Toxicity of chemicals (Part 1), principles and methods for evaluating the (EHC 6, 1978)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 6

PRINCIPLES AND METHODS FOR EVALUATING THE
TOXICITY OF CHEMICALS
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
status of any country, territory, city or area or of its authorities,
or concerning the delimitation of its frontiers or boundaries. The
mention of specific companies or of certain manufacturers' products
does not imply that they are endorsed or recommended by the World
Health 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.

CONTENTS

PREFACE

REFERENCES

1. SOME GENERAL ASPECTS OF TOXICITY EVALUATION

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
testing
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
1.3.3.1 Species differences and related factors
1.3.3.2 Safety factors
1.3.3.3 Low-dose extrapolation
1.3.3.4 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
benefits
1.5.3. An example of toxicological information used in
standard setting
1.6. Limitations of safety evaluation
ANNEX
REFERENCES

2. FACTORS INFLUENCING THE DESIGN OF TOXICITY STUDIES
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
2.3.2.1 Rate of absorption
2.3.2.2 Site of action
2.3.2.3 Biotransformation
2.3.2.4 Species
2.3.2.5 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
2.4.2.1 Selection of species
2.4.2.2 Animal models representing special
populations at risk
2.4.3. Cyclic variations in function or response
2.4.4. Environmental variables
2.4.4.1 Temperature
2.4.4.2 Caging
2.4.4.3 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
injury
2.7.4. In vitro test systems
REFERENCES

3. ACUTE, SUBACUTE, AND CHRONIC TOXICITY TESTS
3.1. Introduction
3.2. General nature of test procedures
3.2.1. Housing, diet, and clinical examination of test
animals
3.3. Acute toxicity tests
3.3.1. Underlying principles
3.3.2. Experimental design
3.3.2.1 Selection of species
3.3.2.2 Selection of doses
3.3.2.3 Method of administration
3.3.2.4 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
3.4.2.1 Selection of species and duration of
studies
3.4.2.2 Selection of doses
3.4.2.3 Method of administration
3.4.2.4 Biochemical organ function tests
3.4.2.5 Physiological measurements
3.4.2.6 Metabolic studies
3.4.2.7 Haematological information
3.4.2.8 Postmortem examination
3.4.2.9 Controls
3.4.3. Alternative approaches in chronic toxicity
3.4.3.1 Perinatal exposure
3.4.3.2 Use of nonrodent species
3.5. Evaluation and interpretation of the results of toxicity
tests
REFERENCES

4. CHEMOBIOKINETICS AND METABOLISM
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
4.6.1.1 Microsomal, mixed-function oxidations
4.6.1.2 Conjugation reactions
4.6.1.3 Extramicrosomal metabolic transformations
4.6.1.4 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
(TCDD)
ANNEX
REFERENCES

5. MORPHOLOGICAL STUDIES
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
tissues
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
REFERENCES

6. INHALATION EXPOSURE
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
6.7.7.1 Isolation units
6.7.7.2 Head and nose exposures
6.7.7.3 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
6.8.2.1 Heterogeneous aerosols
6.8.3. Monitoring contaminant concentrations
6.8.3.1 Vapour sampling
6.8.3.2 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
6.10.2.1 Measurement of respiratory frequency
6.10.2.2 Measurement of mechanics of respiration
6.10.3. Biochemical end-points
6.10.4. Other end-points in inhalation studies
REFERENCES

7. CARCINOGENICITY AND MUTAGENICITY
7.1. Introduction
7.2. Carcinogenicity
7.2.1. Long-term bioassays
7.2.1.1 Species, strain, and sex selection, and
size of groups
7.2.1.2 Route of administration
7.2.1.3 Inception and duration of tests
7.2.1.4 Dose-level and frequency of exposure

7.2.1.5 Combined treatment and cocarcinogenesis
7.2.1.6 Positive and untreated controls
7.2.1.7 Test material
7.2.1.8 Survey of animals, necropsy, and
histological examination
7.2.2. Short-term tests (rapid screening tests)
7.2.2.1 Metabolic activation, reaction with DNA,
and DNA repair
7.2.2.2 In vitro neoplastic transformation of
mammalian cells
7.2.2.3 Mutagenicity tests
7.2.2.4 Submammalian assay systems
7.2.2.5 Mammalian somatic cells
7.2.2.6 Host and tissue-(microsome) mediated
assays
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
REFERENCES

NOTE TO READERS OF THE CRITERIA DOCUMENTS

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.

PREFACE

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
work.

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
purpose.

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 Health^a;
Dr H. Bartsch, Unit of Chemical Carcinogenesis, IARC, Lyons^b:
Dr J. F. Copplestone, Vector Biology and Control^b; Dr F. C. Lu,
Chief, Food Additives^b; Dr R. Montesano, Unit of Chemical
Carcinogenesis, IARC, Lyons^a,b; Dr H. Nakajima, Drug Evaluation and
Monitoring^a; Dr M. Vandekar, Vector Biology and Control^a; and
Dr G. Vettorazzi, Food Additives^a. 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
treatise.

