INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 30
PRINCIPLES FOR EVALUATING HEALTH RISKS TO
PROGENY ASSOCIATED WITH EXPOSURE TO
CHEMICALS DURING PREGNANCY
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 Orgnization
Geneva, 1984
The International Programme on Chemical Safety (IPCS) is a
joint venture of the United Nations Environment Programme, the
International Labour Organisation, and the World Health
Organization. The main objective of the IPCS is to carry out and
disseminate evaluations of the effects of chemicals on human health
and the quality of the environment. Supporting activities include
the development of epidemiological, experimental laboratory, and
risk-assessment methods that could produce internationally
comparable results, and the development of manpower in the field of
toxicology. Other activities carried out by the IPCS include the
development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and
promotion of research on the mechanisms of the biological action of
chemicals.
ISBN 92 4 154090 7
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CONTENTS
PREFACE
1. INTRODUCTION
2. PROCESSES INVOLVED IN NORMAL AND ABNORMAL DEVELOPMENT
2.1. Basic events underlying normal development
2.1.1. DNA and chromosomes
2.1.2. Transcriptional control
2.1.3. Translational control
2.1.4. Post-translational control and
significance of membrane proteins
2.1.5. Placentation
2.2. Abnormal development
2.2.1. Genetic influences
2.2.2. Nutrition
2.2.3. Critical and sensitivity periods during
development
2.2.4. Abnormalities of placental development
2.2.5. Toxicokinetics and toxicodynamics
2.2.5.1. Placental transfer
2.2.5.2. Biotransformation
2.2.6. Structure-activity relationship
3. METHODS OF ASSESSING PRENATAL TOXIC MANIFESTATIONS
3.1. Human studies
3.1.1. Measures of reproductive outcome
3.1.1.1. Abortion
3.1.1.2. Stillbirths and neonatal deaths
3.1.1.3. Birth weight
3.1.1.4. Congenital malformations
3.1.2. Prenatal diagnostic procedures
3.1.2.1. Invasive intrauterine techniques
3.1.2.2. Non-invasive techniques
(a) Ultrasound investigations
(i) Visualisation
(ii) Fetal heart rate monitoring
(b) X-rays
(c) Analysis of maternal blood
(d) Analysis of maternal urine
3.1.3. Epidemiological methods
3.1.3.1. Hypothesis generating (descriptive) studies
(a) Case reports
(b) Surveillance
(c) Correlation studies
3.1.3.2. Hypothesis testing (analytical) studies
(a) Cross-sectional studies
(b) Case control studies
(c) Cohort studies
(d) Intervention studies
3.1.3.3. Populations at special risk
3.1.3.4. Confounding and complicating factors
3.1.3.5. Statistical power
3.1.3.6. Quantifying studies
3.2. Experimental animal studies
3.2.1. Species
3.2.2. Dosages and dose levels
3.2.3. Positive control groups
3.2.4. Historical controls
3.2.5. Dosing regimen
3.2.6. Route of administration
3.2.7. Numbers of animals and statistical analyses
3.2.8. Observations on pregnant animals
3.2.9. Observations on the progeny
3.2.10. Animal husbandry, and laboratory practices
4. POSTNATAL MANIFESTATIONS
4.1. Introduction
4.2. Behaviour
4.2.1. Strategy of testing
4.2.1.1. Behavioural functions to be assessed
4.2.1.2. Tests to be used in behavioural studies
4.2.1.3. Choice of species
4.2.1.4. Variables to be controlled
4.2.2. Methods of assessment of specific functions
4.2.2.1. Physical development
4.2.2.2. Reflex development
4.2.2.3. Sensory functions
4.2.2.4. Motor function
4.2.2.5. Activity, reactivity, and emotionality
4.2.2.6. Cognitive development
4.2.2.7. Social behaviour
4.2.3. Relevance of behavioural studies for human risk
assessment
4.3. Reproduction
4.3.1. Normal gametogenesis and development of the genital tract
4.3.2. Mechanisms of abnormal development
4.3.3. Testing procedures
4.3.3.1. "Fertility test" on progeny
following prenatal exposure
4.3.3.2. Multigeneration studies
4.3.3.3. Choice of species
4.3.3.4. Doses, route, and duration of treatment
4.3.3.5. Presentation of results
4.4. Transplacental carcinogenesis
4.4.1. Principles and mechanisms of action
4.4.1.1. Comparative sensitivity of the adult
and fetal organism to carcinogens
4.4.1.2. Dependence on the stage of
prenatal development
4.4.1.3. Species and strain-specificity
4.4.1.4. Mechanisms of organotropism
4.4.1.5. Metabolism of chemical carcinogens in the
maternal organism, the placenta, and the
embryo
4.4.2. Relationship between teratogenesis and carcinogenesis
4.4.3. General principles of transplacental carcinogenicity tests
4.4.3.1. Species
4.4.3.2. Stages of pregnancy
4.4.3.3. Dose and route of administration
4.4.3.4. Evaluation of results
4.4.4. Methods with potential for the future
4.4.4.1. Transplacental host-mediated cell culture
4.4.4.2. Pre- and postnatal exposure to carcinogens
and promoting factors
4.4.4.3. Intraamniotic and intrafetal injection
technique
5. IN VITRO DEVELOPMENTAL AND NON-MAMMALIAN ANIMAL SYSTEMS:
CURRENT AND FUTURE APPLICATIONS
5.1. Scope of in vitro developmental systems
5.2. Essential and desirable features of short-term selection tests
5.3. Validation of short-term selection tests
5.4. Available developmental systems
5.4.1. Whole-embryo culture (warm-blooded animals)
5.4.1.1. Ovum maturation and preimplantation stages
5.4.1.2. Postimplantation mammalian embryos
5.4.1.3. Chick embryo in culture
5.4.1.4. Chick embryo in ova
5.4.2. Organ cultures
5.4.2.1. Organ culture of limb buds
5.4.2.2. Organ culture of the pancreas
5.4.2.3. Organ culture of palatal shelves
5.4.2.4. Organ culture of tooth anlagen
5.4.2.5. Organ culture of the embryonic lens
5.4.2.6. Organ culture of the embryonic kidney
5.4.2.7. Organ culture of embryonic gonads and
accessory duct systems
5.4.2.8. Organ culture of thyroid tissue
5.4.2.9. Other organ culture systems
5.4.3. Culture of non-mammalian or non-avian embryos
5.4.3.1. Studies on the development of fish
5.4.3.2. Studies on the development of amphibia,
lurchae, sea-urchins, and other
invertebrates
5.4.4. Tissue culture systems
5.4.5. "Micromass" culture of dispersed cells from
the embryonic limb and lung
5.4.6. Studies with non-embryonic tissues
6. HUMAN RISK EVALUATION
6.1. Introduction
6.2. Interpreting in laboratory animal studies
6.2.1. Endpoints
6.2.2. Interspecies variations
6.2.3. Statistical limitations
6.2.4. Quantitative risk assessment
6.3. Interpretation of human data for risk assessment
7. CONCLUSIONS
8. RECOMMENDATIONS FOR FUTURE ACTIVITIES
9. EXPLANATION OF TERMS USED IN THE DOCUMENT
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 Manager of the
International Programme on Chemical Safety, 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.
IPCS TASK GROUP ON PRINCIPLES FOR EVALUATING HEALTH RISKS TO
PROGENY ASSOCIATED WITH EXPOSURE TO CHEMICALS DURING PREGNANCY
Members
a,b,e Dr V.A. Alexandrov, N.N. Petrov Research Institute
Oncology of the USSR Ministry of Health,
Leningrad, USSR (Vice-Chairmanb,e)
b,e,f Dr E.1. Anderson, Office of Health and
Environmental Assessment, RD-689, US Environmental
Protection Agency (EPA), Washington, DC, USA
a,b,e Dr V.N. Anisimov, N.N. Petrov Research Institute of
Oncology of the USSR Ministry of Health, Leningrad, USSR
b,c,e Professor C.L. Berry, Department of Morbid Anatomy,
The London Hospital Medical College, London, England
e Dr C. Cortinas de Nava, Institute of Biomedical
Investigations, National Autonomous Universtiy of
Mexico, Mexico
a,b,c Dr R.L. Dixon, Laboratory of Reproductive and
Developmental Toxicology, Department of Health and
Human Services, National Institute of
Environmental Health Sciences, Research Triangle
Park, NC, USA (Chairmanb )
e Professor A.P. Dyban, Department of Embryology,
Institute of Experimental Medicine, Leningrad, USSR
b,e Dr S. Fabro, Colombia Hospital for Women, Washington,
DC, USA
b,e Dr W.G. Flamm, Division of Toxicology, Food and Drug
Administration, Washington, DC, USA
b Dr S. Frankova, Institute of Psychology, Czechoslovak
Academy of Sciences, Prague, Czechoslovakia
b,e Dr E. Guerrero, National Institute of Health, Bogota,
Colombia
b Dr O.P. Heinonen, Public Health Laboratories,
Helsinki, Finland
b,e Dr R. Jelinek, Institute of Experimental Medicine,
Czechoslovak Academy of Sciences, Prague,
Czechoslovakia
b,e Dr J.Ev. Jirasek, Research Institute for the Care of
Mother and Child (UPMD), Prague, Czechoslovakia
Members (contd.)
b,c,e,f Dr K.S. Khera, Health Protection Branch,
Department of Health and Welfare, Tunney's
Pasture, Ottawa, Canada (Rapporteur)
b,e Professor E. Klika, Department of Histology, Faculty
of General Medicine, Charles University, Prague,
Czechoslovakia (Vice-Chairmanb )
e Dr J. Ku?cera, Research Institute for the Care of
Mother and Child (UPMD), Praque, Czechoslovakia
a,b Professor V. Kusak, Institute of Experimental
Medicine, Czechoslovak Academy of Sciences,
Prahue, Czechoslovakia
b,d,e Dr B.V. Leonov, Laboratory for Experimental
Embryology, All-Union Research Institute of
Obstetrics and Gynecology, Moscow, USSR
b Professor M. Marois, Faculté de Médecine
Saint-Antoine, Laboratoire d'Histologie, Paris,
France (Vice-Chairman)
b,e Dr L. Martson, All-Union Scientific Research
Institute of the Hygiene and Toxicology of
Pesticides, Polymers, and Plastics, Kiev, USSR
e Dr N. Matsumoto, Department of Public Health, Faculty
of Medicine, University of Tokyo, Tokyo, Japan
b,e Dr R.V. Merkurjeva, A.N. Sysin Institute of General
and Community Hygiene, Academy of Medical Sciences
of the USSR, Moscow, USSR
a,c,e, Professor N.P. Napalkov, N.N. Petrov Research
Institute of Oncology of the USSR Ministry of
Health, Leningrad, USSR (Chairmana )
b,c,e,f Professor D. Neubert, Institute of Toxicology and
Embryo-Pharmacology of the Free University of
Berlin, Berlin 33 (West) (Chairmane,f )
b,e Dr A.I. Nikitin, Laboratory of Early Human
Embryogenesis, Institute of Obstetrics and
Gynecology of the USSR Academy of Medical
Sciences, Leningrad, USSR
b,e Dr A.K. Palmer, Department of Reproductive
Toxicology, Huntingdon Research Laboratories,
Huntingdon, England
b,e Dr A. Pavlik, Institute of Physiology, Czechoslovak
Academy of Sciences, Prague, Czechoslovakia
Members (contd.)
b,e Dr R.M. Pratt, Experimental Teratogenesis Section,
Laboratory of Reproductive and Developmental
Toxicology, NIEHS, Research Triangle Park, NC, USA
b Dr J.M. Rice, Laboratory Comparative Carcinogenesis,
National Cancer Institute, Frederick Cancer
Research Facility, Frederick, MD, USA
b Dr L. Rosival, Centre of Hygiene, Research Institute
of Preventive Medicine, Bratislava, Czechoslovakia
b,c,e Dr L. Rossi, Institute of Oncology, Genoa, Italy
b Dr Z. Rychter, Institute of Physiology, Czechoslovak
Academy of Sciences, Prague, Czechoslovakia
b,e Dr J.G. Scandalios, Department of Genetics, North
Carolina State University, Raleigh, NC, USA
b,e Professor T.H. Shepard, Central Laboratory for Human
Embryology, University of Washington, Seattle,
Washington, USA (Vice-Chairmane )
a,b,c, Dr S. Tabacova, Institute of Hygiene and
e,f Occupational Health, Medical Academy, Sofia,
Bulgaria (Rapporteur)
e Dr H.A. Tilson, Laboratory of Behavioural and
Neurological Toxicology, National Institute of
Environmental Health Sciences, Research Triangle
Park, NC, USA
b Dr M. Vargova, Research Institute of Preventive
Medicine, Bratislava, Czechoslovakia
b,e Dr T. Vergieva, Institute of Hygiene and Occupational
Health, Medical Academy, Sofia, Bulgaria
e Dr B.R. Vitvitskaya, I.M. Sechenov First Moscow
Medical Institute, Chair of Communal Hygiene,
Moscow, USSR
Secretariat
a,e Dr M.M. Buslajeva, Group of International Research
Programmes, N.N. Petrov Research Institute of Oncology
of the USSR Ministry of Health, Leningrad, USSR
a,b Dr M. Draper, Medical Officer, International
Programme on Chemical Safety, Division of
Environmental Health, WHO, Geneva, Switzerland
Secretariat (contd.)
e Dr M. Gounar, Scientist, International Programme on
Chemical Safety, Division of Environmental Health,
WHO, Geneva, Switzerland
e Dr Z.P. Grigorevskaja, Centre of International
Projects, USSR State Committee for Science and
Technology, Moscow, USSR
b Dr V. Kodat, Department of Hygiene and Epidemiology,
Ministry of Health of the Czech Socialist Republic,
Prague, Czechoslovakia (National Focal Point for IPCS)
b,e Dr A.I. Kucherenko, Scientific Officer, UNEP/IRPTC,
Geneva, Switzerland
b,e,f Dr A. Lihachev, Programme on Mechanisms of
Carcinogenesis, IARC, Lyons, France
b Dr M. Mercier, Manager, International Programme on
Chemical Safety, Division of Environmental Health,
WHO, Geneva, Switzerland
e Dr M. Mikheev, Regional Officer for Workers' Health,
WHO Regional Office for Europe, Copenhagen, Denmark
e Dr L.A. Moustafa, IPCS Interregional Research Unit,
WHO, Research Triangle Park, NC, USA
a,b,c, Dr J. Parizek, Scientist, International Programme
e,f on Chemical Safety, Division of Environmental
Health, WHO, Geneva, Switzerland (Secretary)
e Mr I.A. Rozov, Programme Support Service, Division
of Public Information and Education for Health,
WHO, Geneva, Switzerland
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a Preparatory meeting, Leningrad, 28-30 June 1981.
b First Task Group meeting, Prague, 30 November - 4 December
1981.
c Steering Committee.
d Alternate for Professor N.P. Napalkov at the Steering
Committee.
e Second Task Group meeting, Leningrad, 8-15 June 1983.
f Editorial Committee.
PREFACE
This monograph was prepared by an international Task Group of
experts from thirteen countries. The generous contributions and
the personal involvement of every member of the group during
successive stages of the preparation of the document are
gratefully acknowledged.
The aim of the document and the plans for its development were
discussed at a preparatory meeting, chaired by Professor N.P.
Napalkov with Dr R.L. Dixon, as Vice-Chairman. The meeting, hosted
in Leningrad on 28-30 June 1981 by the N.N. Petrov Institute of
Oncology of the USSR Ministry of Health, was attended by
representatives from IPCS institutions that had expressed their
particular interest in the project. These included, in addition to
the Petrov Institute, the Institute of Experimental Medicine,
Czechoslovak Academy of Sciences, Prague, the National Institute of
Environmental Health Sciences (NIEHS), Research Triangle Park, and
the Institute of Hygiene and Occupational Health, Medical Academy,
Sofia. It was agreed that these Institutions would prepare four
background papers for the first plenary meeting of the Task Group.
The first meeting of the Task Group took place in Prague,
Czechoslovakia, hosted by the Ministry of Health of the Czech
Socialist Republic, on 30 November - 4 December 1981. Dr R.L.
Dixon was elected Chairman of the meeting and Dr V. A. Alexandrov,
Professor M. Marois, and Professor E. Klika, Vice-Chairmen. Dr K.
S. Khera and Dr S. Tabacova were designated Co-rapporteurs for this
and subsequent Task Group meetings. The experts identified and
discussed in detail the problems to be covered in the monograph and
established three working groups for further development of the
draft sections dealing with prenatal and postnatal manifestations
of embryotoxic effects, and with non-mammalian and in vitro tests.
Professor C.L. Berry, Dr L. Rossi, and Professor D. Neubert were
elected leaders of the respective working-groups. The Steering
Committee, consisting of the Chairmen of the preparatory and first
Task Group meetings, leaders of the working groups, and the Co-
rapporteurs, co-ordinated the work of the working groups, after the
first Task Group meeting, and prepared a consolidated draft, which
was sent for comments to all the members of the Task Group.
Comments were also received from the OECD Chemicals Division.
A revised draft was prepared by Professor C.L. Berry, with the
assistance of the Steering Committee, and submitted to the Task
Group before its second meeting, held in Leningrad on 8-15 June
1983. Professor D. Neubert was elected Chairman of the Leningrad
meeting, Dr V. A. Alexandrov and Professor T.H. Shepard, Vice-
Chairmen, and Dr K. S. Khera and Dr S. Tabacova again acted as Co-
rapporteurs.
The second Task Group meeting, hosted by the Petrov Institute
and the USSR Commission for UNEP, completed the document, including
those sections dealing with human risk assessment (on the basis of
a draft prepared by Dr R.L. Dixon and Professor D. Neubert), the
conclusions and recommendations of the Task Group, and explanation
of terms used in the document. A small Editorial Committee
consisting of Professor D. Neubert, Chairman, Dr E.L. Anderson,
leader of the working group responsible at the Leningrad meeting
for the human risk assessment section, and both Co-rapporteurs, was
designated by the Task Group to finalize the document.
The International Programme on Chemical Safety would like
to express deep gratitude to all the members of the Task Group
for their work.
1. INTRODUCTION
This document is intended to aid in the design and assessment
of studies concerned with exploring the association between
exposure to chemicals during pregnancy and defective development.
Personal tragedies are involved when defects are present at birth
or appear later in life and the need for improved methods for the
detection of embryo/fetal toxic agents, the assessment of health
risks, and the prevention of unfavourable outcomes of pregnancy, is
evident.
It is not possible, at the moment, to give a precise estimate
of the extent to which chemicals contribute to the induction of
abnormalities in human development. Though hundreds of chemicals,
which have been listed by Shepard (1983), have induced embryotoxic
effects in laboratory animals, very few have been shown to be
teratogenic (for definition see section 9) in human beings.
Differences between animals and man in metabolism, inherent
sensitivity, and levels of exposure may be responsible for this
apparent discrepancy. Among the problems involved in conducting
epidemiological studies is the detection of chemically-induced
anomalies against a background of sporadic defects. The size of an
epidemiological study necessary to detect changes in the rates of
some abnormalities, is so great that such studies are seldom
conducted. Smaller studies may result in inaccurate risk
estimates.
There are many difficulties involved in extrapolating human
risk from animal studies. For example, if a chemical, tested in
animals at high doses, alters maternal homeostasis, it is possible
that the conclusions drawn will be inappropriate for human beings
exposed to lower levels of the same chemical and that such studies
may over-predict the incidence of teratogenic events.
Gross structural defects are an obvious area of concern.
However, in recent years, it has become clear that consideration of
developmental toxicity following prenatal exposure must be expanded
to include chemically-induced embryolethality, reduced fetal
growth, and functional alterations that may not be expressed until
late after delivery. Some chemicals may decrease fertility by
causing a loss of the early conceptus, even before pregnancy is
suspected.
The processes of cellular and morphogenetic differentiation,
though poorly understood, offer many potential targets for toxic
chemicals. The chemicals that can alter these processes have the
potential to induce adverse developmental effects. Thus, increased
understanding of the underlying processes will aid in using
laboratory studies to identify potential human health effects and
to quantitatively estimate risks resulting from exposure to
chemicals.
Current test methods are generally carried out using whole
animals. Although the number of animals tested might be reduced
and the studies designed more efficiently, there is no substitute
for animal tests, at present. In the future, the potential of
biochemical, cellular, and other in vitro approaches might be more
effectively exploited.
This document includes a description of the use of laboratory
data in defining the potential embryotoxic hazards of chemicals and
methods of assessing the human risks associated with occupational
or environmental exposure to such chemicals. Three major areas are
covered including: prenatal toxic manifestations, postnatal
manifestations, and short-term tests. Prenatal toxic
manifestations are discussed in relation to human epidemiological
studies and laboratory studies on experimental animals. In the
discussion on postnatal manifestations, the emphasis is placed on
test methods for the assessment of alterations in behaviour,
reproduction, and of transplacental carcinogenesis following
gestational chemical exposure. Short-term tests are discussed in
the context of understanding the mechanisms of developmental
toxicity and their potential for future application in
developmental toxicity testing.
In each area, basic data and processes were considered by the
Task Group in relation to three questions, i.e.,
(a) What is the value of currently used toxicity tests
for predicting human risk?
(b) What are the difficulties involved in applying test
results to the estimation of human health risks and
hazards?
(c) How can testing strategies be improved?
The document is divided into nine sections, which differ in
structure. The Introduction is followed by Section 2, which
provides background information on the mechanisms of development.
Section 3 is devoted to methods of assessing prenatal toxic
manifestations and represents the overall views of the Working
Group, in this complex field. Sections 4 and 5 include detailed
discussions on postnatal events and short-term tests, respectively,
since these sections present a new field and the Task Group wished
to review the potential of available tests. Section 6 on human
risk assessment, was considered by the Task Group to be
fundamental. The Task Group's conclusions are given in Section 7
and their recommendations in Section 8. Section 9 comprises an
explanation of terms used in the document.
The Task Group, aware of the existence of several guidelines
published by national or international bodies (FDA, 1966, 1970;
NHW, 1973; NAS, 1977; US EPA, 1978; OECD, 198l; CMEA, 1982), wish
to state that the present document is not intended to replace,
complement, or discuss these guidelines.
For detailed information on methods of studying prenatal
toxicity, reference can be made to a number of publications
including those of Nishimura et al. (1968), Shepard et al. (1975),
Ebert & Marois (1976), Neubert et al. (1977b), Wilson & Fraser,
(1977-78), Benesova et al. (1979), Klingberg et al. (1979), Porter
& Hook (1980), and Kimmel & Buelke-Sam (198l).
This document primarily deals with chemical exposures during
pregnancy and should be useful to all those concerned with the
evaluation of chemical safety. Evaluation of the effects of
exposure to drugs is not within the scope of this document and has
been addressed in other publications (WHO, 1967; CIOMS, 1983).
There are a number of reference sources dealing with chemicals
reported to be teratogenic for man and laboratory animals
(Schardein, 1976; Heinonen et al., 1977; Shepard, 1983). The
embryotoxic effects of chemicals are regularly assessed in the IPCS
Environmental Health Criteria documents, available from the World
Health Organization. The Environmental Teratology Information
Center (ETIC) operated by the Oak Ridge National Laboratory (ORNL)
maintains computer-stored teratological scientific literature on a
large number of chemicals, listed according to CAS (Chemical
Abstract Service) registry numbers. Advice on access to this
information is available from the International Register of
Potentially Toxic Chemicals, United Nations Environment Programme
(UNEP), Geneva.
The International Clearinghouse for Birth Defects Monitoring
Systems covers nineteen monitoring programmes from various
countries. The results are published, annually, by the Swedish
National Board of Health and Welfare, Stockholm and, quarterly, by
the March of Dimes Birth Defects Foundation, White Plains, New
York.
2. PROCESSES INVOLVED IN NORMAL AND ABNORMAL DEVELOPMENT
The purpose of this section is to give an outline so that those
working in different fields can see the background against which
new developments might occur. For more detailed discussions of
processes involved in normal and abnormal development, see, for
example, Hicks & D'Amato (1966), Thomas (1968), Langman et al.
(1975), Scandalios (1979), Page et al. (198l), and Krowke & Neubert
(1977).
2.1. Basic Events Underlying Normal Development
Embryogenesis is a finely balanced programme of cellular
events, including proliferation, migration, association,
differentiation, and cell death, precisely arranged to produce
tissues and organs from genetic information, present in each
conceptus. Underlying the morphological development of the embryo
is a progressive unfolding of biochemical potentialities,
determined by temporally- and spatially-regulated transcription and
translation of genetic messages.
Embryogenesis involves complex interactions occurring in both
time and space. In the earliest stages, rapid cell multiplication
is the rule. The cells of the early embryo have vast developmental
potential, depending on their relative positions in the embryonic
mass. The development of primordial tissue depends strongly on the
interactions of adjacent cell groups, apparently mediated by
endogenous chemicals or growth modifiers. Complex processes of
cell migration, pattern formation, and the penetration of one cell
group by another, characterize the later stages of organogenesis
(Johnston & Pratt, 1975). Final morphological and functional
development occurs at different times in different species and is
sometimes completed after birth.
2.1.1. DNA and chromosomes
DNA is the target of several mutagenic, growth-inhibiting, and
carcinogenic chemicals. The eukaryotic chromosome is a complex
structure mainly composed of DNA and protein (Lewin, 1980). A
large part of the chromosomal protein is made up of a small group
of basic proteins known collectively as histones, which may be
important in controlling the functional state of DNA. All
eukaryotic cells contain essentially the same types of histones. A
chromatin fibre contains a double strand of DNA and associated
proteins. Between cell divisions, chromatin is normally dispersed.
