-methylation of several physiologically active compounds and drugs
    (Axelrod, 1971). They are widely distributed in different organs, but
    only a small amount of catechol- O-methyltransferase ( and
    almost all of the phenol- O-methyltransferase ( (Axelrod &
    Daly, 1968) activity is localized in the microsomes of the liver. Only
    microsomal transferases are induced by benzo(a)pyrene and inhibited by
    SKF 525Aa. The methods used for the determination of
    catechol- O-methyltransferase activity are based on the principle
    that the enzyme catalyses the transfer of methyl groups to catechols
    in the presence of  S-adenosylmethionine as a methyl donor. The
    substrates employed include adrenaline (Axelrod & Tomchick, 1958),
    3,4-dihydrobenzoic acid (MacCaman, 1965), 3,4-dihydroxybenzeneacetic
    acid (3,4-dihydrophenylacetic acid) (Assicot & Bohuon, 1969; Broch &
    Guldberg, 1971) as well as I-(3,4-dihydroxyphenyl)ethanone
    (3,4-dihydroxyacetophenone) (Borchardt, 1974). The end products of the
    enzymatic reaction are measured either spectrofluorimetrically
    (Axelrod & Tomchick, 1958; Borchardt, 1974; Broch & Guldberg, 1971),
    or radiometrically using labelled methyl groups in the coenzyme
    (MacCaman, 1965).


    a  Diethyl aminoethanol ester of diphenyl-propyl acetic acid.

          Acetylation reactions of the amino group of foreign compounds are
    catalysed by acetyltransferases (Weber, 1971). Substrates of these
    enzyme reactions, localized in the soluble part of the cells, are
    arylamines, hydrazines, and certain aliphatic amines. Coenzyme A is an
    essential factor in these acetylations. Acetylation of arylamines has
    been studied quantitatively,  in vivo, in human beings and animals
    (Williams, 1967b).

          Methods for the determination of  N-acetyltransferase (
    activities  in vitro summarized by Weber (1971) include colorimetric
    (Brodie & Axelrod, 1948; Maher et al., 1957; Marshall, 1948; Shulert,
    1961; Weber, 1970), spectrophotometric (Jenne & Boyer, 1962; Tabor et
    al., 1953; Weber & Cohen, 1968; Weber et al., 1968) as well as
    radiometric procedures (Stotz et al., 1969).

          Conjugation of aromatic carboxylic acids (benzoic acid,
    substituted benzoic acids, and heterocyclic carboxylic acids) with
    amino acids by means of acetyl coenzyme A and ATP is called peptide
    conjugation. Glycine is the most generally involved amino acid in this
    reaction resulting in the formation of  N-benzoylglycine (hippuric
    acid). Indole-3-acetic acid, benzeneacetic acid, as well as
    4-aminosalicylic acid, can conjugate with glutamine in man, and
    several mammals. Determination of hippuric acids (Ogata et al., 1969)
    enables the quantitative investigation of this conjugation reaction.

          Conjugation of glutathione with foreign compounds, catalysed by
    at least ten different glutathione  S-transferases, is an important
    pathway for the elimination of these compounds (Boyland, 1971).
    Following the conjugation of foreign compounds with glutathione, the
    conjugate is most frequently hydrolysed to the cysteine conjugate
    which is excreted in the urine. Furthermore, the cysteine conjugate
    may be acetylated and the resulting mercapturic acid excreted. The
    significance of the mercapturic acid biosynthesis in man, however, is
    difficult to assess.

          Determination of glutathione  S-transferase activities are based
    on spectral change of the substrate (1,2-dichloro-4-nitrobenzene) due
    to conjugation (Booth et al., 1961), or loss of glutathione content
    (Boyland & Chasseaud, 1967; Boyland & Williams, 1965; Johnson, 1966)
    or release of labile groups (Al-Kassab et al., 1963; Boyland &
    Williams, 1965; Johnson, 1966) as well as on chromatographic
    separation of the products (Suga et al., 1967). The determination of
    the activity of gamma-glutamyltransferase (, catalysing one
    intermediary step of the overall mercapturic acid synthesis may also
    be informative.  Extramicrosomal metabolic transformations

          Foreign compounds, either transformed by oxidation or initially
    having characteristic groups (hydroxyl, amino) may resemble normal
    constituents of physiological metabolism. Thus, they may undergo
    metabolic transformations similar to those of normal body
    constituents: oxidation, reduction, deamination, hydrolysis. The
    enzymes catalysing these reactions are localized in the cytosol or are
    intrinsic compounds of the mitochondria.

          In contrast to the extensive data in the literature on
    enzyme-chemical interactions (MacMahon, 1971; Zeller, 1971) only a few
    enzyme activities are commonly used to monitor toxicological events.

          The alcohol dehydrogenase ( of the liver is one of the
    most important enzymes which catalyses the NAD-mediated oxidation of
    various aliphatic and aromatic primary and secondary alcohols.
    Determination of the activity of alcohol dehydrogenase is based on the
    spectrophotometric measurement of the amount of NAD being reduced in
    the presence of excess alcohol (Bonnichsen & Brink, 1955).

          Among the amine oxidases, monoamine oxidase (, localized
    in the mitochondria, regulates the balance of the biogenic amines and
    probably does not participate in the metabolism of foreign amines to a
    great degree (Zeller, 1971). However, the fact that a large number of
    substances (substrates and substrate analogues, alkyl and arylamines,
    hydrazine derivatives, sulfhydryl reagents, etc.) inhibit this enzyme,
    enables monoamine oxidase to be used as a tool in studies of the
    toxicity of these inhibitors.

          Monoamine oxidase activity can be measured manometrically
    (Creasey, 1956) based on oxygen-consumption, by determination of
    ammonia production (Cotzias & Dole, 1951), spectrophotometrically
    (Dietrich & Erwin, 1969; Obata et al., 1971; Weissbach et al., 1960),
    fluorimetrically (Takahashi & Takahara, 1968; Tufvesson, 1970) as well
    as radiometrically (Otsuka & Kobayashi, 1964).

          Hydrolysis by carboxylesterases (ali-esterases or arylesterases)
    of foreign compounds containing ester groups may be important in
    assessing their toxicity (La Du & Snady, 1971). Determination of
    esterase activities using different substrates in the presence of the
    chemical to be tested can disclose its possible inhibitory potency.  Nonenzymatic reactions

          Although the foregoing sections have discussed enzymatic
    modifications of chemicals, the investigator should not overlook
    nonenzymatic, spontaneous reactions between chemicals and natural
    constituents in the body that lead to the formation of metabolites,
    e.g. the reaction of an alkylating agent with glutathione.

    4.6.2  Species variability

          A serious problem facing every research worker using an animal
    species to study the metabolism of a foreign compound is whether or
    not the metabolic pathway in the animal is similar to the metabolic
    pathway in man. The problem is not only important in metabolic
    studies, but is of utmost importance in using animal toxicity studies
    to predict toxicological phenomena in man. Conney et al. (1974)
    illustrated that the use of an animal species that metabolizes a
    foreign compound in a similar manner to man will give a more precise
    prediction of the type of toxicological phenomena to be expected in

          Different animal species have been shown to metabolize foreign
    compounds at different rates. Quinn et al. (1958) has shown that
    benzeneamine (aniline) has a metabolic half-time in the mouse of 35
    minutes and in the dog of 167 minutes. In the same study it was
    demonstrated that the metabolic half-time of an antipyrine in the rat
    was 140 minutes, whereas in man it was 600 minutes.

          Considerable species differences in metabolic pathways have also
    been demonstrated. In the rat, mouse, and dog the carcinogen,
     N-2-fluoranylacetamide (FAA), is  N-hydroxylated to  N-hydroxy-FAA
    which is a more potent carcinogen than FAA. In the guineapig little or
    no hydroxylation of FAA occurs. In toxicity studies, Miller & Miller
    (1971b) and Weisburger et al. (1964) demonstrated that the rat, mouse,
    and dog are susceptible to the carcinogenic activity of FAA, whereas
    the guineapig is not. Thus, a difference in the metabolic pathways of
    a foreign compound may greatly influence its toxicity.

          Species variability in metabolism has been related to other
    factors such as species differences in protein binding, and enzyme
    concentration and type. Hucker (1970) described, in detail, species
    differences in chemical metabolism and some of the factors responsible
    for these differences.

    4.6.3  Enzyme induction and inhibition

          For some time it has been known that chemicals can increase the
    activity of metabolizing enzyme systems. These chemicals have been
    termed enzyme "inducers". Inducers exert their action by
    quantitatively increasing the enzymes and components responsible for
    the metabolism of foreign compounds. The importance of induction to
    the toxicologist is two-fold. If metabolism leads to the formation of
    excretable or nontoxic metabolites, induction will enhance
    detoxification and excretion of the compound. However, if metabolism
    leads to the production of a more toxic metabolite, induction will
    increase the toxicity of a compound.

          Many chemicals are known to increase metabolizing enzyme systems.
    The reviews by Conney (1967), Kuntzman (1969), and Mannering (1968),
    depict the large number of chemicals which induce metabolizing enzymes
    and comprehensively review the factors involved in enzyme inductions.

          Most inducers give maximum effects rather quickly -- within 2-3
    days (Fouts, 1970). However, some require 2 weeks or longer (Gillette
    et al., 1966; Hart & Fouts, 1965; Hoffman et al., 1968, 1970;
    Kinoshita et al., 1966). Frequently, the degree of induction after
    obtaining a maximum level may decline despite continuing treatment of
    the animal with a chemical (Gillette et al., 1966; Hoffman et al.,
    1968; Kinoshita et al., 1966).

          Drug-metabolizing enzymes can also be depressed by foreign
    chemicals, and these compounds are termed inhibitors. 2-(Diethylamino)
    ethyl-alpha-phenyl-alpha-propyl benzeneacetate hydrochloride
    (SKF-525A) is the best known of the inhibitors and is used routinely
    in determining the effect of enzyme inhibition on the metabolism of

    4.6.4  Metabolic saturation

           In vivo saturation of metabolic pathways can play an important
    role in determining the toxic profile of a chemical. A recent article
    by Jollow et al. (1974) demonstrated the effect of enzyme saturation
    on the metabolism and toxicity of bromobenzene. Bromobenzene was first
    metabolically transformed to an epoxide which is hepatotoxic. After a
    small nontoxic dose, approximately 75% was converted to the
    glutathione conjugate and excreted as bromophenylmercapturic acid.
    After a large toxic dose, only 45% was excreted as the mercapturic
    acid. It was established that, at the toxic dose, the metabolic
    conjugation pathway was overwhelmed due to lack of glutathione, which
    resulted in an increased reaction of the epoxide hepatotoxin with DNA,
    RNA, and protein.

          It is very important to elucidate dose-dependent metabolism to
    assess the hazard of a chemical. Frequently, the doses of a chemical
    used to characterize toxicity are many times those encountered in the
    environment. Toxicity incurred at these large doses may be influenced
    by relative changes in metabolism and therefore must be interpreted
    with caution and judgment in assessing the hazard of low doses.

    4.7  Experimental Design

          Since, for the most part, toxicity is a function of the
    concentration of the toxicant in the tissues and cells, this
    information together with its dynamics provides for inter- as well as
    intra-species extrapolation of the results of toxicological effects.

          The overall objectives of a chemobiokinetic study are to
    determine the amount, rate, and nature of absorption, distribution,
    metabolism, and excretion of a chemical. The approach to meeting those
    objectives must be flexible and designed to meet the specific needs of
    each chemical.

          It is difficult to predict, without prior data, an animal species
    that will metabolize a chemical similarly to man. Usually, initial
    studies are performed in the rat and one other nonrodent species, such
    as the dog or monkey, in an attempt to determine species variability.
    If there are significant differences among species, it is important to
    determine whether differences in the chemobiokinetic parameters
    correlate with differences in toxicity or pharmacological activity.
    Animals should be acclimatized to the environment of the metabolism
    cage prior to the experiment. Light cycle, temperature, humidity, and
    time of feeding should be standardized. The physical condition,
    weight, and food and water consumption of each animal should be
    monitored and recorded throughout the study.

          There are advantages in using radioactively-labelled chemicals in
    initial studies because of the ease with which radiochemical methods
    (Chase & Rabinowitz, 1968) can be applied to chemobiokinetic studies.
    An important advantage of using a radioactively-labelled chemical is
    that it allows the establishment of the total recovery of the parent
    chemical and its metabolites, i.e. the mass balance. To obtain this
    the total radioactivity eliminated via the urine, faeces, and exhaled
    air as well as that remaining in the carcass following termination of
    the experiment should be determined. Until a reasonably good recovery
    is obtained, 90% or greater, one can never be sure whether other
    chemobiokinetic parameters obtained from the study are accurate.
    Furthermore, the isolation and ultimate identification of unknown
    metabolites is greatly enhanced by using radioactively-labelled

          When using a radiolabelled chemical, the measurement of
    radioactivity confirms the presence of the radioisotope, not the
    chemical or its metabolites. In order to determine the identity of the
    radioactively-labelled compound, the parent chemical and its
    metabolites, analytical methods such as gas, high-pressure liquid, and
    thin-layer chromatography and a combination of gas chromatography and
    mass spectroscopy are frequently employed.

          Until it is established that the radioactivity being monitored is
    from the chemical in question, kinetic parameters apply to the
    radioactivity only, not to the chemical studied. Difficulties can
    arise if the radioactive atom does not remain an integral part of the
    molecule under study. Tritium and carbon-14 are often incorporated
    into the body pools of normal tissue components (Griffiths, 1968;
    Rosenblum, 1965). Once the radioactivity is incorporated into these

    compartments, its clearance depends on their rates of turnover.
    Therefore, by monitoring radioactivity only, it can be falsely assumed
    that a compound is being retained in the body.

          Another very important reason for differentiating the parent
    chemical from its metabolite is to assure that toxic effects that may
    be present are associated with the parent chemical and not a
    metabolite. Also persistence of a metabolite in the body rather than
    the parent chemical may constitute the ultimate hazard.

          In initial studies, consideration should be given to the
    administration of the compound by intravenous injection as well as via
    the route by which man is exposed to the chemical. The intravenous
    route is used to provide a more definite assessment of the earlier
    phases of distribution and/or elimination. Also, large variation in
    rates of absorption will in some cases make the differentiation of the
    early phases of distribution and elimination difficult. At least two
    doses should be used. One dose should be equivalent to the dose
    required to cause signs of toxicity. The second dose should be well
    below the toxic dose and, if possible, equivalent to anticipated human
    exposure levels.

          Most frequently, kinetic parameters for elimination of a chemical
    are established by sequential sampling of blood plasma and excreta,
    following its administration. A preliminary probe study using one or
    two animals is often needed to establish the time at which samples
    should be collected, because this will vary with the species and the
    chemical in question. After collection and until prepared for analysis
    of the chemical or its metabolites, samples should be stored in a
    manner that will preclude the breakdown of the chemical or its
    metabolites. The data required from the initial chemobiokinetic
    studies can be used to design further studies which may include the
    following: distribution studies using autoradiography; the isolation
    and identification of metabolites; studies to determine the
    chemobiokinetic profile of metabolites; biliary excretion studies;
    bioconcentration; and  in vitro metabolism studies. The methods and
    techniques needed to perform these studies are documented by La Du et
    al. (1972).

    4.8  Chemobiokinetics

          Chemobiokinetics aims at quantification of the processes
    discussed previously in this chapter. Thus, chemobiokinetics provides
    quantitative information on the absorption, distribution,
    biotransformation, and excretion of chemicals (including drugs and
    endogenous substances) as a function of time. Since the classical
    introduction of this discipline by Teorell (1937a,b), the concepts and
    methods have been developed extensively, principally for application
    to the clinical evaluation and/or use of drugs (Levy & Gibaldi, 1972,
    1975; Wagner, 1968, 1971). The reader is also referred to Gehring et
    al. (1976) who discuss the subject in greater detail.

          One difficulty of many toxicologists and biologists on first
    exposure to chemobiokinetics is the concept of compartments. The body
    is composed of a large number of organs, tissues, cells, and fluids,
    any one of which could be referred to morphologically and functionally
    as a compartment. However, in chemobiokinetics, a compartment refers
    collectively to those organs, tissues, cells, and fluids for which the
    rates of uptake and subsequent clearance of a chemical are
    sufficiently similar to preclude kinetic resolution. The rapidly
    equilibrating compartment, referred to as the central compartment, may
    be comprised of all those tissues with a profuse blood supply whereas
    the slow or peripheral compartment may include tissues with a more
    limited blood supply, i.e. fat and bone.

    4.8.1  One-compartment open model

          The simplest chemobiokinetic model is a one-compartment, open
    model as shown in Fig. 4.1. In using this model, it is assumed that
    the chemical equilibrates with all tissues to which it is distributed
    sufficiently rapidly to preclude kinetic differentiation by the
    techniques being used to characterize its movement in the body. For
    example, if it requires 30 min for a chemical to attain equilibration
    in the body after entering the blood stream, and if samples of blood,
    tissues, and excreta are taken at 30 min intervals, it will appear
    that the body consists of only one compartment.

          Assuming that the rate of elimination of the chemical is
    proportional to its concentration in the plasma, the concentration in
    the plasma will be described by apparent first-order kinetics. The
    rate of change of concentration in the plasma may be expressed in the
    form of the linear differential equation

                               =  - ke C(t)                              (1)

    where  C(t) is the concentration at time  t, and  ke is the rate constant
    for elimination. Solution of this differential equation with initial
    condition  C(t) =  C(0) at time zero gives

           C(t) =  C(0) exp(- ke t)     (exponential form)                 (2)



    FIGURE 7

    In these equations,  C(0) is the concentration of the chemical in the
    plasma at time zero. A plot of  C(t) versus time on semilogarithmic
    paper will yield a straight line (Fig. 4.2) with slope - ke and
    intercept  C(0).

    FIGURE 8

          Having determined  ke, which is measured in units of reciprocal
    time, the time required to reduce the plasma concentration by one-half
    is estimated; this time is referred to as the  t or half-time. It
    can be determined from the equation

                               ln 2     0.693
                          t =        =                                 (5)
                                ke       ke

    When the chemical is not absorbed instantaneously, the mathematics
    needed to describe the concentration in plasma as a function of time
    become somewhat more complicated. Assuming apparent first-order
    absorption as well as elimination, the concentration  C(t) in plasma
    is given by the expression

              contour integral* D0* ka
      C(t) =                            {exp (- ke t) - exp (- ka t)}         (6)
                      Vd( ka -  ke)

    In this expression, the terms not previously mentioned are  D0, the
    dose; contour integral, the fraction of dose absorbed; Vd, the
    apparent volume of distribution; and  ka, the apparent first-order
    absorption rate constant.

          The elimination rate constant,  ke, is determined as described
    previously using that portion of the solid line representing the
    plasma concentration after absorption is essentially complete. In Fig.
    4.2, this occurs when the dotted line blends into the solid line. The
    rate constant for absorption,  ka, may be estimated by projecting
    the solid line backward to the origin. The difference between the
    experimentally-determined values used to characterize the dotted line
    are subtracted from those predicted by the backward projection at
    corresponding times. Subsequently, the values obtained by this "curve
    stripping" procedure are plotted producing a curve like the dash-dash
    line in Fig. 4.2. Using this procedure, the  t for absorption and
     ka are determined.

          The volume of distribution,  Vd, is a term used to describe the
    apparent volume to which a chemical is distributed when it is assumed
    that the affinity of the plasma and all tissues is equivalent. An
    analogy is placing a known amount of a dye in a liquid contained in a
    system of unknown volume. After the concentration of the dye has
    attained a constant value, the volume of the system can be determined
    by dividing the dose,  D0, by the concentration to give the volume
    of distribution,  Vd.

          In the plasma, the concentration of the chemical declines because
    of elimination as well as distribution to tissues. Therefore, to
    estimate  Vd, it is necessary to project the elimination phase of
    the curve back to the origin. The value obtained at the time zero
    intercept by this projection is divided into  D0 to obtain the
    volume of distribution,  Vd, in ml/kg.

          The value of  Vd provides some important information about the
    distribution of the chemical in the body. As the distribution to the
    tissues increases, for whatever reason, physicochemical affinity,
    active transport into cells,  Vd increases. If the distribution of a
    chemical in the human body is limited to plasma, extracellular fluid,
    or total body water, the respective values of  Vd will be
    approximately 40, 170, and 580 ml/kg. If a chemical has a high
    affinity for a particular tissue, for example, the affinity of a
    lipophilic chemical for fat,  Vd may exceed significantly 1000 mg/kg.
    When the volume of distribution is known, the amount of chemical in
    the body at any time  t, A(t), can be calculated from the equation

                      A(t) =  C(t)Vd                                       (7)

    Until now, concepts relating only to the concentration of the chemical
    in the plasma have been discussed.

          However, these concepts are equally applicable to other tissues
    or, for that matter, to excreta, expired air, or urine. In the case of
    urine, the concentrating power of the kidney must be accounted for to
    normalize the data. If the affinity of the chemical for the various
    tissues and excreta is equivalent and if rapid equilibration is
    assumed, the concentration curves will be superimposable. However,
    this would be an unusual occurrence. Because of the differences in
    affinity, it is more likely that a family of parallel concentration
    curves will be obtained. It is emphasized that these curves will be
    parallel only after an apparent steady state has been achieved between
    the tissues.

          In addition to concentration, the same concepts apply if one
    desires to characterize the total amount of chemical in the body,
     A(t), as a function of time following exposure. For example, if a
    dose  D0 is ingested and apparent first-order kinetics is assumed,
    the amount of the chemical in the body is given by the expression

                    A(t) =  D0 exp (- ke t)                                (8)

    Using equation (7), equation (8) can be shown to be equivalent to

                 C(t) =  C(0) exp (- ke t)                                 (9)

    Logarithmic transformation of equations (8) or (9) may be used to
    obtain curves like those in Fig. 4.2. The dotted curve would apply if
    the chemical were applied to the skin and subsequently absorbed.

          One caution must be emphasized in resolving the kinetics of the
    amount of an agent in the body. Usually, it is not adequate to
    determine the amount of the agent excreted and calculate the amount
    remaining in the body by subtracting the cumulative amount excreted
    from the original dose. This can be done if, and only if, the agent is
    metabolically transformed to a very limited degree and, essentially,
    all of the original dose is recovered. This seldom happens.

          To circumvent the problem just described, the amount of the
    chemical excreted over designated time intervals is determined until a
    significant amount can no longer be detected. Assume that the rate of
    excretion is proportional to the amount of chemical in the body,
     A(t). Let  B(t) be the cumulative amount excreted to time  t after
    administration. Then

                              =  ke A(t)                                (10)


                        A(t)  =  D0 exp(- ke t)                           (11)


                                =  kex A(t)                             (12)

                  dB(t)     kex
                       =          D0 exp(- ke t)                         (13)
                   dt       ke


                  B(t) =  D0      {1 - exp (- ke t)}                      (14)

    In these equations,  ke represents the apparent first-order overall
    elimination rate constant and  kex is the rate constant for
    excretion via the route being analysed. If  Ei is the amount
    excreted in the  ith time interval of duration deltat then

               Ei =  B( ti) -  B( ti - delta t)                             (15)

    where  B( ti) is the amount excreted between administration and  ti,
    the time at the end of the  ith time interval.

          In terms of the dose administered  D0, and the rate constant

            Ei =  D0      exp(- ke ti) {exp( kedelta t) - 1}                 (16)

    the logarithmic form of which is



    A semilogarithmic plot of  Ei versus  ti will give a straight line
    with slope - ke. The above expression can be modified to accommodate
    unequal time intervals, but in doing so graphic insights are lost.

          In using excretion data to resolve kinetic parameters, it is
    desirable to keep the collection intervals as short as practical.
    Ideally, the collection intervals should be shorter than the  t for
    elimination of the chemical; otherwise resolution of a biphasic
    excretion pattern may be precluded. Biphasic refers to two kinetically
    distinct excretion phases. For a volatile chemical excreted to some
    degree by exhalation, determination of the chemical exhaled as a
    function of time may be particularly useful for resolving its

          As already stated, the excretion rate of a chemical by one route
    of excretion may be different from its overall rate of elimination.
    This is true because the agent may be eliminated by other routes
    and/or metabolically transformed. The following scheme may be used to
    depict a chemical that is eliminated by a metabolic pathway as well as
    by excretion in the urine and exhalation:

            / ku     excretion in urine
          C - kr     excretion via exhalation
            \ kmx    metabolic transformation to compound y

    In this case, the overall elimination constant will be  ke =  ku +
     kr +  kmx.

          The various metabolic transformation and excretion rates may be
    estimated using the following equations:

                         ku=  U infinity( ke/ D0)                         (18)

                         kr =  R infinity( ke/ D0)                        (19)

                         kmx =  X infinity( ke/ D0)                       (20)

     Uinfinity and  Rinfinity are the total amounts of the parent chemical
    excreted in urine and expired air.  Xinfinity is the total amount of
    metabolite,  X, recovered from excreta. For excretion of the chemical
    in the urine, the differential equation is:

                      =  ku D0 exp(- ke t)                                (21)

    The solution of the equation (21) yields

                    U inifnity =  ku D0/ ke                               (22)


                    ku =  U infinity ke/ D0                               (23)

    When the urinary excretion of a chemical is determined, it is
    frequently desirable to determine its renal clearance in order to
    ascertain whether the chemical is actively secreted, reabsorbed, or
    only passively filtered by the kidney in the excretion process. Renal
    clearance is defined as the urinary excretion rate, delta  U/delta
     t, divided by the plasma concentration,  C:

                    Rc = (delta  U/delta  t)/ C                           (24)

    If the plasma concentration is changing during the urinary collection
    interval, the concentration at the midpoint of the interval is used
    frequently. It may also be shown using equations (9), (21), and (24)

                    Rc =  ku Vd                                          (25)

    which precludes the necessity of knowing the plasma concentration.
    Renal clearance values for inulin measure excretion via glomerular
    filtration. For man, the normal value is 125  15 ml/min (Pitts,
    1963). If the renal clearance of a chemical exceeds this value in man,
    it constitutes evidence that the chemical is actively secreted. If it
    is less, it indicates the chemical is actively reabsorbed. If the
    compound is bound to a significant degree to protein, it may be
    necessary to determine and use the concentration of unbound chemical
    in plasma in order to obtain a realistic value for renal clearance.