^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.

REFERENCES

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.

PRINCIPLES AND METHODS FOR THE TOXICITY EVALUATION OF CHEMICALS

Editorial Group

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

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,
Netherlands

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

PRINCIPLES AND METHODS FOR THE TOXICITY EVALUATION OF CHEMICALS

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'Hygiène
industrielles, Faculté des Sciences pharmaceutiques et
biologiques. Université René 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. SOME GENERAL ASPECTS OF TOXICITY EVALUATION

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
evaluation.

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
Environment^a, 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
probabilities^a 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 man^a. 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
concern.

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.,
1966).

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
applicable.

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
from:

(a) administered dose or intake;

(b) measurement of the concentrations in tissues and organ
samples;

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
therapy

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
methods

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 tumour^a.

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" (Z_sp) which is the ratio
between the threshold^b 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 Z_sp > 1, the toxic action is specific;
if Z_sp < 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
Appendix.

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 ct^b = 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;

(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 integral_A contour integral_B contour integral_C
= + +
ED₅₀ (A,B,C) ED₅₀ (A) ED₅₀ (B) ED₅₀ (C)

where contour integral_A, contour integral_B and contour integral_C 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 action^a. 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
Ariëns 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" limits^a
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
µ ± 1.96sigma where µ 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
that:

"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,
1970);

(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.

1.3.3.1 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,
1975).

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).

1.3.3.2 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.

1.3.3.3 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 ED_0.000001 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 ( d_e) that would produce a response
which is, with a 99% probability, lower than R is calculated from
the equation d_e = 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 (ß) equal to 1 (i.e. one probit per 10-fold
dose-range). The choice of the slope (ß) is critical in this
procedure. ß = 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.

1.3.3.4 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
(1976b).

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
techniques.

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
exceeded.

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
monograph.

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 1.3.3.2). 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
setting

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.

Annex

MATHEMATICAL EXPRESSIONS OF DOSE-EFFECT AND DOSE-RESPONSE
RELATIONSHIPS

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 D^K2.

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
susceptibilities.

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.

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 transformation^a. Many
toxicologists use log-probability graph paper to express dose-response
relationships as a linear function for log-normal distributions of an
effect.

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.

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.

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2. FACTORS INFLUENCING THE DESIGN OF TOXICITY STUDIES

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
chemicals.

^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
points.

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

2.3.2.1 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.

2.3.2.2 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).

2.3.2.3 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.

2.3.2.4 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.

2.3.2.5 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.

2.4.2.1 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 (3.1.1.7)) 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.

2.4.2.2 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.

2.4.4.1 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.

2.4.4.2 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
4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-1,2-benzenediol
(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.

2.4.4.3 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-ß (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 LD_{50 (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
distinctions.

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
(3.1.1.8) 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
applicable.

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,
1974).

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
exposure.

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.,
1973).

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
results.

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
metabolites.

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. ACUTE, SUBACUTE, AND CHRONIC TOXICITY TESTS

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
procedures.

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
substances.

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

3.3.2.1 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).

3.3.2.2 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.

3.3.2.3 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
animal.

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.

3.3.2.4 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

3.4.2.1 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
peculiarities.

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.

3.4.2.2 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).

3.4.2.3 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.

3.4.2.4 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
(1.1.1.14) 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 (2.6.1.1), lactate
dehydrogenase (1.1.1.27), 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.

3.4.2.5 Physiological measurements

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

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).

3.4.2.6 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
evaluated.

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.

3.4.2.7 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
biopsy.

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.

3.4.2.8 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).

3.4.2.9 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

3.4.3.1 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.

3.4.3.2 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. CHEMOBIOKINETICS AND METABOLISM

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 Å (intestinal epithelium and mast cells) to 30 Å
(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
degree.

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 Å, 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
chemical.

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.
(1971).

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.,
1975).

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.

4.6.1.1 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 (1.6.2.4) 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 (1.1.1.49) 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,
1964a).

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 (Borgå 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).

4.6.1.2 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
mechanism.

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
derivatives);
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 (1.1.1.70) (Marselos & Hanninen,
1974), by enhancement of D-glucuronolactone and aldehyde dehydrogenase
(1.2.1.3) 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 (2.4.1.17) 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 (2.1.1.6) and
almost all of the phenol- O-methyltransferase (2.1.1.25) (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 525A^a. 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 (2.3.1.35)
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 (2.3.2.2), catalysing one
intermediary step of the overall mercapturic acid synthesis may also
be informative.

4.6.1.3 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 (1.1.1.1) 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 (1.4.3.4), 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.

4.6.1.4 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
man.

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
chemicals.

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
chemicals.

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

dC(t)
= - k_e C(t) (1)
dt

where C(t) is the concentration at time t, and k_e 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(- k_e t) (exponential form) (2)

or,

(3)

(4)

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 - k_e and
intercept C(0).