Most chromatin can undergo the transition between dispersed
(euchromatic) and condensed (heterochromatic) states. The
information encoded in DNA is transferred by a multistage process
to RNA (see below).
The amount of a particular mRNA, and hence the rate of specific
protein synthesis, can be regulated by controlling the number of
genes specific for its production. Gene amplification is one means
by which a cell can produce large quantities of a specific gene
product.
Microscopically visible excesses, deficiencies, or
rearrangement of chromosomes and chromatids, occurring
"spontaneously" or induced by chemicals, have been found to be
associated with human developmental defects. Similar cytogenetic
changes may result from viral infections or irradiation. Many
cytotoxic agents act by inhibiting the synthesis of DNA, thereby
slowing or preventing mitosis. Other chemicals interfere with the
polymerization of tubulin into the microtubules of the spindles,
which prevents cell division by arresting the formation of the
mitotic spindle.
2.1.2. Transcriptional control
Each of the two steps (transcription and translation) involved
in the synthesis of a protein is controlled. In eukaryotic cells,
transcription occurs in the nucleus and translation outside the
nucleus. These processes may also occur in mitochondria.
Differential gene activation or transcriptional control is the
principal mechanism for gene control in prokaryotic cells. The
finding that three distinct forms of RNA polymerase (EC 2.7.7.6)
are present in eukaryotic cells, transcribing different sets of
genes, points to transcriptional control as a predominant mechanism
regulating gene expression in eukaryotic cells.
Transcriptional controls are well documented for highly
specialized single-copy genes, the products of which make up a
large proportion of cellular mRNA and protein. Examples are genes
for globin, ovalbumin, and metallothionein.
Eukaryotic genes seem to be transcribed from large regions of
DNA, producing RNAs with up to about 20 000 nucleotides, which are
then processed into smaller mRNAs, before leaving the nucleus. The
process is not a conservative one, however, for a considerable
fraction of the RNA is degraded, without ever leaving the nucleus.
This degraded RNA represents linkage regions between structural
genes. The extent of gene expression emerges as the controlling
factor in differentiation, as different gene products are produced
at specific developmental stages or in response to hormones.
2.1.3. Translational control
The generally long lifetime of eukaryotic mRNA might imply that
translational control is especially important; merely turning off a
gene does not stop the synthesis of its enzyme, as long as the
appropriate mRNA is intact and available for translation.
The translation of mRNA after the fertilization of an oocyte is
an example of translational control that seems to involve
activation of a stable mRNA. The advantage of translational
control is the speed with which protein synthesis can be turned on
and off. The longer an mRNA can survive intact, the more important
it becomes in controlling the rate of its translation. The
lifetimes of mRNAs are highly variable. Enzymes that increase or
decrease in quantity in response to external signals have mRNA with
shorter lifetimes than enzymes that are not closely regulated. The
half-lives of most mRNAs in eukaryotic cells range from about an
hour to several days. The extreme case of stability of mRNA is
found in an unfertilized egg, in which masked mRNA may remain
untranslated for very long periods, even years, until fertilization
occurs.
The amount of translation of a given message can be controlled
by regulating the lifetime of the mRNA. However, little is known
of the reason why some mRNAs are more stable than others or how
exogenous chemicals affect this stability. Complementary sequences
within the same mRNA molecule result in a specific folding,
consisting of loops and hairpins. An alternative scheme suggests
that the number of times some mRNAs are translated, is
predetermined.
2.1.4. Post-translational control and significance of
membrane proteins
Post-translational control involves the modification of
synthesized proteins (e.g., sub unit interactions,
compartmentation, turnover, conjugation of prosthetic groups,
etc.).
The plasma membrane of eukaryotic cells is composed of lipid
glycoproteins. Controlled membrane permeability is essential to
cell maintenance and survival. The plasma membrane also serves as
a site for receptors with a high affinity for specific hormones.
Interactions of hormones with receptors trigger the chain of
molecular events necessary for gene expression and protein
synthesis. Furthermore, plasma membranes provide cell-specific
antigens, and differentiated cells within an organ provide organ-
specific antigens. Antigenic specificity is essential for the cell
recognition required in morphogenesis and embryogenesis.
Therefore, any alteration of plasma membranes by toxic chemicals
can be expected to alter permeability, receptors, and immunological
cell specificity, both qualitatively and quantitatively, as well as
cell-cell interactions.
2.1.5. Placentation
The placenta is a temporary organ that establishes a functional
union between mother and fetus. This unique organ transfers all
the nutrients needed for development, eliminates fetal metabolic
waste, synthesizes hormones essential to pregnancy, and carries out
many other anabolic and catabolic functions. Placental morphology
varies with species and with the stage of fetal development.
During and immediately after implantation, before the placenta
has developed, the embryo depends on histiotrophic nutrition,
consisting of digested endometrial cells mixed with a secretion of
the endometrial glands. The histiotrophic material resorbed by the
trophoblast reaches the embryonal anlage by diffusion. Another
source of early embryonal nutrition is the content of the yolk sac.
In mammals, the significance of the yolk sac varies considerably.
It usually functions as a non-selective structure allowing transfer
to the fetus of most chemicals resorbed by the trophoblast from the
chorion and chorionic cavity until allantoic (embryochorionic,
feto-placental) circulation is established. The yolk sac cavity,
enclosed by endodermal cells and visceral mesoderm, is of
nutritional importance for the embryo for up to 9 1/2 days in the
hamster (Boyer, 1953), up to 10 days in the rat, and for
approximately one month in man (Langman, 1969). Though its role
becomes less significant with the progress of pregnancy, the yolk
sac in the rabbit and rodent species continues to function in the
maternal-fetal transfer of chemicals until later in pregnancy
(Lambson, 1966; Seibel, 1974), when the yolk sac is separated from
the gut.
The embryonal yolk sac mesoderm is the source of the first
haematopoietic tissues supplying the blood cells of the embryo-
chorionic circulation. During implantation, as the trophoblast
penetrates into the endometrium, maternal capillaries come into
contact with the trophoblastic shell, and consequently open into
the trophoblastic lacunae. As the trophoblastic shell changes into
the villous chorion, maternal blood begins to circulate within the
intervillous space.
The chorion is vascularized by allantoic vessels. As reported
by Jirasek (1980), the human embryochorionic circulation begins
about day 28-30, there being a gap of about 14 days between the
time when the maternochorionic (maternal) circulation and the
embryochorionic (fetal) circulation are established.
The allantois is an endodermal diverticulum from the yolk sac
(later hind gut) into the connecting stalk located between the yolk
sac, the amnion, and anchored to the chorion. The allantois is
accompanied by umbilical vessels connecting the intraembryonal
vessels with the chorionic vessels. As the amnion expands, the
yolk sac duct, and the connecting stalk containing the allantois
and umbilical vessels are pushed together, giving rise to the
umbilical cord. After establishment of the embryochorionic
circulation, embryonal tissues are nourished from the embryonal
blood.
Placentation is highly species-specific with important
physiological and anatomical peculiarities in different mammals.
The relationships between the structure and function of placental
membranes have not been satisfactorily clarified.
2.2. Abnormal Development
The complicated series of developmental events offers a variety
of time-specific targets for toxicity. Even temporary retardation
in the growth of one group of cells may have serious consequences
for overall development, because each step in embryogenesis may
depend on a previous one and the development of numerous tissues
and organs is interrelated.
The development of an organism concerns several levels of
biological organization. The targets for chemical agents occur at
the molecular level; chemicals may interact with DNA, RNA, membrane
constituents, enzymes, receptors, etc. It does not mean, however,
that the primary events resulting from these interactions need
either be specific for the cells of developing systems, or lead
inevitably to some kind of embryotoxicity manifestation. In
teratogenicity studies, the outcome of the primary events depends
on the properties of the affected morphogenetic systems (Jelinek &
Rychter, 1979). Occurrence of defects depends, in principle, on the
coincidence of the critical and sensitivity periods (defined in
Section 9). Some factors can cause embryonic death directly by
interrupting the basic life functions of the conceptus, e.g., blood
circulation.
Teratogenic research was stimulated by the observation that
congenital malformations in human beings could be induced by
maternal exposure to thalidomide, pelvic irradiation, or by
maternal infection with rubella.
Teratogenesis, mutagenesis, or carcinogenesis may occur
spontaneously or may be induced by external physical or chemical
treatment.
Teratogenic mechanisms have mainly been studied in laboratory
animals; human data have been obtained from either case reports or
epidemiological studies. The mechanisms of action of embryotoxic
agents are still poorly understood, even though rapid advances are
being made. Only by increasing knowledge concerning the
biochemical and biological events in normal embryogenesis can the
processes of teratogenesis be better understood.
Chemicals that interfere directly with embryonic or fetal
development at exposures lower than those that cause apparent
maternal toxicity are most likely to be of concern. Many of the
most widely-recognised embryotoxic or teratogenic chemicals meet
this criterion of selective action; the difference between doses
that induce malformation and doses that cause maternal death or
serious intoxication, may be considerable (Khera, 1983).
Embryotoxic evaluation of such chemicals requires more serious
attention than evaluation of chemicals that induce toxic effects in
the embryo only at doses toxic to the mother. With the latter,
recognition of maternal toxicity aids in controlling the risk of
embryotoxic dosages. There are cases in which both embryonic and
maternal effects in experimental animals are elicited within the
range of human exposure levels (for instance with alcohol or methyl
mercury) and the dual danger to mother and fetus should be
recognized.
Many chemicals, including teratogens, when administered at high
doses or very early in embryonic development, can cause embryonal
or fetal death followed by resorption or abortion of the fetus.
However, substances that can kill fetuses selectively in
experimental animals do not necessarily induce teratogenic effects
in the survivors. The association between intrauterine death,
spontaneous abortion, still birth, and congenital malformation in
human beings needs to be explored further.
Although very detailed studies of the teratogenic effects of
drugs have been carried out on developing mammals, birds, and other
submammalian species, relatively little information is available
regarding biochemical changes in the human embryo. Any knowledge
of these effects is the result of limited clinical observations
made some time after accidental or incidental ingestion of
chemotherapeutic or other agents. As yet, there is not sufficient
knowledge of the associations between the various manifestations of
developmental toxicity or, more precisely, the reasons for the lack
of consistency in the associations. This may partly be because
information on the biochemical changes in the human embryo is
lacking, thus leaving a gap in the link with the apparent final
common pathway that results in a marked reduction in the number of
cells, as suggested by Connors (1975), or cell products, which then
fail to support either full morphogenesis or complete functional
maturation.
2.2.1 Genetic influences
As previously mentioned, morphogenesis involves precisely
coordinated interactions of cell groups. Each embryonic cell
depends entirely on its genome and signals coming from the
exterior. Both of these can be disturbed by chemical agents
transported and transformed between entry into the maternal
organism and arrival at the target cells. Thus, the response to
embryotoxic agents strongly depends on embryonic and maternal
genotypes.
Genetic alterations can be the result either of hereditary
defects, already present in the zygote, or of an acute effect on
the embryonic genome. However, the importance of somatic mutations
in development is not clear. Chromosomal aberrations contribute
substantially to early embryonic loss and may persist in the form
of proliferation mosaics.
The mechanisms that control the timing of gene expression or
repression during development and result in the orderly development
of the embryo, are not understood. It is not known how the
products of gene expression control development, though it is
obvious that an altered gene product may affect developmental
selectivity. The achondroplastic gene induces dwarfism of a
particular type; abnormal meiotic divisions with the production of
aneuploid cells may result in embryonic death or phenotypic
anomalies such as Down's syndrome or Patau's syndrome. If the
genetic defect is present in the germ cells (as with defects
characterized by Mendelian inheritance such as fibrocystic
disease), then it will be transmitted to future generations.
However, the majority of human malformations are not inherited in
this way.
The considerable variation in the susceptibility of individuals
to teratogens, is well documented for animals. For example, some
strains of mice are markedly more susceptible to cleft palate
induction by glucocorticoids, than others. Mice, in general, are
more susceptible than other species, but, results of studies with
highly potent fluorinated corticosteroids indicate that it would be
unwise to presume that other species including rats and primates
are completely resistant to cleft palate induction. The
differences in susceptibility could be related to such
toxicokinetic features as the rate at which the corticosteroid is
absorbed, distributed, eliminated, or transformed by the maternal
animal or its rate of passage across, or biotransformation by, the
placenta. The nature of the chemical's interaction within the
cells and tissues of the embryo (toxicodynamics) is also an
important determinant of toxicity. The basic rate of embryonic
development may be important. There is evidence that the number of
glucocorticoid receptors in the developing palate of sensitive mice
is higher than the number found in resistant strains. However,
further studies are needed to establish a cause and effect
relationship.
In man, epidemiological studies on the families and relatives
of the malformed children have shown evidence of the influence of
genetic factors in the human response to teratogens. The
pathogenetic mechanism appears to be the interaction of
environmental factors with several genes. This hypothesis, the
polygenic or "multi-factorial" theory, has been described in detail
in the human setting by Edwards (1969) and Carter (1976). For
discussion concerning other factors causing malformations see
Neubert et al. (1980) and Berry (198l).
In animals, the interaction of both genetic and environmental
factors determines the "liability" of a given congenital
malformation to become overt. Genetic predisposition is usually
due to the additive effects of many genes. Thus, the various genes
regulate the expression of heritability and in doing so, are
influenced by the environment.
2.2.2. Nutrition
Malformations can be induced experimentally by nutritional
imbalance. Thus, the relationship between nutrition and human
congenital malformations needs further study. Contrary to the
long-held view that the fetus has first claim on available
nutrients, vitamin deficiencies or a reduced availability of trace
metals, such as zinc (Hurley & Shrader, 1972), appear to
selectively damage the animal fetus. Teratogenic effects may be
caused by deficiencies that are too slight to harm the mother and
may be a reflection of the nutritional requirements of the growing
fetus and its lack of stored reserves. On the other hand, general
malnutrition has not been found to induce teratogenic effects.
The results of recent studies in the United Kingdom have
suggested that occult vitamin deficiency may be a factor in the
pathogenesis of neural tube anomalies. This finding tends to
emphasize that the nutritional status of the mother is an important
consideration in developmental toxicity (Smithells et al., 1982,
1983).
2.2.3. Critical and sensitive periods during development
It is widely accepted that the developing conceptus is
particularly sensitive to toxic agents during certain periods,
generally related to the development of particular organ systems or
types of cells. A critical phase for the induction of structural
malformations usually occurs during the period of organogenesis.
In the rat, for example, this critical period extends from about
day 6-15 (day of finding sperms in vaginal smear counted as first
day of pregnancy) of the 22-day gestation period. In man, 20-70
days after conception is perhaps the comparable period. It may be
unwise to rely absolutely on these time periods.
Before implantation, specific malformations cannot be induced
in the conceptus. Malformations induced with actinomycin D or
cycasin administered during the preimplantation periods are
probably due to the persistance of these compounds in the maternal
or fetal organism until a later stage of development.
With physical agents such as X-rays, exposure can be limited
exactly to a period of seconds or minutes. However, with chemical
teratogens, the situation is more complicated because of the time
courses of absorption, metabolism, and excretion. In addition, the
actual (proximate) teratogen may be a metabolite rather than the
compound administered.
If the time in gestation, when the differentiation of a
particular organ is complete, is known with certainty, then a
teratogen must be present prior to or at that time, if it is to be
a causative agent of malformation.
It might be thought that all chemicals operating at a
particular critical time in organogenesis would induce the same
pattern of abnormalities, but this is not the case. Differences
between species in fetal development, toxicokinetics, and
toxicodynamics account partly for this variability in response
caused by interplay of critical and sensitive periods.
Other forms of developmental toxicity may have the same or
different periods of peak susceptibility. For example, functional
(behavioural) deficits and other latent defects may originate more
commonly during the period of cellular differentiation that follows
organogenesis. This susceptibility extends into the period of
postnatal development to a greater or lesser extent, according to
species.
2.2.4. Abnormalities of placental development
Certain diseases (syphilis, toxoplasmosis, or viral infections)
may affect the embryo indirectly by damaging the placenta. Certain
placental anomalies (placenta praevia, accreta) are associated with
a relatively poor pregnancy outcome, but few cases of abnormal
development, entirely due to placental causes, can be singled out
(Benirschke, 1975). However, the placenta is a crucial organ in
development, and abnormal fetal growth and development may be
related to placental insufficiency. It is also active
metabolically and, for this reason, it is important, both in the
biotransformation of chemicals and in determining how chemicals are
distributed between fetus and mother.
2.2.5. Toxicokinetics and toxicodynamics
The term toxicokinetics is used in this document to emphasize
the fact that the toxicity of environmental chemicals is the
subject of concern in this document.
The rates of absorption, distribution, and elimination of any
chemical are interrelated and collectively determine the level and
duration of the presence of the compound or its metabolites in the
maternal and fetal body compartments (Mirkin & Singh, 1976). The
toxicokinetics of a chemical in the maternal organism determine, to
a large extent, the availability of the chemical for the embryo,
though the placenta may exert modifying effects.
The kinetics of a chemical vary with species, the route of
administration, length of exposure, the physiological status of the
animal, etc. Differences in kinetics, especially in
biotransformation, account for a considerable number of species
differences in response to chemicals. It has been, and continues
to be, suggested that prior performance of toxicokinetic studies
would make it possible to choose the most appropriate species for
toxicity testing. But toxicokinetic similarity is only one of
several aspects that have to be considered in the choice of a model
system.
Biological responses in tissues are initiated by an interaction
between a chemical and a receptor, as, for example, interactions
between enzymes and substrates, oxygen and haemoglobin,
catecholamines and neuroreceptors. Agents such as pharmaceutical
compounds and pesticides are often designed to compete with the
natural substance for the receptor or to prevent a response by
altering or blocking the receptor. This process may be labelled
pharmacodynamic, if desirable, and toxicodynamic if undesirable.
It is generally assumed that pharmacodynamics are similar for most
species and while this may be the case for individual receptors,
species-and strain-differences can arise, because of the different
numbers of receptors present during development.
2.2.5.1. Placental transfer
Concentration gradients and physicochemical factors, such as
lipid solubility, relative molecular mass, and protein-binding,
determine the rate at which chemicals cross biological membranes
and also largely determine the rate of placental transport. The
binding of a substantial fraction of a chemical to maternal plasma
proteins will reduce the concentration gradient of free (unbound)
molecules and thereby diminish the rate of their passage across the
placenta. The fetal plasma protein may act as a sink for the
chemical molecules after they cross the placenta; therefore, a
relatively large amount of a chemical may have to be transferred,
against a relatively low concentration gradient, in order to
establish maternal-fetal equilibrium. If the protein-binding
capacity in the fetal compartment is smaller than that in the
maternal organism, the free concentration of the xenobiotic may be
higher in the fetus.
Substances that are very lipid-soluble (e.g., the anaesthetic
gases) diffuse across the placental membranes so rapidly that their
overall rates of equilibration are probably limited only by organ
blood flow. Though some steroids cross the placenta readily, their
glucuronide conjugates penetrate at much slower rates. In general,
the placenta does not seem to represent a barrier to chemicals with
relative molecular masses of approximately 300 or less, but
chemicals of higher relative molecular mass appear more slowly in
fetal blood after maternal exposure, because the rate of transfer
is lower. However, even substances of comparatively high relative
molecular mass, such as antibody globulins, viruses, cellular
pathogens, and erythrocytes, pass from the maternal circulation
into the fetus.
2.2.5.2. Biotransformation
A number of chemicals are biotransformed into metabolites by
the enzyme systems in the mother, placenta, or fetus. The exact
role of genetically-determined biotransformation enzymes in
influencing the expression of teratogenicity has not been
ascertained for most substances. Important enzymes, in this
respect, are localized in the endoplasmic reticulum and
constitute a family of cytochrome P-450-dependent
mono-oxygenases. Environmental chemicals that enhance or
inhibit the activities of these mono-oxygenases (and thus the
rate of metabolism of certain chemicals), are referred to,
respectively, as inducers or inhibitors. The inducers include
phenobarbital, chlordane, DDT, polycyclic aromatic
hydrocarbons, flavons, dioxins, indoles, and polyhalogenated
biphenyls, many of which are combustion products, industrial
chemicals, and pesticides. The inhibitors include carbon
monoxide ( in vitro), imidazole, methylene blue, aniline,
amines, and some more or less specific inhibitors. The
ability of chemicals to induce or inhibit the activity of the
microsomal enzyme systems is an important factor to be taken
into account in devising experimental protocols.
2.2.6. Structure-activity relationship
The invention of new pharmaceutical compounds or pesticides is
based on the knowledge of chemical groups that will interfere with
specific enzyme reactions or act on specific receptors. The
development of new chemicals is intended to enhance the desired
activity and reduce undesired effects. However, a similar strategy
cannot yet be extended to developmental toxicity, since it is not
possible to predict, with certainty, the effects of chemicals on
developmental processes, on the basis of their structure alone.
3. METHODS OF ASSESSING PRENATAL TOXIC MANIFESTATIONS
3.1. Human Studies
Human data are considered to be of great potential value. The
present casual attitude to documenting abortions and examining the
products of conception is a serious handicap to acquiring knowledge
concerning the influence of environmental chemicals on human
development. Epidemiological studies are important for identifying
environmental hazards and for providing human data as a basis for
validating animal models that might predict human disease before it
occurs. Therefore, well-designed studies are extremely valuable
and should be encouraged.
When it is suspected that the environment is contaminated from
a known or unknown source, the exposed area and population should
be monitored. The chemical should be identified and the levels of
exposure quantified; contamination of the soil, air, water, food,
animals, and people should be assessed. Specimens from these
sources should be retained for future study. Information on
exposure levels and/or the body burden of exposed persons can
sometimes be obtained by analysis of body fluids, such as blood,
urine, or breast milk, biopsy or autopsy tissues, placental
tissues, hair, or extracted teeth.
3.1.1. Measures of reproductive outcome
Because of the complex integrated nature of reproduction in all
mammalian species, abnormal reproductive performance provides a
sensitive indicator of chemical effects. Widely-used measures of
reproductive performance include abortion, still birth, birth
weight, and peri- or neonatal death. These measures are discussed
below.
3.1.1.1. Abortion
An area of concern for the Task Group was the lack of general
awareness of present data on human reproductive wastage, even among
those who deal with the effects of chemicals on development. In
addition, the Task Group considered that the acquisition of new
data in this field would be of major importance in estimating
putative risks from chemicals.
"Spontaneous" or induced impairment in development often
results in embryonic death during early pregnancy. A spontaneous
wastage of early conceptuses (15-20% abortion rate) is documented
by clinical observations of embryonic loss after implantation.
However, the results of studies using the Beta-HCG radioimmunoassay
for detecting trophoblast differentiation and implantation, as
well as observations on the development of transplanted pre-
implanted human embryos, have indicated that, even in a group of
women who might be expected to have good reproductive performance,
more than half of early conceptuses are lost within the first weeks
of gestation (Miller et al., 1980; Edmonds et al., 1982; Lopata et
al., 1982).
Important in its own right, the death of the conceptus becomes
even more relevant, because of its frequent association with
congenital malformations. Thus, while 2.5-3% of live births show
major malformation, numerous studies have shown more than 90% of
conceptuses with anomalies as diverse as cebocephaly, Turner's
syndrome, and neural tube defects are aborted in early pregnancy
(Table 1, Berry, 1981). It is becoming clear that many abnormal
embryos are aborted. In spontaneous abortions occurring in the
first trimester, about 60% exhibit chromosomal aberrations, mostly
trisomy, monosomy, or triploidy (Boue et al., 1975).
Nishimura (1969) reported that 3.7 - 4.7% of nearly a thousand
late embryos and early fetuses obtained from surgical abortions had
externally visible malformations. This relatively high background
of "spontaneous" abnormalities makes it difficult to detect a small
increase in the overall incidence that might be caused by a single
agent.
Curetted materials of 5-12 weeks gestation were collected by
Watanabe (1979) from a private hospital in Nigata city, in
collaboration with attending physicians, and the chromosomes of
embryonic cells and chorionic villi analysed. All of the material
was taken from women whose pregnancies had been terminated for
socio-economic reasons only. During May 1977, 1250 cases were
analysed from which 80 chromosome abnormalities were detected,
giving an incidence of 6.4%. An increased incidence was found with
increasing maternal age, particularly in the occurrence of complete
aneuploidy.
Table 1. Malformations in aborted fetuses and newborn
------------------------------------------------------------
Prevalence per 1000
Malformation At abortion At birth Loss %
------------------------------------------------------------
Neural tube 13.1 1.0 92
Cleft lip and palate 24.4 2.7 89
Polydactyly 9.0 0.9 90
Cyclopia and cebocephaly 6.2 0.1 98
------------------------------------------------------------
From: Nishimura (1969) and Berry (1981).
In order to ascertain the frequency of chromosome aberrations
among newborn infants in Japan, Higurashi (1979) screened 12 319
newborn babies, 6382 male and 5937 females, for clinical
manifestations of autosomal aberrations and for sex chromatin and
sex chromosome aberrations. The incidence of chromosomal
aberrations was 1/725 (0.14%) for autosomal trisomy and 1/766
(0.13%) for sex chromosome aberrations. Any potential human
teratogen must be evaluated against the above background.