    4.8.2  Two-compartment/multicompartment open systems

          Rapidly equilibrating compartments in which the chemical has
    reached equilibrium with plasma before the first blood samples are
    taken will appear kinetically as one compartment, but a "deep" or more
    slowly equilibrating compartment will give rise to a plasma
    concentration curve that appears biphasic. The model used to describe
    this system is a two-compartment open model (Fig. 4.1). The central
    and the "shallow" or rapidly equilibrating compartments are considered
    as one. The major sites of metabolic transformation and excretion are
    the liver and the kidneys. Since these organs are perfused with blood,
    it can be assumed, generally, that they are part of the central
    compartment and that elimination occurs from the central compartment.
    Fig. 4.3 is a simulated plasma concentration curve representing a
    two-compartment system following rapid intravenous administration of a
    chemical. The chemical has first been rapidly distributed to
    well-perfused tissues, then more slowly to other tissues comprising
    the deep compartment.

          Assuming all the transfer processes are first order, the system
    of linear differential equations describing the two-compartment model
    shown in Fig. 4.1 is as follows:

                dC(t)                           k21 VD CD (t)
                     = - k12 C(t) -  ke C(t) +                            (26)
                 dt                                Vd

                dCD( t)      k12 Vd C( t)
                       =               -  k21 CD( t)                    (27)
                 dt          VD

    FIGURE 9

    where  C(t) and  CD (t) are concentrations of the chemical in the
    central and deep compartments respectively. The apparent volumes of
    distribution for these compartments are  Vd for the central
    compartment and  VD for the slow exchange compartment. If the
    apparent volumetric flow rates between the two compartments are the
    same, i.e.  k12 Vd =  k21 VD, the differential equation system can be
    solved with initial conditions  C(0) =  D0/ Vd and  CD(0) = 0 at time
    zero to give the following mathematical representation for the solid
    curve in Fig. 4.3:

                  C(t) = phiexp(-alpha t) + psi exp(- t)                (28)

     is the slope of the line for the slow phase of elimination and alpha
    is the slope for the rapid phase of elimination. The value of  is
    determined as previously described and a technique called feathering
    is used to obtain alpha. This technique constitutes projecting the
    solid line for the slow phase backward to the origin (dash-dash line)
    and subtracting the respective projected values from the experimental
    values used to delineate the rapid phase of clearance. These values
    are replotted (dotted line). The slope of this line is alpha. The
    values for phi and psi are the intercepts at the ordinate for the
    rapid and slow elimination phases, respectively.

          The rate constants  k12,  k21, and  ke (Fig. 4.1) may be
    determined as follows:

                        phi + psi alpha
                 k21 =                                                 (29)
                        phi + psi

                 ke =                                                  (30)

                 k12 = alpha +  - ( k21 +  ke)                          (31)

     k12 is of particular importance because from it the amount of
    chemical in the deep compartment  (AD (t)) is readily calculated
    from the equation

                  k12 D0
          AD( t) =            {exp(-alpha t) - exp(- t)}                 (32)
                  - alpha

    Using this information, toxicologists can ascertain whether there may
    be correlations between the effect of a chemical and its presence in a
    deep compartment. Indeed, for the toxicologist, a prominent slow phase
    for the elimination of a chemical is a red flag suggesting that with
    repeated administration cumulative toxicity may constitute a problem.

          These concepts developed for the plasma concentration of a
    chemical conforming to a two-compartment open-model system can be
    extended to describe the amount of the agent in the body or the amount
    excreted. Also, an absorption component may be added which would give
    a function involving the sum of three exponential terms:

     C( t) = phiexp(-alpha t) + psi exp(- t) + (phi + psi)exp (- ka t)     (33)

    4.8.3  Repeated administration or repeated exposure

          The concentration of a chemical in the plasma or tissues or the
    amount of chemical in the body following repeated administration or
    exposure is illustrated in Fig. 4.4 for a one-compartment open system.
    Mathematical representation of these concentrations is obtained by
    addition of the exponential terms for each dose so that the
    concentration of the chemical at time  t following the  nth dose is
    given by

    FIGURE 10


    where tau is the interval between doses. After a large number of doses,
    the term exp  (-nketau ) approaches zero, and the value for the
    concentration of chemical becomes


    Once the plateau concentration is reached, further exposure to the
    same dose at the same frequency will not result in any further
    increase in concentration. At the plateau, the maximum concentration
    which will occur immediately following the last exposure is given by:

    The minimum concentration will occur immediately before the next
    exposure and is given by:


    The expression defining the average concentration after the plateau
    has been attained is:

                               contour integral D0
               C(av)infinity =                                            (38)
                                       Vd ketau

    If the exposure or the route of administration is such that the
    first-order rate of absorption, ka, must be considered, the plasma
    concentration following  n repetitive doses at a dose interval tau is
    given by:


    The rate constant for absorption, ka, may be replaced by the rate
    constant for delivery of a substance being inhaled.

    4.8.4  Kinetics of nonlinear or saturable systems

          Dose-response curves for an effect arising from the
    administration of a range of dose levels of a toxic agent usually
    follow a log-normal distribution. Extrapolation of the logarithmic
    probability transformation of these curves predicts that some
    individuals will respond at an infinitesimally small dose, while
    others will never respond, no matter how large the dose. The
    assumption inherent in such extrapolation beyond the range of observed
    data is that the chemobiokinetic profile of the compound in question
    is independent of the dose level administered.

          Assuming dose-independence, a 10-fold increase in the plasma
    concentration of a chemical will result from a 10-fold increase in the
    administered dose. However, many metabolic and excretory processes are
    saturable and, as the dose of chemical begins to overwhelm these
    processes, it may be expected that there will be a disproportionate
    increase in toxicity. Therefore, nonlinear chemobiokinetics is of the
    utmost importance in toxicology.

          Many metabolic and active transfer processes as well as some
    passive protein-binding processes have a finite capacity for reactions
    with a chemical. The rate of these nonlinear processes can be defined
    by the Michaelis-Menten equation

                 - dC(t)       Vm C(t)
                        =                                             (40)
                    dt       Km +  C(t)

    where  C(t) represents concentration of the chemical at time  t,
     Vm is the maximum rate of the process, and  Km is the
    concentration of chemical at which the rate of the process is equal to
    one-half of  Vm. Although this equation has been found useful in
    delineating  in vivo nonlinear kinetics, the constants should be
    referred to as  apparent in vivo constants, since they are
    undoubtedly influenced by many other biological processes. Two
    important limiting cases for this equation are as follows. When the
    concentration of chemical is much smaller than  Km( C( t)   Km) then
    equation (40) reduces to

                   - dC( t)     Vm
                          =         C( t)                               (41)
                    dt        Km

    and the ratio of  Vm/ Km will approximate an apparent first-order
    rate constant. However, when the concentration is much greater than
     Km  (C(t)   Km) then the rate is described by

                     - dC( t)
                             =  Vm                                     (42)

    In this case, the rate is no longer dependent on the prevailing
    concentration, but has become zero order and thus independent of

          Fig. 4.5 displays a typical concentration versus time curve for a
    chemical the elimination of which follows nonlinear or
    Michaelis-Menten kinetics. As long as the concentration remains
    significantly less than Km, the log-linear portion of the plot is
    applicable and all the principles of apparent first-order kinetics
    apply. But, as the concentration approaches and then exceeds Km, the
    semi-logarithm plot becomes nonlinear. In this region of zero-order
    kinetics, the plot will be linear if rectangular coordinates are used.

    FIGURE 11

    4.9  Linear and Nonlinear One Compartment Open-model Kinetics of
         2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)

          To illustrate the use of chemobiokinetics in toxicology, some
    results obtained from studies with 2,4,5-T are presented below.
    2,4,5-T, a herbicide, has been reported to be teratogenic, fetotoxic,
    and embryotoxic at doses of 100 mg/kg/day during the period of
    organogenesis (Collins & Williams, 1971; Courtney & Moore, 1971;
    Courtney et al., 1970; Roll, 1971; Sparschu et al., 1971).

    FIGURE 12

          To elucidate the potential hazard of this compound, 5 mg/kg of
    14C ring-labelled 2,4,5-T was administered as a single oral dose to
    rats and dogs (Piper et al., 1973). The plasma concentration versus
    time curves (Fig. 4.6) indicated compliance with a one-compartment
    open model system having apparent first-order rates of absorption and
    clearance; the  t values for the clearance of 2,4,5-T from the
    plasma of rats and dogs were 4.7 and 77 h, respectively. For
    elimination from the body via the urine (Fig. 4.7), the  t values
    were 13.6 and 86.6 h. Since clearance of 2,4,5-T from the plasma of
    rats was more rapid than its elimination in the urine, the compound
    may have been actively concentrated in the kidneys prior to excretion
    in the urine. Also, the much slower elimination by dogs than rats
    correlates with the higher toxicity in dogs; the single oral LD50 is
    100 mg/kg and 300 mg/kg for dogs and rats, respectively (Drill &
    Heratyka, 1953; Rowe & Hymas, 1954).

          Another species difference was demonstrated by the fact that
    virtually all the 14C excreted by the rats was through the urine
    while approximately 20% of that excreted by dogs was through the
    faeces. Also, no breakdown products of 2,4,5-T could be detected in
    the urine of rats given 5 mg/kg, but about 10% of the 14C activity
    in the urine of dogs was attributable to breakdown products.

          If an active secretory process in the kidney was the primary
    elimination process in rats, then this nonlinear process should be
    saturable by the administration of higher doses. Figs. 4.8 and 4.9
    show that this is the case, since the  t for both the clearance
    of 2,4,5-T from plasma and its urinary elimination increase with
    increasing dose. At doses of 100 or 200 mg/kg, the process was
    saturated and the rates of elimination from the plasma and from the
    body were the same. Further evidence of nonlinear kinetics was the
    fact that a larger percentage of the 14C administered as
    14C-2,4,5-T was excreted through the faeces as the dose was
    increased. Also, degradation products of 2,4,5-T were found in the
    urine of rats given 100 or 200 mg/kg, but not 5 or 50 mg/kg.

          The nonlinear chemobiokinetics of 2,4,5-T were further
    characterized following intravenous doses in rats of 5 or 100 mg/kg
    (Sauerhoff et al., 1975). Clearance from the plasma of rats given
    100 mg/kg followed classical Michaelis-Menten kinetics (Fig. 4.10).
    The values for  Vm and  Km were calculated to be 16.6  1.82 g/h/g of
    plasma and 127.6  25.9 g/g of plasma, respectively. During the
    log-linear phase of excretion the  t was 5.3  1.2 h.

    FIGURE 13

    FIGURE 14

          In the experiments of Sauerhoff et al. (1975), the volume of
    distribution increased from 190 to 235 ml/kg in rats given 5 and
    100 mg/kg, respectively. This increase in the volume of distribution
    indicates that with increasing dose a larger fraction of the dose is
    distributed into various tissues and cells. Thus, a disproportionate
    increase in toxicity may be expected. The fate of 2,4,5-T following
    oral doses of 5 mg/kg has also been investigated in man (Gehring et
    al., 1973). The elimination of 2,4,5-T from the plasma and in the
    urine followed apparent first order kinetics with  t1/2 of 23.1 h
    (Figs. 4.11, 4.12, 4.13). A comparison of the elimination rates in man
    with those in rats and dogs indicates that the toxicity of 2,4,5-T to
    man would lie somewhere between that to rats and dogs. The peak plasma
    levels attained with a dose of 5 mg/kg, which are higher in man than
    in either rats or dogs, are associated with a greater degree of plasma
    protein binding in man. Also, the volume of distribution in man of
    80 ml/kg is attested to the retention of 2,4,5-T in the vascular

          Fig. 4.14 illustrates simulated levels of 2,4,5-T that would be
    attained in the plasma of man with repeated ingestion. If 0.25 mg/kg
    were ingested daily, a level equalling that attained by ingesting a
    single dose of 5 mg/kg, as in this study, would never be reached.

    FIGURE 15

          Additional studies on 2,4,5-T have demonstrated that it is
    actively secreted by the kidney (Hook et al., 1974). This process of
    elimination is saturable at high doses and the capacity for excretion
    in dogs is more limited than in rats. As indicated previously, when
    doses of 2,4,5-T are given that exceed the capacity for renal
    excretion, the compound finds its way into more tissues and cells, is
    eliminated more slowly, and undergoes a greater degree of metabolic
    transformation. Thus, to use the toxicity incurred by high doses of
    2,4,5-T to make statistical estimates of the toxicity that may be
    incurred at low doses violates a basic  a priori assumption.

    FIGURE 16

    FIGURE 17

    FIGURE 18

    FIGURE 19

    FIGURE 20

          The nonlinear chemobiokinetics of toxic doses of 2,4,5-T is an
    example for many other compounds (Gehring et al., 1976). Indeed, it is
    likely that for most compounds, toxicity may coincide with the
    saturation of the detoxification process, operative at low doses.
    Recently, Gillette (1974a,b) has given special consideration to the
    chemobiokinetics of reactive metabolites of chemicals that react with
    macromolecules (DNA, RNA, and protein) causing toxic effects. The
    concepts presented in these papers are very important to the
    toxicologist because they indicate possible threshold mechanisms for
    toxicity, in particular chronic toxicity.

    4.10  Linear Chemobiokinetics Used to Assess Potential for
          Bioaccumulation of 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD)

          TCDD is a highly toxic compound formed as an unwanted contaminant
    in the manufacture of 2,4,5-trichlorophenol (Schwetz et al., 1973).
    Use of trichlorophenol to manufacture 2,4,5-trichlorophenoxyacetic
    acid may result in contamination of 2,4,5-T with TCDD. The
    physicochemical properties of TCDD suggest that exposure to small
    amounts may result in the persistent accumulation of the highly toxic
    material and, eventually, in toxic effects. To elucidate the
    propensity of TCDD to accumulate in the body, a series of
    pharmacokinetic studies was conducted (Rose et al., 1975). In these
    studies, one group of rats was given a single oral dose of 14C-TCDD
    at 1 g/kg and the excretion of 14C activity in urine, expired air,
    and faeces was determined. Other groups of rats were given orally
    0.01, 0.1 or 1.0 g of 14C-TCDD/kg/day, from Monday to Friday, for
    up to 7 weeks. In addition to determining the amounts of 14C
    activity excreted in the urine and faeces of these rats, the amounts
    remaining in the body were calculated as a function of time and the
    levels of 14C-activity residing in various tissues after 1, 3, and 7
    weeks of administration were determined.

          Since the overall recovery of 14C in rats given a single oral
    dose of 14C-TCDD was 97  8%, the amounts of 14C activity
    remaining in the bodies of the rats as a function of time was
    calculated by subtracting the cumulative amount excreted from the
    original dose. The resulting body burdens of 14C are depicted in
    Fig. 4.15. The halftime for elimination of 14C from the body ranged
    from 21 to 39 days. All of the 14C activity was eliminated via the

          The concentration of 14C activity in the bodies of rats given
    0.1 or 1.0 g/kg/day, from Monday to Friday, for 7 weeks as a function
    of time are shown in Fig. 4.16. The data show clearly that, with
    repeated exposure, the concentration of 14C activity in the body
    increases but the rate of increase decreases with time and the amount
    in the body begins to plateau, even though exposure continues.

    FIGURE 21

    FIGURE 22

          The average overall recovery of administered 14C was 97.7  9%
    of the cumulative dose of 14C. Mathematical analyses of the data
    presented in Fig. 4.16 revealed a rate constant for excretion of TCDD
    of 0.0293  0.0050 days-1 which corresponds to a half-time of 23.7
    days. The fraction of each dose absorbed was 0.861  0.078. Using
    these values, it may be calculated that the ultimate steady state body
    burden would be 21.3 D0 for rats given a daily dose of D0, 5
    consecutive days weekly for an infinite number of weeks. If D0 were
    administered every day for an infinite time, the ultimate steady state
    body burden would be 29.0 D0. Within the 7 weeks of this study, the
    rats had attained 79.1% of the ultimate steady state body burden. The
    time required to reach 90% of the ultimate steady state body burden
    would be 78.5 days.

          The concentrations of 14C-activity in the liver and fat of rats
    given 14C-TCDD at concentrations of 0.01, 0.1, or 1.0 g/kg/day,
    from Monday to Friday, for 1, 3, or 7 weeks are illustrated
    graphically in Figs. 4.17 and 4.18, respectively. Just like the body
    burden levels, the levels in these tissues increase at a decreasing
    rate and begin to plateau. It should also be noted that at each time
    of measurement, there is a direct relationship between the dose being
    administered and the level in the tissue. This latter observation is
    illustrated more clearly in Figs. 4.19 and 4.20, where the
    concentrations of 14C-activity in the liver and the fat have been
    divided by the dose. This shows that over the range of doses given,
    0.01 to 1.0 g/kg/day, the relative degree of accumulation of
    14C-TCDD by these tissues is not influenced by dose.

          Mathematical evaluation of the data presented in Fig. 4.17-4.20
    revealed that the rates for the clearance of TCDD from liver and fat
    were 0.026  0.000 and 0.029  0.001 days-1 respectively. These
    rates are essentially the same as the rate of elimination from the
    body  in toto, which is not unexpected because these tissues
    contained the bulk of the 14C-TCDD in the body. The ultimate steady
    state concentrations in liver and fat that would be attained with an
    infinite duration of exposure are 0.250  0.000 and 0.058  0.003
    D0 g TCDD/g where D0 equals the dose being administered in g/kg.
    The times required to reach specified fractions of the ultimate steady
    state concentrations would be identical no matter what dose, D0, is
    being administered.

          The 14C-activity in liver tissue from rats given 0.1 or
    1.0 g/kg/day, from Monday to Friday, weekly for 7 weeks, was
    demonstrated by gas chromatography and by a combination of gas
    chromatography and mass spectrometry to be due to 14C-TCDD. Also
    important was the finding that the 14C-TCDD present in the liver was
    readily extractable, indicating that TCDD does not bind irreversibly
    with tissue. With regard to the assessment of the hazard of repeated
    exposure to very small amounts of TCDD, the results show that TCDD

    would not continue to accumulate in the body with prolonged repeated
    exposure. In rats, 93% of the ultimate steady state level of TCDD in
    the body would be attained within 90 days. Recently, a toxicological
    evaluation of TCDD was conducted in rats given doses of 0.001, 0.01,
    0.1 and 1.0 g TCDD/kg/day, from Monday to Friday, for 13 weeks
    (Kociba et al., 1975). Perceptible adverse effects did not develop in
    rats given 0.001 or 0.01 g TCDD/kg/day. Adverse effects including
    hepatic pathology and functional changes, atrophy of the thymus, and
    haematological alterations were observed in rats receiving 0.1 or
    1.0 g TCDD/kg/day. Indeed, some rats receiving 1.0 g TCDD/kg/day
    died. The results of the studies on the fate and accumulation of TCDD
    in rats given repeated daily doses showed clearly that even with more
    prolonged exposure those rats which received 0.01 g TCDD/kg/day would
    not continue to accumulate TCDD in the body and its tissues to the
    extent leading to the toxic manifestations as seen in those rats
    receiving 0.1 or 1.0 g/kg/day. Since the levels of TCDD in the
    tissues had essentially plateaued within 90 days, more prolonged
    exposure would not be expected to lead to the attainment of toxic
    amounts of TCDD in the body or its tissues.

    FIGURE 23

    FIGURE 24

    FIGURE 25

    FIGURE 26


    Table 4.1  Different types of drug-metabolizing reactions


     (a)  Microsomal oxidations

    (Ciaccio, 1971; Dahm, 1971; Daly, 1971; Gillette, 1971b, Gram, 1971;
    Smuckler, 1971; Weisburger & Weisburger, 1971.)

    Aliphatic oxidation    RCH3  ---->  RCH2OH


                                                     / \
    Epoxidation      R - CH2 - CH2 - R  ---->  R - CH - CH - R


                            CH3               H
                           /                 /
     N-dealkylation   R - N      ---->  R - N      + CH2O
                           \                 \
                            CH3               CH3

     O-dealkylation   R - O - CH3  ---->  R - OH + CH2O

     S-dealkylation   R - S - CH3  ---->  R - SH + CH2O

    Table 4.1 (contd.)

    Metalloalkane dealkylation    Pb(C2H5)4  ---->  PbH(C2H5)3

                     R             R
                      \             \
     N-oxidation   R - N  ---->  R - N = O + H+
                      /             /
                     R             R





    Desulfuration        R               R
                          \               \
                           C=S   ---->     C=O
                          /               /
                         R               R



     (b)  Nonmicrosomal oxidations

     Monoamine and diamine oxidation

                               O2             H2O
                     RCH2NH2  --->  RCH = NH  --->  RCHO +  NH3

    Table 4.1 (contd.)

    Alcohol dehydrogenation RCH2OH + NAD+ ----> R - CHO + NADH + H+

    Aldehyde dehydrogenation  R - CHO + NAD+ ----> R - COOH + NADH + H+


     (a)  Microsomal reductions

    Nitro reduction   RNO2 ----> RNO ----> RNHOH ----> RNH2

    Azo reduction    RN = NR ----> RNHNHR ----> RNH2 + RNH2

    Reductive dehalogenation  R - CCl3 ----> R - CHCl2

     (b)  Nonmicrosomal reductions

                         R             R
                          \             \
    Aldehyde reduction     C = O ---->   CHOH
                          /             /
                         R             R


    Ester hydrolysis   R - CO - O - R1 ----> R - COOH + R1 - OH

    Amide hydrolysis   R - CO - NH2 ----> R - COOH + NH3

    Table 4.1 (contd.)


     (a)  UDPGA-medicated conjugations

     O-glucuronide formation ether type:


    ester type:


     N-glucuronide formation


    Table 4.1 (contd.)

    IV. CONJUGATION (cont'd)

     S-glucuronide formation


     (b)  PAPS-medicated conjugation

    Sulfate ester formation


    Table 4.1 (contd.)

     (c)  Methylations





     S-methylation           C2H5SH  ----->  C2H5S-CH3

    Table 4.1 (contd.)

     (d)  Acetylations


     (e)  Peptide conjugations


     (f)  Glutathione conjugations


    Table 4.2  Methods for the determination of several mixed-function
               oxidase activities

    (a)    Aryl hydrocarbon hydroxylation (using 3,4-benzpyrene as
           (Nebert & Gelboin, 1968a,b; Wattenberg et al., 1962)
    (b)    Aliphatic side-chain hydroxylation (of pentobarbital)
           (Cooper & Brodie, 1955)
    (c)    4-hydroxylation (of aniline)
           (Brodie & Axelrod, 1948; Chabra et al., 1972; Gilbert &
           Golberg, 1965; Henderson & Kersten, 1970; Hilton & Santorelli,
           1970; Imai et al., 1966; Kato & Gillette, 1965; Schenkman
           et al., 1967; Sternsen & Hes, 1975)
    (d)     N-hydroxylation (of aniline)
           (Herr & Kiese, 1959)
    (e)     N-oxidation (determination of amine oxides)
           (Fok & Ziegler, 1970; Ziegler et al., 1973)
    (f)    Nitro reduction
           (Fouts & Brodie, 1957; Hietbrink & DuBois, 1965)
    (g)     N-demethylation (of aminopyrine)
           (Brodie & Axelrod, 1950; Chrastil & Wilson, 1975; Cochin &
           Axelrod, 1959; Dewaide & Henderson, 1968; Feuer et al., 1971;
           Kinoshita et al., 1966; Klinger, 1974; La Du et al., 1955;
           MacMahon, 1962; Nash, 1953; Pederson & Aust, 1970; Poland &
           Nebert, 1973; Schoene et al., 1972)
    (h)     N-demethylation (of benzphetamine)
           (Hewick & Fouts, 1970a,b; Liu et al., 1975; Lu et al., 1969;
           Nash, 1953)
    (i)     N-demethylation (of ethylmorphine)
           (Anders & Mannering, 1966)
    (j)     O-demethylation (of  O-nitroanisole)
           (Christensen & Wissing, 1972; Kinoshita et al., 1966; Netter,
           1960; Netter & Seidel, 1964; Schoene et al., 1972;
           Zannoni, 1971)
    (k)     O-dealkylation (of ethylumbelliferone)
           (Ullrich & Weber, 1972)


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    5.1  Introduction

         Morphological studies are often the corner stones of toxicity
    experiments. The variety of such studies leads to many different
    approaches from the viewpoint of pathology, and skill and flexibility
    in working procedures seem to be far more important than a strict

         On the other hand, it is advisable to have some general
    guidelines on pathological procedures for routine quality testing for
    toxicity, even though specific questions may often be posed, special
    experiments may have to be carried out, and special animals,
    techniques, and examinations used in order to elucidate certain

         This chapter deals with the various phases of morphological
    studies with a view to providing some general recommendations
    concerning the procedures to be followed. It must be emphasized,
    however, that these recommendations are only a guide, and that it will
    be the pathologist's special responsibility to see that studies are
    carried out in the way most likely to ensure optimum results.

    5.2  General Recommendations

         Gross necropsy facilities should be in close proximity to those
    of the pathologist. The autopsy room must be equipped with adequate
    dissection tables, dissection materials, running water, drains,
    lighting, ventilation, and facilities for disinfection. In addition,
    gross photography facilities are necessary. Cooling facilities must be
    available for the storage of dead animals until necropsy, but the
    animals should not be frozen (Sontag et al., 1975). To carry out
    proper experiments and to prevent the loss of a considerable number of
    animals by cannibalism or autolysis, it is essential that animals be
    observed at least once a day, including Saturdays and Sundays. Animals
    in moribund condition should be killed.