Having determined k_e, 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)
k_e k_e

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* k_a
C(t) = {exp (- k_e t) - exp (- k_a t)} (6)
V_d( k_a - k_e)

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

The elimination rate constant, k_e, 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, k_a, 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
k_a are determined.

The volume of distribution, V_d, 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, V_d.

In the plasma, the concentration of the chemical declines because
of elimination as well as distribution to tissues. Therefore, to
estimate V_d, 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, V_d, in ml/kg.

The value of V_d 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, V_d 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 V_d 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, V_d 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)V_d (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 (- k_e t) (8)

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

C(t) = C(0) exp (- k_e 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

dA(t)
= k_e A(t) (10)
dt

or,

A(t) = D₀ exp(- k_e t) (11)

And

dB(t)
= k_ex A(t) (12)
dt

dB(t) k_ex
= D₀ exp(- k_e t) (13)
dt k_e

or,

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

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

E_i = B( t_i) - B( t_i - delta t) (15)

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

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

k_ex
E_i = D₀ exp(- k_e t_i) {exp( k_edelta t) - 1} (16)
k_e

the logarithmic form of which is

(17)

A semilogarithmic plot of E_i versus t_i will give a straight line
with slope - k_e. 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
biochemokinetics.

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:

/ k_u excretion in urine
C - k_r excretion via exhalation
\ k_mx metabolic transformation to compound y

In this case, the overall elimination constant will be k_e = k_u +
k_r + k_mx.

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

k_u= U infinity( k_e/ D₀) (18)

k_r = R infinity( k_e/ D₀) (19)

k_mx = X infinity( k_e/ D₀) (20)

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

dU
= k_u D₀ exp(- k_e t) (21)
dt

The solution of the equation (21) yields

U inifnity = k_u D₀/ k_e (22)

and

k_u = U infinity k_e/ 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:

R_c = (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)
that

R_c = k_u V_d (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 V_D C_D (t)
= - k₁₂ C(t) - k_e C(t) + (26)
dt V_d

dC_D( t) k₁₂ V_d C( t)
= - k₂₁ C_D( t) (27)
dt V_D

where C(t) and C_D (t) are concentrations of the chemical in the
central and deep compartments respectively. The apparent volumes of
distribution for these compartments are V_d for the central
compartment and V_D for the slow exchange compartment. If the
apparent volumetric flow rates between the two compartments are the
same, i.e. k₁₂ V_d = k₂₁ V_D, the differential equation system can be
solved with initial conditions C(0) = D₀/ V_d and C_D(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(-ß t) (28)

ß 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 ß 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 k_e (Fig. 4.1) may be
determined as follows:

phiß + psi alpha
k₂₁ = (29)
phi + psi

alphaß
k_e = (30)
k₂₁

k₁₂ = alpha + ß - ( k₂₁ + k_e) (31)

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

k₁₂ D₀
A_D( t) = {exp(-alpha t) - exp(-ß t)} (32)
ß - 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(-ß t) + (phi + psi)exp (- k_a 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

(34)

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

(35)

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:

(36)
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)
V_d k_etau

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

The rate constant for absorption, k_a, 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) V_m C(t)
= (40)
dt K_m + C(t)

where C(t) represents concentration of the chemical at time t,
V_m is the maximum rate of the process, and K_m is the
concentration of chemical at which the rate of the process is equal to
one-half of V_m. 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 K_m( C( t) « K_m) then
equation (40) reduces to

- dC( t) V_m
= C( t) (41)
dt K_m

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

- dC( t)
= V_m (42)
dt

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

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 K_m, 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 K_m, the
semi-logarithm plot becomes nonlinear. In this region of zero-order
kinetics, the plot will be linear if rectangular coordinates are used.

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).

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 V_m and K_m were calculated to be 16.6 ± 1.82 µg/h/g of
plasma and 127.6 ± 25.9 µg/g of plasma, respectively. During the
log-linear phase of excretion the t_´ was 5.3 ± 1.2 h.

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 t_1/2 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
compartment.

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.

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.

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 µg/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 µg 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
faeces.

The concentration of ¹⁴C activity in the bodies of rats given
0.1 or 1.0 µg/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.

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 µg/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 µg/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 µg TCDD/g where D₀ equals the dose being administered in µg/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 µg/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 µg 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 µg 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 µg TCDD/kg/day. Indeed, some rats receiving 1.0 µg 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 µg 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 µg/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.

Annex

Table 4.1 Different types of drug-metabolizing reactions

I. OXIDATIONS

(a) Microsomal oxidations

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

Aliphatic oxidation RCH₃ ----> RCH₂OH

O
/ \
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

N-hydroxylation

Sulfoxidation

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

Dehalogenation

(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⁺

II. REDUCTIONS

(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

III. HYDROLYSIS

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

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

Table 4.1 (contd.)

IV. CONJUGATION

(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.)

N-methylation

O-methylation

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
substrate)
(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. MORPHOLOGICAL STUDIES

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
schedule.

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
problems.

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
required.

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 facilitie