Hereditary factors, irradiation, certain drugs and chemicals, and
some viruses are all known to cause malformations. However, few
data are available to define the incidence of malformations induced
by them. Precise evaluation of relative risks is difficult in any
group with a high background incidence of abortion and a wide
variation in malformations.
3.1.1.2. Still births and neonatal deaths
Though still births and neonatal deaths are well recorded in
some countries, their low frequencies make them weak indicators of
developmental toxicity related to environmental chemicals.
Clusters can be identified and studied only by using statistical
data on prenatal mortality. Studies of cohorts after exposure to a
chemical may include laboratory examinations to determine the body-
burden of the chemical, cytogenetic studies, and examination of
tissues obtained by biopsy or at autopsy.
3.1.1.3. Birth weight
Birth weight is an appealing variable for study, because it is
sometimes obtainable from birth certificates and is known to be
markedly affected by several factors such as cigarette-smoking
during pregnancy (Abel, 1980). Certain malformations are
associated with low birth weight, and a few others with high birth
weight (Ciba Foundation Symposium, 1974). Some agents, such as
aminopterin or methotrexate, cause low birth weight, when
administered early in pregnancy (Milunsky et al., 1968). Before
lower birth weight for gestational age can be associated with
exposure to a chemical, alternative explanations, such as
differences between cases and controls in prepregnancy weight,
social class, diet, and ethnicity must be excluded. With care,
birth weight can be analysed as a continuous variable. Thus,
subtle effects of chemicals may be detected by differences in the
mean and variance of birth weight, which would not be noticed by
measuring the frequency of birth weights under a fixed value (e.g.,
2.5 kg).
3.1.1.4. Congenital malformations
Congenital malformations are recorded to a certain extent on
some birth certificates, but generally the omissions are too
numerous for useful study. To be detected from registry data, a
teratogen or embryotoxic chemical would, of course, have to be
present in the area covered by the registry. Furthermore, the coded
classification of anomalies in the registry must be of sufficient
detail to reveal an excess of the particular birth defect observed;
e.g., the diagnosis should be listed as "limb-reduction deformity"
rather than "other skeletal anomalies". Often, the use of plain
language giving accurate detail can provide more reliable
information than the use of strictly scientific terms. Also, the
registry staff must review its data on a regular basis to determine
if clustering has occurred. If a cluster is observed, the
diagnoses should be verified, and a case-control study conducted to
seek the cause. Rarely, if ever, do all of these events occur in a
way that would make it possible for a new teratogen to be detected
from a registry.
In a geographical area where chemical contamination has
occurred, surveillance for congenital malformations and other
effects on development may be of value. If surveillance is deemed
necessary, a full description of the malformations observed should
be recorded. Photographs should be taken of patients with unusual
findings, to assist in documenting the abnormality.
When a potential teratogen is studied, exposed persons should,
if possible, be compared with a similar group of people who were
not exposed. If circumstances prevent the selection or study of
such a cohort group, internal comparisons, such as high versus low
exposure, may be possible.
Another approach is to study all the children of exposed
mothers in an effort to identify clustering of specific types of
malformations. If a cluster is found, knowing the nature of the
exposure may suggest a relationship to a particular chemical. The
malformation observed should be classified according to other
possible aetiologies such as infection, trauma, or heredity. For
example, webbed toes in a family with a history of this disorder is
obviously hereditary rather than due to environmental exposure.
There are observations that suggest that offspring vary
markedly in their susceptibility to embryotoxic chemicals. This is
demonstrated by the occurrence of the "fetal hydantoin syndrome" in
only one of four siblings whose mother continued to take
phenylhydantoin during each of her four pregnancies (Allen et al.,
1980).
When classifying single and multiple malformations, each child
should appear in a tabulation only once. Patterns of multiple
malformation should be noted, since they may provide evidence of
the time at which developmental disturbances occurred.
3.1.2 Prenatal diagnostic procedures
These methods can be invasive or non-invasive. The detection
of malformation by these methods can only be made after conception
and, since many patients and health personnel do not approve of
planned termination of pregnancy, their value in the prevention of
malformations is limited.
3.1.2.1. Invasive intrauterine techniques
Though invasive intrauterine techniques are valuable in well-
defined clinical situations for diagnosis, they are not suitable
for use in population studies. Invasive techniques include: (a)
amniocentesis, by which it is possible to sample the amniotic fluid
and obtain fetal cells for cytogenetic studies and enzyme assays as
well as fluid for chemical analysis; (b) fetoscopy by means of
which the fetus can be inspected directly; (c) combined fetoscopy
and biopsy or d) ultrasound guided biopsy for obtaining samples of
fetal blood, skin, muscle, or other tissue (such as choriomic
villi) for special analysis. These methods may be combined with
molecular biological techniques in the future. Such techniques
include restriction enzyme fragmentation of genomic DNA, in which
altered DNA restriction sites can be detected when a large
percentage of the cells have the altered genotype. Specific probes
can also be derived from structural gene sequences or from highly
repetitive genomic DNA sequences. cDNA probes for the globin have
been used to detect defects in the structural gene regions, coding
for the haemoglobin proteins. Deletion of specific globin gene
fragments has been found in the case of thalassaemia. Defects have
been found in genes coding for the Beta-globin protein. Some of
these approaches have already been applied in clinical prenatal
diagnosis.
3.1.2.2. Non-invasive techniques
(a) Ultrasound investigation
(i) Visualisation
Visualisation of the conceptus is possible as early as 15-20
days after conception, and heartbeat can be detected at around 35
days. Subsequently, the conceptus can be "inspected"
morphologically, its size and growth determined, and some
malformations diagnosed. This is a valuable technique for
determining fetal growth in the mid and final trimesters. When
three or four subsequent measurements of standard variables, such
as the biparietal distance, are performed around weeks 18, 22, 26,
and 32, a reliable estimation of the prenatal growth is obtained.
Gross abnormalities, such as anencephaly, omphalocoele, and cystic
kidneys can be diagnosed long before term.
(ii) Fetal heart rate monitoring
Fetal heart rate can be determined with ultrasound, and
recorded in the prenatal period with special monitors. The
technique permits the follow-up of acute changes, produced when the
mother has been exposed to a toxic agent.
(b) X-rays
X-rays are generally only used to confirm fetal death.
(c) Analysis of maternal blood
By measuring alpha fetoprotein (AFP) in maternal plasma, it is
possible to detect certain malformations, especially CNS
dysraphias, without amniocentesis. The advantages and
disadvantages of maternal serum AFP monitoring are being studied in
several countries.
(d) Analysis of maternal urine
Various indicators of development are found in maternal urine.
Levels of HCG, or its B-subunit, monitor trophoblastic (placental)
development. Levels of estriol are quantified to estimate fetal
"well-being" towards the end of pregnancy. The quantity of
maternal urinary estriol reflects the capacity of hydroxylation of
C-19 steroids at the 16 position in the fetal adrenals and liver
and of the transport of these steroids into the placenta, where
they are aromatized. Improved chemical tests involving analysis of
maternal urine are being developed for the detection of fetal
metabolic abnormalities.
3.1.3. Epidemiological methodsa
The epidemiological approach contributes to the attainment of
three main goals:
(a) identification of environmental teratogens;
(b) monitoring of poor pregnancy outcome in populations
exposed to chemicals; and
(c) evaluation of the usefulness of preventive measures.
The following approaches can be used to assess the toxic
effects of chemicals (Tables 2 and 3).
3.1.3.1. Hypothesis-generating (descriptive) studies
Descriptive studies are useful for generating a hypothesis,
but, alone, they are not a means of testing an aetiological
hypothesis. The main aspects considered in these studies include:
(a) Case reports
The description of cases of malformation, in which a birth
defect coincides with known chemical exposure, rouses suspicion
that the chemical is a potential human teratogen.
(b) Surveillance
Data collection systems for the surveillance of congenital
malformations have been established at both regional and national
levels, e.g., the International Clearinghouse for Birth Defects
Monitoring Systems, already mentioned in section 1. The value of
these monitoring activities, which should be continuous programmes
with rapid reporting and periodic reviews and analyses of data, is
to provide background for testing hypotheses.
In the Scandinavian countries, workers are registered according
to occupation (Erickson et al., 1979; Hemminki et al., 1981). This
computerized registry can be linked to the occurrence of
developmental hazards such as abortions and malformations, to
identify populations at risk. Once a registry has been
established, such data analysis is rapid and relatively
inexpensive. The shortcomings of this approach are that not all
women go out to work and some may have been in their recorded
occupation for only a short time.
---------------------------------------------------------------------------
a See also IPCS Environmental Health Criteria 27: "Guidelines on
Studies in Environmental Epidemiology" Geneva, World Health
Organization (1983)
Table 2. Hypothesis-generating studies and observations
----------------------------------------------------------------------
Methods Advantages Limitations
----------------------------------------------------------------------
Case report No special resources needed; Relationships with
calls attention to possible causal agent cannot be
potential teratogens established; lack of
information on
variables of interest
Surveillance Documentation on background Controls not included;
registers and time trends information sparse;
occupational exposures
missing; slow;
limitations on further
contacts
Correlation Useful pointers to the Non-causal correlations
studies etiological factor; cannot be ruled out;
Easy to design confounding variables
difficult to control
----------------------------------------------------------------------
With standardized records, it is possible to compare data
collected over a certain period. Such a registry would be useful
for the chemical industry. The technique can be supplemented by
special epidemiological methods including analytical studies.
These methods will produce more reliable results if population-wide
surveillance systems for monitoring congenital malformations are
established. The longer these systems operate, the more reliable
will be the information concerning both the incidence of
spontaneous malformations in a population (Kucera, 1961a,b, 1977),
and more will also be known of the temporal and spatial variations
in the frequency of congenital malformations (Kucera, 1971a). The
occurrence of unusual malformations (Kucera, 1968); and possible
environmental impacts on fetal formation (e.g., maternal
occupation) can be identified (Kucera, 1968b, 1971b).
A surveillance system is generally considered to be a
descriptive method, that has been formally justified and is
generally accepted. However, surveillance should be regarded as an
essential element in further epidemiological investigation rather
than a separate entity for evaluating chemical hazard and/or the
subsequent risk.
(c) Correlation studies
Correlation studies are concerned with the pattern of
distribution of any health condition or malformation, as well as
variations in the occurrence of a malformation in relation to time,
space, and personal characteristics, in populations with various
levels of exposure to chemicals. The advantages and disadvantages
of hypothesis-generating studies are summarized in Table 2.
Table 3. Hypothesis-testing studies
-----------------------------------------------------------------------
Type of study Advantages Limitations
-----------------------------------------------------------------------
Cross-sectional Fast to execute; No incidence rate is
studies Relative low cost possible, only a
prevalence rate
Control of confounding Poor for cause-effect
variables possible relationships
Case control Specific; Difficult design;
studies fast to execute; difficult
inexpensive; reasonable interpretation;
data base; control of exposure data usually
confounding variables obtained after birth
possible
Cohort Exposure data prior Large, slow to execute;
studies to birth; less difficult expensive; less specific
to design and interpret; information; many
control of confounding confounding variables to
variables possible be controlled
-----------------------------------------------------------------------
3.1.3.2. Hypothesis-testing (analytical) studies
These studies help in establishing cause-effect relationships
and in the estimation of their magnitude.
(a) Cross-sectional studies
The objective of cross-sectional or prevalence studies is to
determine the prevalence rate of a malformation in a given
population. The burden and distribution of a malformation are
described and its association with a potential causal factor is
determined. Often a prevalence study constitutes the first phase
of a prospective study and is usually conducted on a representative
sample of the population, within a short period of time, to provide
an "instantaneous" image about diseased and healthy groups within
that population. The population and the variables to be considered
must be very well defined.
(b) Case-control studies
In case-control studies, which supplement hypothesis-generating
studies, the frequency of malformations and extent of exposure to a
potential teratogen are compared with those in an unaffected
control group. The affected individual is the starting point,
while the control group may be selected from either unaffected
children or those affected with another type of malformation. The
advantage of such a control group is that the so-called memory bias
in pregnancies with abnormal outcomes is reduced. This approach is
of great value when rare outcomes, such as a specific malformation,
are examined. Case-control studies are feasible, only if previous
exposure is ascertained. A major problem is lack of adequate
exposure data. Other advantages and disadvantages are shown in
Table 3.
(c) Cohort studies
These are prospective studies in which two or more groups of
people exposed to different levels (including no exposure) of a
potential teratogen are followed up, and the pregnancy outcomes
recorded. The prospective method tends to be more informative
about the quantitative risk associated with exposure to a potential
agent, and is a very valuable method for analysing the association
between exposure to chemicals and subsequent developmental effects.
The value of epidemiological studies in associating environmental
agents with health effects is greatly increased by carefully
defining the study group and its cohort. Women of child-bearing
age may be grouped according to occupational exposure, prescribed
drugs, age, diseases, smoking and drinking habits, and other
variables of interest, but the difficulties involved in the
evaluation remain. The size of study populations is a major
problem. The use of epidemiological studies for risk estimation
can be improved by combining study populations from several
countries, an approach used successfully in the Scandinavian
countries. Other advantages and disadvantages of the method are
shown in Table 3.
(d) Intervention studies
In these studies, the frequency of a specific disorder in
groups where corrective measures have been applied is compared with
that in controls. Intervention studies can be conclusive, if they
are carefully designed and there are adequate controls.
3.1.3.3. Populations at special risk
As with carcinogens, most human teratogens have been identified
in studies on therapeutically- or occupationally-exposed
populations (Sokal & Lessmann, 1960; Shepard & Fantel, 1981). This
emphasizes the necessity for identifying sub-populations at high
risk of producing abnormally developed offspring.
Both men and women exposed to chemicals at the workplace should
be followed up to evaluate the health of their progeny.
Occupational health programmes, in particular, should include
reproductive data as an index of workers' health. Special
attention should be paid to pregnant women who have been exposed
accidentally or over a long period to high levels of chemicals,
even if they appear to be in good health.
3.1.3.4. Confounding and complicating factors
All of the previously described methods have their limitations,
if used as the only source of data for risk assessment. Of
particular interest, in the design of epidemiological studies, is
the consideration of confounding variables. One of the most
troublesome factors is lack of agreement about what constitutes a
developmental defect. Structural defects are emphasized more than
abnormalities that develop later in life. As mentioned earlier,
endpoints include growth retardation, functional disorders,
malformation, and death. Ascertainment of malformation is
incomplete, even by the end of the first year of life, while
examination for minor structural changes may be haphazard or
virtually non-existent. In addition, one case of malformation
could be reported several times by different medical doctors,
sometimes with a different diagnosis.
The accuracy of incidence rates is further complicated by
differences in the frequency of malformations known to occur in
different populations. The rate for the same malformation may vary
significantly according to state or country. Some of this can be
attributed to ethnic differences, but probably other more subtle
factors are also involved. Little is known regarding the
susceptibility of women of different ethnic groups to chemicals
that induce birth defects. Thus, it is difficult to extrapolate
epidemiological findings from one geographical area to another.
Age and parity of the mother and sex of the offspring are
variables that need to be recorded. Smoking and drinking habits
may play a role with regard to induction of embryotoxic effects.
Thus, many variables appear capable of influencing the rates of
abnormal development. Other variables include the time and method
of evaluation and the interpretation of manifestations considered
to be adverse. Furthermore, no woman is exposed to only a single
agent, and the effects of exposure to other environmental agents
and other factors such as eating and drinking habits, drug use, and
diseases acquired during pregnancy should be taken into
consideration.
3.1.3.5. Statistical power
Statistical power is largely determined by the frequency of
unfavourable pregnancy outcomes and the size of the population
under study. To define the probability that a recently introduced
chemical will double the frequency of normally observed birth
defects, large numbers of mother/child pairs will have to be
studied, and even larger numbers will be needed, if a specific type
of relatively common abnormality (e.g., ventricular septal defect)
is to be investigated. Conversely, slightly lower numbers may
suffice, if an increased incidence of an extremely rare abnormality
coincides with exposure to a specific agent (e.g., phocomelia and
thalidomide), though, in such cases, a consensus of opinion
accepting the association, usually precedes completion of formal
statistical analysis.
3.1.3.6. Quantifying studies
As already mentioned, the main drawback of most epidemiological
studies is the lack of a quantitative assessment of exposure.
The environment contains numerous chemicals to which people are
continuously exposed. It is therefore difficult to isolate an
exposure to a single agent, and a dose-response relationship is
rarely established. The best conditions appear to be in the use of
prescribed drugs, where use, dose, and dosage regimen are recorded
on the prescription. Computerized linkage between prescribed drugs
and pregnancy outcome has been used to establish associations
between drug exposure and pregnancy outcome (Jick et al., 1982).
Occupationally-exposed groups constitute another appropriate model
for observational health risk studies, in particular, if
complemented by analysis of the potentially toxic chemical(s) or
the metabolites in body fluids.
3.2. Experimental Animal Studies
Developmental toxicity studies should be designed and performed
in such a way as to be compatible with other types of toxicological
investigation. Designing, performing, interpreting, and
extrapolating specific studies in isolation rather than as part of
this wider background makes it more difficult to estimate risk from
extraneous agents.
For historical rather than objective reasons, the main focus of
a range of reproductive toxicity tests (section 4.3.3.1 - 4.3.3.8),
necessary for investigating new chemicals, is the teratogenic
study, which may be more aptly designated as a test for selective
embryo/fetotoxicity. These initial investigations should not be
termed "teratogenicity tests" since they aim to identify a variety
of effects on the mother and developing conceptus in addition to
malformations (Palmer, 1976). The tests are conducted to
determine: (a) deviation in prenatal variables (resorptions, fetal
deaths, fetal anomalies, and fetal weight); and (b) a minimal or
no-observed-adverse-effect dose that does not induce any
significant deviation from normal development. The period of
administration should cover the period of organogenesis, which is
usually equated with daily dosing on pregnancy days 6-15 and 6-18
for rats and rabbits, respectively. During pregnancy, maternal
responses should be recorded at least daily and body weight
recorded regularly; a record of food consumption may also be
required. Just prior to parturition, dams should be killed, litter
values (number of live and dead young, fetal weights) recorded, and
fetuses examined for external, visceral, and skeletal
abnormalities. There are variations employing different dosing
periods and some designs allow for some litters to be reared so
that offspring can be examined for postnatal manifestations of
prenatal toxicity.
Basically, experimental animal studies should be subdivided
into two types (Palmer, 1967): (a) studies in which an endpoint is
sought with a material of uncertain potential; and (b) studies in
which a defined endpoint has already been established. The latter
are usually second-stage investigations that might be regarded as
follow-up studies after initial tests and most of the literature
published in this field deals with this type of investigation. The
aims and approaches of the two types of examination are quite
different (Table 4).
When used in the initial testing of materials of unknown
potential, the aims and approaches of the developmental toxicity
study are quite different from the subsequent study in which the
activity of a known and potent agent needs to be defined. For
initial testing, the main priorities are: (a) to detect the lowest
dosage causing an adverse effect; and (b) to determine whether the
effects on the developing conceptus are selective, occurring at
doses lower than those causing maternal effects.
The purpose of an initial test is accomplished if results
indicate that the probability of selective effects is unlikely.
Elucidating the nature of the selective action, if necessary, is
reserved for second-stage studies, because, with a more precise
target to investigate more appropriate studies, because, with a
more precise target to investigate, more appropriate studies can be
designed. The design of such secondary studies must be flexible
and will vary according to circumstances, to such an extent that a
general account would be inadequate. The following comments are
therefore confined to initial tests.
Table 4. Types of experimental animal studies
---------------------------------------------------------------------------
Initial testing Second stage
investigation
---------------------------------------------------------------------------
Aims: To determine the lowest To determine optimum
dosage causing any adverse conditions of dosage
embryo/fetal response and timing for maximizing
the frequency of specific
malformations or other
endpoints
Means: Chemicals of unknown but Chemicals of known or
expectedly low terato- expected high embryo-
genic potential, adminis- toxic or teratogenic
tered in repeated doses potential administered
to determine a broad as a single dose
range of possible effects
Usual results: Low rates of malformations High rates of embryotoxic
not clearly effects and malformations,
distinguishable from clearly distinguishable
control values from control incidences
---------------------------------------------------------------------------
3.2.1. Species
Animal studies have mainly been carried out on mice, rats,
hamsters, and rabbits because of high fertility, short gestation
period, ease of determining the onset of pregnancy, and economy of
cost and housing of animals. Other species such as ferrets, cats,
dogs, pigs, and non-human primates have also been used when
toxicokinetic similarities between the test species and man or a
longer gestation period was thought important (Palmer, 1978).
The suitability of a test species is determined by a multitude
of factors including cost, availability, ease of handling and
housing, life span, and similarity to human beings in respect of
both developmental processes and toxicokinetics. However, the
choice is too often based on a limited and unbalanced set of
factors, for example, on the assumption that sub-human primates are
more similar to man than other species, because of evolutionary and
toxicokinetic factors. This reasoning is valid only when the test
is used to confirm a positive response to a potent material such as
thalidomide.
The use of primates in prenatal studies has frequently been
advocated by various authors, stressing that these animals can be
used to good effect to provide a closer approximation to the likely
human response to a material with a known or strongly suspected
teratogenic effect (Delahunt & Lassen, 1964). As a means of
demonstrating or confirming the absence of an effect, they are
impractical, even when toxicokinetic similarity to man can be
defined. The reason for this deficiency is that malformations
occur at a low frequency, so that to detect a ten-fold increase in
malformations occurring naturally at a rate of 0.1% would require
100 or more monkeys per group. Moreover, primates tend to abort
defective fetuses fairly readily, so that, particularly with
monotocous species, the number of endpoints that can be used may
often be restricted to the two "all or none" responses of abortion
or malformation.
It can be expected that, if alternative species are used,
investigators will naturally exploit species that they already use
in other studies because of the valuable background data already
available.
3.2.2. Dosages and dose levels
The selection of doses and dose regimens, and prediction
concerning the types of effect most likely to occur, are difficult
issues. Errors in choosing dose levels are most often made because
data from other studies are not used (Palmer, 1978). Cooperation
between the general toxicologist and reproductive toxicologist is
essential. The usual requirement is for 3 test groups and 1
control (vehicle) group; the highest dosage should induce signs of
minimal maternal toxicity. It is rarely necessary to use more than
3 dose levels of a test compound, when it is administered during
organogenesis to females of 2 (or more) mammalian species via a
route similar to that in human exposure.
Very high doses may alter the toxicokinetics. An effective
alternative is to use a single "limit" dose (equivalent to a high
dosage) for chemicals with low toxicity. The high dose is best
arrived at following a preliminary study in which females of the
same strain and species are administered the same dose of the test
material, with the same dosage regimen under conditions similar to
those to be used for the main study (Palmer, 1978). Conversely,
with many industrial chemicals, food additives, and pesticides, as
well as materials administered by unusual routes, it may be
impractical to seek a maternally toxic dose. Under these
circumstances, the maximum practical dosage or a limit dosage is
usually employed. The extent to which the vehicle affects
absorption of the test compound or to which it could exert an
independent toxic effect on the fetus or dam should be known.
3.2.3. Positive control groups
Inclusion of a group of animals dosed with a known teratogen is
sometimes recommended. However, it would be better to use these
animals for increasing the size of test groups or a negative
control group rather than for creating a positive control group.
3.2.4. Historical controls
Control values should be collected and permanently recorded.
They provide qualitative assurance of the nature of spontaneous
malformations that occur in control populations. Such records also
monitor the ability of the investigator to detect various subtle
structural changes that occur in a variety of organ systems
(Palmer, 1977b).
Historical control values for spontaneous abnormalities are
difficult to use in assessing the relevance of test group
observations, because they show marked temporal fluctuations and
clustering, particularly for malformations, anomalies, and variants
(John et al., 1982). Thus, it is often more appropriate to express
historical values in terms of different blocks denoting the
frequency of various effects in control groups of different average
size. However, extensive historical control observations may be of
value in the case of very rare (low frequency) malformations.
3.2.5. Dosing regimen
As mentioned, the dosing regimen employed is usually daily from
days 6 to 15 of pregnancy in studies on rats, and from days 6 to
18, in studies on rabbits. The day of mating or of finding
spermatozoa in vaginal smears is designated as day 0 of pregnancy.
For routine testing, specified dosing periods need not be
considered sacrosanct; as long as the dosing period includes the
entire duration of organogenesis. The dosing periods selected
represent a compromise between a short period of exposure (one day
or less), which with a potent agent will usually provoke a specific
response, and a longer dosing period which, because it covers a
greater number of potential responses, induces a less precise
response. When testing agents of unknown toxic potential, the
latter is recommended as a first step.
Sometimes, the test agent may be administered more than once a
day, if, for example, the results of toxicokinetic studies indicate
that it has an extremely short half-life. Minipumps have been used
to maintain constant blood levels (Nau et al., 1981). Conversely,
for materials with a long half-life, a dosing interval of more than
one day might be needed to avoid accumulation of the agent in the
animal. Appropriate intervals should be based on the biological
half-life of the compound.
Two or more complementary studies, each employing a short
overall dosing period of 4 or 5 days, should be considered when
testing compounds with either pronounced cumulative toxicity or
ability to induce tolerance associated with enzyme induction or
inhibition. Such refinement of the dosage regimen is particularly
important, when there is only a slight margin between human
exposure levels and animal test dosages (Palmer, 1978).