         For the trimming of fixed tissues, a well-ventilated area,
    preferably with an exhaust hood, and running water and drains, is

         The histology laboratory should be separated from the autopsy
    room and should be equipped with tissue-processing equipment,
    microtomes, cryostat, embedding and staining facilities, and supplies
    (Sontag et al., 1975). Storage facilities are necessary for the fixed
    tissues, as well as for the tissue block and histological slide files.
    The facilities should be vermin-proof and temperature controlled or at
    least cool.

         Veterinary or medical pathologists with experience in laboratory
    animal pathology or others with appropriate training and experience
    should be responsible for all pathology procedures. In addition, the
    pathologist should participate in the design and conduct of

         Histology technicians with appropriate training and experience in
    the histological field will guarantee good histotechnical work,
    whereas technicians trained and experienced in laboratory animal
    dissection will be of great help in performing necropsies. Technicians
    must be able to recognize and adequately describe gross abnormalities.

         Personnel should be available at the weekends to perform
    necropsies on animals found dead or killed  in extremis.

    5.3  Gross Observations

         Gross pathology can be performed in most cases by experienced
    technicians under the guidance of the pathologist. They should follow
    a certain scheme of necropsy technique and must be able to recognize
    abnormalities. A checklist should be used to ensure that all organs
    and tissues are inspected and dissected. Information on clinical signs
    must be available. Abnormalities found must be recorded on autopsy
    cards. The descriptions must be clear and provide details such as
    location, size, colour, texture, number, etc.

         A great number of lesions will be within the scale of known
    abnormalities for the animal species and breed used. Lesions found
    beyond this normal scale should always be examined by the pathologist
    himself. It is advisable for all new lesions, especially those
    attributable to the treatment, to be photographed.

         Dead animals should be necropsied unless cannibalism or autolysis
    precludes this. Autolysis should not readily be accepted as an excuse
    for not performing an autopsy. An inadequate gross necropsy cannot be
    replaced by microscopic examination, no matter how well performed. On
    the other hand, a well-performed gross necropsy may provide optimum
    information for microscopic examination and may, in certain cases,
    facilitate more selective microscopic examination.

         Several ways of killing animals are available and the methods
    depend on the facilities, the purpose of the experiment, and the
    species used. Sacrifice of animals by anaesthesia (by ether or
    barbiturates) is widely used as well as asphyxiation by carbon
    dioxide. In rodents, the blood of the body can be removed by cardiac
    puncture or puncturing the abdominal aorta, and in dogs by puncture of
    the common carotid artery. Alternatively in small rodents decapitation
    can be performed, though this may result in blood aspiration and

    damage to certain tissues or organs. On the other hand decapitation
    will probably give more constant results with respect to the total
    blood loss, and consequently lead to fairly consistent organ weights.

    5.3.1  Autopsy techniques

         It is difficult, if not impossible, to prescribe working
    procedures or techniques that will be adequate in all conditions.
    Often, observations made when the animals were alive or experience
    with other structurally-related compounds may focus attention on
    certain organs or tissues. Sometimes fixation by perfusion is
    necessary for proper examination of specific tissues (for example, the
    central nervous system).

         Large numbers of animals should not be sacrificed at any one
    time, if too few technicians are available to perform the necropsies.
    Animals should be killed in sequence, especially where it is necessary
    to sample tissues for biochemical, enzyme-histochemical, or analytical
    studies. However, organs and tissues should be weighed as soon as
    possible after death to ensure that they have not dried out and that
    they can be fixed quickly. Special procedures may shorten the time
    needed for full autopsy especially in the case of small rodents. The
    exact organization of working procedures depends largely on the
    facilities and labour available, but good preparation and teamwork are
    crucial factors.

         Sacrifice should be carried out so that animals of the
    experimental and control groups are killed at approximately the same
    time of day, thus preventing the introduction of variations in
    physiological status dependent on circadian rhythm. In certain cases,
    however, this may not be feasible.

         Special care is necessary when test elements or compounds are
    analysed after sacrifice. At times it may be necessary to kill the
    animals in a given order to prevent contamination (e.g. sacrifice of
    the control first, followed by the lowest dose level, then the
    intermediate dose level, and finally the high-dose level).

         Autopsy techniques have already been described (Roe, 1965) and
    only a general outline of the procedures is given.

    5.3.2  Rat, mouse, guineapig, rabbit, monkey

         The necropsy starts with external examination including the body
    orifices. Thereafter, the animals are fixed on their backs. After a
    median incision, the skin is partly removed and the subcutis,
    superficial lymph nodes, salivary glands, and mammary glands can be
    inspected and dissected. The abdomen can be opened and the negative
    pressure of the thorax may be checked and the thorax opened.

         Thyroid and thymus can then be inspected and dissected.

         Trachea, lungs, and heart are removed en bloc. The heart is
    detached, but the trachea and lungs remain intact to provide easy
    handling for inflation of the lungs with fixative (see under fixation

         Abdominal organs are removed. The examination of liver and kidney
    should include making a routine number of parallel slices of the
    organs to examine their cut surfaces. The gastrointestinal tract
    should be removed from the abdominal cavity and opened. In chronic
    toxicity experiments, the entire gastrointestinal tract must be
    opened, the mucosal surface examined, and representative parts taken
    for histological examination. The oesophagus and parts of the
    intestine can be rolled according to the Swiss roll technique in order
    to obtain a considerable length of mucosa musculature and serosa in
    the microscopic section. Brain, peripheral nerves, skeletal
    musculature, and bones (including a joint) are removed. The spinal
    cord may best be fixed  in situ.

    5.3.3  Carnivores, swine

         After external examination, necropsy is performed when the animal
    is lying on its right side. Left front and hind legs are removed
    without opening the apertura thoracis superior. The skin is partly
    removed after a median ventral incision from mouth to os pubis.
    Subcutis, fat, mammary glands, salivary glands, and superficial lymph
    nodes may be inspected and dissected. In addition, the different
    joints of the legs are opened and inspected. After opening the
    abdomen, the thorax is checked for negative pressure and opened by
    removing the left side of the thoracic wall. Then the right side of
    the pleural cavity is inspected. The oesophagus is removed by cutting
    it cranially. The thoracic and abdominal organs are removed and
    individually inspected and opened. The nasopharynx is then opened and
    inspected, and the tongue, larynx, pharynx, tonsils, brain, spinal
    cord, peripheral nerves, and musculature collected. Most organs should
    be sliced in parallel slices to examine the cut surfaces.

    5.4  Selection, Preservation, Preparation, and Storage of Tissues

    5.4.1  Selection of tissues

         Which organs and tissues should be collected for fixation is
    determined by the type of toxicity experiment and the compound being
    tested. Sufficient material should be collected to prevent the
    necessity of having to repeat a certain experiment. Since it is
    impossible to provide strict rules for selection, only a general
    outline will be given.

    5.4.2  Oral toxicity tests

         In acute toxicity experiments, restrictive microscopic
    examination may be necessary in certain cases. Some laboratories fix
    liver and kidneys, others do not. The additional information obtained
    from histological examination of these organs in acute experiments is,
    usually, limited.

         In range-finding tests, restrictive pathology is common practice.
    Normally, the heart, liver, spleen, and kidneys and all grossly
    abnormal tissues are collected for fixation. When the compound tested
    is given by stomach tube, it is advisable to include lungs,
    oesophagus, and stomach in the histological study. In subacute
    (90-day) and chronic studies, it is advisable to select all tissues
    and organs for fixation in buffered formal saline. This normally
    includes the following organs and tissues:

    brain                         gall bladder (if present)
    pituitary                     oesophagus
    thyroid (parathyroid)         stomach
    thymus                        duodenum
    (trachea)                     jejunum
    lungs                         ileum
    heart                         caecum
    sternum (bone marrow)         colon
    salivary glands               rectum
    liver                         urinary bladder
    spleen                        lymph nodes -- mandibular or mesenteric
    kidneys                       lymph nodes -- popliteal or axillary
    adrenals                      mammary glands
    pancreas                      (thigh) musculature
    gonads                        peripheral nerve
    accessory genital organs      (eyes)
    aorta                         (femur -- incl. joint)
    (skin)                        spinal cord (at three levels)
                                  (exorbital lachrymal glands)

    The tissues mentioned between brackets are sometimes considered to be
    optional. In addition, all tissues containing grossly observed lesions
    should be fixed.

         The same selection is usually made in carcinogenicity tests. In
    both chronic toxicity and carcinogenicity experiments, it is also very
    important to collect all organs and tissues of animals that have died
    or have been killed  in extremis. Prompt examination of the tissues
    of these animals may provide valuable information leading to a more
    meaningful pathological examination of animals, that die or are
    sacrificed later on, or at the end of the experiment. In cases where
    only part of an organ or tissue is taken, it is important that the
    same part of that organ or tissue be selected at approximately the
    same site in all animals.

    5.4.3  Inhalation toxicity studies

         In acute inhalation studies, it may be worthwhile, in some cases,
    to select lungs for fixation and subsequent microscopic examination,
    as well as liver and kidneys. In most cases, however, this seems
    unnecessary since the pulmonary lesions are usually of the same type
    (hyperaemia and oedema) and can be detected by gross inspection.

         Examination of the entire respiratory tract is necessary in
    subacute inhalation studies. This includes nasal cavity, pharynx,
    larynx, trachea, main bronchi, and lungs. The exact orientation of
    these organs for trimming, embedding, and sectioning is of prime
    importance and needs special attention. More organs and tissues may be
    selected in the inhalation studies, selection usually being effected
    in the same way as for oral studies.

    5.4.4  Dermal toxicity studies

         In acute dermal toxicity studies, it is advisable to perform
    microscopic pathology on liver and kidneys. The presence of
    degenerative changes in these organs indicates that the compound is
    active transdermally. Examination of the skin is also advisable.

         In subacute dermal toxicity studies, the organs and tissues are
    selected in the same way as that described for oral tests. Of course,
    the skin deserves special attention in that both treated areas and
    normal skin in comparable areas are selected.

    5.4.5  Special studies

         In the case of sensitization or irritation studies, the selection
    of tissues depends completely on the type of test. In eye irritation
    studies, the examination of eye and conjunctivae seems adequate
    whereas in a Landstainer-Draize test, and especially in a maximization
    test, the skin may be examined. In all these cases, it may, or may
    not, be necessary to select more tissues.

    5.5  Preservation of Tissues

         Preservation of tissues can be performed by immersion, inflation
    or distension, and perfusion. Immersion is most commonly used, but in
    some circumstances may not result in satisfactory preservation.
    Microscopic examination of the lungs, for example, cannot be done
    adequately if they have not been inflated or have not been fixed by
    perfusion. When the central nervous system is a target organ, it is an
    absolute necessity to use perfusion to ensure proper fixation, as
    fixation by immersion leads to many artifacts that are
    indistinguishable from certain degenerative changes. When animals in
    long-term tests are killed  in extremis, it is advisable to use
    fixation by perfusion. Perfusion is usually essential for electron
    microscope studies of tissues.

         With perfusion, the best conditions for microscopic examination
    are obtained while effective gross examination remains possible. All
    tissues should be fixed in 10% neutral buffered formalin or another
    appropriate fixative.

    5.5.1  Immersion

         Immersion is the most used and usually the most appropriate
    method of preservation in toxicity experiments. The tissues are placed
    in the preservative and fixed for 24-48 h. Fixation at higher
    temperatures (40-50C), in a vacuum, or by use of microwaves (Gordon &
    Daniel, 1974) may shorten the fixation time considerably. Tissues
    thicker than 0.5 cm should not be fixed. The preservative/tissue ratio
    is very important and must be greater than 10:1 (volume fixation
    fluid/volume tissue) to obtain acceptable fixation. The fixation of
    intact animals with opened abdomen should not be carried out. All
    tissues and organs must be fixed separately, and some may need special
    attention. Skin and peripheral nerves must be fixed in a straight (but
    not stretched) and flat position. To achieve this, these tissues can
    first be attached to a piece of thick filter paper. The spinal cord
    can best be fixed  in situ before it is taken out. Special fixing
    solutions may be used in certain cases such as Bouin's fixative for
    the fixation of ovaries, testes, thyroid, and adrenals or Zenker's
    solution for the fixation of the eyes.

         As certain organs (pituitary, thyroid, ovaries, adrenals, and
    lymph nodes) of some of the smaller rodents are rather small, they
    should be fixed separately in smaller jars to prevent loss; various
    fixatives may be used.

    5.5.2  Inflation

         Inflation is used to preserve lungs effectively. To prevent the
    formation of artifacts that may be misjudged as emphysema, it is
    necessary for inflation to be carried out at constant pressure
    (Chevalier, 1971; Fawell & Lewis, 1971). In certain cases, however,
    even fixation of the lungs by inflation may not be adequate and
    perfusion will be preferred (for example, in inhalation studies).

         Inflation with fixative is also necessary for correct fixation of
    the urinary bladder. If the bladder is distended, urine must be
    replaced by fixative via the urethra using a syringe with a blunt
    needle. Contracted empty bladders should be partly distended with
    fixative. The reflux of fixative in the bladder is prevented by
    ligation of the urethra. Inflation may also be used for fixation of
    the digestive tract. Here again, ligation is necessary. In these
    cases, the organs have to be bisected after fixation and the interior
    surface inspected.

    5.5.3  Perfusion

         The best way to preserve tissues is by perfusion, which is
    usually effected by infusion in the left ventricle of the heart and by
    opening the sinus venosus to provide for proper circulation. Perfusion
    of isolated organs (liver, kidneys) is another possibility. Before
    perfusion, the animals are anaesthetized with a barbiturate,
    administered in combination with nitrate and heparin to ensure
    vasodilation and prevent clotting. A solution consisting of 77.5 ml
    sodium nitrite (1.25%) in water, 10 ml heparin (5000 IU/ml) and 12 ml
    pentobarbital (60 mg/ml), of which 10 ml is administered per kg body
    weight, is satisfactory. Then the blood is removed with an isotonic
    saline solution using slight overpressure. When all the blood has left
    the body via the sinus venosus or right atrium, the body may be
    perfused with the fixative.

         Perfusion is sometimes not possible especially in experiments
    where the recording of organ weights is important (i.e. in subchronic
    toxicity studies). This difficulty can be solved by increasing the
    number of experimental animals, though this may lead to a considerable
    increase in costs. Perfusion of large numbers of animals is possible
    using simple facilities at low cost (Fig. 5.1).

    5.6  Trimming

         It is important that the trimming be carried out by well-trained
    people. It must be emphasized that knowledge of pathological phenomena
    is necessary, as it frequently happens that the person responsible for
    trimming cuts out the "good looking" areas and discards the tumours
    present in the organs. The tissues must be sliced in such a way that
    the cut surfaces present the largest possible area for examination.
    The use of a special trimming scheme during the procedure may be

         The kidneys should be sectioned through the cortex and medulla,
    one kidney mid-longitudinally, the other mid-transversely.

         The brain must be cross-sectioned at, at least, three sites: the
    frontal cortex with basal ganglia, parietal cortex with thalamus, and
    cerebellum with pons.

         The lungs should be sectioned transversely, parallel to the long
    axis of the body. These sections must include the main bronchi and

         The hollow organs should be trimmed in such a way that a
    cross-section from mucosa to serosa is obtained.

    FIGURE 28

         Tumours or tumorous masses usually need to be trimmed in several
    portions. Preferably, tissues surrounding the tumour should be

         When intestines are fixed as a roll, the roll can best be
    embedded as such, since, trimming of these rolls is practically
    impossible, even after fixation.

         For certain organs, special trimming procedures are needed. For
    example, the nasal cavity should be sectioned transversely at three
    sites. The trimming of the larynx/pharynx is crucial in order to
    obtain a section that can be properly interpreted.

         Trimmed tissues should have a maximum thickness of 2-3 mm for
    satisfactory processing.

    5.7  Storage

         Material not used for processing should not be discarded, but
    should be stored in airtight jars or plastic bags to ensure that the
    tissues do not dry out. Plastic bags are an excellent way of storing
    tissues in minimum space. The bags should be clearly and permanently
    labelled. The tissues should be stored, at least, until the
    microscopic examination has been completed and the findings adequately
    evaluated. If at all possible, the tissues should be stored for a long
    period (e.g. material from 90-day studies should be stored for 2 years
    and that from chronic experiments for 5-10 years), but storage
    facilities may prevent this. Tissue blocks and sections can be stored
    in a cool area for a considerable time (10 years or more).

    5.8  Histological Techniques

         Embedding in paraffin or polymer-containing paraffins or waxes is
    advisable. The embedding procedures may be shortened considerably by
    using automatic tissue-processing equipment and special frames in
    which the paraffin blocks can be made in large numbers (Fig. 5.2).
    Proper and exact labelling of the blocks is a necessity. The blocks
    may be prepared in such a way that they can be placed as they are in
    the microtomes.

         Tissue sectioning can be performed at a thickness of 4-6 mm and
    sections can be stained routinely with haematoxylin and eosin or a
    comparable routine stain. Serial sectioning can best be done with the
    help of an engine-powdered microtome.

         The use of semithin sections (1 m) is of considerable importance
    for specific organs such as bone-marrow, kidneys, lymph nodes, spleen,
    and endocrine glands. These sections can now be made on special

    FIGURE 29

    microtomes, that cut the normal paraffin blocks with glass knives. Of
    course, this procedure should only be followed when specific details
    have to be followed during the microscopic examination. The use of
    semithin Epon sections yields even better results.

         It will often be necessary to use special staining techniques on
    tissues in order to provide more information on the presence of
    carbohydrates, proteins, fats, elements, or certain structural
    organizations. Special fixation is sometimes necessary for appropriate
    staining. Bones and calcified tissues have to be decalcified. Eyes
    usually need to be fixed and embedded differently. Blood vessels may
    be stained with Sudan black in order to study vascular lesions.

    5.9  Special Techniques

    5.9.1  Enzyme histochemistry

         Enzyme histochemistry is a technique used to detect the activity
    or presence of an enzyme in a tissue by incubating the fresh tissue in
    an appropriate medium, so that a fine coloured granular precipitation
    forms wherever the enzyme is present.

         The use of the enzyme-histochemical technique is not common in
    toxicity testing. Nevertheless, the technique is a valuable one, since
    it provides information about the metabolic activity and function of
    the tissues. Furthermore, it introduces the possibility of correlating
    biochemical with histological findings that may lead to a more correct
    interpretation. Enzyme-histochemical investigations also make it
    possible to visualize certain differences in enzyme activity within
    the structural organization of tissues.

         In some cases, a decrease in enzyme activity in certain cells is
    compensated for by an increase in other cells within the same organ.
    Such biological differences can only be detected by
    enzyme-histological investigation, when the results of biochemical
    determinations are within normal limits and no alterations are seen
    using conventional histological techniques. Enzyme-histological
    investigations can easily be incorporated in routine toxicity testing,
    if necessary.

         Relatively small pieces of tissue are quickly frozen in an inert
    liquid (i.e. isopentane), or cooled in liquid nitrogen or a mixture of
    solid carbon dioxide and methanol. Storage of the tissues is effected
    at -70C. Cryostat sections are prepared and incubated in specially
    prepared media. Good reference works for the methods have been
    published (Barka & Anderson, 1965; Pearse, 1968, 1972). To obtain
    optimum information, the prepared section should be examined by
    semiquantitative methods.

         An enzyme histochemical investigation can also be performed at
    the electron microscope stage (Geyer, 1973). In this case, the
    activity or presence of an enzyme is determined by the deposition of
    electron-dense material at the sites where the enzyme is located. It
    is evident that these delicate techniques are not easy to apply in
    routine toxicity testing. They may, however, be of great importance in
    specific studies on the biological effects of a compound on cellular
    components, or to detect early damage.

    5.9.2  Autoradiography

         Autoradiography is based on the principle that radioactive
    substances present in tissues are able to produce an image on a
    photographic film or plate. The radiations emitted by the radioactive
    substances must be of relatively weak energy, so that they will have a
    short range in tissues and emulsions. When the range is too long,
    developed silver grains can be found in the emulsion far away from the
    radioactive source.

         In this respect only alpha particles, beta particles, and Auger
    electrons are useful since electromagnetic radiations such as gamma
    and X-rays give poor results due to their penetrating power.

         Tritium (3H) has been most frequently used in autoradiographic
    studies, although 14C and 32P are also being used as tracers.

         Autoradiography, at both light microscopic and ultrastructural
    levels, can be used in kinetic studies on tissues by incorporating
    radioactive-labelled bases in DNA or RNA resulting in the production
    of silver grains on a photographic film covering the section. The
    different methods have been extensively described for nondiffusible
    substances (Baserga & Malamud, 1969; Rogers, 1967) as well as for
    diffusible substances (Roth & Stumpf, 1969).

         Apart from studying the kinetics of tissues, autoradiographic
    techniques can also be used to study the distribution of radiolabelled
    compounds and their metabolites. This, again, can be done at light
    microscopic and ultrastructural levels.

         The technique of whole-body autoradiography as developed by
    Ullberg et at. (1972) is a valuable tool, in this respect, since the
    distribution of a compound can qualitatively (or even
    semiquantitatively) be studied in the whole body. In addition, pieces
    can be cut out to use for microautoradiographic research.

         Substances may also be labelled with fluorescent chemicals
    permitting their detection by illumination of the sections with
    ultra-violet radiation. The technique of fluorescence microscopy can
    also be used for the detection of certain dyes that possess
    autofluorescing properties. Some substances, such as the
    catecholamines, can be made visible by reaction with formaldehyde

    which converts mines into fluorescent substances (Ernko & Risnen,
    1966). Fluorescence in tissues can be measured quantitatively, thus
    facilitating controlled conditions (Ploem et al., 1974).

         Autoradiographic studies cannot be incorporated easily into
    routine toxicity experiments. However, the technique may be a very
    important tool in special studies.

    5.9.3  Immunofluorescence and immunoenzyme techniques

         Immunofluorescence techniques are primarily used in determining
    the presence or absence of certain antibodies or antigens in tissues.
    Antigens are detected by binding them to specific antibodies. If a
    specific antiserum is conjugated with a fluorescent dye, the specific
    antigen-antibody complex formed can be seen by studying the tissue
    section using ultraviolet radiation microscopy. This direct
    immunofluorescent technique can be replaced by a more sensitive
    indirect method in which the substance actually rendered fluorescent
    is not the antigen under consideration but an intermediate material
    the distribution of which corresponds precisely to that of the antigen
    being studied. Information on the principle of these techniques is
    available (Goldman, 1968; Nairn & Marrack, 1964).

         The use of enzyme conjugates has recently been developed
    (Sternberger, 1974). This system is used when antigen-antibody
    complexes have to be localized at ultrastructural levels.

         Immunofluorescence or immunoenzyme techniques will not be used in
    most routine toxicity experiments, but they may be of importance in
    special studies such as those described in Chapter 7.

    5.9.4  Electron microscopy

         Transmission electron microscopy is the most commonly used
    technique for studying the ultrastructure of tissues. Different
    fixation, embedding, and cutting techniques have to be used to obtain
    ultrathin sections of tissues (Flauert, 1973, 1974, 1975; Hayat, 1970,
    1972, 1973). At the ultra-structural level, very minute changes can be
    detected and they have to be distinguished from the many artifacts
    that can be introduced during the different processing procedures.
    Under optimum conditions, tissues can be examined at high

         As tissue examination by electron microscopy is very time
    consuming and costly, it is important only to select tissues from
    those experiments that justify it. Ultrastructural studies have
    contributed enormously to our knowledge of molecular biology. In the
    case of toxicology, ultrastructural studies are always carried out as
    a secondary investigation, and are usually applied to
    ultrastructurally, specific, pathological changes already studied in
    detail at the light microscopic level, in order to secure a better

    judgment of the importance of the lesion. Ultrastructural studies are
    also used to confirm that target organs that appear normal at a
    certain close level using light microscopy, do not in fact show
    pathological changes.

         Scanning electron microscopy (SEM) techniques, that have come
    into use in recent years, provide 3-dimensional images of complete
    biological units and also chemical information (Hayes, 1973). Here
    again, special processing procedures may be needed, although, in
    certain cases, formalin-fixed and paraffin-embedded material can be
    used. Natural surfaces, dissected material, sectioned tissue, and
    living specimens may be studied. SEM is a valuable tool in biology
    but, for toxicological investigations, its use is still rather
    restricted, apart from the possibility of obtaining quantitative
    chemical information. The development of analytical electron
    microscopy, using the principles of X-ray microanalysis, also makes it
    possible to correlate tissue ultrastructure and chemistry (Hayes,
    1973; Weavers, 1973).

    5.10  Microscopic Examination

         Routine histopathological examination is very important and
    should be carried out correctly. First of all, the sections to be
    studied should be of good quality. Additional sections and special
    stains must be prepared if necessary. Special stains are used to study
    and describe individual lesions; they may also be used to examine
    certain organs or lesions to permit better judgment of
    semiquantitative comparison. For example, when haemosiderosis is found
    to be an important effect, special stains, based on the reaction of
    the dye with iron, facilitate semiquantitative analysis of the degree
    of the lesion.

         Good microscopic equipment is necessary to perform optimum
    microscopic examination. Sources of ultraviolet radiation and
    polarized light should be available.

         The microscopic examination must be carried out by well-trained
    pathologists or other persons trained and experienced in the field of
    laboratory animal pathology. The use of parapathologists for routine
    microscopy in toxicity and carcinogenicity experiments is extremely
    valuable and is important for overcoming a shortage of personnel,
    trained in laboratory animal pathology (Toxicol. appl. Pharmacol.,
    1975). Experience has shown that well-trained parapathologists can
    become very skilful in microscopy and in screening sections for
    abnormalities. With further experience, they are also able to describe
    certain lesions. It is the pathologist's duty to see that all lesions
    observed are described and interpreted correctly, and to check the
    sections for any lesions that may have been overlooked.

         It is a serious mistake, in an experiment, to undertake
    microscopic examination before the results of other examinations such
    as biochemical determinations, haematology, and organ weights, are
    available, for the information obtained from these procedures may give
    important directions for the microscopic study. For example, an
    increased thyroid weight may lead to more careful examination of the
    thyroid for hyperplasia and may prompt histometric determinations.
    Lower lymph node weight may point to semiquantitative examination of
    these organs with regard to reduced immunocapacity (Cottier et al.,
    1972). Differences or changes in the blood picture call for more
    detailed study of the bone marrow, for example, by preparing and
    studying 1 m sections, to detect suspected haemopoietic disturbances.

         Gross observations should always be correlated with microscopic
    findings. It is a false assumption to think that a microscopic
    examination will be more objective when performed blindly; knowledge
    of clinical signs and macroscopy are indispensable for a meaningful
    examination. Of course, the pathologist must be careful not to let
    knowledge of clinical effects influence his objective evaluation of
    the tissues; thus, sections of the tissues considered to be involved
    may have to be re-examined blindly. If such a re-examination is
    carried out, semiquantitative scoring of the extent of the lesion will
    also help to detect a possible dose-effect relationship. Should there
    be any doubt in interpreting the significance of a lesion, it is
    advisable to consult other pathologists, all of whom should re-examine
    the slides blindly.

    5.10.1  Number of animals and number of organs and tissues studied

         In acute and rangefinding tests, the organs and tissues are
    usually fixed and examined microscopically after processing. In
    subacute and long-term studies, and, sometimes, in rangefinding tests,
    it is customary initially to examine only the tissues of the highest
    dose group, the control group and the target organs, if known. In
    addition, all grossly observed lesions are processed in the
    intermediate and low-level groups. If the results of the microscopic
    examination of the highest dose group indicate a need to examine
    certain organs at lower levels, this can be done at a second stage.

         In chronic toxicity experiments, all males and females of the
    highest dose group should be examined. It is also advisable to examine
    all control animals completely, since this will be the only way to
    ascertain the incidence of tumours and other "lesions" normally
    occurring in the strain of animals used. Such information is
    indispensable for correctly evaluating the significance of changes
    observed in exposed animals.

         In carcinogenicity experiments, where larger numbers of animals
    per group are usually used, some laboratories restrict microscopic
    examination mainly to the grossly observed abnormalities and tumours,

    and perform complete histological examinations on only 15-20 male and
    15-20 female survivors of the highest dose group and of the control
    group, on the assumption that a well-performed gross examination will
    detect most of the tumours present. Histological examination of all
    tissues of 15-20 animals of each sex will give some information on the
    possible presence of certain pre-malignant hyperplastic or neoplastic
    changes not grossly observed. If such lesions are found, the
    histological examination should cover all animals, while small organs
    which may bear tumours that cannot be grossly observed (thyroid,
    pituitary, adrenals) must be examined histologically.

    5.10.2  Description of the lesions

         For the microscopic examination, it is essential to use a
    check-list to detect losses, especially of small organs lost in the
    processing procedure. All organs and tissues examined should be
    listed, even though no abnormalities are seen, while all lesions found
    should be described clearly and accurately. It is essential to
    describe lesions in a semiquantitative way using such words as slight,
    moderate, strong, and very strong to indicate the extent of the
    lesion; it is also essential to indicate the criteria used to
    investigate and classify abnormalities. It will often be necessary to
    re-examine certain lesions in order to obtain a well-balanced
    semiquantitative judgment. Several reference works may be of help in
    the classification of tumours and other lesions (Beveridge & Sobin,
    1974; Cotchin & Roe, 1967; Ribelin & McCoy, 1965; and Turusov, 1973).

         Certain lesions found by microscopic examination may, in fact,
    consist of several entities. In such cases, it may be important to
    classify these entities independently. For example, perilobular liver
    degeneration may be combined with bile duct proliferation, but one of
    the two phenomena may be more extensive than the other with respect to
    dose level and time. For a better interpretation, it is preferable to
    describe and classify such lesions independently.

    5.11  Presentation, Evaluation, and Interpretation of Pathological

         A well-performed and well-described microscopic examination is
    not complete if the results cannot be studied and compared easily and
    adequately. The lesions found in the different groups should be
    tabulated according to organ, group, and sex in order to facilitate
    comparison of the incidence of such lesions in the different groups.
    If, in addition, a semiquantitative estimate of the extent of the
    lesions is made, it will be easy to see whether they are
    compound-induced and increase with time and dose.

         It is important that these tables include details such as the
    number of animals necropsied and the number examined microscopically,
    per group and sex. In addition, it is advisable to state, the exact
    number of organs or tissues examined microscopically, since it is well

    known that, in every toxicity experiment, organs and tissues get lost
    during the processing procedure. In this way, the loss can be
    adequately estimated and the lesions found can be expressed against
    the real number of organs or tissues examined.

         For correct interpretation of the tumour tables, it is advisable
    to include hyperplastic and preneoplastic lesions as individual
    groups. The table should also include the number of days that elapsed
    from the start of the experiment until the observation of each rumour.
    This first observation may be clinical, for example, when mammary, ear
    duct, or skin tumours are involved and the time that has elapsed can
    then be called the "induction time". However, usually, tumours are
    found after death or killing  in extremis, or at sacrifice at the end
    of the experiment. Thus, in these cases, it seems most appropriate to
    use the term "time of observation". The inclusion of the "time of
    observation" of tumours in the table is of help to the investigator
    since it provides information on shorter or prolonged latency periods
    (e.g. time that has elapsed between initiation and the appearance of
    tumours). In addition, it reminds the reader to take into account the
    survival differences when expressing the results.

         In long-term experiments, the number of lesions found may be
    quite large. Furthermore, some lesions may have been noticed during a
    certain period in life, when the animals died or were sacrificed
     in extremis, while others will have been noticed at the end of an
    experiment. When such lesions are tabulated in the same table, this
    information may get lost. It seems, therefore, most appropriate in
    such long-term experiments to pool the results of the pathological
    examination for certain periods, for example the first 12 months of
    life and then for 3- to 6-monthly intervals, and to give the results
    of the examination undertaken at the end of the experiment separately.

         It is obvious that in preparing such tables, the use of an
    automated data acquisition and computer-based system will be of great
    help. These systems have been described for application to
    toxicological studies, especially for animal weights, food and drug
    consumption, organ weights, and biochemical data (Munro et al., 1972).
    In pathology, such systems offer considerable potential. The necessity
    for the correct coding of pathological changes, and the use of a code
    system set up so that detailed information about lesions can still be
    obtained, is obvious. Several systems have been described (Becker,
    1973; Enlander, 1975; Smith et al., 1972), and may be adapted for this

         Pathological examinations must always be evaluated and
    interpreted in connexion with other phenomena found. It must be
    decided whether lesions noticed are in fact pathological, and whether
    they are found in connexion with changes in other variables such as
    organ weights, biochemical tests, etc. If compound-induced lesions are
    found, they may not only be present at the highest dose level, but
    also at an intermediate dose level. In the latter case, it is

    important to determine whether there is a dose-dependent increase in
    the severity and extent of the lesion, a finding which would
    strengthen the assumption that the lesion is compound-induced.
    Moreover, a clear dose-response relationship permits better evaluation
    of the potential risk of a compound. Whenever lesions are found only
    at the highest dose level, the sections at all dose levels should be
    re-examined blindly to remove any doubt. Often the two sexes will show
    a different sensitivity to the action of a compound.

         Problems may arise when certain lesions are found more frequently
    and to a greater extent in the treated groups compared with the
    control group and compared with the common incidence of the lesion in
    the strain used. The usual attitude towards this phenomenon is to
    consider it as an effect or response.

         In the evaluation of tumour incidence, criteria such as increased
    tumour incidence, decrease in latency period, and the appearance of
    tumours in organs where they do not occur spontaneously, have to be
    considered. For this reason, detailed information on the spontaneous
    occurrence of tumours in the species and strain used is essential,
    particularly when tests indicate that the compound possesses a weak
    carcinogenic action. Simple comparison of tumour incidence in control
    animals and experimental animals at one point in time may lead to
    erroneous conclusions, especially when only one of the above three
    criteria is met. Many factors other than treatment may influence the
    incidence of spontaneous tumours.

         Statistical evaluation of the results is often necessary, but, as
    yet, there are no agreed procedures for comparing statistically the
    incidence of malignant tumours in controls and experimental groups.


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         micro-spectrofluorometer with epi-illumination operated under
         computer control.  J. Histochem. Cytochem., 22: 668-677.

    RIBELIN, W. E. & MCCOY, J. R. (1965)  The pathology of laboratory
          animals. Springfield, Charles Thomas.

    ROE, F. J. C. (1965) Spontaneous tumours in rats and mice.  Food
          Cosmet. Toxicol., 3: 707-720.

    ROGERS, A. W. (1967)  Techniques of autoradiography. Amsterdam,
         London, New York, Elsevier.

    ROTH, L. J. & STUMPF, W. E. (1969)  Autoradiography of diffusible
          substances. New York, London, Academic Press.

    SMITH, J. E., MCGAVIN, M. D, & GRONWALL, R. (1972) English-language
         computer-based system for storage and retrieval of pathological
         diagnosis.  Vet. Pathol. 9: 152-158.

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         Cancer Institute.

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         Prentice-Hall.  Toxicol. appl. Pharmacol. (1975) 31: 1-3, A
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    6.1  Introduction

         Advances in technology, throughout the world, have increased the
    number and amount of chemicals in the atmosphere. The health effects
    of inhalation of these chemicals can be predicted to some extent by
    experimental investigation. In order to simulate environmental
    conditions, a special technology has evolved relating to the design
    and operation of inhalation chambers and the generation and
    characterization of aerosols and vapours. This chapter will introduce
    this technology and review the significance of certain biological
    end-points commonly measured in inhalation studies.

         This chapter is not intended to be all-inclusive, nor to provide
    complete instructions on inhalation technology. No toxicological
    protocol is sufficient to cover all situations with all materials.
    Hence, the expertise of the individual investigator and the objectives
    of the investigation will govern the selection of the final protocol
    and the biological end-points relevant to the particular compound

         The costs associated with evaluation of toxicity by the
    inhalation route are considerably higher than for toxicity studies
    using other routes of exposure because of the cost of the specialized
    equipment used in inhalation studies and the time required for its
    calibration. In general, it can be estimated that the total cost of
    any type of inhalation study, short- or long-term, will be two to
    three times that of a comparable oral study. Because of this, it is
    important to determine when inhalation studies should be performed and
    when, and if, studies by other routes would suffice. Finally, in
    addition to costs, other resources should be considered. In most
    countries, the number of laboratories capable of performing inhalation
    studies is limited. Thus, it is essential to develop priorities for
    the selection of compounds for evaluation of inhalation toxicity.

    6.2  Need for Inhalation Studies

         The effect of a compound depends on its concentration at the
    receptors of the affected organ or system. The concentration at
    different sites and times is a function of the route of entry. Thus
    the route of entry is an important factor with regard to toxicity. No
    good substitute model for inhalation exposure as a means of direct
    exposure of the lungs has been developed, although intratracheal
    exposure has been frequently used, particularly in pulmonary 


    a  The assistance of Dr J. O'Neil, Dr M. Amdur, and Dr W. Busey in
       the preparation of this chapter is gratefully acknowledged.

    carcinogenesis studies. The distribution kinetics are different with
    pulsed exposures compared with constant inhalation exposures. With
    pulsed exposures, such as oral, intravenous, intraperitoneal, etc.,
    the concentration of the test material will reach a peak and then
    usually fall off, depending on the distribution coefficients for each
    compound and organ in question. With continuous exposure,
    concentrations in many body compartments will attain an equilibrium,
    depending again on the concentration of the test material and the
    distribution coefficient. Thus, quantitative toxicity is often quite
    different, depending on the route of entry.

         With the exception of certain drugs, man's exposure to
    environmental agents comes either from skin contact, ingestion, or
    inhalation. Inhalation is of particular importance in occupational
    exposure and in exposure to air pollutants. For airborne substances,
    the lung is the first organ that foreign chemicals encounter and it is
    one of the body's first lines of defence. Chemicals that enter the
    lung can either exert a direct effect on the cells of the lung, or be
    absorbed into the systemic circulation. Blood passing from the lung to
    the heart and then into the peripheral circulation can carry agents
    directly to other organs without passing through the detoxication
    processes of the liver. This contrasts with oral exposure where
    chemicals may be absorbed into blood that immediately passes through
    the liver and can be metabolically transformed into either more or
    less toxic compounds.

         Direct contact with irritants causes local inflammation in the
    respiratory system, the degree of which may depend on the local
    concentration and not on the total dose. For example, Kljackina (1973)
    demonstrated that inhaled bromine acts as a specific irritant of the
    respiratory system whereas bromine administered orally results in
    changes in the nervous system.

         Thus, specific reasons for performing inhalation studies include:
     (a) determination of specific responses of the respiratory tract;
     (b) assessment of the toxic hazard of agents whose principal route
    of exposure is via inhalation;  (c) investigation into the mechanism
    of toxicity of inhaled materials; and  (d) study of the comparative
    toxicity of agents administered by different routes.

    6.3  Fate of Inhaled Materials

    6.3.1  Nature of aerosols

         The nature and characteristics of aerosols have been treated in
    detail elsewhere (Cadle, 1965; Mercer, 1973a,b; Raabe, 1970), but it
    may be useful to review a few general concepts. Aerosols consist of
    finely divided particles ranging in size from about 0.01 to 100 m in
    diameter. The particles in a cloud are usually of many sizes and the
    size can be expressed as a size-frequency distribution curve which
    usually best fits a log-normal distribution. There are several ways to

    express the diameter of a particle, the common ones being count median
    diameter, mass median diameter, and aerodynamic mass median diameter.
    The last term is important as it considers each distribution as if it
    were made up of unit density spheres and measures its diameter as if
    it were acting aerodynamically like a unit density sphere. Thus,
    aerosol behaviour can be compared, regardless of the individual shape
    and density of the particles.

         The division of a solid into fine particles that become airborne
    results in a large increase in the surface area of the material. The
    consequence is a generally increased chemical reactivity of the
    material, thus accelerating all physical and chemical processes, such
    as oxidation, dissolution, evaporation, absorption, and electrical
    activity. The physiological activity of these particles also
    increases. The sizes of interest from a biological standpoint range
    from about 0.1 to 10 m. Larger particles do not usually enter the
    respiratory tract or, if they do, they are deposited in the nose.

    6.3.2  Deposition

         Material that enters the respiratory tract with the inspired air
    can either be deposited or exhaled. Many factors affect the deposition
    of particles or the absorption of vapours in the respiratory system.
    Retention of vapours is governed by the diffusion rates of the vapour,
    the solubility of the vapour in the various body compartments, and the
    degree to which these compartments have attained the equilibrium. This
    in turn, depends on the duration of the exposure, the concentration,
    and the rate of removal of the vapours. This subject has recently been
    reviewed by Stupfel & Mordelet-Dambrine (1974).

         Many more factors govern the deposition of particulates in the
    respiratory tract. Roe (1968) has divided these into physical and
    chemical characteristics of the particle, anatomical, physiological,
    and pathological factors. The size, density, and shape of the
    particles are the physical variables that determine their aerodynamic
    behaviour. The Task Group on Lung Dynamics (Morrow et al., 1966) has
    discussed deposition as a function of particle size.

         Three distinct physical processes act on particles suspended in
    the atmosphere to cause them to be deposited in the respiratory tract.
    Inertial impaction results from the tendency of particles to move in
    straight lines. The repeated branching and subdivision of the
    respiratory tract causes particles to be impacted on surfaces,
    particularly near the bifurcations. Inertial forces are greater with
    larger particles. Sedimentation due to gravity also causes particles
    to strike the surface of the respiratory tract. Finally, Brownian
    movement, particularly of smaller particles, causes them to be
    deposited in the lung. The effectiveness of these mechanisms depends
    on the anatomy of the respiratory tract, the size of the particle, and
    the breathing pattern. During artificially induced hyperventilation in

    rabbits, more material was deposited in comparison with controls
    (Velickovskij & Kacnelson, 1964). Normal deposition and deposition in
    diseased states are discussed in detail by Albert et al. (1973), Brain
    & Valberg (1974), Goldberg & Laurenco (1973), Macklem et al. (1973)
    and Stuart (1974).

         Aerosols are deposited all along the respiratory tract. Large
    particles (5-10 m) are mainly deposited in the upper respiratory
    tract, including the nasal cavity. The depth of penetration increases
    as particle size decreases and particles in the 1-2 m range are, for
    the most part, deposited in the alveoli. As a first approximation, 25%
    of inhaled particles are exhaled, 50% are deposited in the upper
    respiratory tract, and 25% are deposited in the lower respiratory
    tract (Morrow et al., 1966).

    6.3.3  Clearance

         Soluble particles readily dissolve at the site of deposition.
    Generally, they enter the bloodstream and then behave as if they had
    been intravenously injected. Insoluble particles can be removed from
    the respiratory tract by several mechanisms depending on the site of
    deposition. Particles that are deposited on the mucous blanket are
    carried towards the pharynx by the cilia and are usually swallowed or
    expectorated. Hence, it is impossible to completely separate
    respiratory exposure from gastrointestinal exposure. The rate of
    clearance by the mucociliary escalator has been measured in man by a
    number of investigators (Albert et al., 1969, 1973; Camner et al.,
    1972, 1973; Morrow, 1970; Sanchis et al., 1972) and animals (Albert et
    al., 1973; Thomas, 1969; Velickovskij & Kacnelson, 1964; Watson et
    al., 1969).

         Particles that are deposited in the deep non-ciliated portion of
    the lung clear more slowly. One mechanism responsible for alveolar
    clearance is phagocytosis. Although alveolar macrophages engulf
    particles within a few hours, there is evidence that macrophages do
    not carry them actively to the mucociliary escalator in the first few
    days following inhalation (Camner et al., 1977). Recent studies
    indicate that there are other mechanisms of alveolar clearance. Tucker
    et al. (1973) have shown alveolar clearance by normal interstitial
    drainage pathways. Casarett (1972) and Morrow (1973) described a
    possible mechanism whereby particles could move from the alveolus up
    to the terminal bronchiole, and then to the ciliated epithelium for
    removal on the mucus. Very fine particles can also enter the blood
    directly through the lymphatic system. Finally, particles can dissolve
    and be absorbed into the bloodstream. Some particles remain in the
    alveolar region of the lung for considerable periods of time, during
    which dissolution forces can operate.

    6.4  Dose in Inhalation Studies

         The term dose has many meanings depending on the background and
    expertise of the investigator. In toxicology, dose is usually defined
    as the mass of material introduced into the animal, and it is often
    divided by the body weight. Hence, toxicologists often speak of dose
    in mg/kg. Even this is misleading, to some extent, because the
    material may not interact in any way, and because of other reasons
    that become apparent in species comparisons. The actual dose or amount
    of a substance entering the internal milieu of the body depends on the
    concentration and the particle size of the inhaled material, the
    duration of the exposure, and the breathing variables of the test
    species. Thus, the actual absorbed dose is difficult to determine and
    inhalation toxicologists often refer to exposure conditions instead of
    dose. The exposure must be defined both in terms of concentration  (C)
    and time  (t), and is sometimes expressed as the product of these two
     (Ct) (MacFarland, 1968) (see also Chapter 1). While the  Ct product
    is not a true dose, it can be used in a similar fashion. Haber (1924)
    recognized this and Haber's rule states that, for a fixed  Ct
    product, the response will be the same. This rule holds reasonably
    well over limited ranges of  C and  t, but deviations occur when
    extreme values of the variables are examined.

         More frequently, inhalation toxicologists keep the time constant
    and vary the concentration of the test material. Hence, they measure
    an LC50, i.e. the median concentration to which animals are exposed
    for a specified time that will kill 50% of the animals within a fixed
    period of time after exposure. It is implicit in LC50 data that the
    durations of both the exposure and post-exposure period are
    standardized. Comparative LC50 data are often obtained for 4-h
    exposures and a 14-day post-exposure observation period (Carpenter et
    al., 1949; Pozzani et al., 1959).

    6.5  Choice of Species

         The ideal subject for studies relevant to man is man himself.
    However, human volunteers can only be used where the toxicological
    hazard is already reasonably well defined and accepted. Human studies
    have been conducted recently with chlorinated hydrocarbon solvents and
    common air pollutants (Andersen et al., 1974; Hazucha et al., 1973;
    Stewart, 1972; Stewart et al., 1970a,b, 1973). These experiments were
    well controlled and monitored and the exposure levels were low.

         Rats, dogs, and monkeys have been the species most used for
    inhalation toxicity studies, although investigators have also used
    mice, hamsters, guineapigs, rabbits, cats, miniature swine, and
    donkeys. The choice of species should be made, primarily, with a view
    to extrapolating the experimental results to man. However, choice on
    this basis alone is difficult since the validity of such an
    extrapolation is often uncertain. When selecting the particular

    species for study, the following factors must be considered: the
    comparative morphology of the respiratory tract; the presence or
    absence of lung disease or susceptible states; and the similarity of
    biochemical and physiological responses to those in man.

    6.5.1  Anatomical differences

         The respiratory systems of various laboratory animals and man
    differ widely. As man is a primate, it is sometimes erroneously
    assumed that the monkey is a good model for inhalation studies, but
    marked differences are noted when comparing human and sub-human
    primate respiratory systems. In the monkey, the end airway is always a
    respiratory bronchiole, whereas this is rarely the case in man.
    Furthermore, the monkey's lung is not lobulated.

         The most commonly used laboratory animals are quadrupeds, and
    their respiratory systems are horizontal and not vertical as in the
    primate. This is important because the aerodynamics of a horizontally
    arranged lung are different, and particle deposition will also be
    different. In human subjects, maximum dust deposition is in the upper
    portion of the lung; in experimental animals, maximum deposition in
    the more ventral portions of the lung is usual.

         The gross anatomy of the respiratory systems of the various
    laboratory animals and man is quite different. In animals such as the
    rat and guineapig, the nose contains highly developed tortuous
    turbinates. In monkeys and man with relatively smaller noses, the
    turbinates are less complex. These differences in nasal anatomy are
    especially important in studies involving exposure to particulates.
    More particles impinge in more complex turbinate systems and will not,
    therefore, reach the deeper portions of the respiratory system.

         The subgross anatomy of the lungs of various laboratory animals
    and man has been detailed and compared by McLaughlin et el. (1961a,b,
    1966). The authors have grouped the common laboratory animals and man
    in three basic categories based on subgross pulmonary anatomy
    (Table 6.1). They grouped the various animals on the basis of lung
    lobulation, the presence or absence of respiratory bronchioles, the
    presence or absence of terminal bronchioles, pulmonary artery/
    bronchial artery shunts, and the termination of the bronchial
    arteries. Their studies indicate that the pulmonary anatomy of the
    horse most closely resembles that of man.

    6.5.2  Physiological considerations

         Pertinent differences in the lung physiology of various species
    must be considered in inhalation toxicology. Normal values or ranges
    for several species, including man, are summarized in Table 6.2
    (Sanockij, 1970a).

        Table 6.1  Comparative subgross anatomy of the lunga
    Species      Lung    No. of lobes     Lobulation          Respiratory        Terminal      Pulmonary/artery    Termination        Pleura
                 type                                         bronchioles        bronchioles   Bronchial/artery    of bronchial
                         left    right                                                         shunts              arteries

    Cow          I       3       5(4)     well developed      extremely poor     present       present             distal airway      thick
    Sheep        I       3       4        well developed      extremely poor     present       present             distal airway      thick
    Pig          I       3       4        well developed      extremely poor     present       not present         distal airway      thick
    Monkey       II      3       4        not present         very well          absent        not present         distal airway      thin
    Dog          II      3       4        not present         very well          absent        not present         distal airway      thin
    Cat          II      3       4        not present         very well          absent        not present         distal airway      thin
    Guineapig    IIa     3       4        not present         fairly well        absent        present             distal airway      thin
    Rat          IIa     1       4        not present         fairly well        absent        few present         distal airway      thin
    Rabbit       IIa     3       4        not present         fairly well        absent        present             tertiary           thin
                                                              developed                                            bronchus
    Horse        III     (3)     (4)      imperfectly         poorly             present       present             distal airway      thick
                                          developed           developed                                            and alveoli
    Man          III     2       3        imperfectly         poorly             present       present             distal airway      thick
                                          developed           developed                                            and alveoli

    a  From: McLaughlin et al. (1961a,b, 1966).

    Table 6.2  Some physiological indices of man and animalsa
                                                 Man       Dog       Cat       Rabbit    Guineapig    Rat        Mouse

    Body surface (m2)                            1.8       0.528     0.2       0.18      0.040        0.030      0.006
    Relation body surface to body weight         0.0257    0.044     0.066     0.072     0.12         0.15       0.3
    Basal metabolism (kJ/kg)                     105       222       --        188       360          615        711
    Frequency of respiration (min)               14-18     10-30     20--30    50-100    80-135       110-135    140-210
    Size of alveoli (m)                         150       100       100       --        --           50         30
    Surface of lungs (m2)                        50        100       7.2       5.21      1.47         0.56       0.12
    Relation of lung surface to body weight      0.7       8.3       2.8       2.5       3.2          3.3        5.4
    Inhaled air (ml)                             616       40-60     --        --        1.75         0.865      0.154
    Lung ventilation (ml/min)                    8732      --        1000      600       155          73         25
    Relation of lung ventilation to body         0.13      --        0.30      0.29      0.33         0.05       1.24
    weight (ml/min/g)
    Consumption of oxygen (ml/kg/hr)             203.1     3600      9420      522.7     2180         2199       3910
    Elimination of CO2 (ml/kg/h)                 168.8     --        --        --        --           2650       4240
    Coefficient of respiration                   0.82      --        --        0.83      --           0.82       0.85-1.33
    Pulse frequency for 1 min                    70-72     90-130    120-180   150-240   206-280      300-500    520-780

    a  From: Sanockij (1970a).
        6.5.3  Disease and susceptibility states

         Most toxicological investigations are performed on healthy
    animals. However, epidemiological studies have indicated that during
    air pollution episodes the populations at greatest risk are the young,
    the aged, and those people with pre-existing cardiopulmonary disease.
    One task of the inhalation toxicologist is to identify those segments
    of the population that are particularly susceptible to the presence of
    airborne contaminants. Certain animal models of diseased or stressed
    states have been described (Boyd et al., 1974; Drew & Taylor, 1974;
    Silver et al., 1973; Taylor & Drew, 1975; see also Chapter 2) which
    could be used or could be adapted for use in inhalation studies. The
    most commonly used model of this nature is the papain-induced
    emphysematous animal (Gross et al., 1965; Martorana et al., 1973;
    Niewoehner & Kleinermann, 1973; Snider et al., 1974). Both aged and
    neonatal animals could be used as models of high susceptibility
    groups. Disease and susceptibility states are important considerations
    in the selection of the species and, in some cases, even the strain of
    animal to be investigated since it is well known that the incidence of
    cancer differs considerably among certain species and strains.