3.2.6. Route of administration
In routine tests, administration should be by the anticipated
route(s) of human exposure. This is logical, since the amount and
rate of a chemical that reaches the embryo varies according to the
route of administration. However, it is not always true that routes
allowing administration of the greatest amount of material will
induce the greatest effects. For example, application of
hexachlorophene to the vagina or mucous membranes results in a
greater systemic reaction in mother and fetus than larger doses
administered dermally or by incorporation in the diet (Thorpe,
1967; Kimmel et al., 1974).
Though administration by the intended human exposure route(s)
is usually best for the prediction of the human situation, it may
not always be possible to achieve other important objectives such
as the administration of adequate doses or the avoidance of
unnecessary stress, which in itself may induce embryo/fetal
toxicity. In this respect, inhalation studies with "face only"
exposure are more stressful than whole body exposure and should be
used only when there is no alternative. A study in which a more
practical route of administration is coupled with an awareness of
species differences in toxicokinetics will often provide more
meaningful data. An understanding of comparative toxicokinetics
may reduce the need to examine a large number of routes of
administration.
3.2.7. Numbers of animals and statistical analyses
Group sizes generally considered acceptable have been
determined by tradition and empiricism, rather than by sound
reasoning. Usually, the larger and more expensive the species, the
smaller the group, in spite of the fact that, on a purely
scientific basis, this can be questioned. For example, the number
of primates per group should be higher than that of rabbits because
the primate is monotocous and offers fewer endpoints, all of which
are of an "all or none" type such as abortion or malformation.
Similarly, the number of rabbits per group should be higher than
that of rats, because of greater variations in the incidences of
maternal and fetal death, abortion, malformations, and anomalies in
the rabbit. The scientific principle holds, even though it is
somewhat compensated for by the fact that it is easier to examine
primate and rabbit fetuses for both visceral and skeletal
anomalies.
The advantage of using polytocous species such as mice, rats,
or rabbits is not because there are more sample units (litters) for
analysis, but because a higher number of endpoints can be recorded
and a series of responses can be obtained within litters. This
makes it possible to use statistical methods, other than
contingency analysis, to detect significant differences with
smaller sample (group) sizes. Any comprehensive investigation of
values employed in screening tests (e.g., litter size, incidences
of embryonic death, malformation, anomaly, or variation) shows a
litter based, non-normal distribution for which non-parametric
analysis is safest (Weil, 1970; Palmer, 1974; Staples & Haseman,
1974; Haseman & Hogan, 1975; Palmer, 1976; Palmer, 1977a; Cooke,
1976, 1981; Woo & Hoar, 1979, 1982; Shirley & Hickling, 1981).
There has been and remains great resistance to accepting the
viewpoint that the litter, not the fetus, represents the valid
sample unit and that litter values are not normally distributed.
3.2.8. Observations on pregnant animals
Observations on dams should include examination for signs of
toxicity and regular measurement of body weight. Examination of the
dam contributes to the study in two important ways: (a) by
providing a reference point so that any effects on the conceptus
can be judged as selective and specific, rather than secondary to
toxic effects on the dam; and (b) by making it possible to
ascertain whether the agent is more toxic for pregnant animals.
Such a phenomenon has been observed for anti-inflammatory agents
and iron dextrans (Flodh et al., 1977).
Records of food and water consumption are often requested, but
rarely provide more precise information than body weights and/or
clinical signs, provided dosages were correctly chosen in the first
place.
In initial testing, the emphasis is logically placed on using
observations on the pregnant animal to determine the selectivity of
action on the conceptus. However, in second stage investigations
there may be, in specific situations, a need to pay greater
attention to maternal toxicity as a cause of embryo/fetal toxicity.
Maternal ill-health and disease conditions in the mother have been
associated with malformations in human beings (Kalter & Warkany,
1983) and toxic effects in the dam with malformations in the mouse
(Khera, 1983). Thus, where exposure to chemicals at, or near,
maternally toxic levels is unavoidable, greater attention should be
paid to the possible consequences, for the conceptus, of maternal
disorder.
3.2.9. Observations on the progeny
When examining the litter, fundamental requirements include the
recording of the number of live young, and embryonic and fetal
deaths; the number of implantations can be recorded directly or
deduced. It may be useful to count corpora lutea of pregnancy
(except with mice, because of the difficulty of counting) to insure
against incorrectly attributing reduced litter size to treatment.
With studies in which dosing is initiated at, or before
implantation, or when a species, such as the pig or the sheep, that
has a long gestation period is used, corpora lutea counts can
contribute directly to determining embryonic loss.
Viable fetuses should be examined for external malformations,
and skeletal and visceral anomalies and variations, and their sex
should be determined. Other suggestions, some of which are of
value while others are less useful, include:
(a) The weighing and examination of dead fetuses for
abnormalities. This usually provides data that are difficult to
interpret, because dead fetuses undergo decomposition.
(b) Distinguishing between early and late embryonic deaths
(resorptions). This is generally a useful exercise as observations
of dose-related effects can help in determining the sensitive
period.
(c) The recording of individual fetal weights (or crown-rump
length) of rats or mice. This is generally of little value in
initial testing, unless individual fetal weight can be associated
with altered fetal development, such as delayed ossification or
malformation. Most investigators who record individual fetal
weights rarely attempt to associate these with fetal anomalies or
variations. In routine tests, it is just as effective and less
tedious to weigh the entire litter and calculate the mean fetal
weight. The occasional abnormal or obviously oversized or
undersized fetus may be weighed individually and the result related
to abnormalities.
Another disadvantage of weighing fetuses individually is that
it may lead to inappropriate statistical analysis, which, in turn,
would provide "statistically significant" differences between
groups, which would be of little biological relevance (Palmer,
1978). Similar difficulties occur when individual crown-rump
length is measured in rodents or lagomorphs. This variable is
tedious to record and has little value on its own, though it may
complement measurements of fetal weight and skeletal development.
However, this level of effort and specificity is best reserved for
second-stage studies rather than initial testing.
(d) Comparison of litter weights. This is a particularly
useful parameter in initial tests, because it reflects both litter
size and fetal weights. A non-specific toxic response to an agent
may be seen as reduced fetal weight, at low doses, and as reduced
litter size through increased deaths, at higher doses. In such
circumstances, a dose-related reduction in litter weight provides
useful information.
(e) The weighing of the uterus and contents (gravid uterus).
Some investigators weigh the gravid uterus and subtract this from
the total maternal body weight to obtain a corrected maternal body
weight in the belief that this corrected weight, together with
maternal weights measured during pregnancy, facilitate distinction
between the maternally-mediated and direct effects of chemicals on
the fetus. Other workers are not convinced that such data are
enough to determine the direct effect of a chemical on the fetus.
(f) Allocation of a percentage of fetuses for either visceral
or skeletal examinations. It has been suggested that 66%, 50%, or
33% of fetuses should be allocated for one or other type of
examination, but this applies only to rats, mice, and hamsters. It
is quite feasible to examine the fetuses of rabbits and most other
species for both skeletal and visceral anomalies, thus,
considerably increasing the amount of information obtained from a
single study. With fetuses of the smaller species, the safest
procedure is to allocate half of the fetuses in each litter for
visceral examination and the other half for skeletal examination.
However, the most important criterion is to ensure that sufficient
fetuses and litters are examined for each technique, to make a
valid statistical analysis.
Two suggested methods of visceral examination are hand
sectioning (Wilson technique) and microdissection. When performed
well, the Wilson technique (Wilson, 1965; Beck, 1977) is
acceptable. However, good hand-sectioning is difficult to achieve
and high quality is rarely attained. For the vast majority of
investigators, microdissection is a safer technique for small
fetuses and a better technique for large fetuses.
(g) Reporting units. Data should be reported on each endpoint
in the form of a test group value (mean, median, or incidence) and
an individual litter value. Reporting of malformations and
anomalies should clearly indicate the specific fetuses and litters
affected. Accurate interpretation and correct analysis is not
possible, if anomalies are listed by type, without indicating their
distribution among fetuses and litters. If developmental toxicity
is recorded at dosages considerably lower than those eliciting
maternal toxicity, the effect may be considered selective, and a
direct effect on the embryo or a specific result of interference
with its nutrition.
In concluding whether or not selective action has occurred, the
answer should be positive, negative, or inconclusive. When the
answer is positive or negative, the process of extrapolation can
begin. When the results are inconclusive, further investigations
should be carried out.
3.2.10. Animal husbandry and laboratory practices
Animal studies should be performed according to the highest
standards of care, welfare, and husbandry, for both ethical and
humane reasons. It is equally important from the practical point
of view, as animals in poor condition will provide unreliable
results. Furthermore, all animal studies are conducted under
conditions with a large number of potential confounding variables
(Chance, 1957; Biggers et al., 1958; Russell & Burch, 1959; Kalter,
1965; Laroche, 1965; Dearborn, 1967; Ellis, 1967; Kallai, 1967;
Palmer, 1969). Studies involving pregnant animals may be especially
susceptible to these variables because of the rapid changes in
maternal physiology and in interactions between the maternal
organism and the developing conceptus.
Laboratory practices necessary for scientific acceptability of
any work are, honesty, care, consistency, and responsibility, in
addition to those mentioned or implied in this and other sections
of this report. Principles of good laboratory practice (GLP) are
detailed in various regulatory requirements and presented for
instance, with the guidelines mentioned in Section 1.
4. POSTNATAL MANIFESTATIONS
4.1. Introduction
The production of offspring able to adapt to postnatal life
requires the development of functionally normal organs and systems.
Since organs and systems may often be at greater risk of damage
from the adverse effects of a chemical during the period of their
development and growth, functional abnormalities in various organs
and systems have at times been linked with exposure to toxic
chemicals during the prenatal period. The consequences of such
damage may not be readily evident, unless functional or behavioural
tests are performed.
The period of organ and system maturation extends beyond the
period of organogenesis and even beyond the prenatal period.
Therefore, the susceptible period for the induction of insults that
may lead to functional deficits is much longer than that for the
induction of gross structural defects.
Functions that have been shown to be affected by prenatal and
early postnatal exposure to chemicals include behaviour,
reproduction, endocrine function, immune competence, xenobiotic
metabolism, and various other physiological functions. Late
manifestations of toxicity may include neoplasia and shortened life
span.
Fetal damage by chemicals has been shown to modify enzyme
ontogeny. Examples of active compounds are PCBs, TCDD,
organomercurial compounds, and formaldehyde and its derivatives
(Andrew & Lytz, 1981; Merkurjewa et al., 1983). Since organ-
specific profiles of enzymes continue to evolve and undergo
pronounced changes, long after the organs have become
morphologically distinct, enzyme systems are an easy target for
chemical insults. Alterations in enzyme activity indicate
disturbance in the fundamental biochemical processes (Battaglia &
Meschia, 1978) and in the maturation of organ functions (Stave,
1975).
It has been reported that the level of drug-metabolising
microsomal monooxygenases is altered following prenatal exposure to
chemical inducers, and such an effect may persist in adult life
(Illsey & Lamartinière, 1979; Yanai, 1979; Salganik et al., 1980).
As a result, the metabolism of chemical carcinogens, e.g.,
aflotoxin B1, may be altered (Faris & Campbel, 1983).
Toxic agents might exert their effects by causing alterations
in endocrine function and hormone levels. Hormones are the natural
stimuli that evoke the synthesis of many enzymes, especially those
required for adaptation to extrauterine life (Greengard, 1973;
1975a,b). Major disorders may result from impairment or imbalances
in developmental endocrinology, if they occur at critical stage,
when hormones normally exert discrete effects on maturation. So
far, few studies have dealt with the effects of hormonally-active
xenobiotics on the developmental programming processes (Illsley &
Lamartinière, 1979; Lamartinière & Lucier, 1981).
Deviations in functional development may also be mediated by
induced adverse effects on liver function. Prenatal rat liver does
not acquire full competence until some weeks after weaning, which
makes it a suitable model for studying developmental delays. There
are data suggesting that in utero exposure to environmental agents
may result in altered liver function (Lucier et al., 1975; Chahoud
& Eggert, 1978; Lucier & McDaniel, 1979).
There are indications that compounds that interfere with adult
immune systemsa may have effects on developing immune systems
(Roberts & Chapman, 1981). It has been reported that exposure to
toxic substances during immune ontogenesis may induce functional
defects that remain dormant until adulthood (Spyker & Fernandes,
1973), and that the stage of immune development at the time of
exposure may affect the nature, magnitude, and duration of the
immune defect (Pinto-Machado, 1970).
Methods of assessing organ function in progeny do not differ
from those conventionally used in toxicology and are not discussed
in this document. Evaluation of behaviour, reproduction, and
transplacental carcinogenicity requires more specific methods and
these are discussed in the following subsections. The evaluation
of postnatal growth and survival has been incorporated in the
subsection on behaviour (section 4.2).
4.2. Behaviour
The argument has been advanced that behavioural tests may be
useful in the risk evaluation of prenatal exposure to chemicals,
because behaviour is a functional indicator of the net sensory,
motor, and integrative processes occurring in the central and
peripheral nervous system (Mello, 1975). Thus, alterations in
behaviour following exposure to environmental agents might be a
relatively sensitive indicator of nervous system toxicity (Spyker,
1975). Because behaviour can also be influenced by the functioning
of other organ systems, specific organ toxicity might also be
reflected as a change in behaviour (Weiss, 1975; Tilson & Mitchell,
1980).
Two separate phenomena underly behavioural deviations that
appear as a manifestation of embryotoxicity. The first, an
afferent phenomenon, involves the interaction of a chemical with a
molecular entity of the developing organism, such as a receptor or
a specific enzyme. The second, an efferent phenomenon, represents
the consequences of this interaction at the molecular, subcellular,
cellular, or cell population leve1. Malformations are the outcome
of altered development at the cell population level and are due to
disturbances in basic morphogenic processes.
---------------------------------------------------------------------------
a Principles for assessing immunotoxic effects will be dealt
with in a separate IPCS document.
An embryotoxic agent may induce behavioural deviations by:
(a) disturbing the morphogenesis of the central nervous
system and consequently influencing its functional
development (Langman et al., 1975; Pellegrino &
Altman, 1979; Rodier et al., 1979); and
(b) affecting functional development by interfering with
the processes of axogenesis, dendrogenesis,
synaptogenesis, and myelination (Jacobson, 1978) or
with the function of already differentiated cell
populations in the brain.
The relationship between morphogenesis and functional
development is far from clear. Specific components of the nervous
system may be affected to induce neurobehavioural deviations, in
the absence of any apparent morphological changes in the brain
(Reinisch & Karow, 1977; Lundborg & Engel, 1978; Rosengarten &
Friedhoff, 1979). However, this view may be too simple, since an
altered rate of cell acquisition in the developing brain and other
morphological changes may escape detection (Patel et al., 1977;
Rodier, 1978; Bendek & Hahn, 1981). Altered structural and/or
biochemical components must be present to account for functional
abnormalities.
In some cases, structural defects of the central nervous system
have been found not to be accompanied by behavioural changes
(Brunner et al., 1978). However, the manifestations of behavioural
defects may be minimized by plasticity and compensatory processes
or may escape detection, because of inadequate testing.
If behavioural deviations are considered to be embryotoxic
phenomena, then dose-response, stage-response, and stage-effect
relationships should be demonstrated. The latter characteristics
are determined by a pattern of behavioural deviations that may
change in a day-to-day fashion, according to the time of chemical
exposure (Rodier et al., 1979). The sensitive period for inducing
behavioural deviations in animals appears broader than the period
for inducing malformations of the central nervous system (Vorhees
et al., 1979). Behavioural changes can be induced at certain times
in the early postnatal life of mice and rats that correspond to the
late prenatal and perinatal periods of human brain development
(Langman et al., 1975; Lundborg & Engel, 1978; Pavlik & Jelinek,
1979; Pellegrino & Altman, 1979; Pavlik et al., 1980). The
development of the human brain is characterized by intensive
morphogenic and functional developmental processes during the
perinatal period, which continue, even during the first and second
years of life (Lewis & Patel, 1980). Thus, the relevant period of
brain development in the subject must be considered in a
behavioural study.
Testing procedures to be employed for assessing the mechanisms
of behavioural effects should be decided on the basis of the
mechanisms underlying embryotoxicity. One possible approach, may
include the use of the primary interaction of chemicals with
molecular entities of the developing brain, such as receptors or
specific enzymes. Affinities of chemicals for the following classes
of receptors could also be assessed in vitro: steroid hormone
receptors, classical neurotransmitter receptors, peptidergic
receptors, neuromodulator receptors. Furthermore, the rate-
limiting enzymes in the synthesis and/or degradation of different
mediators could be investigated. The disturbances of morphogenic
and functional developmental processes that are triggered by
exposure to test chemicals during brain development can also be
quantified. A third approach deals with assessing behavioural
functions.
4.2.1. Strategy of testing
Study of the behavioural effects induced by prenatal exposure
to chemicals is a new field which has, so far, dealt mainly with
the detection and description of behavioural deviations induced by
environmental agents in experimental animals rather than with the
elucidation of the mechanisms of their induction (Coyle et al.,
1976; Kimmel, 1976; Rodier et al., 1979; Vorhees et al., 1979). As
stated by Zbinden (1981), "there are no direct and unambiguous
measures of behaviour; ... even the simplest behavioural response
results from an interplay of many parts of the whole nervous
system, and it is always modulated by a unique set of information
stored in the memory of the experimental subject".
The incomplete knowledge of normal behaviour and its underlying
mechanisms, together with the fact that the study of altered
behaviour has only developed very recently, account for the lack of
uniformly established and accepted strategies and methods for the
evaluation of behavioural effects. No single approach or
behavioural test battery has been identified as the most reliable,
sensitive, and economic means of detecting behavioural disfunction
following developmental insult, and, with the current state of
knowledge, it is not possible to select a battery of tests from the
procedures available. Thus, it is considered premature to decide
on a fixed protocol to be included in routine experimental animal
studies for testing chemical substances (Mitchell, 1978; Tilson &
Cabe, 1978; Adams & Buelke-Sam, 1981; Zbinden, 1981).
Behavioural testing is performed after something is known about
the teratological and embryotoxic effects of the agent and at
exposure levels lower than those inducing structural abnormalities.
It is suggested that tests be performed at two levels of
sensitivity/complexity: initial and secondary. Initial testing of
chemicals of unknown potential should ensure that behavioural
effects are not missed. It should be broad and comprehensive with
regard to the types of functions assessed. This is necessary
because many types of functions may be adversely affected by an
agent and these effects cannot be reliably predicted. Using test
methods that monitor a number of functions simultaneously is
appropriate at this stage, in order to save time, cost, and effort
(Butcher, 1976; Dews & Wenger, 1979). The behavioural study should
preferably be conducted in combination with a developmental
toxicity study on the same progeny, so that data from the toxicity
study can be used to support and clarify the behavioural findings.
Secondary testing should be performed if behavioural and/or
neurological alterations are observed in initial testing, or
suggested by human data, or by structure-activity relationships.
More sophisticated and selective behavioural techniques, perhaps in
other species, should be used to help delineate the type(s) and
extent of effects produced and the possible mechanisms involved.
Also, at this stage, more selective exposure regimens and testing
schedules should be employed, as well as procedural controls to
distinguish a direct neurotoxic action from an indirect effect.
Secondary behavioural evaluations should be flexibly designed with
agent specificity in mind (Adams & Buelke-Sam, 1981).
In behavioural testing, several aspects should be taken into
account including the choice of functions to be assessed, the tests
to be employed, the species to be used, and the factors to be
controlled.
4.2.1.1. Behavioural functions to be assessed
The following functional categories should be evaluated:
(a) physical growth and maturation;a
(b) reflex and motor functions;
(c) sensory functions;
(d) affective functions (activity/reactivity);
(e) cognitive functions (learning and memory);
(f) social behaviour; and
(g) reproductive behaviour.
Ideally, behavioural functions should be assessed as they are
initiated, as well as during their development, maturation, and
decline, since behavioural deficits might be expressed by: (a)
retardation or abnormal development of certain functions; (b)
alterations in adult function; (c) premature decline in functional
capacity or induction of premature senescence (Grant, 1976). At
present, most behavioural testing is done on adolescent or young
adult animals, with the exception of reflex and motor development
tests, which are performed in early neonatal life (Buelke-Sam &
Kimmel, 1979).
---------------------------------------------------------------------------
a Although not behavioural parameters, physical growth and
maturation are so integrated with behavioural development that
they must be taken into account in any interpretation of
alterations in behaviour.
4.2.1.2. Tests to be used in behavioural studies
Tests used in behavioural studies are basically derived from
experimental ethology, psychology, neurology, neurophysiology, and
neurotoxicology. The decision regarding the tests to be used
depends on several factors related to the characteristics of the
animal model, the availability of technology for measuring a
specific function, and on cost-effectiveness/time-efficiency
factors (Tilson et al., 1980). Within a particular study, the
choice of tests depends basically on the desired level of analysis.
At the initial level of behavioural analysis, procedures are sought
that sample a wide range of functions (e.g., apical tests), are
inexpensive to perform, require little or no training of
experimental animals, and make possible the assessment of large
numbers of animals under study. These tests are used to provide a
tentative assessment of behavioural deficits and to quantify them
as precisely as possible. However, such procedures frequently
require subjective measurements, often yield quantal data, and may
not be sensitive enough to detect subtle neurobehavioural
perturbations.
When a more detailed evaluation is necessary, tests that
require extended or special training, special behavioural
instrumentation, and refined procedures are used. At this stage of
analysis, behavioural tests can be supplemented with other methods
to determine the mechanism(s) of action, such as neurochemical
assays and sophisticated morphological examinations.
The present state of development of behavioural tests requires
that these methods be validated and standardized to be maximally
used in any neurobehavioural-testing programme. However, before
recommending a particular testing method for routine use, it must
be certain that the test actually measures the desired parameters.
In this context, very few of the present behavioural test methods
have been validated (Tilson et al., 1979). The choice of a test
method can be greatly facilitated by knowing more about its
inherent variability in normal populations. As yet, such data are
scarce (Phillips et al., 1980).
Another related problem is the choice or determination of the
testing sequence. In general, the testing sequence should progress
from the least stressful to the more stressful procedures, for
instance, from natural to manipulated behaviour (Vorhees et al.,
1979). However, the possible "carry-over" effect from one test to
another presents problems, for which there is no easy solution.
Possibly, in initial testing, the risk of carry-over should be
accepted, in order to reap the benefits of being able to correlate
the responses of an individual through different procedures. If
there is a suggestion of a real problem associated with carry-over,
this may be resolved in second-state studies by allocating separate
sub-sets of animals to each test procedure.
4.2.1.3. Choice of species
The selection of suitable species obviously involves practical
and economic factors. Animals with pregnancies of short duration
and high fecundity are preferred. It is also important that
adequate embryological, toxicological, and behavioural data are
available for the species chosen. As it is preferable to study
species with well-investigated normal behavioural profiles, rats
and mice would be the first choice. Other species or strains may
be used to investigate specific behavioural deficits only at the
secondary testing leve1. The choice of test species at this level
will greatly depend on the specific function(s) to be measured.
For instance, albino rats are inappropriate for assessing visual
function. The use of species that are phylogenetically close to
man, such as primates, may sometimes be justified.
4.2.1.4. Variables to be controlled
Control of variables is essential in behavioural studies, since
the integrated and subtle nature of behaviour may make it more
susceptible to factors that have little impact on other endpoints
of developmental toxicity.
In the current context, this control must be exercised at the
time of exposure, at the time of testing, and in between exposure
and testing. The list of potential influential factors is so
extensive that it cannot be dealt with adequately in this document.
As brief examples, psychosocial factors such as litter size, number
of animals per cage, type of cage, and frequency of contact
(handling by the attendant technician or investigator) can affect
performance considerably. The circadian rhythm may markedly affect
performance in activity tests, so it is important not to test
animals from different groups at different times of the day or
night (assessment of possible effects of chemicals on circadian
rythm should also be considered) (Chahoud et al., 1975).
There are pronounced maternal and litter influences operating
from conception to weaning that can exert a lasting impression on
maturity. Fostering and cross-fostering techniques can be applied
in second-stage investigations to separate direct treatment effects
from postnatal maternal influences (Spyker & Spyker, 1977). Litter
size is an important factor that determines not only the level of
nutrition but also the amount of social stimulation (Frankova,
1968). Variations in the litter size have been shown to affect the
responses to open field and T-maze tests (Robinson, 1976; Akuta,
1979). Reducing litters to a certain standard number, in order to
avoid nutritional differences, has been widely practised, but there
are discrepancies in the methods of standardizing litter size for
postnatal behavioural stud-ies. However, according to some
authors, standardization of litter size could introduce bias into
the study (Tesh, 1977).
As in other aspects of developmental toxicity, time of exposure
may be critical in the determination of the type of effect induced.
Timing in the performance of behavioural tests may also be critical
in the determination of the effects detected. As well as being
delayed in appearance, behavioural manifestations may be
multiphasic in character (Sobrian, 1977; Tabacova, 1981).
Furthermore, the integrated nature of behaviour is such that the
consequences of damage may be overcome by the development of
adaptive or compensatory mechanisms. Ideally, therefore, behaviour
should be investigated as a dynamic progression. This could be
uneconomic and impractical on a routine basis, but it should be
borne in mind that differences in behaviour recorded at any one
time may be misleading. The research worker should also be aware
that the "carry-over" effect of repeated testing may interfere with
the evaluation of borderline differences.