         For example, Kuschner et al. (1975) recently reported a high
    incidence of respiratory tract tumours in rats after exposure to
    oxybis[chloro-methane] (bis(chloromethyl)ether) but only a few tumours
    in hamsters. These tumours were about equally divided between
    esthesioneuro-epitheliomas and bronchogenic squamous cell carcinomas.
    Leong et al. (1975) repeated these experiments; however, all the
    tumours in Leong's study were nasal tumours with no bronchogenic
    carcinomas. The only difference noted in the protocol was that Leong
    et al. used specific-pathogen-free, caesarean-derived rats, whereas
    Kuschner et al. used Sprague-Dawley rats, that were not

    6.6  Duration of Exposure

         Acute inhalation toxicity studies usually consist of a single
    exposure (or occasionally a few exposures) of not more than 8 h.
    Repeated exposure studies consist of a number of daily exposures for
    fixed periods of time. Occasionally, investigators terminate exposure
    after several days or weeks, and then maintain the animals in the
    colony to observe delayed development of long-term effects (Kuschner
    et al., 1975).

         The duration of chronic studies varies considerably. One logical
    proposal is based on the life span of the test species (Sanockij,
    1970b). If, for example, one considers that toxic signs will appear in
    man after an exposure over 10% of his life span (7 years), animals
    should also be exposed for 10% of their life span. Thus, rats should
    be exposed for 3-4 months, and larger animals for a somewhat longer
    period. Some authors (Sidorenko & Pinigin, 1970) consider that even
    continuous exposure for 3-4 months is insufficient to simulate

    lifetime exposure in man, and many studies last longer, some up to 5
    years (Lewis et al., 1974). Powell & Hosey (1965) consider the minimum
    duration of a chronic study to be one year, the animals being exposed
    6 h a day for 5 days a week. This corresponds to a significant portion
    of a rodent's lifetime.

         Chronic studies are conducted to determine the effects of
    long-term exposure to compounds and particularly (in the USSR) to
    establish minimum effect levels (Limch). In order to evaluate the
    effects of long-term exposures, such as elevated incidences of
    infection, emphysema, or the induction of cancer, inhalation controls
    should be run concurrently. Two species are usually used. Occasionally
    the toxic effects and the mechanism of chronic toxicity are entirely
    different from those manifested in acute exposures. Benzene, for
    example, is a central nervous system depressant at high
    concentrations, while, at low concentrations over long periods of
    exposure, it affects the hematopoietic system.

         The selection of concentration for chronic studies is difficult.
    For example, in the USA, concentrations are chosen that do not produce
    mortality and produce only minimal changes in other biological indices
    of toxicity during limited studies. In the USSR, the concentration
    selected is below the Limac. In order to investigate dose-response
    relationships, it is advisable to use at least three different dose
    levels, hoping that the highest level chosen will produce quantifiable
    effects and that the lowest level selected will produce minimal or
    even no effects. In the USSR, research workers often use as the
    highest level the concentration which, in single short-term exposures
    of human subjects, does not produce any effect during the study of
    reflex reactions.

         When investigating the various biological indices of toxicity, it
    is necessary to carry out measurements more frequently during the
    early stages of the study (Camner et al., 1972, 1973). Studies carried
    out at the Institute of Labour Hygiene and Occupational Medicine of
    the USSR show that the time to the display of the first signs (period
    for initial decompensation) varies considerably. Thus, the variables
    are recorded at 1, 4, 8, 15, and 30 days and monthly thereafter. After
    terminating the exposure, the frequency of measurements may be
    increased again to record any early changes.

    6.6.1  Intermittent versus continuous exposures

         Long-term exposures are usually patterned on projected industrial
    experience, giving the animals a daily exposure of 6-7 h, 5 days a
    week (intermittent exposure), or on a possible environmental exposure,
    with 22-24 h of exposure per day, 7 days a week (continuous exposure),
    with about an hour for feeding the animals and maintaining the
    chambers. In both cases, the animals are usually exposed to a fixed
    concentration of test materials. Thus, neither situation approaches
    actual human experience, where concentrations of atmospheric

    pollutants are continuously fluctuating by one or two orders of
    magnitude. A major difference to consider between intermittent and
    continuous exposure is that with the former there is a 17-18 h period
    in which animals may recover from the effects of each daily exposure,
    and an even longer recovery period at weekends. The recovery period in
    some cases is extremely important as in the case of continuous versus
    intermittent exposure to dichlormethane (Haun, 1972).

         The choice of intermittent or continuous exposure depends on the
    objectives of the study and on the human experience that is to be
    simulated. However, certain technical difficulties must be considered.
    For example, the advantages of continuous exposure for simulating
    environmental conditions may be offset by the necessity of watering
    and feeding during exposure, and by the need for more complicated (and
    reliable) aerosol and vapour generation and monitoring techniques.
    Intermittent systems require simpler chambers, since provision for
    food and water is not necessary. The contaminant dispersal systems are
    also simpler as they need to operate for only 6-7 h per day.

    6.7  Inhalation Systems

    6.7.1  Facilities required

         It is more advantageous to build facilities designed specifically
    for inhalation studies than to modify existing buildings. High
    cellings are necessary for housing exposure chambers and related
    equipment, such as aerosol and vapour generators, filters, flowmeters,
    etc. A constant supply of clean filtered air with temperature and
    humidity controls should be available for both the chamber rooms and
    the chambers themselves. Adequate floor space should provide access to
    at least two sides of the chambers, and the chambers themselves should
    be separated by a small space to avoid heat transfer between chambers.

    6.7.2  Static systems

         Inhalation systems can be characterized as static, when the agent
    is introduced into a chamber as a batch and then mixed, or dynamic,
    when airflow and introduction (and removal) of agent are continuous.
    The duration of static exposure is limited by:  (a) the gradual
    depletion of oxygen;  (b) the accumulation of carbon dioxide;
     (c) the accumulation of water vapour; and  (d) the gradual increase
    in temperature inside the chamber. In spite of these limitations,
    static systems are of great practical usefulness in assessing acute
    toxicity, particularly when the supply of material is limited.
    Procedures similar to those described by Draize et al. (1959) are in
    use today for screening commercial products. Another use of static
    systems is for the exposure of animals to biological aerosols. It is
    difficult to generate a viable biological aerosol particle
    continuously because of the limited amount of material available;
    thus, material is dispersed in a large chamber, mixed, and the animals
    exposed through nose tubes connected to the test atmosphere (Jemski &
    Phillips, 1965).

    6.7.3  Dynamic systems

         Today, most inhalation facilities use dynamic systems where the
    airflow and introduction of agents are continuous. The theoretical or
    nominal concentration of chemicals in a chamber can be calculated as

                              flow of chemical
              concentration =                  
                                 flow of air

    Many factors, including wall loss, losses on the skin and fur of
    animals, and uptake by the test animals, cause the actual
    concentration to be somewhat less than the nominal concentration.
    Thus, the concentration should always be measured by an appropriate
    instrument or technique rather than reporting the nominal

         When material is introduced into a chamber, the concentration
    builds up exponentially according to equations originally described
    and verified by Silver (1946). If perfect mixing occurs, the
    concentration can be calculated according to the following equation:

                         C =  (w/b) (1 - exp (-bt/a))                     (1)

    where  C = the concentration of material at time  t; w = amount of
    material introduced per minute;  a = volume of the chamber;
     b = flow of air through the chamber.

         The fraction of equilibrium concentration  (w/b) attained in
    time  t is:

                                 = 1 - exp (1 bt/a)                     (2)

    Thus, the time required to reach 99% (t99) of the equilibrium
    concentration is:

                        0.99 = 1 - exp (-bt99/a)                        (3)


                             t99 = 4.6052 a/b

    This equation may be given the general form:

                               tx =  Ka/b                                (4)

    where  x equals % nominal concentration attained in time  t and


    Values of  K for various values of  x are tablulated below:

                            x                K
                           99              4.6
                           95              3.0
                           90              2.3
                           85              1.9
                           80              1.6

         There are several features of interest in the above equations.
    Since the concentration build-up is exponential, the concentration
    will theoretically never reach a constant value. However, in practice,
    the concentration is not detectably different once the equilibration
    time is equal to t99 or longer. The clearance curve of removal of
    chemical from the chamber after the flow of chemical is discontinued
    also follows an exponential curve (Fig. 6.1). Thus, to ensure that
    little or no material remains in the chamber, it should be operated
    for t99 after discontinuing the flow of chemical. This procedure
    also compensates for the time required for build-up of material in the
    chamber. Finally, it should be noted that in the general equation,
     tx is only a function of the volume of the chamber and the flow of
    air through the chamber. Thus, if the ratio of  a/b = 1,
    t99 = 4.6 min, t95 = 3 min, etc. A 150-litre chamber operated at
    30 litre/min would have  a/b = 5 and t99 = 5 (4.6 min) = 23 min.
    MacFarland (1976) has recently reviewed these principles and has also
    discussed ways of decreasing t99 by manipulating flow rate.

    6.7.4  Typical whole-body systems

         Inhalation exposure technology has been recently reviewed by Drew
    & Laskin (1973) and earlier descriptions and requirements have been
    given by Frazer et al. (1959), Hinners et al. (1966, 1968), and Roe
    (1968). The simplest inhalation system would be a box with facilities
    for air intake and exhaust. This concept has been used for many years
    in chambers similar to that described by Drew & Laskin (1973)
    (Fig. 6.2). In this system, a cylindrical glass battery jar is mounted
    in a horizontal position; a frame holds the jar and provides support

    FIGURE 30

    for a panel which is mounted against the open end and serves as a
    closure. Various openings can be cut in the panel for the introduction
    of the pollutant and for monitoring the concentration.

         Studies at the University of Rochester on the toxicity of
    radioactive materials contributed significantly to the development of
    the technology of inhalation exposure. The original test chamber was
    cylindrical with cones on the top and bottom. A modified chamber in
    the shape of a hexagon with pyramidal ends (Wilson & Laskin, 1950) is
    known as the "Rochester Chamber". A final modification with a square
    cross-section as shown schematically in Fig. 6.3 is known as the "NYU
    Chamber" (New York University Chamber) (Laskin et al., 1970). These
    two shapes are currently in use in several laboratories, although
    cubes, cylinders, spheres, modified hemispheres, and even chambers
    with an elliptical cross-section have all been used (Drew & Laskin,
    1973). The two major considerations that influence the shape of the
    chamber are uniformity of distribution and wall loss of the test
    substance (MacFarland, 1976) with secondary importance placed on
    caging supports, accessibility, and costs.

         A typical chamber is shown schematically in Fig. 6.3. The unit
    has a volume of 1.3 m3 and is about 3 m in height. The body is made
    of stainless steel and the windows can be made of either glass or
    lucite. Clean air is supplied at the top with the pollutant being
    injected in a perpendicular direction to the incoming airstream.
    Chambers with tangential pollutant introduction are also common.
    Airflow is usually down through the chamber and the air is removed
    through the side arm of a Y fitting at the bottom. Animal wastes are
    removed at the bottom via the building drains, usually through a trap
    or a valve. The trap also maintains the integrity of the system and
    allows operation at a pressure slightly (1-2 cm H20) below ambient.
    Chambers of this general shape have been built with volumes ranging
    from 128 litres up to 5 m3, although MacFarland (1976) suggests that
    the pollutant concentration in chambers of less than 1 m3 may not be

         The size of the chamber depends on the number and size of the
    animals to be exposed. The total animal volume should not exceed
    approximately 5% of the total chamber volume. Experience has shown
    that above 5% surface losses begin to cause excessive concentration
    losses and thermal considerations also begin to play a limiting role.

    6.7.5  Construction materials

         Inhalation chambers should be constructed of materials that do
    not react with the test material and are easily cleaned. The most
    versatile materials are stainless steel and glass. However, many other
    materials have been used, including aluminium, wood, wood coated with
    epoxy paints, lucite, and various fibre panels. The chamber should
    have at least two sides made almost completely of transparent material

    FIGURE 31

    FIGURE 32

    in order to view the animals during exposure. The inside surfaces
    should be as free as possible from perturbations and rough surfaces
    and edges, in order to facilitate cleaning. Openings should be
    included to monitor the variables needed to characterize the exposure
    (section 6.7.8).

    6.7.6  Engineering requirements

         Accurate control of airflow in the chambers is essential. The
    usual procedure is to supply filtered, conditioned air in excess of
    that required and then tap off the common supply for each chamber. In
    most experiments, the ratio of chamber volume to airflow ranges from 1
    to about 6.

         When handling hazardous materials, special safety precautions
    must be taken to protect operating personnel and the surrounding
    environment. The chambers are operated at slightly negative pressure
    to ensure that any leaks draw air into the system. Chamber effluents
    must be cleaned, usually by filters or scrubbers, or at least diluted,
    before being released into the environment. Stack effluents should be

         Food and water must be provided in the chambers, when animals are
    being exposed continuously. Facilities for cleaning the chambers,
    while the animals are in them, are also necessary, though in many
    laboratories the animals are removed from the chamber for 45 min-1 h
    to permit it being serviced. Racks for supporting animal cages must be
    included and exposure cages should have all six sides made of wire
    mesh (stainless steel) to ensure good mixing.

    6.7.7  Special systems  Isolation units

         When handling particularly hazardous materials, additional
    precautions are necessary. These can be fairly simple, such as
    operating a battery jar in a fume hood, or complex, such as the
    chamber-within-a-chamber concept described by Laskin et al. (1970). In
    this system (Fig. 6.4) the aerosol is separated by two barriers from
    the operator with separate glove boxes for generation and removal of
    the aerosol. In addition, living quarters are provided behind a
    barrier with pass boxes for food and animal wastes to be moved into
    and out of the chamber.  Head and nose exposures

         Early in the development of inhalation exposure systems,
    investigators realized the value of head or nose only exposures
    (Saito, 1912). Stokinger (1949) described chambers with openings for
    head-only exposures for several species, and Henderson (1952)

    FIGURE 33

    described an apparatus consisting of a cylindrical chamber with
    openings arranged along two sides to enable nose exposure of mice to
    biological aerosols. Nose exposure systems are used in situations
    where: (a) skin absorption is not desirable; (b) the amount of test
    material is limited; (c) the material is hazardous; and (d) there is
    no need for a large chamber. They are most commonly used for acute
    exposures, since it is difficult to restrain the animals for long
    periods of time.

         Nose exposure systems have been used extensively for two
    particular situations -- exposure to radioactive aerosols and exposure
    to cigarette smoke. Investigators at the Lovelace Foundation for
    Medical Education and Research in Albuquerque have developed a series
    of nose exposure units (Boecker et al., 1964; Raabe et al., 1973;
    Thomas & Lie, 1963). The technical difficulties of exposing animals to
    cigarette smoke has prompted the development of several systems
    designed specifically for this purpose (Hoffman & Wynder, 1970; Stuart
    et al., 1970). One device has been developed by Homburger et al.
    (1967) and two have been described by Dontenwill (1970). Albert et al.
    (1974) have developed a device to expose donkeys to cigarette smoke
    via nose tubes. Devices for exposing dogs to cigarette smoke have also
    been described (Cahan & Kirman, 1968).

         A nose exposure system for exposure to dusts has been developed
    at the Institute of Hygiene and Occupational Medicine in Moscow
    (Valeznev et al., 1970) (Fig. 6.5). It consists of a completely
    enclosed system in which up to 40 rodents can be exposed
    simultaneously. A very complex system adaptable to both head-only and
    whole-body exposures is routinely used at the Medical Institute in
    Kiev (Balasov et al., 1968). A schematic diagram of this system is
    shown in Fig. 6.6.  Instantaneous exposure systems

         Occasionally, it is necessary to expose animals to a
    concentration of material, while avoiding the time required to attain
    uniform concentrations. In this case, the air in the chamber is
    equilibrated with the test material and the animals rapidly inserted
    into the chamber. Sometimes double chambers are used for this purpose.
    The animals are placed in the upper half and the pollutant is
    introduced into the lower half. When the desired concentration is
    reached in the lower half, a trap door opens and the animals fall into
    the lower half, while the mechanism immediately closes. Other
    investigators have described various drawer arrangements for rapid
    insertion of animals into test atmospheres.

    FIGURE 34

    FIGURE 35

    6.7.8  Variables to monitor

         It is necessary to monitor several variables during the operation
    of an inhalation chamber. The most obvious is the concentration in the
    chamber of the pollutant in question. This can be done continuously by
    automated samplers and recorded on a strip chart recorder or manually
    at periodic intervals using a variety of sampling techniques. When
    aerosols are involved, particle size should be determined. The flow of
    pollutant, the flow of air, and the chamber pressure should all be
    recorded frequently. Chamber temperature and humidity are other
    variables that should be monitored. It is also useful to measure the
    pressure drop across intake and exhaust filters in order to know when
    to replace them.

    6.7.9  Human exposure facilities

         Ethical principles will always be of first concern, when
    considering the exposure of human subjects to materials for toxicity
    evaluation. However, there are situations where controlled exposure of
    human beings can provide useful information with minimum risk. One of
    the more modern facilities has been described by Stewart et al.
    (1970a,b). It consists of a room approximately 6 m  6 m  2.7 m,
    completely air conditioned and operated at slightly negative pressure.
    Activity within this chamber is strictly sedentary and comfortable
    chairs and study desks are provided. Meals are served during
    exposures, with coffee and soft drinks available continuously. All
    subjects are under continuous surveillance by medical personnel and
    all activities are visually monitored by closed circuit television.
    Such facilities are particularly useful for studying psychomotor
    effects and other sensitive indicators of exposure to low
    concentrations of organic vapours.

         The odour threshold concentrations used in the USSR for
    establishing maximum permissible concentrations for single exposures
    are determined in human subjects over short periods (5-10 min).
    Obviously such studies can only be carried out at very low
    concentrations considered to be safe. The minimum concentration sensed
    by the most sensitive individual is accepted as the odour threshold
    (Rjazanov, 1964).

         Reflex reactions produced in man by irritating the receptive
    zones of the respiratory organs with subsensory concentrations of
    atmospheric pollutants, were established by measuring the light
    sensitivity of the eye, the bioelectric activity of the cerebrum, etc.
    (Bustueva et al., 1960; Rjazanov, 1964). Changes in encephalographic
    responses resulted from exposure to small concentrations of these
    substances. The maximum concentration that did not produce an effect
    on the bioelectric activity of the cerebrum was, in most cases, 3-4
    times lower than the odour threshold concentration (Krotov, 1971;
    Sidorenko & Pinigin, 1972).

    6.8  Contaminant Generation and Characterization

         Extensive reviews have been published by several investigators on
    methods of generating and characterizing vapours and particles (Bryan,
    1970; Cotabish et al., 1961; Drew & Lippmann, 1971; Lodge, 1968;
    Mercer, 1973a,b; Nelson, 1971; Raabe, 1970).

    6.8.1  Generation of vapours

         Vapours can be generated by using one of several flow-dilution
    devices (Cotabish et al., 1961; Drew & Lippmann, 1971; Nelson, 1971;
    Saltzman, 1971; Saltzman & Warburg, 1965). If the contaminant is a
    liquid at room temperature, a vaporization step must be included. One
    procedure is to use a motor-driven syringe and to apply the liquid to
    a wick or heated plate in a calibrated stream of air (Nelson & Griggs,
    1968). Another method is to saturate the airstream with vapour and
    then dilute it with air to the desired concentration (Cotabish et al.,
    1961). A third technique, originally described by O'Keefe & Ortman
    (1966), consists of using permeation tubes. These are especially
    useful when using low concentrations of test materials for
    standardization procedures. In theory, there is no reason why they
    cannot be scaled up for use in inhalation studies.

    6.8.2  Particle generators

         The generation of particulate contaminants is usually more
    difficult than vapour generation. The contaminant may be generated
    from a dry powder or from a liquid and the particles generated may be
    of uniform size (monodisperse) or may vary greatly in size
    (heterogeneous).  Heterogeneous aerosols

         The Wright dust feed (1950) is one of the better known
    instruments for generating aerosols from a dry powder. A gear drives
    the surface of a packed cylinder of finely ground powder against a
    scraping mechanism. A high velocity airstream disperses the powder.
    Proper use of the Wright dust feed is dependent upon the control of
    the relative humidity of the airstream and the packing density of the
    powder. Other devices for producing aerosols from dry powders have
    been described (Crider et al., 1968; Deichman, 1944; Dimmick, 1959;
    Stead et al., 1944). Since the particle size of the resultant aerosols
    depends upon the size of the original powder, elutriators and cyclones
    are sometimes included to limit the maximum size. Laskin et al. (1971)
    described generators that include such devices for producing freshly
    ground polyurethane aerosols.

         Agglomeration, usually caused by electrical charge, is a serious
    problem with dry dust aerosols. The electrical behaviour of aerosols
    has been reviewed by Whitby & Liu (1966). A mechanical solution to
    agglomeration has been proposed by Drew & Laskin (1971) who described
    a fluidizing dust generator.

         Wet dispersion generators break liquid into droplets. The liquid
    may be a solution or a suspension of the test material. In most cases,
    the liquid is drawn into filaments or films that are broken into
    droplets. A number of compressed air-driven generators (nebulizers),
    that produce droplets of many sizes, have been described (Dautrebande,
    1962; Laskin, 1948; Lauterbach et al., 1956; Raabe, 1970). The
    resulting aerosols are polydisperse although relatively narrow size
    distribution can be attained with some nebulizers.

         Monodisperse aerosols are occasionally needed by investigators,
    especially when studying regional deposition. The most popular device
    for dispensing uniform droplets is the spinning disc generator first
    described by Walton & Prewett (1949). Primary droplets thrown off the
    perimeter of a spinning disc are uniform in size. The liquid is fed on
    to the centre of the disc and accumulates at the edge until broken off
    by centrifugal force. Some secondary, smaller droplets are also
    produced but these can be separated dynamically. Several investigators
    have successfully used the spinning disc for inhalation studies
    (Albert et al., 1964; Kajland et al., 1964; Lippmann & Albert, 1967;
    Philipson, 1973). Another device employing a controlled condensation
    process originally described by LaMer & Sinclair (1943) has been found
    to be suitable for inhalation studies. A third principle, consisting
    of size specific collection, resuspension and subsequent dispersion,
    has also been used (Kotrappa et al., 1972).

    6.8.3  Monitoring contaminant concentrations

         The techniques and equipment for monitoring contaminant
    concentrations have been reviewed by a number of authors (Lippman,
    1971; Powell & Hosey, 1965). In many cases, the characteristics of the
    contaminant determine the sampling technique. Sometimes a number of
    techniques are available and the method of choice may depend upon the
    availability of equipment, cost of reagents, time for analysis, or
    other factors. Automated instrumentation is currently available for a
    number of contaminants. However, when using automatic devices,
    a second method, usually chemical, should be used to verify the
    instrument performance.  Vapour sampling

         Two basic methods for the collection of gaseous samples are
    employed. The first involves the use of a gas collector, such as an
    evacuated flask or bottle, to obtain a definite volume of air at a
    known temperature and pressure. The second method involves the passage

    of a known volume of air through a collecting medium to remove the
    desired contaminants from the sampled atmosphere. The samples are then
    analysed by appropriate analytical techniques.

         Since the assays are related to the volume of air sampled, the
    instrumentation for monitoring airflow or volume should be accurately
    calibrated. It is also important that there are no leaks in the
    sampling train, thus assuring that all the air measured has passed
    through the collecting medium.

         A number of devices, currently available, measure the
    concentration of vapours continuously; many record the result
    graphically. Many detection principles are used including
    conductivity, colorimetry, and spectrophotometry. Recent commercial
    instrumentation using the principle of infrared spectrometry are
    especially useful. Most of these instruments have been described by
    Nader (1971).  Particulate sampling

         When monitoring particulate atmospheres, mass concentration and
    particle size must be determined. The mass concentration can be
    measured by techniques similar to those used for monitoring vapours.
    The material can be collected, then assayed by appropriate chemical
    methods. Gravimetric analysis can also be performed by weighing the
    filter paper before and after collecting a sample. The resulting mass
    can be related to the volume of air that was sampled.

         Particle collection techniques include filtration, impingement,
    thermal and electrostatic precipitation, and sedimentation. The basic
    principles for these techniques and specific examples of each method
    have been reviewed (Lippmann, 1971). These principles should be
    thoroughly understood prior to selection of a method for a specific

         The techniques for measuring particle size and numbers have been
    discussed in detail by Mercer (1973a,b). Direct methods consisting of
    both conventional and electron microscopy can be used. In both cases,
    proper sampling methods to ensure collection of a representative
    sample should be followed. Electrostatic and thermal precipitators or
    an impinger can be used. After using an impinger, the dust can be
    counted in a standard haematology counting cell or counted directly
    with a Coulter Counter.

         There are two indirect methods of assessing particle size; the
    use of cascade impactors and the use of light scattering devices. The
    theory of cascade impaction has been described by Mercer (1963, 1964,
    1965). The theory of light scattering devices has been reviewed by
    Hodkinson (1966) and commercially available instruments have been

    listed by Swift (1971). Many factors, including shape, opacity, and
    others, some unknown, affect the amount of light scattered and the
    measured number and size of the particles. These devices should,
    therefore, be used cautiously.

    6.9  Other Methods of Respiratory Tract Exposure

    6.9.1  In vivo exposures

         In addition to direct inhalation, several other methods of
    exposing the respiratory tract have been described (Roe, 1968).
    Andervont (1937) originally described a thread implantation technique
    whereby a thread impregnated with a test compound was literally passed
    through a lobe of the lung. This technique was modified by Kuschner et
    al. (1957) who devised a pellet that could be impregnated with the
    test material or coated with radioactive materials. This pellet had
    hooks which held it in the lumen of the bronchus or bronchiole. This
    technique has been used to demonstrate the carcinogenicity of a number
    of compounds including benzo(a)pyrene, 3-methylcholanthrene, and
    calcium chromate (Laskin et al., 1970). A technique whereby hamsters
    are anaesthetized and then intratracheally intubated with various
    materials is in use in a number of laboratories (Laskin et al., 1970;
    Little & O'Toole, 1974; Saffiotti, 1970; Schreiber et al., 1972). In
    two laboratories, this technique has been coupled with inhalation
    exposures to study the potential cocarcinogenicity of various agents
    (Laskin & Nettesheim, 1974, personal communication).

    6.9.2  In vitro exposures

         A few  in vitro techniques that show promise as regards
    elucidation of the mechanisms of toxicity of inhaled materials should
    be mentioned. A number of investigators (Niemeier & Bingham, 1972;
    O'Neill & Tierney, 1974; Orton et al., 1973) are using various,
    isolated, perfused lung preparations to study toxicity. Such
    preparations could be particularly useful in studying the transfer of
    materials from the lung to the blood. These preparations are also used
    to study pulmonary metabolism as are lung tissue slices (O'Neil &
    Tierney, 1974). Finally, several laboratories are developing methods
    of culturing tracheal rings and sections (Griesemer et al., 1974; Lane
    & Miller, 1975). These preparations show great promise for
    toxicological evaluation (section 2.7.4).

    6.10  Biological End-points and Interpretation of Changes in these

         Throughout the course of an inhalation study, there are a number
    of biological indicators to observe. The classical indices of toxicity
    include weight change and mortality, organ-to-body weight ratios, and
    both gross and microscopic changes in the morphology of the various
    tissues and organs. While the study is in progress, respiratory,

    physiological indices can be monitored and the excretion of certain
    chemicals in breath, urine, and faeces can also be followed. Some
    procedures particularly useful in inhalation toxicity studies are
    discussed below.

    6.10.1  Morphological changes

         The primary function of the lung is the exchange of oxygen and
    carbon dioxide between the blood and alveolar air. The walls of the
    alveoli are lined by a single, flattened layer of epithelial cells
    that are in close proximity to endothelial cells lining capillaries.
    The mean distance from the lumen of the capillary to the lumen of the
    alveolus is less than one micron. Because this distance is so small,
    any thickening or inflammation of the alveolar wall will severely
    disrupt the diffusion of gases.

         The air in the alveolus arrives through a series of branching
    bronchi and bronchioles the specialized epithelial lining of which is
    instrumental in removing particulate matter from the lung. Any lesions
    that result in narrowing of the lumen of these bronchi and bronchioles
    cause a disruption in the ventilation of that portion of the lung.
    Chemicals affecting the specialized ciliated epithelial lining, the
    bronchioles, and the bronchi may interrupt the clearance mechanism,
    resulting in a build-up of inhaled particulates in the lung.

         The type of morphological change observed in the lung as a result
    of the inhalation of materials depends upon the concentration of the
    inhaled material and the length of time for which animals are exposed.
    Short-term inhalation of high concentrations of certain materials may
    result in acute changes in the lung such as oedema, necrosis, and
    purulent inflammation. However, the inhalation of the same material at
    lower concentrations for longer periods of time may result in chronic
    changes such as fibrosis and even neoplasia. Table 6.3 lists a number
    of chemicals and the response elicited in the lung following their

         Techniques for quantifying morphological changes are, at best,
    limited. The usual practice is to assign some number to the degree of
    damage and then to apply statistical procedures to these numbers.
    These procedures are, however, subject to individual interpretation.
    Assessment of the degree of fibrosis is possible (Roe, 1968), although
    such measurements should be checked by special staining procedures.

         Interpretation of any morphological changes in the lung must take
    into consideration the concentration and duration of exposure.
    Exposure to certain materials will elicit immediate acute
    morphological changes which, with time, tend to resolve and disappear.
    This phenomenon suggests that the animal is able to adapt to the
    effects of some chemicals, if the concentration is not too high. An

        Table 6.3  Responses of the lung to various chemicals
    Chemical                      Bronchiolar    Loss of      Oedema     Epithelial      Fibrosis    Emphysema    Granuloma
                                  epithelial     goblet                  metaplasia
                                  hyperplasia    cells

    nitrogen dioxide                  x                          x           x              x            x
    ozone                             x             x            x           x              x
    allergens                                                                                                         x
    sulfuric acid                     x                                      x
    bis-chloromethyl ether
    silica                                                                                  x                         x
    coal dust                                                                               x            x
    beryllium                         x                                      x                                        x
    cotton dust
    nickel compounds

    Table 6.3 (cont'd)
                                  Necrosis    Neoplasia    Bronchial        Alveolar        Nonsuppurative
                                                           constriction     epithelial      alveolitis

    nitrogen dioxide                 x
    allergens                                                                                      x
    sulfuric acid                    x                                           x
    bis-chloromethyl ether                        x
    coal dust
    beryllium                                     x
    cotton dust                                                 x
    nickel compounds                              x
    example of this adaptive phenomenon is seen with ozone. Nonlethal
    exposures to ozone have a protective effect against subsequent lethal
    exposures which would result in marked pulmonary oedema (Alpert &
    Lewis, 1971).

    6.10.2  Functional changes

         There are certain advantages in using alterations in respiratory
    functions as indices of toxicity: (a) quantitative measurements can
    usually be made at concentrations far below those needed to produce
    morphological effects; (b) many effects may be measured at the level
    of reversible rather than irreversible changes; (c) such measurements
    may elucidate mechanisms of action of materials, and (d) in many
    instances similar data can be obtained from experimental animals and
    man.  Measurement of respiratory frequency

         Alteration of respiratory frequency in mice may be used as a
    simple screening test for assessing irritant potency (Alarie, 1973).
    The changes produced are dose-related, which permits construction of
    dose-effect curves and calculation of the concentration required to
    produce, for example, a 50% decrease in respiratory frequency. Some
    irritants, mainly those affecting the upper respiratory tract,
    decrease frequency. Other irritants, mainly those which penetrate to
    the deeper areas of the lung, increase frequency. Measurement of
    respiratory frequency alone is a very sensitive measure of effect for
    those irritants that increase frequency (e.g. ozone and nitrogen
    dioxide), but it is a relatively insensitive measure of effect for
    those irritants that decrease frequency (e.g. sulfur dioxide and
    formaldehyde).  Measurement of mechanics of respiration

         Alterations in pulmonary flow resistance or pulmonary compliance
    may be used to assess irritant potency. The method of Amdur & Mead
    (1958) required three basic measurements: intrapleural pressure, tidal
    volume, and the rate of flow of gas in and out of the respiratory
    tract. Intrapleural pressure is estimated by placing a fluid-filled
    catheter in the pleural space, while the animal is under anaesthesia.
    Once the catheter is positioned, no further anaesthesia is necessary.
    Tidal volume is obtained by placing the animal in a body
    plethysmograph and measuring the pressure changes as the animal
    breathes quietly. Rate of gas flow is obtained by electrical
    differentiation of the volume signal. This method provides data on
    both resistance and compliance, but is generally limited to single
    exposure studies of a few hours duration. The guineapig has been the
    species most commonly used for this method; however, plethysmographic
    studies have been successfully carried out on several other rodents
    including mice (Alarie, 1973), rats (Palacek, 1969) and rabbits
    (Davidson et al., 1966).

         The method of Murphy & Ulrich (1964) does not involve surgical
    intervention and, thus, makes it possible to perform measurements
    repeatedly on the same animals. The animal is placed in a body
    plethysmograph to which an oscillating sine wave pressure is applied.
    The animal's face is fitted with a mask containing a pneumotachograph
    screen for measuring flow. Changes in resistance are calculated for
    the alterations in flow produced by the superimposed pressure
    oscillations. This technique has been used in toxicological studies
    without making compliance measurements. However, by measuring
    oesophageal pressure reflecting intrapleural pressure, it should be
    possible to estimate compliance changes. A compliance measurement is
    described by Mead (1960) that involves no surgical intervention.

         The observed changes in respiratory mechanics are dose-related
    and permit the construction of dose-effect curves. These may be used
    to compare irritant potency or to study such things as the effect of
    inert particles on the reaction to irritant gases. The deep lung
    irritants that increase respiratory frequency tend to show a decrease
    in compliance as the primary alteration in the mechanical behaviour of
    the lung. Resistance changes with such irritants are minimal. The
    irritants that slow respiratory frequency tend to show an increase in
    resistance accompanied by a smaller decrease in compliance, as the
    primary alteration in pulmonary mechanics.

         An increase in pulmonary flow resistance can result from a
    narrowing of the lumen of the bronchi mediated by constriction of the
    smooth muscle. The effect, in this situation, usually occurs rapidly
    on exposure to the irritant. In the case of a gaseous irritant, the
    effect is fairly rapidly reversed when irritant exposure ceases. In
    the case of a particulate irritant which remains in the lung, the
    effect is less readily reversible. Swelling of the respiratory mucosa
    or an increase in mucus secretion can also cause an increase in flow
    resistance. The effect, in this situation, usually takes some time to

         Much of the damage to the lung is to the small airways which
    change either in calibre or stability or both. It would be very useful
    if this damage could be detected early during an exposure before more
    serious disease develops. There are three tests which are promising
    and have been useful in human subjects. They are the maximal
    expiratory flow volume curve, reviewed by Hyatt & Black (1973), the
    alveolar closing volume (Dollfuss et al., 1967) and the frequency
    dependency of compliance (Woolcock et al., 1969). Animal models that
    provide the same data have not yet been fully developed.

    6.10.3  Biochemical end-points

         In the last decade, much research has centred around the
    elucidation of some of the biochemical aspects of the lung. It has
    only recently been demonstrated that the lung is capable of

    biotransforming many foreign chemicals and several authors have
    investigated the role of cytochrome P-450-dependent enzyme systems in
    pulmonary microsomes (Bend et al., 1972; Chhabra & Fouts, 1974;
    Chhabra et al., 1974; Fouts & Devereux, 1972; Harper et al., 1975;
    Hook et al., 1972). The importance of intermediary metabolism is now
    being considered (Tierney, 1974). The biochemistry of pulmonary
    surfactants and their role in pulmonary defence mechanisms has been
    reviewed by King (1974). Investigations of the lung collagen have
    recently been reviewed by Crystal (1974). Witschi (1975) has provided
    an excellent summary of biochemical approaches for the evaluation of
    pulmonary toxicity.

    6.10.4  Other end-points in inhalation studies

         Exposure by inhalation is only a means by which the substance
    enters the animal. This chapter has concentrated on the lung as the
    critical organ. However, there are end-points that are not necessarily
    related to the lung. Inhalation studies have recently been reported to
    assess the teratological effects of inhaled materials (Schwetz et al.,
    1975). They have also been used in behavioural studies (Weiss &
    Laties, 1975; Xintaras et al., 1974).


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    7.1  Introduction

         This chapter deals with the two related but separate processes,
    heritable mutations and cancer. Cancer involves the conversion of
    normal cells to malignant cells and the development of what is usually
    an irreversible malignant disease process in the present generation
    frequently leading to a fatal outcome for the bearer of the
    malignancy. Heritable mutations are mutations that are transmissible
    to later generations and the target cells are the germ cells of either

         Developments in the last two decades make it possible to discuss
    certain aspects of mutagenesis and carcinogenesis in parallel. In
    other words, there is now increasing evidence that, in most cases,
    somatic mutations are probably involved in the conversion of normal to
    malignant cells. Thus, the ability of a chemical or physical agent to
    produce mutations is relevant both to the question of heritable
    mutations (mutations of the germ cell) and carcinogenicity.

         There is a well-established body of knowledge that shows a close
    correspondence between cancer of the whole animal as studied in the
    laboratory and the occurrence of cancer in man; this is especially
    true of occupational cancer. More recently, a fairly close correlation
    has been shown between tests of isolated, simple, biological systems,
    e.g. reverse bacterial mutations and the response to carcinogens of
    whole animals, and human beings.

         A similar situation does not exist with respect to patterns in
    human heritable mutations. Thus, although there is good evidence of
    correlation between  in vitro tests for mutagenicity and heritable
    mutations in insects and experimental mammals, it can only be
    inferred, at present, that this also applies to man. The inference,
    however, is extremely strong and not subject to serious doubt. Thus,
    there is every reason to expect, that, in general, man will respond
    biologically in a manner quite similar to that of other species when
    exposed to mutagens that reach the germ cells. The lack of full
    correlation stems more from the lack of appropriate studies in man
    than from any uncertainty concerning the underlying biological
    considerations. At present, methods for the detection of mutations in
    man are difficult, cumbersome, and insensitive, and it is imperative
    to operate on the assumption that agents capable of producing germ
    cell mutations in laboratory studies would also be capable of
    producing similar mutations in man.

         Thus, two quite different disease processes can have one common
    level of consideration, namely, their ability to produce alterations
    in the genetic material of cells. The following discussion is
    concerned with methods for carcinogenicity testing. First, the
    traditional lifetime exposure of the entire organism to the test

    compound is considered. This is followed by a discussion of various
     in vitro systems for the examination of mutagenicity including DNA
    damage, mutagenicity in bacteria and eukaryotic organisms, and
    transformation of cell cultures. (The first three of these are
    particularly relevant to mutagenicity of the germ cell or of the
    somatic cell.) Following this, the general problem of examination for
    heritable mutations is considered. This includes inquiries at three
    levels: injury to DNA, point mutations, and chromosome alterations.
    Whole animal and isolated test systems are available to make relevant
    determinations. However, the whole field of mutagenicity testing is
    now in a period of rapid evolution with advances being made in a
    number of areas. It can, therefore, be anticipated that present
    procedures will undergo alteration and, it is hoped, will improve
    within the near future.

    7.2  Carcinogenicity

    7.2.1  Long-term bioassays

         The fact that environmental factors have a direct effect in
    producing cancer in man is shown by:  (a) the unequivocal evidence of
    the chemical origin of occupational cancer, for example, urinary
    bladder tumours in workers exposed to aromatic amines, lung cancers in
    workers exposed to bis(chloromethyl)ether, etc.;  (b) the
    well-documented cases of iatrogenic cancer;  (c) the positive
    correlation between cigarette smoking and lung cancer;  (d) the
    differences in cancer incidence in urban and rural populations and
     (e) the results of studies on migrants showing that for some types
    of cancer they acquire incidences similar to those found in the host
    countries. Results obtained in experimental studies on carcinogenesis
    confirm the direct carcinogenic effect of chemicals. However, there is
    not necessarily a correlation between acute toxicity and
    carcinogenicity; a chemical with a high acute toxicity may not have
    any, or only a very low carcinogenic potential. Conversely, a chemical
    that produces a very minor, or no evident toxic effect in acute or
    subacute experiments may be a very powerful carcinogen. Knowledge of
    the acute and subacute toxicity of the test chemical however, is
    important in order to permit better planning of the experiment. For
    instance, the premature death of the test animals, due to unforeseen
    side-effects, may thus be avoided.  Species, strain, and sex selection, and size of groups

         The description of methods and recommended procedures in this
    chapter applies to long-term testing in rodents and specifically to
    the mouse, rat, and Syrian golden hamster (FDA, 1971; NCI, 1976; Weil,
    1972; Weisburger, 1975; Weisburger & Weisburger, 1967). Rodents have
    been preferred to other species because of their susceptibility to
    tumour induction, their relatively short life span, the limited cost
    of their maintenance, their widespread use in pharmacological and
    toxicological studies, the availability of inbred strains and, as a

    consequence of these characteristics, the large amount of information
    available on their physiology and pathology. Nonrodents, in particular
    dogs and primates, have rarely been used in the past; though used more
    at the present time, their much higher cost of maintenance, the long
    period of observation, and the impracticability of using sufficiently
    large numbers put nonrodents at a disadvantage for long-term
    bioassays. Also, comparative studies on the metabolism of various
    chemicals have not indicated necessarily that primates and dogs are
    closer to man in this respect than rodents. Nevertheless, many of the
    procedures for long-term bioassays in rodents are applicable to assays
    in nonrodents.

         Long-term bioassays of the carcinogenicity of environmental
    chemicals are carried out to assess a possible risk to man and to
    estimate the need for primary preventive measures. Because of the
    urgent nature of these problems, it is recommended that a compound of
    unknown activity should be tested on two animal species. Although a
    positive (that is, carcinogenic) effect in one species is considered
    as adequate warning, only negative findings in two species can be
    regarded as adequate negative evidence.

         Among rodents, the species of choice are undoubtedly the mouse,
    the rat, and the Syrian golden hamslet. The European hamster has
    recently been introduced, particularly for studies in lung pathology,
    but its use cannot be recommended for routine testing. Guineapigs and
    rabbits have been used occasionally but they have few of the
    advantages of smaller rodents combined with the disadvantages implied
    by a much longer life span, higher cost of maintenance, and the
    scarcity or absence of inbred strains.

         Of the three rodent species of choice, the mouse and the rat have
    been more widely used than the hamster. However, the last species has
    proved to be an excellent tool for revealing carcinogenic effects in
    the respiratory tract and urinary bladder. Furthermore, hamster cells
    are widely used for  in vitro transformation assays, and, since in
    most cases these assays require the back transplantation of the cells
    in syngenic hosts, the use of the hamster has recently been extended.
    The hamster is generally randomly bred but a pure strain is becoming

         The use of inbred strains undoubtedly has the advantage of making
    available animals with known characteristics such as an average life
    span, and a spontaneous tumour rate with little variability.
    Random-bred animals or, even better, animals bred with maximum
    avoidance of inbreeding, are considered more resistant to infections
    and perhaps more liable than many inbred strains to reveal any
     carcinogenic effect on an unsuspected target organ. It is common
    experience that  carcinogenic activity is more easily recognized by
    the increased frequency and earlier appearance of tumours at sites
    where tumours occur spontaneously. Inbred strains are often known to
    have one or two particularly susceptible organs and the induction of

    tumours in these organs could, in part, be regarded as an
    intensification of whatever underlies the spontaneous occurrence of
    tumours in that organ. A carefully planned experiment must take this
    possibility into account, and preference should be given to strains
    with a low incidence of tumours. Noninbred strains are inconvenient in
    that, in many cases, background tumour incidence in untreated controls
    is unpredictable. Moreover, experimental groups initiated at different
    times can rarely be compared with each other since inter- as well as
    intragroup variation may be considerable.

         Hybrid mice of two known inbred strains are excellent as they are
    particularly robust and long-lived, but they are, of course, more
    difficult to obtain and are likely to be more expensive than inbred

         In selecting the species, it is important to be aware that there
    are, within each species, particular susceptibilites; for instance, it
    is easier to induce liver tumours in the mouse than in the rat, and
    conversely it is much easier to induce subcutaneous tumours in the rat
    than in the mouse. The skin of the mouse and the rabbit is possibly
    more sensitive to tumour induction by skin painting than that of the
    rat and the hamster, particularly when polycyclic hydrocarbons are
    used. The hamster is more susceptible to urinary bladder
    carcinogenesis than the mouse and possibly the rat.

         It is essential that experimental groups should be composed of an
    equal number of animals of each sex, since differences in response to
    the carcinogenic activity of chemicals are well documented. Thus, the
    use of one sex only would not show the full range of activity of the
    test chemical.

         The group should be sufficiently large to permit statistical
    evaluation of the results. The UICC Committee on Carcinogenicity
    Testing (UICC, 1969) recommended that the number of animals should be
    such that it "would be likely to yield reasonable (e.g. 90%)
    statistical significance as between the main induced tumour incidence
    and its spontaneous incidence". A sufficient number of animals must be
    alive at the time that the first tumour appears and it is suggested
    that this number should never be less than 25 per sex.  Route of administration

         It has been agreed that the chemical under test should be
    administered by the route of human exposure. This applies to food
    additives and food contaminants in particular, but it is equally
    desirable for drugs. This rule cannot always be applied to
    environmental or industrial chemicals. It is well known that the three
    main routes of exposure in man are inhalation, ingestion, and skin
    absorption. Inhalation is often predominant in man but in experimental
    carcinogenesis the inhalation route, while highly desirable in

    principle, has rarely been used in the past because of lack of
    adequately equipped laboratories. However, it is now being widely used
    in programmes concerning smoking and health.

         When the route of exposure is  per os, the chemical is
    administered either mixed in the diet or drinking water, or by gavage,
    the choice depending on the specific characteristics of the test
    chemical; a volatile compound, for example, should never be mixed in
    the diet while, for a drug, the route by which it will be used in man
    should be selected, i.e.  per os, or by intravenous, subcutaneous or
    topical application.  Inception and duration of tests

         Most commonly, tests are initiated in young adult animals from 7
    to 9 weeks of age. The start of treatment soon after weaning is
    recommended as a routine procedure. One objective of carcinogenicity
    testing is to obtain the maximum possible carcinogenic effect of the
    test chemical in order to compensate for the limited number of
    individuals at risk. This is accomplished by employing high levels of
    exposure (see 7.1.4) and by starting the treatment when the animals
    are at their most sensitive age. For some years following the pioneer
    work of Pietra et al. (1961), treatment immediately after birth and
    within the first 24 h of life was thought to be the most sensitive
    model. Careful reviews of the data available have indicated that
    neonatal treatment alone can be recommended in some instances, but not
    as a general routine procedure (Della Porta & Terracini, 1969). For a
    single neonatal administration, only a very limited quantity of the
    chemical to be tested is needed and this could be advantageous, when
    only a small amount of the chemical is available.

         The main limitation of the use of a single neonatal treatment is
    that the newborn animal may not have developed sufficient metabolic
    competence to metabolize the test chemical. Another limitation is that
    not all chemical carcinogens are active after one, or even a few

         More recently, prenatal exposure has received considerable
    attention, since it has been demonstrated that some tissues, notably
    nervous tissue, are more susceptible to certain carcinogens during
    fetal life than later (Druckrey et al., 1967; Napalkov, 1973; Napalkov
    & Alexandrov, 1968; Tomatis, 1973). At present, however, it can only
    be speculated that prenatal exposure will reveal the carcinogenic
    effect of a chemical that would not have been revealed had treatment
    started at a later age. A sensitive procedure might be to integrate
    prenatal exposure with long-term, postnatal exposure, in order to
    obtain maximum sensitivity of the bioassay system. To achieve this,
    animals mated at the age of 8-9 weeks, should be exposed to the test
    chemical during the second half of pregnancy. The parents so treated
    can constitute a group under conventional long-term testing, if the
    treatment is continued after delivery. Their offspring, already

    exposed  in utero, and possibly after birth through their mother's
    milk and excreta, should be exposed directly to the test chemical from
    the age of weaning onwards. In this way, two different experimental
    groups can be obtained: one for which exposure was started at the
    young adult stage (the parents) and the other for which exposure was
    started prenatally (the offspring) (Tomatis, 1974b).

         The duration of a positive test, i.e. where exposure to the test
    chemical is followed by an increased incidence of tumours, depends on
    the time of appearance and the rapidity of growth of the induced
    tumours. To agree in advance on a given duration of exposure is of
    prime importance for negative tests. Some research workers have
    adopted a duration for carcinogenicity tests of 2 years in rats and 18
    months in mice, while others have preferred an observation period
    extending over the entire life span of the animals. A long finite
    period -- that is, not less than 2 years -- is recommended in
    preference to the entire life span of the animals, for the following
    reasons:  (a) induced tumours usually occur within this observation
    period;  (b) "spontaneous" tumours appear with highest frequency late
    in life and their appearance may make it more difficult to evaluate
    the carcinogenicity of a compound, particularly if of low potency;
     (c) a few animals may far exceed the normal life span of the species
    and extend unnecessarily the duration of the experiment;  (d) tests
    are very expensive and any justifiable abbreviation means good economy
    (Health & Welfare, Canada, 1973; Tomatis, 1974a; UICC, 1969; WHO,
    1961).  Dose-level and frequency of exposure

         If only one level is to be used, the highest dose allowing long
    survival of the majority of the animals should be chosen. The
    selection of this dose should be made from results obtained in
    subacute toxicity tests. Information on the LD50 of certain
    compounds may be available and this can be useful in deciding the
    level of the dose for the subacute studies.

         While it would be very difficult to give a definition of the
    maximum tolerated dose, it seems quite reasonable to insist that it
    allows adequate survival of the animals and does not produce a
    reduction in weight of more than 10% compared with the controls
    (Friedman, 1974). If two dose levels are chosen, the second should be
    one quarter or one third of the first dose.

         If the goal of the test is merely to ascertain the possible
    carcinogenicity of a compound, then the assay protocol should achieve
    the maximum sensitivity of the test; in this case, the highest
    tolerated dose is the most appropriate. If, however, additional
    information has to be collected, in particular regarding the
    establishment of a possible minimum effect or no-observed-effect
    level, then initiation of a dose-response experiment should be
    envisaged. In this case, a minimum of three doses, and preferably

    four, should be included. An advantage of including several dose
    levels, the lowest of which does not decrease the life span of the
    animals, is the possibility of collecting other data on chronic
    toxicity not otherwise available through a carcinogenicity test at
    high dose levels.