4.2.2. Methods of assessment of specific functions
4.2.2.1. Physical development
Growth and survival are considered to be among the most
important indicators of functional normality (Wilson, 1973a;
Palmer, 1976; Zbinden, 1981). From the point of view of
screening, a difference in body weight gain may be a significant
observation of developmental toxicity, since most physical
landmarks correlate well with body weight (Adams & Buelke-Sam,
1981).
Physical development is routinely assessed by recording body
weight and survival at weekly intervals from birth until weaning,
as well as by registering the time of appearance of certain
physical landmarks (Table 5), which should be looked for daily,
starting at least one day prior to their expected appearance, until
prevalent in all the test animals. However, behavioural parameters
may be affected by the excessive handling of pups.
Table 5. Physical signs and the approximate time of their
postnatal appearance in ratsa
-------------------------------------------------------------
Physical sign Postnatal age
-------------------------------------------------------------
Pinna detachment (unfolding of external ear) day 2
Primary coat of downy hair day 5
Incisor eruption day 8
Development of fur day 9
Ear opening day 11
Eye opening day 14
Testes descent day 25
Vaginal opening day 30
-------------------------------------------------------------
a From: Alder & Zbinden (1977).
Variations in the times of appearance listed in Table 5 can
result from differences in environment, animal strain, and
investigators recognition level (Hughes & Palmer, 1980). Physical
development may also be influenced by the duration of pregnancy,
maternal health, and litter size.
4.2.2.2. Reflex development
The reflexes most frequently evaluated are the righting
reflexes, negative geotaxic response, auditory startle reflex, and
grasping and placing reflexes (Table 6). Variations from the
quoted values can be expected. Detailed descriptions and suggested
procedures for measuring these reflexes are available in the
literature (Bolles & Woods, 1964; Fox, 1965; Altman & Sudarshan,
1975; Alder & Zbinden, 1977; Vergieva et al., 1981a). The normal
disappearance of certain reflexes may also be important, indicating
that the reflex has been integrated into more complex structures of
behaviour (Lapointe & Nosal, 1979).
Table 6. Reflex development in the rat
----------------------------------------------
Test Postnatal age of
appearancea
(mean value)
----------------------------------------------
Surface righting reflex day 1.8
Negative geotaxis day 4.8
Cliff avoidance day 4.8
Palmar grasp day 6.3 (waned)
Auditory startleb day 11.4
Vibrissae placing day 12.5
Free fall righting day 17.5
Visual placingc day 17.6
----------------------------------------------
a From: Smart & Dobbing (1971). For reflex ontogeny in mice, see
Fox (1965).
b At the indicated age, the test reflects the opening of external
meatus of the ear.
c The test may be unreliable, since the visual acuity of many
albino strains is so limited that confusion with tactile
(vibrissae) placing can readily occur.
4.2.2.3. Sensory functions
Tests to assess sensory functions are based on the localization
or orientation responsiveness to stimuli. These tests are
influenced by a wide range of other variables as they are based on
indirect measurements (Evans, 1978). Procedures to evaluate sensory
functions are simple to perform and require little investment in
time and equipment, but the results are limited to quantal data
(the response is scored as present or absent) and the scores are
subjective (Tilson & Mitchell, 1980). Thus, these tests provide
weak evidence of sensory effects. More precise details regarding
the extent of the impairment require other, more sophisticated
methods, based on operant conditioning (Evans, 1978; Stebbins &
Rudy, 1978). However, these methods are not applicable in the pre-
weaning period. The ages when specific sensory functions can be
assessed are presented in Table 7. For more details concerning
early sensory assessment, the reader is referred to Alder &
Zbinden, (1977), Grauwiler & Leist (1977), Tesh (1977), and
Zbinden, (1981).
4.2.2.4. Motor function
Spontaneous movements can be observed in familiar (home cage,
activity cage) or unfamiliar (open field) environments, and provide
information about the time of appearance and type of different
motor functions. Open field studies provide more precise data and
additional information on the latency and organization of motor
behaviour (Zbinden, 1981).
Table 7. Specific sensory functions (rat)a
------------------------------------------------------------------------
Function Postnatal age at which Test
assessment usually starts
to be possibleb
------------------------------------------------------------------------
Vision 18 days pupillary reflex;
visual placing response;
visual cliff avoidance;
Hearing 11-13 days auditory startlec
Olfaction 3-8 days odour aversion
3-12 days odour preference
Ultrasonic 17-20 days failure of vocalisation
vocalization when removed from nest
------------------------------------------------------------------------
a Adapted from Zbinden (1981).
b For all these tests, different authors have established their own
ranges, a practice recommended to investigators.
c See footnote b to Table 6.
The procedure of normal development of spontaneous motility in
rat is indicated in Table 8.
In the rat, a peak of motor activity (as an integral part of
overall activity) usually originates between 14 and 16 days of age
and falls to adult levels at about four weeks of age (Campbell et
al., 1969; Melberg et al., 1976; Randall & Campbell, 1976; Campbell
& Raskin, 1978). This seems to be correlated with the concurrent
development of the brainstem catecholaminergic system (Campbell &
Mabry, 1973).
Table 8. Development of spontaneous motility in rata
-------------------------------------------------------
Type of movement Postnatal age
of appearance
-------------------------------------------------------
Pivoting (early ambulatory movements, day 1
showing no coordination between
fore- and hindlimbs, head and pelvis)
Head raising day 8
Elevation of forelimbs and shoulders day 9
Crawling substituted by walking days 9-11
Elevation of hindlimbs and pelvis days 13-17
-------------------------------------------------------
a From: Alder & Zbinden (1977).
An open-field procedure can be used to quantify the development
of motor function in the pre-weaning period. The open-field test
has been successfully used in early postnatal life for detecting
motor disturbances after prenatal exposure to environmental toxic
substances (Kavlock et al., 1980; Tabacova et al., 1981, 1983).
When used periodically during the first three postnatal weeks,
disturbances in the developmental sequence of normal motor patterns
can be revealed.
Another group of test methods measures movements elicited by
the research worker or by some kind of mechanical device. Tests
frequently employed (Table 9) assess body posture, muscle tone,
motor coordination, equilibrium, and gait, and provide information
on the time of appearance and achievement scores. Most of the test
procedures are described in detail by Fox (1965), Altman &
Sudarshan (1975), and Alder & Zbinden (1977).
Table 9. Elicited neuromuscular activities
-----------------------------------------------------------------
Activity Reference
-----------------------------------------------------------------
Hindlimb support Alder & Abinden (1977)
Hanging-grip strength Werboff et al. (1961); Altman
et al. (1971)
Clinging to inclined plane Cabe et al. (1978); Sobotka et
al. (1974); Rivlin & Tator (1977)
Climbing vertical rod Altman et al. (1971)
Climbing inclined screen Werboff et al. (1962)
Homing response Altman et al. (1971)
Crossing narrow path Grauwiler & Leist (1977)
Rotating rod performance Jones & Roberts (1968)
-----------------------------------------------------------------
Most of these tests can be performed by the end of the second
and during the third postnatal week. Performance on the rotating
rod is best assessed during the fourth to sixth postnatal week.
The optimum time for testing may vary according to the strain of
rat, the design of the rotating rod, and the operating procedure.
If performed early during development, the animals have neither
sufficient coordination nor strength to provide meaningful scores;
if conducted too late, the individual variation may be so
pronounced that intergroup comparisons are not possible. As an
alternative to the rotating rod, the inclined plane test requires
simpler and less expensive apparatus and can be applied over a
wider age range. In the pre-weaning stages, the same apparatus can
be used for assessing negative geotaxis. Generally, the inclined
plane test shows less variation between individual scores and is
less demanding on animals' coordination than the rotating rod.
Physical strength is assessed using activity wheels or by
swimming ability. However, these tests are not practised during
the pre-weaning period.
It should not be inferred that all of the tests listed must be
used in the same study. A small battery of adequately chosen tests
is quite sufficient for any level of analysis (Tilson et al.,
1979), allows better control, and creates less disturbance for the
animals.
4.2.2.5. Activity, reactivity, and emotionality
Activity is a complex measure of behaviour. It might indicate
various functions of the CNS and of the whole organism, for
example, motivational, affective, cognitive, and physiological
responses. These various aspects of spontaneous activity
demonstrate clearly the necessity for a more precise definition of
what is meant by activity measurements. Prenatal exposure to
toxicants may result in shifting the curve of activity development
to a later period (Michaelson et al., 1974) or in producing changes
in activity that disappear as the animal matures (Culver & Norton,
1976; Reiter, 1977; Tabacova et al., 1981). Longitudinal testing
to uncover possible age-dependent changes in activity would
therefore be advisable.
The most widely-used test for assessing activity is the open-
field procedure, of which there are various modifications (Spyker
et al., 1972; Grauwiler & Leist, 1977; Kavlock et al., 1980, etc.).
For screening, automated methods are preferable to direct
observation by the investigator (Ljungberg & Ungerstedt, 1976;
Norton et al., 1976; Marsden, 1979).
Reactivity is defined as the animal's responsiveness to various
external stimuli. Changes in the excitability of the nervous
system can be measured experimentally as changes in overall
responsiveness to environmental stimulation. Reactivity is measured
as avoidance of aversive stimuli, escape responses, orienting
reflexes, and startle responses. The startle response is one of the
most frequently used procedures to assess the responsiveness of
animals to external stimuli (Adams & Buelke-Sam, 1981). This
reflex can be elicited by a variety of environmental stimuli such
as auditory startle or air puff startle and has a reproducible
short latency. The startle response can be influenced by changing
the variables of the eliciting stimulus, the level of background
stimulation, or the general responsiveness of the animal (Tilson &
Mitchell, 1980). Toxicant-induced alterations in startle
responsiveness can be readily interpreted, because the anatomical
pathways and neurotransmitter systems that mediate this response
have been reasonably well studied (Fechter, 1974; Davis, 1980).
Measurement of the startle response has been highly recommended as
a tool for the screening of potential neurotoxic agents (Adams &
Buelke-Sam, 1981). As mentioned earlier, the startle response can
also be used as a test for the development of the ear (in the
preweaning period) and as a specific test for hearing.
Emotionality is directly related to spontaneous activity. In
psychology, the concept persists that emotionality and exploration
are inversely related. High emotionality is felt to inhibit
exploration while low emotionality facilitates it. Urination and
defecation in the open field are often used as criteria of
emotionality (Rodier, 1978), but other criteria may also be
assessed (Archer, 1973).
4.2.2.6. Cognitive development
Cognitive development is essentially defined as the ability to
learn or respond appropriately to a changing environment.
Associative learning involves the acquisition of a response to a
previously neutral stimulus, as a result of temporal association
and/or reinforcement contingencies (Thompson & Glanzman, 1976).
Non-associative learning is expressed by various other kinds of
behaviour such as habituation, latent learning, play, and
manipulation of objects.
Several experimental designs have been developed to assess
associative learning in young animals (Misanin et al., 1971; Amsel
et al., 1976; Blozovski & Cudennec, 1980; Gemberling et al., 1980).
Although it had previously been thought that associative learning
could not take place until a certain level of postnatal maturation
had been reached (Campbell & Coulter, 1976), subsequent research
has demonstrated associative learning in rat pups as young as l day
old (Johanson & Hall, 1979).
Numerous methods are available for the study of learning and
memory in older animals. Different kinds of learning such as
simple associative learning (Pavlovian conditioning) and more
complex forms of learning (such as problem solving in a Hebb-
Williams maze, puzzle boxes, or tests of reasoning activity) have
been studied (Rodier, 1978). The conventional criteria for
learning include speed of learning, number of correct responses,
and duration of latency. Data from such studies are interpreted
more precisely, if behaviour in the course of learning is well
analysed and spontaneous activity occurring during the testing
period is carefully recorded. Analysis of the inter-trial interval
on freely occurring, non-contingent responses can also provide
valuable information about the presence of a neurobehavioural
deficit (Frankova & Barnes, 1968).
Habituation, an example of non-associative learning, is defined
as a decrease in response following repeated presentation of a
stimulus. The development of habituation reflects the functional
maturation of structures responsible for the development of
inhibitory processes (Bronstein et al., 1974). The ontogeny of
habituation has been studied by Campbell & Stehouwer (1980), who
demonstrated ontogenic changes in habituation, learning, and
retention during the suckling period. Methods for the assessment
of learning and memory in neonatal animals have been discussed by
Adams & Buelke-Sam (1981).
4.2.2.7. Social behaviour
The development of social behaviour has mainly been studied in
primates. Although rodents have not been used traditionally, the
development of social behaviour can be assessed in this species,
even during the preweaning period. The type and duration of
contacts in the home cage or in a novel environment of a group (or
pair) of pups can be recorded. Play activities, early sexual
manifestations, and agonistic activities, are other endpoints that
might prove useful (Frankova, 1973).
Social behaviour in older animals has not been used extensively
to indicate developmental toxicity. Basic information on the type
and characteristics of social interactions may be obtained by
analysing contacts between a pair of animals of the same sex placed
in the novel environment. Over 40 elements (acts and postures)
have been described by Grant & Mackintosh (1963). The two most
common social behaviours investigated are dominance and aggression.
Sex-dependent behaviour represents a complex set of
motivationally different activities, such as courtship, pair
formation, reproduction, and parental activities (Gerall & McCrady,
1970; Frankova, 1977). This category of behaviour depends on the
stage of development, on environmental stimuli, as well as on the
hormonal status of the organism.
4.2.3. Relevance of behavioural studies for human risk assessment
There is a current requirement to screen agents for
developmental neurotoxicity in some countries; similar rules are
also being considered in other countries. In view of the already
enormous cost of safety evaluations (Johnson, 1981), great care
must be taken to ensure that the studies provide meaningful
information regarding potential hazard. The following requirements
must be fulfilled, before routine assessment of the effects of
prenatal exposure to chemicals on behaviour can be generally
accepted:
(a) the test(s) should be sensitive to neurobehavioural
alterations produced by a wide range of test agents
or conditions;
(b) the test(s) should be standardized and validated so
that accurate and reproducible results are provided
within and between laboratories;
(c) the test(s) should be cost effective;
(d) the test(s) should be able to recognize chemical
agents known to be neurotoxic to man.
There are several instances in which the neurobehavioural
function of human progeny has been reported to be altered following
in utero exposure to environmental agents (Tanimura, 1980). The
number of examples in which there is a correspondence between
observed neurobehavioural defects in human beings and effects
predicted from animal studies is limited to a few agents (i.e.,
methylmercury, lead, psychoactive drugs). In the case of
methylmercury, similar types of neurobehavioural deficits were
detected in human beings and experimental animals after prenatal
exposure (Reuhl & Chang, 1979).
The general lack of association reflects the need for more
careful epidemiological examinations and additional basic research
using animal models and agents suspected of being neurotoxic.
As developmental neurobehavioural toxicology is a recently
developed field of research, not enough evidence has accumulated to
convincingly demonstrate a parallel between effects in animals and
human beings. Manifestation of similarities may often be obscured
by inadequate testing or interpretation of data, by interspecies
differences in behavioural patterns or in the plasticity of the
central nervous system. Behavioural techniques have been widely
used to detect and quantify neurobehavioural alterations in adult
animals exposed to many environmental agents. There is good reason
to believe that these techniques can also be used to assess
neurobehavioural changes following prenatal exposure. The fact that
the relevance of animal data to human data is less questionable in
the field of adult neurotoxicity is at least partly due to the fact
that more information has been accumulated in this more traditional
field.
4.3. Reproduction
Reproduction depends on structural and functional integration
of various systems. Consequently, there are many ways in which
reproduction may be impaired directly or indirectly. The
development of the reproductive organs takes place over a period
extending beyond birth, and structural as well as functional
maturation does not occur until puberty. Because of the long
quiescent phase between initial formation of the basic structures
and adoption of their final functional form, defects may not become
apparent until a considerable time after induction, unless they are
so gross as to be considered malformations. Even then, certain
changes may be so extreme that they lead to incorrect determination
of sex rather than to identification of a developmental error. The
error is only discovered later; in man, this can be 14 - 20 years
after induction.
4.3.1. Normal gametogenesis and development of the genital tract
In both sexes of mammalian species, the reproductive organs
consist of gonads, an internal duct system, and external genitalia.
Since these components arise from diverse primordia, and
contributions from these primordia vary qualitatively and
quantitatively in the two sexes, the development of the
reproductive system is particularly complex. A detailed
description of the stages of development is beyond the scope of
this monograph and discussion is restricted to the stages most
vulnerable to chemical action.
Primordial germ cells of both male and female, are clones of
special cells present in the ectoblast at the early bilaminar
stage. In later stages of development, they are transferred into
the endoderm of the yolk sac. From there, the primordial germ
cells migrate into the mesoblastic undifferentiated primordia of
gonads located on the medioventral surface of the urogenital
ridges. In individuals with normal Y chromosome expressing H-Y
antigen, gonads differentiate into testes. In individuals without
the Y-chromosome and H-Y antigen, the gonadal anlagen give rise to
fetal ovaries. The primordial germ cells in the ovaries develop
into oogonia and undergo repeated mitosis differentiating into
oocytes. The latter enter meiotic prophase and become invested by
granulosa cells to produce primary follicles. Thus, the oocytes in
postnatal mammals are at an arrested stage of meiotic division;
diffuse diplotene, dictyate, dictyotene, or resting stage
(Erickson, 1967). A complete oocytic envelopment by granulosa
cells separates the oocytes from the ovarian stroma.
The first meiotic metaphase is completed much later after birth
shortly before ovulation. In human beings, the number of oogonia
and oocytes between fetal weeks 18-22 is estimated at 5-7 million,
in the newborn there are approximately 2 million oocytes, and at
the age of 18-20 years about 200 000. From the whole population of
human oocytes only approximately 400 undergo ovulation, assuming
regular ovulation; all the others degenerate.
The embryonal testis contains testicular cords composed of
primitive Sertoli cells and spermatogonia. The embryonal
spermatogonia originate from the primordial germ cells while
Sertoli-cells are mesodermal in origin. In the prenatal period,
spermatogonia, in contact with Sertoli-cells, divide slowly by
repeated mitoses, they do not differentiate into spermatocytes, and
do not undergo meiosis. Spermatogenesis and the onset of meiosis
in the testis follows maturation of the Sertoli-cells, a step under
hormonal control (follicle-stimulating hormone (FSH), luteinizing
hormone (LH), and testosterone), which begins at puberty.
Synthesis of male hormones by the fetal testis is critical for
the growth and differentiation of male accessory organs during the
prenatal as well as postnatal periods. The male phenotype is
mainly imposed on the embryo by two types of hormones, produced in
the testis, i.e., androgenic steroids and the Müllerian-inhibiting
factor (MIH). Androgens, produced by Leydig cells, stimulate the
development of the male reproductive ducts from the epigenital
portion of the mesonephros and from the Wolfian ducts, and the
development of the male external genitalia from structures located
around the urogenital sinus. MIH, a non-steroid factor, produced
by the embryonal testis causes the Müllerian ducts to regress. The
epigenital mesonephric nephrons and Wolfian duct are forerunners of
the epididymides, vasa deferentia, seminal vesicles, and
ejaculatory ducts. The urogenital sinus differentiates into the
urethra and gives rise to the prostate; derivatives of the genital
tubercle and of the urethral plate become the penis; the
labioscrotal swelling turns into the scrotum. Testosterone plays
an important role in the sexual differentiation of the Central
nervous system (CNS) during the perinatal period.
Testosterone is synthesized in the testis from pregnenolone. A
series of steps mediated by enzymes are involved in the conversion
of pregnenolone to testosterone and 5a-dihydrotestoterone, the
active derivative of testosterone in some organs. Interruption of
any of these steps during the critical period in pregnancy would
lead to a deficiency of androgens which, in turn, might lead to the
development of equivocal secondary sex characteristics
(pseudohermaphroditism) with normal sex chromosomes. Thus, a
decreased amount of androgen can fail to masculinize a male fetus,
which might have malformed ambiguous external genitalia, hypospadia
(pseudovaginal pseudoscrotal in some cases), but normal testes and
the XY karyotype. In severe cases of testicular dysgenesis, if the
testes fail to produce MIH, the Mullerian ducts may persist and
develop into a uterus and vagina. In addition, male efferent ducts
may be present in some cases.
Feminization of a male may be related to the absence of, or
deficiencies in, testosterone receptors. In human males exhibiting
errors in testosterone receptor, the phenotype is female despite a
normal male 46,XY karyotype, and the presence of testes and
regressed Müllerian ducts.
In the female, the Wolfian ducts regress and the Müllerian
ducts differentiate into the oviducts, uterus, and the upper 4/5 of
the vagina. The urogenital sinus gives rise to the vestibule and
the lower l/5 of the vagina, while the genital tubercle and the
urethral plate develop into the clitoris and labia minora. The
labioscrotal swelling turns into the labia majora. Female fetuses
can be virilized or masculinized as a consequence of congenital
defects in the synthesis of glucocorticoids or by the
administration of androgens. In the normal female fetus, gonadal
synthesis of testosterone or estrogens is not apparent and there is
no evidence that the interstitial cells or follicular apparatus of
the fetal ovary play any role in female genital differentiation.
In the absence of male gonadal function, the internal and
external genital anlagen differentiate according to the female
pattern. MIH, a protein of relatively high relative molecular
mass, prevents the development of the uterus and the fallopian
tubes in males. The function of testicular testosterone is (i) to
stimulate the growth of the epididymis, vasa deferentia, and
seminal vesicles, and (ii) to promote the stabilization/
transformation of the genital sinus and urogenital tubercle,
urethral plate and labioscrotal swellings into the prostate, male
urethra, penis, and scrotum, respectively, and (iii) to promote the
descent of the testes. Prior to the male differentiation, both the
urogenital sinus and external genital anlagen acquire the capacity
to convert testosterone to 5a-dihydrotestosterone (DHT) which, in
these tissues, is thought to be the effective androgen (Siiteri &
Wilson, 1974).
Postnatally, in both sexes, normal pituitary gonadotrophin
secretion appears to be required for the maturation of germ cells
and their supporting cells. The pattern of FSH and LH secretion
reflects the gradual maturation of a functional hypothalamic
pituitary unit, responsive to feedback inhibition by sex steroids.
Sexual differences in the pattern of gonadotrophin secretion are
well recognized; cyclic pulsatile in females, tonic in males.
Spermatogenesis continues throughout reproductive life. It
starts with stem cells (type A spermatogonia) which, in the rat,
undergo 5 mitotic divisions followed by two meiotic divisions to
produce spermatids. The latter change into spermatozoa, which move
to, and mature in, the epididymis.
4.3.2. Mechanisms of abnormal development
Adverse effects on the reproductive function of offspring,
resulting from prenatal exposure to environmental chemicals, have
not received sufficient attention. Examples of reproductive
effects that have been related to maternal chemical exposure, are
given in Table 10. Chemicals may affect either the gonadal tissues
directly or antagonize the action of growth-promoting or growth
inhibiting factors. However, the precise mechanisms of action are
not known.
When the complexity of sexual development, gametogenesis, and
reproductive tract differentiation is considered, it becomes
apparent that prenatal and early postnatal chemical exposure is a
special toxicological problem. The gonads, the reproductive
accessory organs, and the neuroendocrine system are all vulnerable
to toxicity induced by chemicals acting through unique mechanisms.
The specificity of male and female developmental processes also
accounts for differences in susceptibility to toxic agents between
the sexes.
Dixon (1982) has recently pointed out that there are a number
of periods during which the developing reproductive system is
susceptible to chemical insults. Interference by xenobiotics can
lead to morphological, biochemical, physiological, and/or
behavioural disorders as well as to the ineffective integration of
the biological processes required for successful reproductive
performance. Though reduced fertility in the offspring may be the
most obvious consequence of prenatal exposure to toxic
environmental chemicals, more subtle effects, such as those
involving secondary sexual characteristics and behaviour, are also
possible.
In mammals, the female fetus is especially vulnerable to agents
that damage the germ cells, because, in most species, all oocytes
develop prenatally; no (or few) primordial germ cells are formed
after birth. Therefore, chemicals that affect oogenesis may be
expected to have a lasting effect on the fertility of the female.
Particular periods of susceptibility during oogenesis with long-
term toxicological effects on fertility have been shown to be
connected with peak oocyte DNA synthesis periods (McLachlan &
Dixon, 1973). Fetal ovaries have been reported to be a target site
for the action of some environmental pollutants, particularly
polycyclic aromatic hydrocarbons (MacKenzie et al., 1979a,b).
Unlike germ cell production in the mammalian female,
spermatogenesis is initiated in the pubertal male, and germ cells
are produced continuously throughout adulthood. Prenatal exposure
to several compounds, including benzo( a)pyrene, 7,12-
dimethylbenz( a)anthracene (DMBA), and diethylstilboestrol (DES),
has been reported to alter male fertility (McLachlan et al., 1975;
Davis et al., 1978; Gill et al., 1979; MacKenzie et al., 1979a).
Some compounds act directly on the germ cells, probably at the
level of DNA, RNA, and protein synthesis; other chemicals act by
interfering with the normal hormonal regulation of spermatogenesis.