         Frequency of exposure may vary according to the route chosen. If
    the chemical is administered in the drinking water or mixed in the
    diet, exposure is continuous. If the chemical is given by gavage, the
    frequency can be two to three times per week. Topical applications may
    be made daily, while subcutaneous or intravenous injections must be
    more widely spaced, e.g. once or twice weekly. The duration of
    treatment should cover almost the entire observation period.  Combined treatment and cocarcinogenesis

         An investigation on the effect of more than one agent, given
    simultaneously, may approximate the actual environmental situation,
    where there is never exposure to a single chemical. In particular, it
    may be useful in revealing the carcinogenic effect of a chemical of
    very low carcinogenic potency and thereby help to identify situations
    or populations that may be exposed to an otherwise unsuspected high

         Response to an environmental chemical may be modified by the
    action of other chemicals that may either alter the rate and/or
    pathway of metabolism of the test chemical, or have a cocarcinogenic
    effect. A cocarcinogenic effect may be additive, when the chemical has
    a carcinogenic effect on its own which adds to the effect produced by
    the test chemical; it may be synergistic, when its effect, combined
    with that of the test chemical when given alone, exceeds the summation
    of the separate effects; or it may act as an incomplete carcinogen,
    that is, only as initiator or promoter in the two-stage carcinogenic
    process (UICC, 1969) (see Chapter 13).

         It is also essential to keep in mind that dietary components may
    influence the incidence of tumours in test animals. This may occur
    either because of the unsuspected presence of carcinogens (such as
    aflatoxins or nitrosamines) or because of substances that may modify
    the response to the test chemical by altering, for instance, the
    hepatic microsomal enzyme activity.  Positive and untreated controls

         An adequate group of untreated animals, serving as controls,
    should always be included in the planning of a carcinogenicity test.
    The size of the control group should never be smaller than the size of
    the treated group and, preferably, should be larger. When more than
    one chemical is tested simultaneously, the same control group can be
    used, provided that its size is appropriately increased.

         Besides these controls, i.e. animals not receiving treatment, and
    which could be called negative controls, the inclusion of positive
    controls in planning an experiment has recently been recommended, i.e.
    inclusion of a group of animals receiving a known carcinogen at a dose
    level that has already produced carcinogenic effects in several
    laboratory studies. Such controls ensure:  (a) more confidence in the
    outcome of tests carried out on compounds of unknown activity
    (Weisburger, 1974);  (b) assessment of the relative carcinogenic
    potency of the test chemical, and  (c) an indirect check on the
    reliability of the test laboratory. While it does not seem essential
    to include a positive control in every test, it is highly advisable
    for every laboratory to check the sensitivity of its bioassay system,
    periodically, with selected known carcinogenic chemicals. It is also
    advisable that a group of animals should receive only the vehicle
    (i.e. acetone, dimethylsulfoxide, etc.) in which the chemical under
    test is eventually administered.  Test material

         Long-term studies should not be started until sufficient
    information on the identity and purity of the test chemical has been
    assembled. The importance of knowing the toxicity of impurities is
    well demonstrated by the case of TCDD present as an impurity in the
    herbicide 2,4,5-T. Drugs or food additives should be tested in the
    form and degree of purity intended for human consumption (Health &
    Welfare, Canada, 1973; National Academy of Sciences, 1960; WHO, 1961,
    1969). In this case, as well as for mixtures of chemicals to which man
    may be exposed, it may be highly advisable to carry out additional
    studies in order to identify positively the carcinogenic component of
    the mixture.  Survey of animals, necropsy, and histological examination

         In order to present results correctly, detailed records of all
    experimental procedures and surveillance of the animals during the
    entire observation period should be maintained. While all pertinent
    details of the experiment may not be published, it is a good rule for
    the investigator to keep them for discussion with other interested
    scientists (UICC, 1969). If the results are to be published, it is
    essential that adequate information be given on the test chemical and
    test animal, as well as on all observations made on the experimental
    and control groups (WHO, 1961). A detailed list of items to be
    considered is given in the UICC (1969) report.

         In the case of neoplasms (Turusov, 1973, 1976), it is extremely
    important to give an exact description of the criteria used to
    classify lesions as hyperplastic, preneoplastic or neoplastic, benign
    or malignant. Many times, pathologists do not agree on certain
    diagnoses and it is far easier to interpret the results when the
    criteria used for classification are known. Terms such as hepatoma,
    which do not indicate the specific tissue of origin, should not be

    used (Reuber, 1974), while terms such as cholangioma or adenomatosis
    require additional qualification. The term should clearly indicate
    whether the lesion is considered malignant. Thus, specific
    descriptions should be given, e.g. in the case of "hepatoma" it may be
    either a liver cell adenoma or a hepatocellular carcinoma, while
    "cholangioma" may be cholangiocellular adenoma or cholangiocellular
    carcinoma. In addition, carcinomas may be subdivided into well,
    moderately, and poorly differentiated carcinemas.

         A lesion with atypical cells or with focal malignant change
    should be classified separately rather than under malignant tumours.

    7.2.2  Short-term tests (rapid screening tests)

         To date, there are no reliable alternatives to long-term
    bioassays for testing the carcinogenicity of chemicals; this means a
    delay of two years or more before a carcinogenicity bioassay yields
    results. However, recent advances in mutagenicity testing hold out
    hopes of a short-term test, at least for selecting, from among the
    many chemicals to be tested, those most in need of early attention. So
    far, about 80% of carcinogens have been shown to be mutagenic and with
    the continuously increasing sensitivity of the models now employed an
    even higher correlation is possible. At present, mutagenicity testing
    represents a valuable system for screening chemicals to be submitted
    to long-term carcinogenicity testing; it cannot replace long-term
    testing, until a satisfactory positive correlation is established and
    the possibility of false negatives eliminated.

         A mutation is any heritable change in genetic material including
    a chemical transformation of an individual gene (point mutation) or a
    change involving rearrangement of parts of a chromosome (chromosome
    mutation). The question of whether or not a mutagenic event is a
    prerequisite for carcinogenesis has long been debated. Many mutagens
    have been shown to be carcinogenic but other known carcinogens are
    known not to be mutagenic. Other mechanisms not involving DNA have
    also been implied in the process of carcinogenesis (Pitot, 1974).

         Recent short-term mutagenicity testing procedures (McCann & Ames,
    1976: McCann et al., 1975; Sugimura et al., 1976) have shown greater
    correlation between mutagenicity and carcinogenicity; consequently,
    some thought is now being given to using these assays as preliminary
    screens for potential carcinogens. However, at the moment, the fact
    that a particular compound has mutagenic activity can only be
    considered, in terms of carcinogenicity, as indicative of potential
    reactivity with DNA.  Metabolic activation, reaction with DNA, and DNA repair

         The synthetic and naturally occurring chemical carcinogens
    include a variety of chemicals having no common structural feature.
    However, it is becoming clear that the ultimate reactive forms of many

    chemical carcinogens are electrophilic (electron-deficient) reactants
    (Miller, 1970). Although some chemical carcinogens, such as direct
    alkylating agents and metal ions, are electrophiles  per se, the
    majority require metabolic activation to reactive forms (ultimate
    carcinogens) (Fig. 7.1).

         The formation of these electrophilic reactants from exogenous
    chemicals, in an animal species or in an organ, is the result of the
    balance between  in vivo activation and deactivation reactions
    carried out predominantly by enzymes localized in the endoplasmic
    reticulum of the cell. The ultimately available concentration of
    reactive metabolites seems to account for some of the organ and
    species specificities shown by several chemical carcinogens.

         During the last decade, much progress has been made in
    understanding the metabolism of various classes of chemical
    carcinogens (polynuclear hydrocarbons, Sims & Grover, 1974;
     N-nitroso compounds, Magee et al., 1976; aromatic amines, Kriek,
    1974; Weisburger & Weisburger, 1973; miscellaneous compounds, Miller,
    1973). The activity of microsomal mixed function oxidases is
    influenced by various drugs or environmental chemicals, that may
    stimulate or inhibit the formation of the ultimate carcinogens (Conney
    & Burns, 1972).

         These ultimate carcinogens bind covalently with cellular
    macromolecules such as DNA, RNA, or proteins, which, directly or
    indirectly, leads to heritable changes in the affected cells. Although
    the critical targets of chemical carcinogens are not known, there is a
    great deal of evidence supporting the theory that DNA is one major
    target in carcinogenesis (Farber, 1973).

         Some specific binding sites for the metabolites of carcinogens
    have been identified in the bases of DNA. The carcinogenic  N-nitroso
    compounds are examples (Magee et al., 1976). The main site of
    alkylation of DNA by alkylnitroso compounds is the  N,7 position of
    guanine, as with other alkylating agents. Other sites are also
    attacked and the significance of these DNA interactions is being
    investigated. The formation of 7-alkylguanine, however, seems to be of
    no obvious importance in the process of tumour induction, since no
    quantitative or qualitative correlation between the occurrence of this
    alkylated base in the DNA of treated animals and the carcinogenicity
    of nitrosamines or alkylating agents has been observed (Magee et al.,
    1976). Alkylation of guanine bases in DNA at the 7-position does not
    seem to produce mutations in bacteriophages (Loveless & Hampton,
    1969), nor does it alter the coding properties of synthetic
    polynucleotides  in vivo (Ludlum, 1970). Various laboratories have
    examined the biological importance of the alkylated  O,6 position of
    guanine residues in DNA which, in consequence, is able to induce
    mutations in phage (Loveless, 1969). It has been shown that there is
    an aberrant base pairing with a polymer containing alkylated

    FIGURE 36

     O,6-guanine residues (Gerchman & Ludlum, 1973). Goth & Rajewsky
    (1974) showed,  in vivo, that the initial degree of alkylation at the
     O,6 position in the DNA was, apparently, not correlated with the
    tissue-specific carcinogenicity of ethylnitrosourea which induces
    brain, but not liver, tumours. However,  O,6-ethylguanine persists
    much longer in brain DNA than  N,7-ethylguanine. The  O,6-alkyl
    elimination is also much slower from brain than from the liver DNA.
    From these findings it becomes apparent that variations in the
    sensitivity of different organs to the carcinogenic action of
     N-nitroso compounds might be attributed to different enzyme
    activities capable of repairing lesions in cellular DNA and/or to
    different rates of cell division in the target and non-target organs
    (Craddock, 1976; Margison et al., 1976; Pegg & Nicoll, 1976; Pegg,

         Possibly these repair systems, which recognize damaged DNA, are
    also impaired in the case of some types of cancer in man. The skin of
    patients suffering from the hereditary disease xeroderma pigmentosum
    is extremely sensitive to sunlight and such persons have a very high
    incidence of skin cancer. It has been shown that the cells of the skin
    of these patients are deficient in the capacity to repair UV-damaged
    DNA and that this deficiency is caused by lack of enzymes required for
    the excision of damaged regions from DNA (Cleaver, 1969).

         Some chemicals cause changes in the biological properties of DNA
    without covalent binding, e.g. by intercalation which could cause
    frameshift mutations. Since radioactive labelled chemicals are needed
    for most of these studies, technical problems associated with these
    methods render the tests impractical for routine screening. However,
    they will continue to contribute considerably to the understanding of
    the mechanism of interaction of a chemical or its activating
    metabolite with specific sites in DNA.

         Initial lesions in DNA can either lead to a permanent change,
    such as a mutation, or can be removed by cellular repair processes. In
    bacteria and mammalian cells, three major repair processes can be
    considered. First, the photoreactivation repair process that repairs
    only UV damage to DNA and involves a direct enzymatic cleavage of
    pyrimidine dimers to monomers upon exposure of cells to visible light.
    This repair process is present in most prokaryotes and eukaryotes and
    it was recently identified in human cells (Sutherland et al., 1975).
    The second process, post-replication repair, which is also called
    recombination repair, is limited to the DNA synthesis period. Although
    its mechanism is not well known, it has been proposed that it is
    caused by the presence of damaged bases on parental DNA strands too
    close to the replication fork to be recognized by the third process,
    excision repair. Unlike post-replication repair, excision repair
    occurs throughout the cell cycle and is present in a large variety of
    organisms. During this process, damaged bases are excised, producing a
    single-strand break. This gap is repaired by replacing the original
    bases with bases complementary to those of the opposite intact strand.

    Various enzymes are involved in this process, namely endonuclease, a
    DNA exonuclease, a DNA polymerase and a DNA ligase (Cleaver, 1974).

         From studies on prokaryotes, the concept arose that excision
    repair is error-free (Kondo, 1973). It is thought that mutations
    originate during semi-conservative replication as a consequence of the
    fact that DNA templates containing damage have not yet been or cannot
    be repaired by excision repair. Since DNA replication on damaged
    templates involves post-replication repair, this repair process is
    considered error-prone and responsible for mutagenesis. Whether
    miscoding and mutagenesis of DNA templates is the result of lack of
    excision repair enzymes or due to error-prone post-replicative repair
    is not clear at present.

         The isolation of xeroderma pigmentosum-variant fibroblasts, that
    have normal excision repair but are deficient in post-replication
    repair (Maher et al., 1976) may lead to a better understanding of the
    role of these repair processes in mutagenesis and carcinogenesis.

         Recent efforts have been devoted to the development in mammalian
    systems of a screening test for the detection of possible mutagens or
    carcinogens based on excision repair. The focus on this type of repair
    is mainly based on the greater availability of procedures thought to
    be appropriate to the problem. There are four different ways of
    examining this form of repair in mammalian systems and all are based
    on the assumption that chemical mutagens or carcinogens interact with
    DNA, inducing molecular alterations which result in DNA repair that
    can be measured as unscheduled incorporation of various DNA precursors
    (Cleaver, 1975; Legator & Flamm, 1973). The common basis of all these
    tests is the differentiation between repair and normal
    semiconservative synthesis of DNA.

         One procedure, "unscheduled DNA synthesis", involves
    autoradiography of cultured cells, and consists of the incorporation
    of precursors during resynthesis of short nucleotide sequences that
    have been eliminated from DNA strands following their damage by
    chemicals (Cleaver, 1973; Stich & San, 1970). This procedure permits a
    quantitative evaluation of DNA repair synthesis in various types of
    cell in a tissue and can be applied to an  in vivo system (Stich &
    Kieser, 1974), thus allowing the detection of indirect mutagens and/or

         Another process uses 5-bromodeoxyuridine (BrdU), which is an
    analogue of thymidine. During normal replication, the incorporation of
    BrdU produces a DNA with a high buoyant density; this does not occur
    when BrdU is incorporated in DNA during the repair synthesis. Thus, by
    using radiolabelled BrdU, or radiolabelled thymidine and non-labelled
    BrdU, it is possible to differentiate, on cesium chloride density
    gradients, semi-conservative replication of DNA from the repair
    synthesis according to the different buoyant densities.

         A third procedure also uses BrdU but the differentiation between
    semi-conservative and repair DNA synthesis is done by different means.
    Cells exposed to the chemicals are incubated with BrdU and the DNA is
    subjected to long wavelength ultra-violet radiation. Breaks appear in
    the DNA at the sites of incorporation of BrdU and cause slower
    sedimentation of the DNA in alkaline sucrose gradients (Smith &
    Hanawalt, 1969). This technique appears to be a very sensitive one for
    measuring single-strand breaks but it still seems to be prone to
    artefacts and cannot be considered suitable for large-scale studies
    (Cleaver, 1975).

         Another method involves the total suppression of normal DNA
    synthesis, for example, by hydroxyurea, thus the incorporation of
    precursors into DNA reflects only repair synthesis and not normal

         Some of the above techniques are time-consuming, costly and not
    yet standardized, so that it is difficult to adapt them for
    large-scale studies. The most promising approach appears to be the
    measurement of unscheduled DNA synthesis, but criticisms have also
    been made of the use of this system as an indicator of mutagenic or
    carcinogenic potential (Cleaver et al., 1975). One limitation is that
    unscheduled synthesis is an average measure of repairable damage in
    all cells of a population or in all sites of the DNA, whereas
    mutagenesis and carcinogenesis seem to depend on the amount of
    unrepaired damage in a small percentage of cells and in a few specific
    DNA sites. Due to these limitations and to the current limited
    understanding of these processes, none of these variables could, by
    themselves, be a reliable indicator of a potential mutagenic or
    carcinogenic chemical. However, these studies associated with studies
    of DNA damage as well as of the expression of this damage are
    essential in the understanding and development of reliable screening

         Metabolic activation of chemicals to electrophiles, DNA damage,
    and subsequent repair processes are important factors in the
    initiation of cancer. The phenotypic expression (tumour development)
    of these initial molecular changes is modulated by a number of factors
    (Farber, 1973) that influence the macroscopic appearance of cancer at
    a later stage (Pitot, 1977). However, the initial molecular changes,
    induced by chemical carcinogens, appear prerequisite for initiation of
    the cancer process. Further studies on the alteration of DNA induced
    by chemical carcinogens and on the repair of such lesions before cell
    division by tissues, whether target or not, are required to evaluate
    the role of DNA repair processes in chemical carcinogenesis.  In vitro neoplastic transformation of mammalian cells

         The term transformation in such studies refers to the  in vitro
    observations of various changes present in the cells treated  in vitro
    with the chemicals, when compared with untreated control cells.

    Transformed cells may differ from control cells in various ways such
    as: (a) alteration of cellular and colony morphology; (b) increased
    plating efficiency; (c) agglutinability by plant lectins; (d) altered
    glycolytic patterns; (e) resistance to toxicity of some chemicals; (f)
    altered surface properties (contact inhibition of movement and growth,
    population density, growth in suspension, ability to grow in agar or
    other semisolid media); (g) sensitivity to activated macrophages and
    lymphocytes; (h) establishment of cell lines with the potential to be
    sub-cultured indefinitely  in vitro; and (i) appearance of new
    antigens (Fedoroff, 1967). However, the unequivocal criterion of
    malignant transformation is the capacity of transformed cells to
    develop a malignant neoplasm when injected into a syngenic,
    immunosuppressed or thymus-free host, or into privileged sites
    (Giovannella et al., 1972; Sanford, 1965).

         Recently, Sanford (1974) critically reviewed the significance of
    these various  in vitro changes in relation to the acquisition of
    neoplastic potential. It was stressed that these criteria of
    neoplastic transformation cannot completely replace the  in vivo
    assay for tumour production. With reference to the problems of the
    application of tissue culture to the rapid detection and
    characterization of neoplastic transformation  in vitro, the reader
    is referred to the proceedings of the symposium "New Horizons for
    Tissue Culture in Cancer Research" ( J. Natl Cancer Inst., 1974).

         In most  in vitro transformation experiments the cells used have
    been fibroblasts. Earle & Nettleship (1943) reported the
    transformation by 1,2-dihydro-3-methyl-benz[j]aceanthrylene
    (3-methylcholanthrene) of a long-term culture of fibroblasts as
    demonstrated by the development of tumours after inoculation of the
    cells into mice. However, this observation was attributed to
    spontaneous transformation, since Sanford et at. (1950) observed that
    the cells were tumorigenic also without treatment with chemicals. The
    first clear demonstration of transformation of cells in culture by
    chemicals was that of Berwald & Sachs (1963) who observed the
    transformation of Syrian hamster embryo secondary cells with
    3-methylcholanthrene and benzo(a)pyrene, but not with urethane or
    solvent. Since this report, various cells types originating from
    different animal species have been used for  in vitro transformation.
    This topic has recently been reviewed by Heidelberger (1973) and
    Kuroki (1975), who critically examined the advantages and
    disadvantages of the various strains and lines of fibroblastic cells,
    namely of the Syrian hamster embryo, fibroblastic cells derived from
    mouse ventral prostate, cell lines derived from embryo cells of Swiss
    mouse BALB/c, C3H, AKR, and C57/B1 strains, Chinese hamster lung
    cells, as well as various tissues from organ cultures. Transformation
    of the above cell lines was obtained with polynuclear hydrocarbons and
    with chemicals that do not require metabolic activation.

         Some chemical carcinogens that are active  in vivo have failed
    to induce transformation, when applied directly to hamster embryo
    cells; this is presumably because such compounds require metabolic
    conversion to active intermediates that are lacking or are present in
    insufficient concentration in these cells. However, neoplastic
    transformation was detected in fibroblasts obtained from embryos whose
    mothers had been exposed to indirect carcinogens during pregnancy
    (Di Paolo et al., 1972, 1973). A similar approach was used by Borland
    & Hard (1974), who cultured kidney cells at various times following
     in vivo treatment of rats with  N-methyl- N-nitrosomethanamine
    (dimethylnitrosamine). The cells isolated from treated rats showed
    various morphological and behavioural changes associated with
    transformation. Laerum & Rajewsky (1975) reported the development of
    glioblastomas following injection of glial cells originating from the
    brain of rat embryos, the mother having been treated with
    ethylnitrosourea during pregnancy.

         Epithelial cultures from rat liver were recently established in
    various laboratories and used for transformation studies (Iype, 1974;
    Katsuta & Takaoka, 1972; Montesano et al, 1975; Weinstein et al.,
    1975; Williams et al., 1973). The transformation of these cells by
    various carcinogens that need metabolic activation was determined by
    the development of carcinomas after their back-transplantation into
    suitable hosts. Carcinomas were observed following inoculation of
    epithelial cells from mouse skin, or rat urinary bladder or salivary
    glands, treated with chemical carcinogens  in vitro (Brown, 1973;
    Fusenig et al., 1973; Hashimoto & Kitagawa, 1974).

         One disadvantage of these epithelial cells is that the
    morphological criteria for transformation of fibroblast cultures
    (piling up of cells, crisscross arrangement of cells etc.) do not
    apply possibly because normal epithelial cells have little or no
    locomotion and they continue to divide even when in close contact with
    other cells (Weinstein et al., 1975). However, the capacity for growth
    in soft agar appears to provide a reliable and reproducible
    correlation with the tumorigenicity of these cells (Weinstein et al.,
    1975; Montesano et al., 1977). Although, at present, this system
    provides only a qualitative and not a quantitative assay of  in vitro
    transformation, it is the only instance of unequivocal production of
    carcinoma. The establishment of reliable criteria, measurable  in
     vitro, for distinguishing normal from transformed epithelial cells
    in culture is essential for the development of quantitative systems
    for the transformation of these epithelial cells. Recently the
    neoplastic transformation of human diploid cells by chemical
    carcinogens has been described (Kakunaga, 1977).

         Cell culture has been extremely useful in elucidating the
    cellular and molecular mechanism of chemical carcinogens and it holds
    great promise as a test for screening for the potential

    carcinogenicity of environmental chemicals. However, some time is
    needed before this test may be used for routine testing in a
    reproducible way.  Mutagenicity tests

         The growing experimental evidence linking the carcinogenic
    activity of numerous chemicals with their capacity to be converted
    into electrophilic derivatives, that may also exert a mutagenic
    effect, has led to the suggestion that a relationship between chemical
    carcinogenesis and mutagenesis may exist (Miller & Miller, 1971a,b).
    Such a correlation has so far been limited to those changes of the
    genotype that appear as a consequence of structural or functional
    alterations of nucleic acids. Not all chemical mutagens have been
    shown to be carcinogenic. However, most chemical carcinogens, several
    of which cause cancer in man, have now been found to be mutagens, when
    tested by one of the mutagenicity test procedures that combine
    microbial, mammalian, or other animal cell systems as genetic targets
    with an  in vitro or  in vivo metabolic activation system. The
    growing empirical relationship between chemical mutagens and
    carcinogens does not imply that the two processes are identical, but
    it offers a promising method for the use of mutagenesis as a rapid
    prescreening assay for carcinogenesis (Bartsch & Grover, 1976;
    Bridges, 1976; Council of the Environmental Mutagenic Society, 1975;
    IARC, 1976; McCann & Ames, 1976; Purchase et al., 1976; Stoltz et al.,
    1974; Sugimura et al., 1976; WHO, 1974).

         The choice of mutagen-detecting assay depends on various
    considerations, i.e. the chemical structure and pharmacological
    activity of the chemical, the type of human exposure, and the nature
    of the population at risk. The same considerations should be taken
    into account in assessing the strength of the evidence of mutagenicity
    and its relevance to man.  Submammalian assay systems

         Bacterial phages have been used to test reactive forms of
    chemical carcinogens. Chemicals inducing point mutations can be
    detected by reacting the test compound with the free phage or with
    phage during its duplication inside bacteria (Corbett et al., 1970;
    Drake, 1971).

         In the bacterial transformation of DNA (Freese & Strack, 1962),
    genetic information contained in the DNA isolated from one strain of
    bacteria can be transformed to that of a recipient strain. Purified
    bacterial DNA is readily accessible to reactive forms of mutagens.
    Thus, this system has been used to quantify the mutagenic potential of
    a number of chemicals. Extensive studies have been made on the
    inactivation of  Bacillus subtilis DNA and transformation of the
    tryptophane-requiring strain T-3 (Herriott, 1971; Maher et al., 1968).

         The reverse mutationa system of  Salmonella typhimurium uses
    the genetically well-defined histidine-requiring mutants developed by
    Ames and his colleagues (Ames, 1971; Ames et al., 1972a,b, 1973;
    McCann et al., 1975).

         These revert to prototrophy by single-base pair substitutions,
    e.g. strain TA1535 or by base pair insertion (frameshift), e.g.
    strains TA1536, TA1537 and TA1538. Most of the theoretically possible
    types of mutation may be detected with a set of these test strains,
    where a mutation of one of the genes responsible for excision repair
    (UVrB) has produced a 100-fold increase in sensitivity. Penetration of
    larger molecules through the bacterial cell walls has also been
    facilitated by the use of deep-rough mutants deficient in the exterior
    polysaccharide coat. Two newly developed test strains TA100 and TA98
    were obtained by transferring an ampicillin resistance factor
    (R factor) to the standard test strains TA1535 and TA1538,
    respectively (McCann et al., 1975). These strains are effective in
    detecting classes of mutagens that were not previously detected with
    the original strains. The tests are usually performed by adding a few
    crystals or a drop of solution of the test chemical in
    sulfinylbis[methane] (dimethylsulfoxide) or water to a uniform lawn of
    one of the histidine-requiring mutants on the surface of a Petri plate
    containing histidine-poor medium, or by incorporating the test
    compound, a postmitochondrial tissue fraction, cofactors (NADPH or
    NADPH+ and glucose 6-phosphate) and the bacterial test strain in
    histidine-poor soft agar. For general mutagenicity screening, a liver
    homogenate (9000 g supernatant) from rats induced with a mixture of
    polychlorinated biphenyls (Aroclor 1254) is recommended as a metabolic
    activation system. For routine testing, the strains TA1535, TA1537 and
    TA1538 can be used in combination with the strains carrying the R
    factor (TA100 and TA98). This method of testing using various groups
    of chemicals and various experimental conditions is described in more
    detail by Ames et al. (1975).

         Using the genetically well characterized  Escherichia coli
    strains, reverse and forward mutation can be scored using nutritional
    resistance or fermentative markers (Bridges et al., 1972; Mohn, 1973).
    Prophage lambda induction in  E. coli strains by chemical mutagens
    activated by liver microsomal enzymes has been described as a
    sensitive test for the detection of potential mutagens and carcinogens
    (Moreau et al., 1976).


    a  Mutations in which the function of a given gene is lost are
       called forward mutations. Mutations that bring about the
       restoration of gene function are called reverse mutations or back

         Mutagenesis has been extensively studied in  Neurospora crassa.
    Heterokaryon has been developed, which is heterozygous for two
    closely-linked loci in the  ad-3 region. Mutants at the  ad-3A and
     ad-3B loci have a requirement for adenine and can be selected
    directly on the basis of accumulation of a reddish-purple pigment in
    the mycelium. The  ad-3 mutations can be characterized by a series of
    simple genetic tests to distinguish point mutations from deletions and
    to obtain a presumptive identification of the genetic alterations in
    the point mutations at the  ad-3B locus at a molecular level (de
    Serres & Malling, 1971).

          Saccharomyces cerevisiae and  S. pombe have been used to
    investigate the effects of carcinogenic and mutagenic compounds in
    these organisms. Mitotic gene conversion, in which a sequence of a few
    hundred nucleotides of one chromosome is replaced by one corresponding
    sequence from a homologous chromosome, can be studied in diploid cells
    of the yeast  S. cerevisiae strain D-4 heteroallelic at the loci
     trp-5 and  ade-2. This strain carries two different inactive
    alleles of two genes ( trp-5 and  ade-2) which are located on
    different chromosomes and the functional defects of which lead to a
    nutritional requirement. Mitotic gene conversion, which is increased
    by many types of mutagenic treatment, can transfer the intact region
    of one of these alleles to the defective region of the other, thus
    producing a heterozygotic diploid cell with full functional activity.
    The mechanism is presumably based on the formation of single-strand
    breaks in DNA and probably involves repair processes. Positive results
    are only obtained with agents or metabolites which either bind with
    DNA covalently or interfere with DNA metabolism. In contrast with most
    microbial mutation systems, mitotic gene conversion does not show a
    response specific to any type of mutagen. In addition to gene
    conversion, forward and reverse mutation can be measured with
     S. cerevisiae (Loprieno et al., 1976; Marquardt, 1974; Mortimer &
    Manney, 1971; Zimmerman, 1973).  Mammalian somatic cells

         The application of cultured cell lines is somewhat restricted at
    present by requirements for karyotypic stability and high plating
    efficiency. A widely used system developed by Chu (1971) employs cell
    lines derived from the lung, ovary, and other tissues of Chinese
    hamster, which usually maintain a near-diploid chromosome number and
    exhibit active growth and high cloning efficiency. Two reviews discuss
    this topic in detail (De Mars, 1974; Thompson & Baker, 1973).
    Selective media have been developed to detect both forward and reverse
    mutations at three genetic loci involving enzymes in the salvage
    pathways of purines and pyrimidines. In Chinese hamster, as in man,
    the use of preformed hypoxanthine and guanine is controlled by an
    X-linked gene. Mutant cells at these loci are deficient in the enzyme
    hypoxanthine-guanine phosphoribosyl transferase and are resistant to
    certain purine analogues. Similarly, mutant cells deficient in
    adenosine phosphoribosyl transferase cannot metabolize preformed

    adenosine or its analogues for incorporation into nucleic acid. The
    third type of drug-resistant mutants currently studied in these and
    other mammalian cells are those deficient in thymidine kinase. Such
    cells exhibit resistance to the thymidine analogue,
    5-bromodeoxyuridine. Cells carrying mutation at these loci can be
    selected from the wild type in an environment containing an
    appropriate purine or pyrimidine analogue. Revertants to the wild type
    can be recovered in selective media in which the cells are supplied
    with a natural purine or pyrimidine while the normal  de novo pathway
    of purine or pyrimidine synthesis is inhibited by an antimetabolite.
    It has been demonstrated that Chinese hamster cells treated with
    physical or chemical mutagens undergo a significant increase in the
    frequency of forward and reverse mutations compared with the
    spontaneous frequency (Huberman & Sachs, 1974; Huberman et al., 1971,
    1972). A liver microsomal activation system can be added for the
    metabolic activation of chemicals (Krahn & Heidelberger, 1975; Kuroki
    et al., 1977).

         Forward mutations from thymidine kinase +/- cells to thymidine
    kinase -/- have been induced by treatment of cells with X-rays or
    chemical mutagens (mouse lymphoma L5178 Y cells). These cells can also
    be grown in the presence of  in vitro or  in vivo metabolic
    activation systems, which makes them suitable for host-mediated
    mutagenicity tests (Nahas & Capizzi, 1974).

         Forward mutations to hypoxanthine-guanine phosphoribosyl
    transferase deficiency (resistance to 8-azaguanine) in diploid human
    fibroblasts in culture have been shown to occur either spontaneously
    or after X-radiation (Albertini & De Mars, 1973). Chemical induction
    of forward mutation from hypoxanthine-guanine phosphoribosyl
    transferase - to + in human lymphoblastoid cell line has also been
    reported (Sato et al., 1972).  Host and tissue-(microsome) mediated assays

         These take into account the conversion of chemicals into
    mutagenic metabolites, and are particularly suitable for the detection
    of chemicals that are not mutagenic  per se but require metabolic
    activation. They can be performed  in vivo (host mediated) or  in
     vitro (tissue mediated) using various indicator organisms, such as
    bacteria, fungi, yeast, or mammalian cells.

         In the host-mediated assay, the indicator organisms are injected
    into the interperitoneal cavity of an animal (Legator & Malling,
    1971), which is then treated with the test compound by another route.
    After a given length of time, the animal is killed and the indicator
    organism is recovered and scored for mutants. Comparison between the
    mutagenic action of the compound on a test strain directly and the
    host-mediated assay indicates whether the host can activate or
    inactivate the test compound. The limitations of the host-mediated
    assay are the high spontaneous mutation rates of the indicator

    organism in the host, the host's effect on cell survival and the
    selection of heterogenic cell populations. In order to measure a
    significant increase over the spontaneous mutation rate, doses well
    above LD50 have to be given to rats or mice. This type of assay is
    also limited since it does not identify the site of conversion to a
    mutagenic metabolite. Modifications have been reported (Mohn, 1973).

         In the tissue-mediated assay, the indicator organisms are
    incubated  in vitro in the presence of a tissue fraction plus
    appropriate cofactors and the test compound. The mutants are
    subsequently isolated and scored.

         Ames et al. (1973a,b) have developed a mutagenicity assay that
    combines a Salmonella strain, a liver microsomal preparation, and
    cofactors in a soft agar layer on a Petri dish. Bartsch et al.
    (1975b), Czygan et al. (1973) and Malling (1974) have described a
    liquid incubation system where bacteria, a tissue preparation, and the
    test compound are incubated in liquid suspension. Test compounds that
    are gases at room temperature or are volatile can be assayed by
    exposing Petri dishes containing bacteria, tissue fraction, and
    cofactors to a mixture of gas and oxygen at 37C (Bartsch et al.,
    1975a). The mutagenicity of urinary metabolites, excreted as
    conjugates in experimental animals treated with an indirect mutagen,
    may be detected by treating the urine with hydrolytic enzymes in the
    presence of a bacterial test strain or yeast, and an  in vitro
    metabolic activation system (Commoner et al., 1974; Durston & Ames,
    1974; Marquardt, 1974). In another modification, Huberman & Sachs
    (1974) used lethally irradiated rat fibroblasts that retain a
    drug-metabolizing capacity and cocultivated them with Chinese hamster
    V79 cells which are used as genetic indicators.

         The mutagenicity tests previously described (,, all have individual advantages and limitations determined
    either by the genetic indicators or by the metabolic activation
    system. The use of submammalian organisms for the detection and
    classification of mutants, induced by chemicals, is greatly
    facilitated by the relatively small genome to which fine genetic
    mapping and biochemical analysis can be applied and by the short
    generation time. Huge populations of some of these organisms can be
    raised and they can easily be handled when analysing multiple

         The relevance of such data from submammalian systems to mammalian
    cells is based on the assumption that the basic principles of
    heredity, and the structure and functions of DNA in terms of
    reactivity, the triplet code, transcriptional, and translational
    mechanisms, are essentially the same for all living cells irrespective
    of their evolutionary level. However, this extrapolation is hampered
    by lack of knowledge of repair processes that play a role in mutation
    fixation and expression and are insufficiently understood in mammals.

    It is obvious that normal human diploid cells are most desirable for
    this test, but current studies suggest that reliable conclusions can
    be drawn from results with non-human mammalian cells.

         The other factor, related to the fact that many chemicals are not
    active  per se, is the metabolic activation of the compounds. In many
    cases, reactive metabolites with a limited life span may fail to reach
    or react with the genetic indicator either because they are further
    metabolized to inactive compounds, or because they react with other
    cellular constituents. For this reason, mutagenicity assays in intact
    animals (host-mediated assays) may give negative results, as in the
    case of  N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) and acridine
    mustard (ICR-170), proven to be extremely potent mutagens. Metabolism
    in animals is affected by exogenous and endogenous factors such as
    chemicals causing enzyme induction and inhibition. Other modifying
    factors are age, sex, and strain of animals, diurnal and seasonal
    rhythms, differences between the fetal and adult state, mode of
    administration, cellular uptake, and distribution and excretion of the

         The tissue-mediated mutagenicity test cannot, with certainty,
    reproduce the  in vivo situation, but obvious advantages are the high
    sensitivity, good reproducibility, low cost, and the possibility of
    testing a large number of chemicals. In addition,  in vitro testing
    with well-characterized genetic indicators allows the use of human
    tissue such as human liver to determine their ability to generate a
    mutagen (Bartsch, 1976).

    7.2.3  Correlation between short and long-term bioassays for

         Short-term tests (rapid screening tests) are procedures that do
    not have the  in vivo production of a visible tumour in animals as an
    end point. The variables used in short-term tests to detect chemical
    carcinogens are based on an interaction of carcinogens and/or their
    metabolites with macromolecules, the induction of chromosomal
    aberrations, mutagenesis, DNA repair, and DNA binding.

         In the evaluation of these methods, the major question is which
    screening tests, singly or collectively, serve as reliable indicators
    or predictors of the potential carcinogenic hazard of the chemical.
    The answer can only be obtained by testing a representative number of
    compounds. A valid test should demonstrate that compounds with known
    carcinogenic properties are positive, within the limit of the test,
    and negative compounds are negative. If a test is required for
    preliminary screening, a small proportion of false negatives or
    positive results may be acceptable, but for a final test, no false
    negative results are acceptable.

         Rapid screening tests should be relatively simple and
    inexpensive. They can be used in three ways: to trace carcinogens
    and/or mutagens in the complex environment of man; as a tool for
    prescreening chemicals to be submitted to a more lengthy set of
    bioassays; or for better extrapolation from experimental animal data
    to man and for improving the relevance of long-term bioassays in
    experimental animals.

         Considering reproducibility, cost, the number of chemicals that
    can be examined in a short time, and the scientific basis of the test,
    tissue-mediated mutagenicity procedures using well-characterized
    genetic indicators and a metabolically defined  in vitro activation
    system, appear at present to be the most promising short-term tests.
    With current methods there is still the chance of false negative
    results, depending on the systems used, either for lack of appropriate
    cofactors for activation or because of the extreme reactivity and/or
    toxicity of the compound or its metabolites. However, the number of
    false negative results in  in vitro tissue-mediated assays is small
    compared with other mutagenicity test procedures (Montesano & Bartsch,

         The increasing evidence of a possible correlation between
    mutagenicity and carcinogenicity certainly does not mean that one
    biological effect may be equated with another. Thus, the mutagenic
    activity of a chemical cannot, at present, automatically be assumed to
    imply a definite carcinogenic effect in man, nor can these results
    replace long-term carcinogenicity testing in animals.

         Furthermore, it is still unknown whether all carcinogens will be
    found to be mutagenic, and all mutagens, carcinogenic. Examples are
    the strong mutagens, nitrous acid, hydroxylamine, and base analogues,
    for which no carcinogenic effect in animals has so far been reported;
    they do not act via electrophilic intermediates, a mechanism that has
    now been recognized for must ultimate carcinogenic forms. Nor have
    steroidal sex hormones, carcinogenic in animals, been reported to be
    mutagenic, as yet. On the other hand, various chromium salts have
    recently been shown to be mutagenic in bacteria (Venitt & Levy, 1974).

         Development of cancer  in vivo is determined by a variety of
    factors that cannot be duplicated in an  in vitro short-term testing
    system because:

         (a)  The concentration of ultimate reactive metabolites available
              to react in organs in animal species with cellular
              macromolecules, which is a consequence of a balance between
              metabolic activation and detoxication processes, is only
              partly reflected by  in vitro testing procedures.

         (b)  Species and organ specificity of a chemical carcinogen might
              be determined, in part, by organ-specific DNA repair.

         (c)  Since chemical carcinogenesis is thought to be a multi-step
              process in which the early, apparently irreversible,
              initiation of a cell is followed by several subsequent
              stimuli provoking cellular replications, leading to an overt
              tumour, a short-term test capable of detecting complete
              carcinogens may be useful to detect initiating agents but
              cannot at the present time detect the action of promoting

         Currently used short-term tests, in particular tissue-mediated
    mutagenicity assays are effective in predicting, with a certain
    accuracy, the carcinogenic potential of chemicals (McCann & Ames,
    1976; Purchase et al., 1976; Sugimura et al., 1976), but give no
    indication of the target organs or species specificity of their
    carcinogenic activity. Furthermore, the relative potency of a chemical
    to induce biological effects, defined as the endpoints, for the most
    frequently used rapid screening tests cannot at the present time be
    reliably correlated with its carcinogenic potency (Bartsch et al.,
    1977; Meselson & Russell, 1977). More data are needed to compare
    dose-response curves obtained in rapid screening tests on a given
    chemical with those of other biological effects obtained  in vivo
    (long-term bioassays).

    7.2.4  Significance of experimental testing for assessing the possible
           carcinogenic risk of chemicals to man

         Experience acquired, so far, in long-term carcinogenicity testing
    has shown that nearly all compounds that are carcinogenic in man are
    also carcinogenic in one or several animal species, even though the
    tumour type may not be the same as in man. The concept that animal
    carcinogenicity data are predictive of a human carcinogenic risk and
    useful in preventing human cancer was accepted in 1941 in the case of
    2-actylaminofluorene (AAF). This chemical was not used on a worldwide
    basis as an insecticide, because some experiments had already shown
    its carcinogenicity before it was marketed (Wilson et al., 1941); its
    restriction was facilitated by the existence of a number of
    substitutes at the time when the results of the first experiments on
    AAF were reported. This cautious attitude has not been consistently
    applied in other situations, on the grounds that the experimental data
    were insufficient or inadequate to evaluate the possible hazard to
    man. This is shown by the various relationships between experimental
    carcinogenicity data and possible human hazard that are considered in
    the adoption of preventive measures.

         The first observation that some aromatic amines were involved in
    the causation of urinary bladder tumours in man dates from 1896; it
    was confirmed in 1907 and reconfirmed many times thereafter (IARC,
    1974). In 1938, Hueper et al. reported the induction of bladder cancer
    in dogs exposed to 2-naphthalenamine (2-naphthylamine). Preventive
    measures were taken only in the late 1950s, when conspicuous
    epidemiological evidence had accumulated. In this case, the human

    epidemiological evidence was judged insufficient, and the experimental
    evidence, when it became available, was also deemed insufficient until
    further epidemiological findings came to hand.

         The delay in taking preventive measures applies also to
    carcinogens on which, contrary to the case of aromatic amines,
    experimental evidence preceded observations of their effect in man,
    e.g. diethylstilbestrol,  bis(chloromethyl)ether, and chloroethylene
    (vinyl chloride). (Tomatis et al. 1978; Montesano & Tomatis, 1977).

         One of the main objections to carcinogenicity tests on animals is
    that the experimental system used tries to produce the maximum
    possible carcinogenic effect of the test chemical, does not reflect
    the human situation, and is therefore misleading. It is difficult,
    however, to accept this argument because, in most instances where a
    chemical was found by epidemiological investigations to be associated
    with cancer in man, the incidence was so high that the association was
    clear without animal studies. This has been the case for high risk
    groups such as occupational cancer groups. However, the risk is not
    confined to these groups but also applies to other populations where
    cancer incidence may be too low for detection by normal
    epidemiological methods, hence the need to carry out animal
    experiments under conditions that permit confident judgment of the
    carcinogenicity or inactivity of a chemical.

         Cancer testing in animals has reached a relatively sophisticated
    stage and an exhaustive study of a chemical in animals is sufficient
    evidence of a potential cancer risk for man. An assessment of the
    validity of experimental results is essential for the successful
    prevention of cancer in man. This does not preclude further research
    for the development of short-term tests, but, at the present time,
    these cannot replace long-term carcinogenicity testing.

    7.3  Heritable Mutations

         As noted in the introduction, direct methods for assessing
    whether a chemical has the potential to cause heritable mutations in
    man do not exist at present. Nontheless, information relative to the
    possible production in man of germ cell mutations can be derived from
    a variety of sources. These fall into three major categories: (a)
    primary DNA damage involving DNA alteration, stimulation of DNA
    repair, gene mutations tests including mutagenicity assessment using
    bacterial or other microorganisms, with and without metabolic
    activation; (b) whole animal tests for point or gene mutations
    including insects, e.g. drosophila recessive, and the specific locus
    test in mice; (c) chromosomal mutations including cytogenetic tests in
    mammals, the dominant lethal test in mammals, and the heritable
    translocation test in rodents. Examples of the first category have
    already been presented. These tests provide information relevant to
    heritable mutations and section 7.2.2 should be consulted for fuller
    details. Obviously, tests employing isolated systems only provide

    information on mutagenic action on the specific systems tested. This
    information cannot be fully interpreted without evidence concerning
    the access of this ultimate mutagen to the germ cells.

         An extensive examination of relevant test procedures for
    heritable mutations has recently been completed (DHEW, 1977).

    7.3.1  Whole-animal tests

          Insects. Drosophila melanogaster is one of the best genetically
    characterized species and is widely used. Drosophila of either sex can
    be treated and mutation frequencies from successive germ cell stages
    may be obtained after observation of three generations. The X-lined
    recessive lethal test is one of the most sensitive tests with
    drosophila, since the X-chromosome represents about 1/5th of the whole
    genome. In contrast to most microorganisms, insects possess an enzyme
    system that appears to metabolize foreign compounds in a fashion
    similar to that of vertebrates (Abrahamson & Lewis, 1971; Fahmy &
    Fahmy, 1972, 1973; Sobels & Vogel, 1976).

          Mouse specific locus test. The specific locus test is a method
    of inducing, detecting, and measuring the rate of mutation at several
    recessive loci. It consists essentially of mating treated or untreated
    wild type mice, either male or female, to a strain homozygous for a
    number of known recessive genes. The recessive genes are such that
    they are readily expressed as visible phenotypes in homozygous state.
    If a mutation occurs in any of the test loci in the germ cells of
    treated animals, it may be detected in the offspring. If no mutation
    has occurred following treatment, the progeny from the cross will all
    be of the wild type (Cattanach, 1971; Russell, 1951).

          Chromosomal mutations. Although the molecular basis is not
    understood, a change in the whole chromosome, i.e. a structural
    chromosome aberration, occurring as a consequence of a misrepair of
    chromosomal breaks, may lead to deletions, duplications, and
    translocations. Changes in the whole chromosome complement, i.e.
    numerical chromosome aberrations, arise through nondisjunction, as in
    failure of a pair of chromosomes to separate during gametogenesis,
    meiotic nondisjunction, or during mitotic division. The resulting
    daughter cells are either trisomic with an extra chromosome or
    monosomic and lacking a chromosome. Anaphase lag occurs during nuclear
    division in the progeny cells, and a chromosome may be either lost or
    gained. In somatic cells, nondisjunction and anaphase lag can lead to

         Chromosomal damage may be studied in a number of test systems
    that are best divided into  in vivo and  in vitro systems, on the
    basis of their capability to metabolize the test compound (Frohberg,
    1973). The short-term human lymphocyte culture  in vitro is commonly

    used to assess the effects of chemicals upon chromosomes. Metabolic
    activation of the test compound can be achieved by addition of a liver
    microsomal system (Bimboes & Greim, 1976).

          The micronucleus test. As an  in vivo cytogenetic method, the
    micronucleus test is a procedure for the detection of aberrations
    involving anaphase chromosome behaviour using bone-marrow
    erythroblasts. The test is based on the formation of micronuclei from
    particles of chromatin material which, due to chromosome breakage or
    spindle disjunction, do not migrate to the poles during anaphase and
    are not incorporated into the telophase nuclei of the dividing cells.
    The procedure is to treat animals with clastogenic agents and, at an
    appropriate time after treatment, to aspirate bone-marrow samples into
    calf serum. This is then centrifuged and smears are made from the
    resuspended pellets of cells. The smears are air-dried and stained.
    The evaluation of the bone-marrow preparations involves examination of
    2000-5000 erythrocytes per specimen; the total number of polychromatic
    and normochromatic erythrocytes with and without micronuclei are
    recorded (Schmid, 1973). Sister chromatid exchanges, induced by
    mutagens  in vivo and  in vitro can be scored in peripheral
    lymphocytes or Chinese hamster cells, using the differential straining
    of chromatids substituted with 5-bromodeoxyuridine in place of
    thymidine (Natarajan et al., 1976; Smythe & Evans, 1976; Stetka &
    Wolf, 1976).

          Dominant lethal tests and other in vivo  systems. The dominant
    lethal test is based on the preimplantation loss of eggs or on the
    formation of dead embryonic implants following the injection of
    mutagens into a male or female mouse at a specific time before mating
    (Bateman & Epstein, 1971). Dominant lethal tests assume that a single
    mutation has occurred in the eggs or sperm, which is lethal to the
    embryo and heterozygous at the affected locus. However, a disturbingly
    high number of mutagens have given a negative response with this test.
    The detection of mutations arising from compounds that are metabolized
    to transient reactive intermediates not produced in germ cells or not
    reaching them from other organs may limit this test. Furthermore,
    chemical agents causing spermatogenic arrest or cytocidal effect on
    the sperms may give false positive results.

          Heritable translocation in male mammals. The heritable
    translocation test has the important feature of measuring sexually
    transmissible germ cell mutations in rodent spermatogonia. Generoso et
    al. (1974) have described details of the technique and discussed its
    usefulness for the routine screening of substances that cause
    chromosomal mutations.

         Young adult male mice are treated and females mated with the
    exposed males on a schedule that can be used for the comparison of the
    sensitivity of the different male germ cell stages. Male progeny from
    these matings are collected and mated for the determination of
    semisterility and sterility. Progeny with reduced fertility are

    subjected to cytogenetic analysis. The cytological examination of
    dividing spermatocytes, from animals treated with test chemicals that
    cause breaks on two nonhomologous chromosomes, yields aberrant
    chromosomal figures. These are recognized as rings or chains of four
    chromosomes as opposed to the normal bivalent chromosomal

         A proper evaluation of this test cannot be made at this time
    because of insufficient data in terms of the variety of chemicals
    tested to date.

    7.3.2  Monitoring of human populations

         In spite of the variety of test systems available for detecting
    the mutagenic effects of chemicals, it is difficult, from the results
    obtained at present, to evaluate with confidence the transmissible
    genetic effects caused by the chemical exposure of man. However, the
    potential hazards of mutations are such that every effort should be
    made to reduce the risk.

         Judgment on the potential mutagenic hazard to man should be based
    on various considerations such as strength of the experimental
    evidence of mutagenicity, the exposure pattern to the chemical, and
    the pharmacological properties of the compounds. This is difficult and
    complex. Although human studies are difficult, expensive, and often
    subject to misinterpretation, only studies which directly estimate the
    extent to which environmental factors change human genetic material
    give definitive answers.

         Proper monitoring of the human population (Crow, 1971; Sutton,
    1972) can be carried out by three main approaches:

         (a)  biochemical: detection of inherited protein variants;

         (b)  cytogenetic: screening of blood of newborn infants or of
              fetuses delivered by spontaneous abortion for chromosomal

         (c)  phenotype: surveillance of genetically determined disease or

    The validity of these monitoring systems can only be properly
    evaluated if, and when, a mutagen is actually discovered.

    7.3.3  Significance of tests for heritable mutations

         As mentioned earlier, there is, at this time, no direct
    correlation between laboratory tests for heritable mutations and human
    experience. However, the available body of information from nonhuman
    mammals and lower life forms clearly points to the ability of
    chemicals to produce alterations in germ cells which are inherited in

    succeeding generations. Accordingly, the implications of such data for
    man are so persuasive that they must be taken into account in
    establishing safety measures for the introduction of chemicals into
    use. This is especially true, at this time, since techniques for
    detecting mutations in human populations are so insensitive that a
    significantly mutagenic chemical could easily escape attention.

         These considerations will almost certainly lead to increasing
    concern in establishing regulatory and control procedures. In fact,
    the Environmental Protection Agency in the USA has recently proposed
    preliminary guidelines for conducting tests for heritable mutations as
    part of the routine registration procedure for pesticides.


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    See Also:
       Toxicological Abbreviations