Table 10. Examples of reported abnormalities of the reproductive system
in the offspring of mothers treated with chemicals during pregnancy
-----------------------------------------------------------------------------------------------
Type of abnormality Species Chemical agent Reference
-----------------------------------------------------------------------------------------------
Degeneration and rabbit cyclophosphamide Gerlinger & Clavert (1964)
reduction of gonocytes
in male and female fetuses
Reduced fertility in males mouse cyclophosphamide Sotomayor & Cumming (1975)
and females
Gonadal dysplasia in rat busulfan Forsberg & Olivecrona (1966)
males and females
Clitoral hypertrophy and rat metyrapone Goldman (1967)
adrenal hyperplasia in female
fetuses
Decreased incidence in rat 2,3,7,8-tetrachloro- Khera & Ruddick (1973)
pregnancy and reduced dibenzo- p
litter size -dioxin (TCDD)
Reduced fertility in females mouse procarbazine McLachlan & Dixon (1973)
Reduced fertility in males and mouse benzo( a)pyrene Mackenzie et al. (1979a)
females and gonadal dysplasia
-----------------------------------------------------------------------------------------------
Though the exact mechanisms by which chemicals can alter
neuroendocrine function are not clearly understood, two possible
neuroendocrine effects have been suggested that may result in
impaired reproduction (McLachlan et al., 1981). The first involves
an action on endogenous catecholamines which, secondarily,
influences the ability of the sympathetic nervous system to
stimulate gonadotrophin release; the second involves the more
direct action of a chemical at specific neuroendocrine centres.
Chemicals may also affect hormone action by altering steroid
synthesis, by affecting the activity of steroid-metabolizing
enzymes, or by interfering with hormone-receptor interactions.
Since reproduction is a highly integrated function that
involves a behavioural component, compounds that alter behaviour
may result in infertile animals, even in the absence of direct
toxic effects on the genital tract or the endocrine system (see
section 4.2 on behaviour). Development of the integrative
behaviour necessary to reproduction may be susceptible to
disruption by psychotropic agents or hormonally-active xenobiotics.
Effects on reproduction may also be mediated by prenatally- or
perinatally-induced disturbances in liver function. Compounds that
alter the hepatic metabolism of gonadal hormones can alter the
reproductive characteristics of the affected individual. The long-
lasting effects of neonatal exposure to polychlorinated biphenyls
(PCB) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on hepatic
function (Lucier et al., 1975, 1978; Lucier & McDaniel, 1979)
suggest that in utero exposure to environmental agents may result
in altered enzyme activity.
4.3.3. Testing procedures
A detailed discussion on methods for assessing effects of
chemicals on reproductive function has recently been published by
the Scientific Group on Methodologies for the Safety Evaluation of
Chemicals (SGOMSEC) established jointly by WHO and SCOPE/ICSU and
supported by IPCS (Vouk & Sheehan, 1983).
Potentially all of the processes in reproduction may be
affected by chemicals. Fecundity tests assess spermatogenesis,
sperm maturation, sperm transport, semen production, and mating, in
the male, and ovulation and oviductal transport, fertilization, and
implantation, in the female. These are examined collectively by
measuring reproductive outcome. Results of chemically-induced
reproductive failure must take into account the health of the
animals after chemical exposure. When evaluating chemicals for
effects on reproduction, it is critical to determine the place of
observed reproductive effects within the chemical's general
toxicity profile. For transplacental toxicity studies, effects on
reproductive performance in offspring are most meaningful in the
absence of maternal toxicity and gross malformations in the young.
Testing of chemicals for specific endpoints is likely to
improve the long-term understanding of the processes affected by
toxicity. This is an important area for further development.
However, one of the easiest measures of reproductive capacity is
the ability of male and females to mate and produce normal
offsprings.
The following is an outline of a few of the reproductive tests
that involve exposure to chemicals during either the prenatal or
pre- and postnatal periods. Many of the comments made in section
3.2 are relevant here and will not be repeated.
4.3.3.1. "Fertility test" on progeny following prenatal exposure
If a "fertility test" is to be conducted in the progeny of mice
or rats following prenatal exposure, the chemical is administered
over a period extending from implantation until the end of
pregnancy. The progeny are allowed to litter normally and their
postnatal development is observed until sexual maturity. At
adulthood, the males and females of the test progeny are mated with
animals that have not been previously exposed to the test chemical.
All pregnant females are killed at term. Data on incidence of
pregnancy, and prenatal values are evaluated for toxic effects. Up
to the present, such tests have only been used for second-stage
testing and have not been performed routinely. This approach is
included in multigeneration studies.
4.3.3.2. Multigeneration studies
There is no general agreement about the most satisfactory
design for multigeneration tests or even the number of generations
to be studied (Palmer, 1981). The FDA Advisory Committee (1970)
reported that with a range of substances, including
organophosphorus anticholinesterases, herbicides, and pesticides,
adverse effects were occasionally seen after two, three, or even
four generations that were not clearly evident earlier. However,
it is unlikely that genuine effects would be recorded in later
generations that were not evident in the first or second
generations (Clegg, 1979; Leeming et al., 1982). Until recently, a
minimum of two litters per generation was usually recommended,
though some workers feel that the maximum possible number of
litters should be obtained in one of the generations, to determine
whether there is any overall effect on the maximum reproductive
capacity. However, supportive data for such large studies have not
been published. In some designs, some of the pregnant dams are
killed in each generation so that the offspring can be examined, at
term, for teratogenic effects. Some of the offspring of the third
generation are subjected to full histopathological examination.
No single ideal test exists for the wide range of possible
compounds that will be tested. For substances that show absolutely
no adverse reproductive effects at doses up to those toxic for the
parent, a two-generation, one litter per generation test, would be
satisfactory. For other compounds that show a small, but
increasing effect with time, more litters and more generations may
be required to establish a no-observed-adverse-effect leve1. In
practice, if only one litter is produced per generation, it is
often very difficult to be certain that no effect at all is
present. The preferred basic study design is therefore a two-
generation, two-litter study with three dose levels of the test
compound, plus a control group. The parental generation should be
treated for at least 9 weeks before the second mating, and
treatment continued throughout the study. One advantage of
deriving the F2 generation from the F1a, rather than from the
traditional F1b generation, is that an opportunity is available to
investigate in detail any effects that may be observed during the
mating of the F1a animals. Reproductive performance from mating
first litters should be normal, if the animals are not too young at
first mating. Normally, not less than 20 males and 20 females
should be treated and mated per group. Mating brothers and sisters
is not recommended in the last generation. For the earlier
generations, larger group sizes should be used to reduce the risk
of a concurrent inbreeding bias (Palmer, 1981). All offspring
should be reared to weaning, though it is acceptable to reduce the
litters to a standard size if desired. At weaning, offspring not
involved in producing subsequent generations should be killed and
examined macroscopically. In the absence of any gross changes, it
is doubtful if there is much to be gained by random measurements of
organ weights and histological examination. However, these
procedures must be performed if any toxic effect is suspected. It
may be advantageous to fix and store the tissues from one male and
one female from each litter, at each killing stage, until the study
is completed, so that these tissues can be more carefully examined
if unexpected toxicity is revealed in the final stages of the
study.
4.3.3.3. Choice of species
For economic and temporal reasons, it is usual to perform the
above reproduction studies on mice or rats. Ideally, in assessing
reproductive toxicity, it should be shown that the species chosen
for the reproductive studies metabolize the substances under test
by the same routes as man. In practice, this is not always
possible (especially with pesticides), but efforts should be made
to obtain this information and perform the tests in this way.
Where equivocal results are found in rodent studies, tests in other
species may be of value. There is no evidence that the results
from one particular species or strain extrapolate best to man.
Thus, there are no rigid requirements regarding choice of species,
but it is very important that adequate control data on reproductive
performance should exist for the species used.
4.3.3.4. Doses, route, and duration of treatment
The choice of suitable dose levels is one of the most difficult
problems in the design of reproduction tests. The highest dose
should induce some signs of general toxicity such as reduced gain
in body weight. The lowest dose should be a no-observed-adverse-
effect level. Since, usually, only a small percentage of animals
are adversely affected, a dose-response relationship is very
important in assessing results. Three dose levels are normally
required, unless the highest practical dose does not induce any
adverse reproductive effect.
The route of administration should be relevant to that in the
human exposure. Inhalation exposure for reproductive toxicology
has not been widely used and can give rise to misleading results,
if undue stress is involved. In such circumstances, alternative
routes of administration may be used.
For environmental chemicals such as pesticides, administration
throughout the whole period of gestation seems to be the most
appropriate, but other treatment regimens are also acceptable.
4.3.3.5. Presentation of results
There is no general agreement on the best statistical method
for the analysis of the results of reproductive studies. In the
presentation of results, appropriate statistical methods, depending
on the distribution of particular endpoint measured, can be used
(Snedecor & Cochran, 1967; Sokal & Rohlf, 1969; Hollander & Wolfe,
1973), but it is essential that all of the data for both individual
fetuses and litters are presented, so that other methods of
assessment can be used if necessary. In all studies, it should be
possible to trace the complete outcome of pregnancy in each female.
4.4. Transplacental Carcinogenesis
Transplacental carcinogenesis is defined as the appearance of
neoplasia in the progeny of females exposed to chemical agents
during pregnancy. This phenomenon has been demonstrated in rats,
mice, hamsters, rabbits, pigs, dogs, and monkeys with approximately
60 chemicals (Table 11) including representatives of the major
classes of chemical carcinogens such as nitroso compounds,
polycyclic aromatic hydrocarbons, aminoazo compounds, mycotoxins,
and others. While practically all sites of the developing fetuses
can be affected by carcinogens administered during pregnancy, the
tissues most susceptible to neoplastic responses are those of the
nervous system, kidney, and lung. Usually, prenatal susceptibility
to carcinogens is greater in the advanced stages of organogenesis;
in rodents, for example, it is confined to the last third of the
gestational period. With some exceptions specific for certain
species or strain (lung adenomas in Swiss mice and some tumours in
patas monkeys), tumours do not usually appear before adult life.
In at least some tissues, effects of transplacental carcinogens may
be enhanced by the application of a carcinogen or promotor to the
progeny. In addition, Tomatis (1979) has reported that increased
tendency to develop neoplasia may persist without additional
exposure, although greatly diminished, in subsequent generations of
animals. The same group (Tomatis et al., 1981) has suggested that
this transmission may occur via the germ cells, and this is
supported by the data of Nomura (1982).
Twenty three compounds and groups of chemicals and 7 industrial
processes have been shown to induce carcinogenic effects in human
beings (IARC, 1982). However, there is convincing epidemiological
evidence of transplacental tumour induction in man for only one
compound, i.e., diethylstilboestrol (Herbst et al., 1979a; Rice,
1981).
The following is a summary of the main features of
transplacental carcinogenesis in laboratory animals, the research
perspectives offered by models under development, and advice on the
need for testing compounds in this field.
Table 11. Substances reported to be transplacental carcinogens
in animalsa
---------------------------------------------------------------------------
Polycyclic aromatic hydrocarbons 7,12-dimethylbenz[ a]anthracene
benzo[ a]pyrene
3-methylcholanthrene
Aminoazo compounds 2-fluorenylacetamide
2-aminoazotoluene
dimethylaminoazobenzene
2-toluidine
benezidine
dichlorobenzidine
2-aminotoluene
Carbamates urethane
zineb
Natural products of plant and aflatoxins
animal origin pyrrolizidine alkaloids
cycasin
safrol
Nitroso compounds nitrosodimethylamine
nitrosodiethylamine
nitrosodipropylamine
2-hydroxypropylpropylnitrosamine
2-oxopropylpropylnitrosamine
methylpropylnitrosamine
nitrosobis(2-hydroxypropyl) =
nitrosamine
nitrosodibutylamine
4-hydroxybutylbutylnitrosamine
nitrosohexamethylenimine
nitrosopiperidine
dinitrosopiperazine
nitrosomethylurea
nitrosoethylurea
nitrosopropylurea
nitrosobutylurea
nitrosoethylbiuret
nitrosomethylurethane
nitrosoethylurethane
nitrosobutylurethane
Table 11 (contd).
---------------------------------------------------------------------------
Nitroso compounds resulting from amidopyrine + sodium nitrite
interaction of the following diethylamine + sodium nitrite
precursors methylurea + sodium nitrite
ethylurea + sodium nitrite
butylurea + sodium nitrite
isopropylurea + sodium nitrite
methylbenzylimidazolcarbamate +
sodium nitrite
citrulline + sodium nitrite
propylhexedrine + sodium nitrite
Other alkylating compounds azoxymethane
azoethane
azoxyethane
dimethyl sulfate
diethyl sulfate
1,2-diethylhydrazine
1-methyl-2-benzylhydrazine
3,3-dimethyl-1-phenyltriazene
5-(3,3-dimethyl-1-triazeno) =
imidazol-4-carboxamid
3,3-diethyl-1-phenyltriazene
3,3-diethyl-1-pyridyltriazene
3,3 dimethyl-1-pyridyltriazene
propane sultone
methylmethanesulfonate
ethylmethanesulfonate
methylazoxymethanol
Drugs diethylstilbestrol
carcinolipin
cyclophosphamide
procarbazine
Miscellaneous tobacco smoke condensate
furylfuramide
4-oxyphenyllactic acid
3-hydroxyxanthine
vinyl chloride
4-nitroquinoline N-oxide
---------------------------------------------------------------------------
a Data taken from the review by Rice (1981) with additional references
for following compounds: nitrosoethylurethane, 3,3-dimethyl-1-
pyridyltriazene (Druckrey, 1973), 2-fluorenylacetamide (Armuth &
Berenblum, 1977), benzidine (Vesselinovitch et al., 1979), zineb
(Kvitnizkaja & Kolesnicenko, 1971), pyrrolizidine alkaloids
(Schoental & Cavanagh, 1972), dinitrosopiperazine (Börzsönyi et
al., 1980), nitrosobutylurea (Maekawa & Odashima, 1975),
nitrosobutylurethane (Maekawa et al., 1980), 5-(3,3-dimethyl-1-
triazeno)imdazol-4-carboxamide (Zeller, 1980), amidopyrine +
sodium nitrite (Alexandrov & Napalkov, 1979), diethylamine +
sodium nitrite (Vesselinovitch & Rao, 1974), methylurea + sodium
nitrite (Alexandrov, 1973), butylurea + sodium nitrite (Maekawa
Table 11 (contd.)
a et al., 1977), isopropylurea + sodium nitrite (Schneider et al.,
1977a), methylbenzylimidazolcarbamate + sodium nitrite (Börzönyi
et al., 1976), citrulline + sodium nitrite (Ivankovic, 1979),
propylhexedrine + sodium nitrite (Schneider et al., 1977b),
ethylmethanesulfonate (Schneider et al., 1978),
diethylstilboestrol (Rustia & Shubik, 1976; Napalkov & Anisimov,
1979), carcinolipin (Sabad et al., 1973), cyclophosphamide
(Roschlau & Justus, 1971), tobacco smoke condensate (Nicolov &
Chernozemsky, 1979).
4.4.1. Principles and mechanisms of action
4.4.1.1. Comparative sensitivity of the adult and fetal
organism to carcinogens
All the transplacental carcinogens that have been studied were
already known to be carcinogenic in adult animals. However, the
ability of a substance to induce tumours transplacentally can
differ in a quantitative way from that observed in weanling or
adult animals. For example, it has been established in rats that
fetal sensitivity to the action of nitrosoethylurea (NEU) is 20-50
times higher than that of adult animals (Ivankovic & Druckrey,
1968). On the other hand, both fetal and adult rats (Alexandrov,
1969) showed similar sensitivity to nitrosomethylurea (NMU), while
sensitivity to the action of nitrosodimethylamine (NDMA) proved to
be 6-10 times lower in the fetus than in the adult animal
(Alexandrov, 1968). Thus, it appears that the action of chemical
carcinogens may be stronger or weaker, after passing through the
placenta. A possible explanation is that physico-chemical
characteristics of chemical agents affect the rate of transport
across the placenta and the biotransformation in the mother and
fetus.
Negative results in testing for transplacental carcinogenic
effects should be evaluated with caution. It should be kept in
mind that the period of development when the rodent embryo is
sensitive to carcinogenic stimuli is rather short (10-12 days) and
that some compounds require more prolonged application in order to
induce tumours.
4.4.1.2. Dependance on the stage of prenatal development
The period of development sensitive to carcinogenesis starts in
the advanced stages of organogenesis and lasts until the end of
intrauterine life. This has been observed in at least 3 animal
species, i.e., the rat (Ivankovic & Druckrey, 1968; Napalkov &
Alexandrov, 1968; Alexandrov, 1974), the mouse (Nomura, 1974), and
the hamster (Mohr et al., 1975). However, some carcinogens
administered to pregnant animals during the early period of
embryogenesis, when the placenta has not yet formed, may also
induce tumours in the progeny (Spatz & Laqueur, 1967; Nomura, 1974;
Börzsönyi et al., 1976; Stavrou et al., 1977).
Generally, transplacental carcinogenesis does not reveal stage
specificity for any site in terms of the development and
histological appearance of tumours. For example, many chemicals
almost exclusively induce tumours of the nervous system and kidney
in the rat and of the lung in the mouse, irrespective of the day of
exposure.
Susceptibility to transplacental carcinogenesis is influenced
by genetic predisposition and appears to be time-specific during
pregnancy. Examples of this phenomenon are to be found in rat and
mouse studies (Druckrey, 1973; Rice, 1973a; Vesselinovitch, 1973;
Alexandrov & Napalkov, 1981). There may be periods of maximal
susceptibility that vary from one organ system to another. In the
rabbit, for example, the progeny of animals given NEU early in
gestation developed brain tumours whereas exposure later in
gestation caused only nephroblastoma (Stavrou & Lübbe, 1974;
Stavrou et al., 1977).
4.4.1.3. Species and strain specificity
Studies with carcinogenic substances have shown that the type
of tumours induced in the progeny differs from species to species.
The same agent may induce tumours at different sites, depending on
the animal species or strain (Druckery, 1973; Napalkov, 1973; Rice,
1973b; Rice et al., 1979; Ivankovic, 1979). The type of tumours
induced transplacentally may or may not necessarily be similar to
that induced in adult animals. Among animal species, the most
pronounced similarity has been demonstrated in mice (Rice, 1969;
Vesselinovitch, 1973; Diwan & Meier, 1974).
Strain dependence has been revealed, even when testing highly
organotropic substances. Urethane, which displayed pulmonotropic
carcinogenic properties in adult mice, induced transplacentally
both lung cancer and tumours at other sites, typical of the strain
tested (Vesselinovitch, 1973). The hepatocarcinogen
orthoaminoazotoluene, which induced liver tumours transplacentally
in certain mouse strains, also often induced tumours of the breast
and lung in others (Gelstein, 1961; Golub et al., 1974;
Kolesnicenko et al., 1978). Thus organotropism in mice is not only
determined by the carcinogenic properties of the compound, but also
by strain-specific susceptibility factors. The stage of
embryogenesis is also sometimes a determining factor.
In rats, the great majority of substances tested transplacentally,
with the exception of symmetrical dialkylnitrosamines, tend to
display a pronounced neurotropic carcinogenic effect (Alexandrov,
1976). This great susceptibility of the fetal rat nervous system
to transplacental tumour induction by different carcinogenic
substances is noteworthy. Kidney tumours also often appeared in
rat progeny as a result of transplacental exposure to a number of
compounds including 7,12-dimethylbenz(a)anthracene (DMBA), NDMA,
N-nitrosodiethylamine, NMU, azoxymethane, methylazoxymethanol,
1-methyl-2-benzylhydrazine, procarbazine, and 3,3-dimethyl-1-
phenyltriazene (Alexandrov, 1976). It should be noted that all
these carcinogenic substances, with the exception of DMBA, are also
able to induce kidney tumours in adult animals.
Results of a few studies on rabbits have indicated that the
nervous system and kidney of rabbits are sensitive to develop
tumours (Güthert et al., 1973; Stavrou & Lubbe, 1974; Beniasvili,
1978). Following transplacental exposure to NEU, monkeys suffered
from kidney, soft connective tissue, lung, and nervous system
tumours (Rice et al., 1977). Hamsters have been used for assessing
the transplacental effects of cigarette condensates and hormones
including diethylstilboestrol (Nicolov & Chernozemsky, 1979;
Rustia & Shubik, 1976).
4.4.1.4. Mechanisms of organotropism
The mechanism of organotropism displayed by the majority of
transplacental carcinogens is poorly understood. Some factors that
are believed to contribute to organotropism are:
(a) The predisposition of somatic cells: Little is known
about the causes of variation in predisposition and
susceptibility among various cell types (Kleihues
et al., 1979a);
(b) Metabolic conversion: The extent of metabolic
conversion of indirect carcinogens in the whole
organism or in the target cells may influence the
carcinogenic outcome (Alexandrov & Napalkov, 1981);
(c) Carcinogen-DNA interaction: Cellular DNA is a
principle target for carcinogenic chemicals. Studies
in transplacental carcinogenesis with ENU, NMU, DMBA,
and 3,3-dimethyl-1-phenyltriazene suggest that
neoplastic transformation may depend on the degree
and persistance of DNA damage in the target tissues
(Goth & Rajewsky, 1974; Cooper et al., 1978; Doerjer
et al., 1978; Kleihues et al., 1979a; Lihachev et
al., 1983). The state of differentiation of target
cells at the time of exposure may be important, since
the capacity for DNA repair has been shown to change
gradually as cells approach the final stages of
differentiation (Counis et al., 1977);
(d) Proliferative activity: A relationship has been
established between the stage-dependence of the
number of lung adenomas induced in fetal mice by
exposure to NEU and changes in the number of
epithelial cells within the population of cycling
cells (Kauffman, 1976). Cells with a high rate of
proliferation may be more vulnerable to the action of
carcinogenic stimuli as the induced damage is, thus,
quickly amplified.
All these factors are closely interrelated since the levels of
the enzymes, cell differentiation, and proliferative activity in
the target organs are genetically controlled. Altogether, these
factors determine both the general sensitivity of the growing
organism and the stage specificity of target organs to the insults
by carcinogenic agents (Alexandrov & Napalkov, 1981).
4.4.1.5. Metabolism of chemical carcinogens in the maternal
organism, the placenta, and the embryo
Tumour development in the offspring is generally determined by
the rate of placental transfer, and the concentration of the
carcinogen or its active metabolites in the embryonic tissues. It
is widely acknowledged that almost all non-ionic organic compounds
with a relative molecular mass of less than 600, can cross the
placenta (Ginsburg, 1971). There are almost no carcinogenic
substances with a relative molecular mass greater than 300 and many
are soluble in both lipids and water, which facilitates their
transfer across the placenta.
There is no absolute correlation between the accumulation of a
given carcinogen in the fetus and the neoplastic response. For
example, following administration to pregnant rats, NDMA, a
metabolism-dependent carcinogen in adult rats, reaches a high
concentration in the fetal tissues, where it remains for almost 24
h (Shendrikova & Alexandrov, 1978). Despite this, however, NDMA is
a weak transplacental carcinogen, which suggests that the compound
is poorly metabolized by embryonal tissues, probably because of the
immaturity of the relevant enzymes. Studies with other carcinogens
(polycyclic hydrocarbons and nitroso compounds) have also shown
that the concentration of parent chemicals in various embryonic
tissues did not correspond with the subsequent incidence of
neoplasia in these tissues (Baranova & Alexandrov, 1978).
From experimental data, it appears likely that a transplacental
carcinogen acts on the target fetal cells in the following ways
(Alexandrov & Napalkov, 1981):
(a) Directly, by reaching the fetus either in its original
form or as a spontaneously-formed proximal carcinogen. Examples
are nitrosamides, methylazoxymethanol, dimethyl- and
diethylsulfate, propane sultone, methyl-methanesulphonate. The
stability of the initial compounds or their proximal derivatives is
the limiting factor. Methylnitrosourethane (MNU) when given to
pregnant rats failed to induce carcinogenic effects in the progeny,
because of its rapid degradation in the mother. However, the
carcinogenic effect was readily induced following intra-amniotic
administration and direct injection into fetal rats (Alexandrov,
1972; Napalkov, 1973);
(b) Indirectly, by generating proximal carcinogens in target
fetal cells through metabolic activation. Examples are
dialkylnitrosamines, l,2-dialkylhydrazines, azo- and azoxy-alkanes,
and procarbazine. In this case, the metabolic capacity of the
target cells is a limiting factor. In particular, NDMA, when
administered to pregnant rats, induced no teratogenic effects and
only a weak transplacental carcinogenic effect. These findings
were ascribed to the incomplete metabolic development of the rat
fetuses (Alexandrov, 1968; Napalkov, 1973). When the active
metabolites of enzyme-dependent carcinogens are short-lived, the
enzymatic capability of the target fetal cells acquires primary
importance;
(c) Indirectly, following maternal metabolism. A stable
proximal carcinogen may be formed and pass through the placenta.
Examples are cyclophosphamide (Hales, 1982), and 3,3-dimethyl-1-
phenytriazine (Kleihues, 1979b), which are metabolized in the
maternal liver, and cycasin, which is metabolized by maternal
intestinal flora (Laqueur & Spatz, 1973);
(d) Indirectly, by the production of proximal and ultimate
carcinogenic metabolites in the placenta. This mode of action is
not well documented though polycyclic hydrocarbons are known to
undergo metabolic activation in the placenta (Welch et al., 1969;
Pelkonen et al., 1972; Sehgal & Hutton, 1977), and may belong to
this class.
Transplacental carcinogenesis can also occur as a result of the
formation of nitroso compounds from their precursors in the stomach
of pregnant females (Table 11). The carcinogenic action on the
fetus occurs via (c) and (a), in the case of ethylurea and nitrite
administration, and according to types (c) and (b), in the case of
amidopyrine and nitrite administration.
4.4.2. Relationship between teratogenesis and carcinogenesis
There are a number of compounds that have both carcinogenic and
teratogenic properties (Neubert, 1980b). This is not surprising
when considering electrophilic substances such as alkylating
agents, which react with many cell components. However,
teratogenic activity has not been observed for every carcinogen,
nor would this be expected. Though the possibility of common steps
in teratogenesis and carcinogenesis exists, there are probably
specific mechanisms pertinent to only one or other of these
phenomena. In studies in which rats were tested with a combination
of NMU and NEU, it was established that the development of
malformations of the hypoplastic type (microcephaly) did not
prevent the appearance of brain tumours, and that the phenomena of
teratogenesis and carcinogenesis could coexist, but were apparently
independent (Alexandrov & Napalkov, 1976).
In most cases, since malformations and tumours are induced in
different organs, there is no reason to assume that the two
processes are related. In clinical/epidemiological observations
(Miller, 1977), diethylstilboestrol is the only compound shown to
have caused malformations (adenosis), and malignant tumours (clear
cell adenocarcinomas) in the same organs, the cervix and vagina.
The sensitive period for the induction of these carcinogenic and
teratogenic effects was the same (Herbst et al., 1979b). The
immature opossum is the only animal model developed to date that
has provided clear-cut indications that malformations can be
produced together with embryonal tumours in the same organs
(Jurgelski et al., 1976).
4.4.3. General principles of transplacental carcinogenicity tests
A compound found to be carcinogenic in adult carcinogenicity
studies does not usually need testing for transplacental
carcinogenicity. However, it may be worth investigating the
relative adult versus fetal sensitivity for chemicals to which many
pregnant women have been, or are, unavoidably exposed. As a
primary task, compounds that are suspected of being human
carcinogens (IARC, 1982), should be tested for transplacental
carcinogenicity.
The general guidelines for conducting transplacental
carcinogenicity studies do not differ considerably from those
adopted for adult animal studies (IARC, 1980). Some substances may
prove inactive in conventional test systems. This could be because
the period of development during which the rodent embryo is
sensitive to carcinogenic stimuli is rather short (during last 10-
12 days of pregnancy) and these compounds may require prolonged
application to induce tumours. Therefore, care should be taken in
extrapolating negative results from animal studies to man, and
appropriate models with non-human primates or other suitable
mammalian species should be developed. Variables that should be
taken into account include: species and strain, stage of pregnancy,
dose and route of administration, period of observation,
registration of biological variables (e.g., body weight) and
statistical evaluation of the results. The following experimental
conditions are peculiar to transplacental studies.
4.4.3.1. Species
It is advisable to use at least two laboratory species,
preferably mice and rats. The sensitivity of fetal rats to direct
and indirect carcinogens varies and is manifested with a rather
peculiar organ specificity that is not typical of adult animals.
On the other hand, the sensitivity of fetal mice to the
transplacental action of carcinogens does not differ significantly
from the sensitivity of adult mice, and neoplasms usually appear at
sites typical for spontaneous tumours.
In some studies, other species, including hamsters, have been
used. For economic reasons, non-human primates should be reserved
for studies on the mechanisms of prenatal carcinogenesis related to
human beings.
4.4.3.2. Stages of pregnancy
The period of optimum susceptibility during pregnancy of a test
species should be chosen as the duration of treatment with the test
agent. The following two experimental designs could be recommended
for mice, hamsters, and rats: (a) single exposure, 1-5 days before
parturition; and (b) multiple exposures, daily from day 11 to the
end of pregnancy (Alexandrov & Napalkov, 1981). In monkeys
(Erythrocebus patas),the period between days 30-50 of pregnancy
may be selected, since this period has been shown to be quite
effective in transplacental studies (Rice et al., 1977).
4.4.3.3. Dose and route of administration
To select an appropriate route, toxicokinetic peculiarities
should be given particular consideration. For example, substances
that are slowly absorbed from the site of administration should be
tested by means of a course of treatment. However, for practical
purposes, it is advisable to use two different routes, namely
intravenous and oral.
Doses should be selected from pilot tests in which mortality,
body weight gain, and other pathological alterations in mothers and
offspring, have been evaluated. Administration of the maximum
tolerable dose may provide early and pronounced carcinogenic
effects, while a series of lower doses will help to reveal a "dose-
effect" relationship, which is important for extrapolating and
evaluating the carcinogenic risk for human beings.
The number of pregnant females in each group should be large
enough to provide for not less than 50 offspring per sex. The
offspring of treated and control mothers should be cross-fostered
to ascertain that the carcinogenic effect of a chemical is
transplacental, unless postnatal transfer from mother to offspring
can be excluded.
4.4.3.4. Evaluation of results
The progeny should be followed up for the entire life-span of
the species under consideration, neoplastic changes being analysed
by histological examination of the affected organs and tissues, at
the end of the study. A reliable source for classifying the
tumours has been provided by IARC (Turusov, 1973, 1976, 1979,
1982). Attention should be paid to other toxic manifestations
including dystrophy, necrosis, atrophy, malformations, dishormonal
status, etc. Methods for the statistical evaluation of results
have been described (IARC, 1980).
4.4.4. Methods with potential for the future
4.4.4.1. Transplacental host-mediated cell culture
This system offers a means for the rapid screening of
compounds, including those requiring metabolic activation, for
possible transplacental carcinogenicity. In cells subcultured from
embryonal tissues, following administration of chemicals to animals
during pregnancy, signs of morphological transformation may be
detected several weeks after in vitro growth (Quarles, 1981). In
another model, the activity of a chemical may be determined
according to the marked induction of mutants resistant to 8-
azaguanine in a culture of embryo cells derived from pregnant
animals treated with the test compound (Endo et al., 1980).
4.4.4.2. Pre- and postnatal exposure to carcinogens and promoting factors
A few studies have shown an increased carcinogenic effect when
an initial prenatal exposure to a carcinogen was followed by
postnatal exposure to either a carcinogen (Lichachev, 1971) or a
promoting agent (Goerttler & Loehrke, 1976), or by hormonal
imbalance (Alexandrov & Anisimov, 1976). This approach could be
useful for determining the carcinogenic potential of chemicals with
marginal effects in adult animals (Napalkov, 1973).
4.4.4.3. Intraamniotic and intrafetal injection technique
This procedure distinguishes between the direct effects of a
test compound on the fetus and the effects induced following
maternal treatment. The high susceptibility of rodent fetal cells
to some of the carcinogens administered directly into the fetus
suggests that intraamniotic or intrafetal injections might find
useful applications in detecting potentially hazardous chemicals
(Alexandrov & Napalkov, 1981; Rossi et al., 1983).
5. IN VITRO DEVELOPMENTAL AND NON-MAMMALIAN ANIMAL
SYSTEMS: CURRENT AND FUTURE APPLICATIONS
Various in vitro developmental systems (cell, organ, and whole
embryo culture) have been used to investigate the morphological and
biochemical basis for normal and abnormal development. Such
systems are extremely useful for this sort of basic and applied
research. The current demands on whole animal testing systems may
prevent testing of all the rapidly increasing number of chemical
agents being introduced into the environment.
Short-term tests using various in vitro and in ovo systems
might offer a future means of selecting chemical agents for
subsequent teratology testing in animals. Short-term in vitro
methods and their applicability have been reviewed by Rajan (1974),
Kochhar (1975a), Saxén & Saxén (1975), Ebert & Marois, ed. (1976),
Wilson (1978a,b), Barrach & Neubert (1980), Neubert (1980a),
Neubert & Merker, ed. (1981), Kimmel et al. (1982), Neubert (1982),
Neubert (1983), and Shepard et al. (1983).
5.1. Scope of In Vitro Developmental Systems
These studies are carried out using a variety of biological
systems (Table 13), capable of reflecting normal developmental
processes.
It is evident that, within the last decade, several culture
techniques, especially those using mammalian tissues, have been
successfully applied to the elucidation of basic mechanisms
occurring at the biochemical and cellular levels during embryonic
development, and to a closer analysis of the modes of teratogenic
action. There is no doubt that in vitro methods have greatly
broadened the possibilities in this field of research. In vitro
systems can be considered from three points of view (Neubert, 1981;
Kimmel et al., 1982; Neubert, 1982): (a) as foods for the
elucidation of mechanisms relevant to normal and abnormal
development; (b) as tools for obtaining information on dose
responses and specific organ toxicity; (c) as foods for the
selection of chemicals for possible future developmental toxicity
testing. In vitrodevelopmental systems are attractive for
toxicological studies as many are relatively inexpensive, and they
are easier to perform and less time-consuming than whole-animal
studies. However, a number of problems need to be solved before
using Short-Term Selection Tests (STST) routinely. In vitro
developmental systems, applicable to studies or problems concerning
basic developmental biology, must be able to mimic certain
processes characteristic of prenatal or perinatal development.
5.2. Essential and Desirable Features of Short-Term Selection
Tests
Essential and desirable features of STST, summarized in Table
12, have been proposed by several investigators (Wilson, 1977;
Kimmel et al., 1982; Neubert, 1982; Shepard et al., 1983).
The injudicious use of in vitro test data could be even more
dangerous than extrapolation from routine in vivo tests. Excessive
false positives could lead to an undesirable situation in which in
vivo test systems in use, would be overburdened. Some false
negatives are bound to occur, but they must be small in number,
otherwise there is little purpose in the STST.
A simpler system is preferable to a whole organism, only if the
underlying toxic mechanism is largely understood, occurs
universally, and can be completely mimicked. In the short-term
tests used in mutagenicity studies, these prerequisites seem to
have been fulfilled to some extent but, even here, no single test
system has been found to be fully adequate. The situation in
prenatal toxicity is more complex than in mutagenicity, since
numerous mechanisms are known to lead to abnormal development.
Thus, no one test system is likely to identify all the abnormal
reactions that may occur.
The culture technique, if chosen, will largely depend on the
problem to be solved. If, for example, cell interactions and their
disturbance by certain agents are to be studied, cell cultures or
techniques using cell suspensions may be the method of choice.
Specific embryonic differentiation processes (palate closure,
limb development, kidney development) may be best studied with
organ culture techniques, whereas processes of cell migration over
long distances within the embryo may be best studied in whole
embryo cultures. In many instances, the results obtained are only
valid within the limits of the model and for the species used.
Table 12. Features of a short-term selection test (STST)
---------------------------------------------------------------
Essential Results must be predictive of in vivo effects
Few false positives
Very few false negatives
Inexpensive and readily-available biological unit
Easily quantificated endpoint
Reproducibility of results between laboratories
Desirable Incorporating as many developmental processes as
possible by using a series or battery of tests
Ability to test water-soluble and
water-insoluble chemicals and gaseous agents
Ability to determine dose-response relationships
Ability to incorporate biotransforming systems,
especially hepatic monooxygenase systems, from
different species including man
---------------------------------------------------------------
In short-term tests, it is difficult or impossible to establish
the relationship between embryo/fetal toxicity and maternal
toxicity, which is essential for determining whether the effects
are selective or not. In addition, many compounds are
metabolically activated to electrophilic substances that react with
cell constituents and thereby cause toxicity. This activation may
occur in the embryo/fetus or the mother, or in both. The role of
enzyme induction in the mother and the conceptus is another
important issue. The addition of metabolizing enzyme systems from
different species may make it possible for investigators to obtain
information on inter-species differences (Fantel et al., 1979;
Neubert, 1982; Shepard et al., 1983). This method might be
especially valuable in extrapolating from experimental animals to
human beings, even though this approach will not fully reproduce
metabolism in vivo.
The influence of the mother on the toxic effects in the fetus
is difficult to reproduce in an in vitro system. This is also
true of chick embryos in ovo, which are used for the assessment of
the embryotoxic potential of chemicals. The maternal factor is
highly relevant if little is known of the toxicokinetics of a given
chemical - as is the case with many chemicals in the environment.
5.3. Validation of Short-Term Selection Tests
To be accepted as part of a routine testing process, STSTs will
have to be validated for their ability to predict the teratogenic
potential of an agent. For the purpose of validation, necessary
criteria will have to be developed, the number and type of
chemicals (representative of diverse structures and biological
activities) required for testing will have to be decided, and an
acceptable percentage of false positives and false negatives will
need to be agreed upon. To carry out validation in a controlled
fashion and to facilitate interlaboratory comparisons, a list of
chemicals to be used should be developed jointly by scientists
experienced in teratology, toxicokinetics, and biochemistry. An
attempt to choose agents that are teratogenic or non-teratogenic
has been undertaken by Smith et al. (in press).
Final validation of tests to be used must be based on the
results of the in vitro tests of known human teratogens. Up to
the present, no STSTs have been fully validated and no
recommendations are made in this document as to which batteries of
STSTs are likely to emerge for possible use in the future. The
STSTs are not in any way intended to replace animal tests, but
merely to supplement them so that they can be made more efficient
and productive.
5.4. Available Developmental Systems
Some developmental systems that are in use at present for basic
mechanism studies, or proposed as STSTs (Table 13) are discussed in
section 5.4.1. None of these developmental systems involves the
chorioallantoic placenta or an intact maternal metabolic system.
In some cases, a mono-oxygenase or other type of bioactivating
system may be added. A summary of many of these developmental
systems has been published by Neubert (1982).
5.4.1. Whole-embryo culture (warm-blooded animals)
There are three types of cultures that have been used
extensively in various laboratories:
(a) culture of mammalian preimplantation stages;
(b) culture of mammalian postimplantation embryos; and
(c) culture of non-mammalian (avian) embryos.
Though not a culture method, the evaluation of the chick embryo
in ovo will also be discussed.
5.4.1.1. Ovum maturation and preimplantation stages
In vitromaturation of the ovum of different mammalian species
(especially rodents) has been intensively studied (Edwards, 1980;
Edwards & Purdy, 1982). With this system it is possible to
investigate the mechanism of action of various chemicals on
chromosomal disjunction, ovum maturation, and activation. It is
possible to use in vitro fertilization of human ova not only as a
method of treating human infertility, but also to obtain a better
understanding of the basic mechanism of human oogenesis (Edwards,
1980; Kurilo et al., 1982, 1983).
Culture of cleaving mammalian ova is being used to study the
mechanism of embryotoxic action of some chemicals, especially those
that are expected to influence proliferation and induce chromosomal
aberrations (Dyban et al., 1976, 1977a,b; Spielmann et al., 1981;
Matsumoto & Spindle, 1982).
Hsu (1979, 1980, 1981) published results of a system in which
it is possible to study mammalian development in vitro from the
preimplantation phase to the late embryonic stages. However, the
success rate of this model has not yet exceeded 10%. In general,
preimplantation embryos cannot be cultured beyond stages
corresponding to implantation or early postimplantation.
Table 13. Developmental systems
-----------------------------------------------------------------------
Period of Developmental
System development process studied
covered and endpoint
-----------------------------------------------------------------------
I. Whole-embryo culture
Mammalian preimplantation fertilization,
embryos cleavage, and
(including human) blastocyst formation
Rodent embryos post-implantation organogenesis
organogenesis over 2 (or 3) days
Table 13 (contd).
-----------------------------------------------------------------------
Period of Developmental
System development process studied
covered and endpoint
-----------------------------------------------------------------------
Chick embryo culture organogenesis organogenesis
Chick embryo in ovo entire whole development
Amphibian embryos entire whole development
Zebra fish entire whole development
II. Organ cultures
Limb buds (avian, late organogenesis morphogenesis
rodents) cartilage formation
muscle formation
Palatal shelves organogenesis epithelial fusion and
cell death, palate
closure
Tooth bud organogenesis dental development,
histogenesis of tooth
buds
Lens organogenesis lens differentiation,
histogenesis, and
protein production
Pancreas organogenesis acinar development,
histogenesis,
biochemical
development
Sex organs late organogenesis gonadal and organ
development, germ cell
maturation, accessory
sex gland,
histogenesis
Kidney late organogenesis nephrogenesis,
histogenesis
Thyroid late organogenesis thyroid,
histogenesis, and
functional development
-----------------------------------------------------------------------
Table 13 (contd).
-----------------------------------------------------------------------
Period of Developmental
System development process studied
covered and endpoint
-----------------------------------------------------------------------
III. Tissue culture
Chick organogenesis migration and pigmen-
neural crest tation, melanin for-
mation or acethyl-
choline transferase
activity
Chick limb bud organogenesis growth and
mesenchyme chondrogenesis
Lung organogenesis pattern formation,
micromass culture cell recognition, and
adhesion
Embryonic heart organogenesis histogenesis and
functions
Skeletal muscle organogenesis myogenesis, cell
fusion,
and histogenesis
Teratocarcinoma organogenesis histogenesis
Mouse tumour cells none cell attachment
Human embryonic organogenesis growth of patatal
cell culture mesenchymal cells
IV. Invertebrates
Hydra attenuae adult and embryonic aggregation and
movement regeneration
of damaged and
disassociated adults
Drosophila entire myogenesis, nerve
melanogaster attachment
Planaria regeneration of adult neurogenesis, eye
development
Sea urchins cleavage cell replications
and contacts
Dictyostelium entire aggregation,
differentiation
-----------------------------------------------------------------------
Table 13 (contd).
-----------------------------------------------------------------------
Period of Developmental
System development process studied
covered and endpoint
-----------------------------------------------------------------------
V. Other approaches
Amphibian limb differentiation myogenesis,
adult regeneration neurogenesis,
and chondrogenesis
Virus unknown (viral replication)
Lipophilic or unknown (cell membrane
electrophilic penetration,
chemical property toxic potential)
-----------------------------------------------------------------------
It is now possible to culture mammalian embryos from the one-
or two-cell stage to the blastocyst stage. Most of the work has
been performed with the preimplantation stages of mice (McLaren &
Biggers, 1958; Brinster, 1963; Runner, 1965; Biggers et al., 1971;
Whitten, 1971; Whittingham, 1971; Eibs & Spielmann, 1977; Speilmann
& Eibs, 1977), but cultivation of preimplanted rat, rabbit, and
human embryos has also been carried out successfully (Brachet,
1913; Chang, 1949; Daniel, 1965; Daniel, Jr, 1968; Mauer et al.,
1968; Onuma et al., 1968; Edwards et al., 1969; Brinster, 1970;
Steptoe & Edwards, 1978; Edwards & Purdy, 1982). A chemically-
defined, serum-free, culture medium is generally used for this
purpose.
Biophysical and biochemical techniques have been used in
addition to morphological and biochemical methods to analyse the
normal and abnormal development of preimplantation stages. The
measuring of membrane potentials has been used as an indication of
functional stage in the eggs of sea urchins and mammals (Crose et
al., 1973; Leonov et al., 1972, 1975; Persianinov et al., 1973).
Most investigators agree that the preimplantation period is not
sensitive to the induction of gross structural abnormalities
(Austin, 1973). Preimplantation embryos, therefore, have not been
used much in teratology. However, abnormal development associated
with early or delayed mortality can be induced before implantation
(cf. Snow, 1973, 1975; Goldstein et al., 1975; Spielmann et al.,
1981; Pedersen, 1981).
Many drugs may reach the preimplantation embryo via the tubal
or uterine fluids, and agents, such as nicotine, may even
accumulate within the blastocyst (Fabro, 1973; Sieber & Fabro,
1974). Lutwak-Mann (1973), has suggested the use of "flat mounts"
of rabbit blastocysts to detect abnormalities induced by
embryotoxic agents, but this technique has not gained acceptance.
5.4.1.2. Postimplantation mammalian embryos
Developed by New and co-workers (New, 1966a,b, 1976, 1978; New
et al., 1976a,b), the culture of whole rodent embryos in vitro is
now an established procedure in many laboratories. Mouse or rat
embryos are cultured with their embryonic membranes intact by the
rotating bottle technique (New et al., 1973; Cockroft & Coppola,
1977), which is generally preferred to more sophisticated
experimental procedures. Rat embryos (8.5 - 10.5-day-old), can be
cultured over a 24-h or even a 48-h period and embryonic stages
corresponding to about 35-somites may be reached. Up to the 30-
somite stage, the development proceeds at roughly the same rate in
vitro as in vivo. Abnormal development may occur in culture, if
conditions are inadequate.
Some studies have been conducted on mice (Clarkson et al.,
1969; Sadler, 1979a,b; Davis et al., 1981) and on hamster embryos
(Givelber & Dipaolo, 1968). Rat embryos have been traditionally
cultured in a medium containing rat serum, but some investigators
have successfully grown them in human serum (Shepard et al., 1969;
Chatot et al, 1980; Klein et al., 1980, 1981; Herken & Anschütz,
1981), dialysed serum (Clarkson et al., 1969; Gunberg, 1976), and
in a chemically-defined serum-free medium (Klee-Trieschmann &
Neubert, 1981). In several studies, the direct embryotoxic
potential of teratogens for the developing embryo was elucidated
following their addition to the culture medium (for bibliography
see Neubert, 1982). Popov et al. (1981) and Fantel (1982) used a
liver microsomal system added to the culture to activate
cyclophosphamide. Dyban as well as Klein and his co-workers
studied the effects on embryos in a culture serum containing a
variety of compounds (e.g., cyclophosphamide), which had been
obtained from animals treated in vivo or from patients (Dyban,
1977; Dyban et al., 1979; Klein et al., 1980, 1981). Only gross
morphological evaluations were performed in most of the studies. A
morphological scoring system (Brown et al., 1980) may be useful for
evaluation, but, for more detailed analysis, histological
examination is essential (Herken & Anschütz, 1981). A technique of
culturing whole embryos in which it is possible to the record the
embryonic heart beat has been developed (Robkin et al., 1972). An
attempt has also been made to use a sequence of whole-embryo
cultures with subsequent organ culturing of, for example, limb buds
(Kochhar, 1976). The applicability of the whole-embryo technique
as a routine method is limited, because it is expensive, too
sensitive to disturbances, often of unknown origin, and quite prone
to abnormal development, even under normal conditions. Furthermore,
a maximum culture period of 48 h is often too short compared with
the entire period of organogenesis. The method is especially
useful for studying certain aspects of developmental biology, such
as the nutrient requirements of embryos during organogenesis, or
for performing biochemical analyses or studies with radioactively-
labelled precursors (Shepard et al., 1970).
5.4.1.3. Chick embryo in culture
Explantation of the early chick embryo is technically more
difficult than culturing embryonic mammals at the preimplantation
stages. The problems involved, however, have mostly been solved in
the technique of New (1955; 1966a,b). It consists of supporting the
explanted blastoderm on a piece of vitelline membrane stretched
across a glass ring. The ring is placed on a watch glass serving
as a reservoir for nutrient medium - thin albumen, obtained perhaps
from the same donor egg. The watch glass is kept in a covered
Petri dish, which forms a moist chamber. Primitive streak
blastoderms explanted by this technique develop at a normal rate
for approximately 48 h, until after the 20-somite stage. Agents
can be tested by direct addition to the medium, and sera from
individuals exposed to the agent can be added to the culture.
Embryos examined for toxic effects are examined for changes in size
and gross morphology, histology, and biochemical variables such as
DNA and protein content, glucose consumption and lactate
production, cellular damage, etc. (New, 1976). This method is
inexpensive and can be used routinely with standard laboratory
equipment. Large numbers of embryos can be processed in a short
time.
Since the embryos can only be maintained for a relatively short
period of time, the usefulness of this technique is limited to the
early developmental stages. Effects caused by substances acting
mainly or entirely at later developmental stages cannot be detected
by this procedure.
5.4.1.4. Chick embryo in ova
Chemicals have frequently been tested in chick embryos, at all
stages of development, by administration into fertile eggs. A
chemical, in solution or suspension, is usually deposited on the
air-sac membrane or injected into the yolk and the chicks examined
before or after hatching. This method has often failed to supply
reliable results as it has been difficult to standardize the blind
injection technique. Moreover, it is doubtful whether the form and
concentration of a chemical, when it reaches the embryo, is known.
A standard window technique for delivering the chemical to the
chick embryo has been developed for embryotoxicity and
teratogenicity studies in which better control of experimental
conditions is possible (Jelinek et al., 1976; Jelinek, 1979;
Jelinek & Peterka, 1981). The test substance is administered in a
single application to the subgerminal portion of the yolk of the
embryo on day 2, and intra-amniotically on days 3 or 4. The number
of dead, malformed, and growth-retarded embryos is counted on the
eighth day of incubation, when the study is terminated. The method
is inexpensive and requires a moderate degree of skill. The effect
of a large number of variables can be assessed and the amount of
the test chemical needed is small. With this test it is possible
to determine dose-response and stage-response relationships and the
affinity of a substance for particular organ systems. The capacity
of the system for the metabolic activation of xenobiotics is ill-
defined. Testing of compounds that are insoluble or soluble in
toxic solvents is difficult and false-positives are common with
surface-active compounds. Though many compounds have so far been
tested in chick ova, the predictive value of these tests has not
yet been established.
5.4.2. Organ cultures
The term organ culture should be used when a whole-organ
anlagen, or its representative part (e.g., a lobe of a lung or
liver), is kept in culture and growth, particularly organ-specific
differentiation, is observed. Two approaches have been used:
(a) the explant, is kept on a filter or other support, and
differentiates at the interface of the culture medium and
the gas phase (Trowell, 1961); and
(b) the explant is submerged in the culture medium, and
shaking or rotation is used to guarantee gas exchange and
diffusion.
Both of these techniques show a number of advantages and
certain disadvantages depending on the embryonic organ or organ
anlage used. The successful use of organ culture systems in
developmental biology and toxicology has recently been reviewed
(Neubert, 1982). Serum-containing medium has been employed in the
majority of cases, but successful attempts have been made to
culture certain tissues in chemically-defined media.
The technique described by Trowell (1961) has been used in the
culture of a variety of tissues and organs from mammalian or avian
embryos or fetuses. It is comparatively easy to perform and 100-
200 explants can be prepared by one person in one day. A
modification of the method, the trans-filter technique, is used for
induction studies (Grobstein, 1956; Saxén et al., 1968; Wartiovaara
et al., 1974; Saxén & Lehtonen, 1978).
5.4.2.1. Organ culture of limb buds
The Trowell technique is used for the culture of limb buds from
rat or mouse embryos (Shepard & Bass, 1970; Kochhar, 1970;
Aydelotte & Kochhar, 1972) but it may also be used for limb buds
from embryos of other species, e.g., rabbit, chick, or ferret
(Lessmöllmann et al., 1975; Beck & Gulamhusein, 1980a). Growth of
limb buds in culture, is retarded compared with in vivo
development, but morphological differentiation of the cartilaginous
bone anlagen and of muscle can be observed. With any of these
techniques, the extent of the morphogenetic differentiation of the
cartilaginous bone anlagen depends largely on the stage of
development at which the cultures are initiated. In earlier
studies, serum-containing medium was used, but more recently a
chemically-defined medium has been preferred (Lessmöllmann et al.,
1975, 1976).
The submerged culture system has also been successfully used
for the study of limb bud differentiation in vitro (Neubert &
Barrach, 1977b; Blankenburg et al., 1981), and it shows many
advantages over the Trowell technique. Dissected limb buds can be
stored at low temperatures for several weeks (Neubert & Bluth,
1981a) and some studies may be facilitated by the use of such an
organ bank. This organ culture technique is one of the best and
most extensively studied. Normal development and differentiation,
predominantly of the cartilaginous bone anlagen, have been studied
and a variety of morphological (e.g., Merker, 1975; Merker et al.,
1978b; Neubert et al., 1974a, 1976) and biochemical (e.g.,
Rautenberg & Neubert, 1975; Neubert & Rautenberg, 1976; Barrach et
al., 1975, 1980, 1981; Blankenburg et al., 1980, 1981, Dusemund &
Barrach, 1981) variables have been evaluated. Although the amount
of protein increases within the explants, there is little or no
increase in the total DNA (Neubert et al., 1976).
It has been possible to induce abnormal development in vitro by
adding a variety of teratogens to the culture medium (Kochhar,
1970, 1975b; Aydelotte & Kochhar, 1972; Neubert et al., 1974b,
1976, 1977a; Barrach et al., 1978; Merker et al., 1978a; Welsch et
al., 1978; Merker & Günther, 1979; Beck & Gulamhusein, 1980b;
Bochert et al., 1981; Neubert & Bluth, 1981b; Stahlmann et al.,
1981, 1983).
It has also been possible to quantify the extent of
differentiation achieved in culture and to establish dose-response
relationships for abnormal development using the organ culture
system. Biochemical variables, such as levels of DNA, RNA,
protein, collagen, etc. can be measured (Neubert et al., 1974a); or
a score system may be used for morphogenetic differentiation
(Neubert et al., 1977a; 1978b). Image analysis (Kwasigroch et al.,
1981) can also be used to quantify abnormal development.
5.4.2.2. Organ culture of the pancreas
Organ culture has been used by Rutter and his colleagues
(Pictet & Rutter, 1972; Rutter et al., 1975) to study the
development of the embryonic rat pancreas. The information gained
using this system has provided the most extensive biochemical
background for any prenatally developing mammalian tissue
investigated so far. However, the pancreas is not considered a
major target for embryo- or fetotoxic agents.
5.4.2.3. Organ culture of palatal shelves
Growth and differentiation of the palatal shelves has also been
achieved in organ culture. This system has been extensively used
to study cleft palate-inducing agents (Lahti & Saxén, 1967, 1972;
Saxén, 1973; Fairbanks & Kollar, 1974; Pratt et al., 1980;
Brinkley, 1980; Pratt, 1983).
5.4.2.4. Organ culture of tooth anlagen
Normal and abnormal development of tooth anlagen has been
studied, both morphologically and biochemically, using the organ
culture technique. Important information on connective tissue
development and on the induction of abnormalities has been obtained
(Ruch & Karcher-Djuricic, 1971; Kollar, 1973; Galbraith & Kollar,
1974; Koch, 1974; Hetem et al., 1975; Kerley & Kollar, 1978;
Thesleff & Pratt, 1980).
5.4.2.5. Organ culture of the embryonic lens
Lens development can be followed using human embryonic
material. The induction of abnormal development by rubella virus
can be mimicked in vitro. Cataract formation was observed by
Karkinen-Jääskeläinen et al. (1975).
5.4.2.6. Organ culture of the embryonic kidney
Organ culture techniques have been developed for the study of
explanted mouse or human embryonic kidney (Saxén, 1965; Saxén et
al., 1968; Crocker & Vernier, 1970; Crocker, 1973; Saxén &
Lehtonen, 1978; Saxén & Ekblom, 1981). This system may prove
useful for investigating the normal mechanisms of renal development
and also testing suspected nephrotoxic agents. The expense and
time involved are both fairly high, since the endpoints require
histological analysis.
5.4.2.7. Organ culture of embryonic gonads and accessory duct systems
Organ culture of embryonic gonads together with assessory ducts
has contributed to the understanding of how hormones and some
hormone inhibitors modify sexual development (Martinovitch, 1938;
Jost & Bergerard, 1949; Price & Ortiz, 1965; Josso, 1974; Lasnitzki
& Mizuno, 1981). Both mammalian and avian embryos have been used.
These techniques could be used to investigate new chemicals that
might have specific effects on the genital system. The method is
moderately expensive and time-consuming.
5.4.2.8. Organ culture of thyroid tissue
Tissue from the thyroid glands or their embryonic primordia
from chick, rodents, and human beings may be explanted into organ
cultures, which may then be exposed to hormones and various
xenobiotics. The effects can be studied biochemically or
histochemically (Shepard, 1965, 1967, 1974). Appearance of colloid
within thyroid follicles, histoautoradiography of incorporated
radioiodine, or biochemical analysis of various labelled organic
compounds are used for evaluation. The expense and time required
are moderate. This test system would be of use in investigating
very specific questions about the effects of new chemicals on gland
maturation and function.
5.4.2.9. Other organ culture systems
The use of organ culture is not restricted to the examples
provided in the preceeding paragraphs and other organ anlagen have
been cultured. Examples include the use of bone from rat or mouse
fetuses (to demonstrate the pathological developments induced by
substances such as glucocorticoids or tetracycline) (Fell & Weiss,
1965; Saxén, 1966; Saxén & Kaitila, 1972), digits (Rajan, 1969,
1974; Rajan et al., 1980), lung anlagen (Riso & Zimmermann, 1981),
intestine (Silano et al., 1981), and liver (Räihä & Schwartz,
1973).
5.4.3. Culture of non-mammalian or non-avian embryos
Embryos of amphibia, lurchae, or fish have been extensively
used in research (New, 1966b), but only a few of these systems have
been suggested as models for the evaluation of embryotoxic risks.
Some of these animal models are listed below, though these species
are much less favourable for the routine testing of potential
embryotoxic agents, as their response to such agents has not been
properly investigated, so far.
5.4.3.1. Studies on the development of fish
Several species of fish have been studied embryologically and
at least two species, Oryzias and zebra fish, have been used for
studies on abnormal development (Smithberg, 1962; Schreiweis &
Murray, 1976; Leung & Bulkley, 1979). Streisinger (1975) proposed
using zebra fish for the study of teratogens. By fertilizing eggs
with genetically-inactive sperm and blocking their first mitotic
division, homozygous diploid embryos can be obtained, some of which
develop into adult forms. Clones of the fish can be established,
which may be useful in dose-response analysis. The raising and
study of the wild form of zebra fish might prove to be inexpensive
and not too time-consuming. So far, not much information on
teratogenicity has been obtained from these studies, but they may
be valuable for the evaluation of the effects of environmental
chemicals on wild life.
5.4.3.2. Studies on the development of amphibia, lurchae,
sea-urchins, and other invertebrates
None of these systems has been adequately analysed for
responsiveness to toxic substances. The species observed in
developmental studies include: amphibia (Schultz et al., 1982),
Drosophila(Demerec, 1950), sea urchins (Lallier, 1965) and worms
(Deppe et al., 1978). Sea urchin eggs have been the subject of
reproduction studies, because synchronically developing embryos can
be obtained in large numbers (Harvey, 1956; Costello et al., 1957;
Mateyko, 1967) and experimental conditions (fertilization and
incubation) are simple (Tyler & Tyler, 1966; Hinegarduer, 1967;
Reverby, 1971; Hörstadium, 1973). It is possible to study
embryotoxic and teratogenic effects (Hagström & Lönning, 1973;
Leonov, 1979) and many chemical substances can easily penetrate the
external coats of eggs and embryos.
Hydra attenuaehas been recommended as a model for
developmental toxicity testing but, in this case, it is not the
development that is studied. Hydra attenuata are grown in trays
and are fed Artemia nauplii(Johnson, 1980; Johnson et al., 1982).
Two endpoints can be measured after addition of a chemical agent to
the liquid medium. One endpoint, representing the adult toxic
dose, is an irreversible morphological alteration called the
"tulip" stage. With the second, artificial "embryos" are formed by
compacting dissociated adult cells (Gierer et al., 1972) and
dissolution of the aggregates is the endpoint of the test. The
expense is small and little time is required. If the system can be
validated, it might prove useful in evaluating large numbers of
substances.
5.4.4. Tissue culture systems
The use of tissue culture systems in developmental toxicology
is limited, but primary cultures from various embryonic tissues
have been extensively used in developmental studies including the
chick neural crest (Greenberg, 1982), heart mesenchymal cells, and
skeletal muscle cells (Holtzer et al., 1958). Cell lines can be
used in developmental toxicology. They may be employed in testing
for the general cytotoxicity of chemical. However, it must be
remembered that some cells may change their characteristics in
culture. Pratt et al. (1980) have used an established cell line
from the human embryonic palatal mesenchyme (HEPM) to aid in
understanding the biochemical basis for the way in which
glucocorticoids inhibit palatal mesenchymal cell growth and cause
cleft palate in the rodent. Furthermore, Pratt et al. (1982) have
suggested that teratogen-induced growth inhibition in these HEPM
cells might be used as part of a battery of assays to screen for
potential environmental teratogens.
It seems possible that, in some instances, primary cell
cultures of embryonic or fetal cells - including those from human
embryos or fetuses - may be used for studies on the drug-
metabolizing capacity of cells during prenatal, and especially
perinatal, development (Nau et al., 1977; Egger et al., 1978;
Liddiard et al., 1978; Merker et al., 1978a; Kremers et al., 1981).
Comparative studies of this kind can be helpful when potential
differences between the capacity of embryonic or fetal cells and
adult cells of various species for metabolizing xenobiotics are
sought.
5.4.5. "Micromass" culture of dispersed cells from
the embryonic limb and lung
On the basis of work performed with cells of chick embryos
(Caplan, 1970; Schacter, 1970; Levitt et al., 1975), a method was
developed for the study of cartilage formation from limb blastema
cells grown at high cell density (Umansky, 1966; Ahrens et al.,
1977; Pennypacker et al., 1978b; Solursh et al., 1978). This
technique is referred to as micromass, spot, or high density cell
culture. Dispersed cells from a number of organ anlagen can be
used for this purpose.
Blastema cells from the limbs of 11-day-old mouse embryos are
disintegrated with trypsin and the dispersed cells cultured in a
drop of culture medium in a Petri dish. At the time of initiation
of the culture, there is no or little indication of the formation
of cartilaginous structures in the anlage. After two days in
culture, nodules of cartilage form in vitro. This system has been
used extensively in several laboratories, and the effect of a
variety of chemical agents on cartilage formation has been
investigated (Hassel et al., 1978; Pennypacker et al., 1978a,b;
Lewis et al., 1978; Flint, 1980; Merker et al., 1980a,b). The
culture system has a major disadvantage; it is not possible to
study typical morphogenetic differentiations, only differentiation
at the cellular level. However, the advantage of this system is
that the technique is easy and results are obtained after a
comparatively short period.
Merker and his group established a system in which it is
possible to observe the morphogenetic differentiation processes in
lung cells (Merker et al., 1981). The tissue of a lung anlage from
a 14-day-old mouse embryo is disintegrated with trypsin and
collagenase and the cells cultured at high density in a drop of
culture medium in a Petri dish. Budding with structures resembling
embryonic lung tissues, can be obtained in culture. Abnormal
development has also been studied using this system (Zimmermann et
al., 1981).
5.4.6. Studies with non-embryonic tissues
It may be asked whether it is feasible to use non-embryonic
tissues for evaluating the possible embryotoxic effects of
chemicals. Basic processes such as cell-cell interactions, cell
migrations, differentiation processes, and proliferation processes,
can be used as end points, as these are certainly involved in
embryonic development. Systems suggested so far include the
investigation of aggregation phenomena with cells of mammalia
(Moscona & Moscona, 1952) or lower invertebrates such as hydra
(Johnson, 1981), the investigation of cell-cell interactions as
studied by the attachment of cells to lectin-coated surfaces (Braun
et al., 1979, 1982), and the possibility of studying induction
processes using hormones or drugs. The use of teratocarcinoma
cells, which are pluripotent cells capable of many types of
embryonic differentiation, might also be considered (Stevens, 1967;
Graham, 1977; Martin, 1978).
The regeneration of the tail of the Xenopuslarvae or of the
crested newt has been suggested as a model for the evaluation of
the teratogenic potential of drugs. The biology of this
regeneration has been well investigated (e.g., Lüscher, 1946, 1955;
Gebhardt & Faber, 1966; Thornton, 1968; Neukomm, 1969; Tsonis &
Eguchi, 1980) and possible means of interfering with this
regeneration have been explored. It has been found possible to
induce abnormal regeneration in this system with thalidomide and
some of its teratogenic and non-teratogenic derivatives (Bazzoli et
al., 1977). There are no systematic studies on the use of such
systems in routine testing for embryotoxicity.
6. HUMAN RISK ASSESSMENT
6.1. Introduction
Human risk assessment is intended to provide an estimate of the
potential for human disease, and of exposure levels for protecting
human health. The dose-effect data from human epidemiological
and/or environmental animal studies are essential for human risk
evaluation. When coupled with estimates of actual exposure levels,
they can be used to estimate the probability (magnitude of the
likelihood) of an effect in the exposed population. Such a hazard
assessmenta requires a quantitative evaluation of exposure for each
sub-population at risk. Since the assessment of hazard for sub-
populations necessarily relies on the level and type of exposure
specific to each circumstance, hazard assessment is not discussed
in this document.
Direct observations in human beings obviously provide the best
information for assessing human risk. These data, however, are
rarely available and may often be difficult to interpret,
particularly with respect to the timing and level of exposure.
Given these factors, scientists most often rely on observations
from laboratory animal studies to estimate potential human risks
associated with environmental exposures.
Most experience with risk assessment has been in the area of
carcinogenesis, although methods are being developed for assessing
risk for other health endpoints (EPA, 1976; Albert et al., 1977;
IRLG, 1979; EPA, 1980; EPA, 1982; NAS, 1983). The assessment of
human risk from prenatally-induced toxicity presents particular
problems, which are described in this chapter. In general, the
risk assessment comprises two steps: (a) a qualitative assessment
of the biomedical data to determine the likelihood that the
chemical may pose human risks of prenatally-induced toxicity, and
(b) on the assumption that the chemical is capable of causing such
adverse effects, a quantitative estimate or prediction of the dose-
effect relationships in human beings, to provide the basis for
recommending safe exposure levels and for further assessing the
potential hazard to sub-populations exposed to the chemical at
various levels.
The first step (i.e., qualitative assessment) relies on
observations in human beings and laboratory animals together
with other pertinent supporting data. Taking a weight-of-evidence
approach, observations in human beings, backed up by animal
studies, provide the most convincing evidence that a chemical is
capable of causing adverse effects in developing progeny. In the
absence of human data, however, observations in laboratory animals
must be relied on. The best animal evidence is provided by
---------------------------------------------------------------------------
a In this context, the term "hazard assessment" 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 (WHO, 1978).
replicated results in laboratory animals and results in multiple
species. Experimental animal data of a more limited nature may
contribute some evidence of potential adverse human effects
provided the design and conduct of the study is adequate; although
the data for each chemical must be evaluated independently. In an
assessment of these data, the nature, severity, and extent of the
biological responses should be considered together with other data
that may have a bearing on the overall evaluation. These include
pharmacokinetic parameters, cytotoxicity, other toxic endpoints,
structure-activity relationships, maternal toxicity, and
information from short-term non-mammalian and in vitro tests.
The second step, that of quantitatively estimating the dose-
effect relationship for human beings, generally requires
extrapolation from higher doses, where effects have been observed
in human beings or in laboratory animals, to lower doses to which
human beings may be exposed in the environment. A dose-response
relationship, observed in human beings is more readily
interpretable for low-dose extrapolation than animal data. Very
often, however, observations in human beings are not accompanied by
very precise estimates of the magnitude and duration of exposure.
In the absence of such data, experimental animal data may be used
to extrapolate dose-effect relations for human beings. For this, it
is usually necessary to fit some shape to the dose-effect curve
outside the observed range, with a view to extrapolating adverse
effects observed at higher doses in experimental studies to
possible effects that might occur at lower human exposure. The
shape of the dose-effect curve should be justified, if possible, on
the basis of mechanisms of action. It is also necessary to
compensate for the biological, especially pharmacokinetic,
differences between animals and human beings. Although a variety
of mathematical models have been suggested for extrapolation
involving other toxic manifestations, e.g., carcinogenicity (Brown,
1980; Anderson & CAG, in press), their applicability for predicting
adverse health effects in progeny following exposure during
pregnancy has not been justified. In the absence of a biological
justification for applying mathematical extrapolation models to
these biological data, safety factors are being used to recommend
safe levels of exposure. Various factors involved in data
interpretation for the purpose of human risk assessments are
discussed in sections 6.2 and 6.3.
6.2. Interpreting Laboratory Animal Studies
For qualitative risk assessment purposes, positive results in
laboratory animals may provide substantial evidence that the agent
may be a potential human teratogen. However, animal studies must
be interpreted in the light of the adequacy of the study and the
nature and extent of the responses, together with all other
biological information that might have a bearing on the weight-of-
evidence to establish potential human teratogenicity.
The primary basis for using animal bioassay systems to predict
prenatally-induced toxicity in human beings is that almost all of
the 15-20 known or suspected human chemical teratogens give
positive results in animal bioassay systems (Schardein, 1983;
Shepard, 1983). However, many agents shown to induce developmental
toxicity in laboratory animals have not been clearly associated
with human effects. Factors such as interspecies differences, and
the use of very high doses, often causing toxic effects in the dam,
in experimental animal studies compared with the low levels to
which human beings are exposed, may account for this. It is
possible that most agents that produce teratogenic effects in
laboratory animals would also be shown to affect human beings, if
the methods for detection were sufficiently sensitive and the human
exposures were high enough.
Several approaches have been proposed for classifying the
qualitative evidence for developmental toxicity. For example,
Neubert et al. (1980) have proposed that agents that cause
developmental toxicity should be classified as inducing non-
specific or specific effects. Non-specific effects can be induced
by almost any agent, if the doses are high enough. These effects
include weight reduction, developmental retardation, and embryo-
mortality. Such effects may often be caused by maternal toxicity
and may, therefore, warrant different quantitative approaches. If
maternal toxicity is the cause of the fetal effect, then the
extrapolation of experimental data is carried out according to the
methods used in adult toxicity. Also lower safety factors are
often used for such agents. On the other hand, agents that cause
specific effects do so by interfering with some stage of fetal
development, independent of maternal toxicity. For specific
effects, any extrapolation must be related to the fetal effect and
be based on the mechanisms of action, if possible, and safety
factors higher than those for non-specific effects should be used.
Similar use of different safety factors for different classes of
effects has been proposed by Khera (in press). One of the
difficulties associated with using any categorization scheme is
that some chemicals may not fall clearly into any one category.
The adequacy of animal data depends in part on experimental
conditions. The route, temporal pattern, and duration of exposure
for experimental studies should simulate those of man for use in
performing risk assessments. Sometimes it might be advisable to
test impure chemicals or chemical formulations to which man is
exposed.
Despite widespread interest in short-term non-mammalian and in
vitrotechniques, pregnant mammals are today the only accepted test
systems for predicting human disease. Currently, some short-term
test systems offer promise for elucidating the mechanisms of action
of embryotoxic agents and some may also be useful for providing
supporting information for interpreting animal bioassay studies.
6.2.1. Endpoints
A wide variety of responses characterize developmental
toxicity. Spontaneous abortion, intrauterine death, prematurity,
low birth weight, birth defects, and postnatal manifestation
following prenatal insults are effects of abnormal human and animal
reproduction. Even offspring that seem perfectly normal at birth
may be afflicted with some degree of mental retardation or with
transplacentally-induced infertility (Khera & Ruddick, 1973) and
cancer later in life. At present, most of the evidence regarding
these types of delayed toxicity is derived from studies on animals,
though a few examples have come to light in human beings. These
are that prenatal exposure to diethyl stilboestrol impaired
fertility in male and female offspring and induced vaginal cancer
in female offspring (Herbst et al., 1979b) and prenatal exposure to
methyl mercury induced cerebral palsy in offspring (Reuhl & Chang,
1979). Thus, risk assessment is complicated by the various
outcomes of altered development that need to be considered.
Mechanisms of action may differ for these different endpoints;
therefore characterization of dose-effect curves, particularly at
low levels of exposure, to quantitatively assess human risk for
different disease endpoints is complex.
The various endpoints of developmental toxicity can occur
spontaneously or be induced. Altered growth consists of reduced
fetal weight, which is widely recognized, and increased fetal
weight which is less recognized. Structural variations include
changes of a permanent nature i.e., extra rib, absent vertebral
segment, retarded ossification, precocious ossification, and open
ductus arteriosus. The role of minor structural deviations
relative to more obvious forms of developmental toxicity in animals
and human beings has not received sufficient attention to determine
their relative significance in human risk assessment (Khera, 1981).
6.2.2. Interspecies variations
Even among laboratory species, the embryotoxic and teratogenic
responses to the same chemical may vary markedly. Such variations
in response complicate the extrapolation of laboratory animal data
to man, because it is not known which is the best animal model.
Interspecies differences are presumably due to differences in the
intrinsic sensitivity of embryonic and fetal processes to chemical
perturbation, the rate and stage of embryogenesis, and various
toxicokinetic factors (Neubert & Chahoud, 1983; Khera, in press).
There is also a difference in the background for developmental
abnormalities between some laboratory animal species and human
beings. For example, the preimplantation and early
postimplantation losses in rats are low, less than 10% (Perraud,
D., 1976; Fritz, H. et al., 1978; Frohberg, 1977), while the fetal
wastage in human beings may be as high as 50% (section 3.1.1.1).
Human beings have an apparent mechanism to naturally abort,
particularly if the fetus is abnormal. At present, not enough is
known to account for these differences in predicting potential
human disease.
If a teratogen acts by means of the same biochemical mechanism
in the animal model and in man, then the concentration of the
chemical at the target site is an important determinant of the
susceptibility of the species. In turn, the concentration of the
chemical at the target cell is determined by pharmacokinetic
variables. By modelling pharmacokinetic constants in the test
species and man, it may be possible to predict the incidence of
teratogenicity based on maternal blood concentrations.
The differences in toxicokinetics make it almost impossible to
extrapolate data from rodents to man with any certainty (Khera,
1976). Nau et al. (1981) demonstrated that, with the use of
"minipumps", concentrations similar to the therapeutic
concentrations obtained in human beings can be obtained in mice for
valproic acid. Such methods may aid an evaluation of teratogenic
effects.
Pharmacokinetic models may greatly aid in assessing the human
risk associated with exposure to potential teratogens. These
procedures, however, must be perfected and validated. In the
future, computer models based on well-validated mechanisms,
structure-activity relationships, pharmacokinetic data, in vitro
data, and whole animal models should greatly enhance the
reliability of data extrapolation from laboratory animals to man.
6.2.3. Statistical limitations
Although many variables are controlled in well-conducted animal
tests, there remains the major problem of the statistical
limitation of the study. A basic aspect of toxicology is the dose-
response relationship relating the incidence of a specific effect
induced by a chemical to increasing exposure levels. The incidence
of an effect and the size of the study population are important in
determining the sensitivity of the study. Because the underlying
mechanisms may be different, it is advisable, in principle, to
evaluate dose-effect relationships for all prenatally-induced toxic
effects, separately. When considering effects as completely
separate entities, it must be recognized that the relevant dose-
range for that effect may be severely restricted. If the endpoints
studied are restricted to "all or none", such as low frequency
events of malformations or abortions, then the power of statistical
tests in studies of conventional dimensions is often extremely
poor. Because a single effect in prenatal toxicity may often be
modulated and obscured by other effects of toxicity, such an
approach can frequently prove unsatisfactory. In such a case, the
evaluation of compiled data concerning several effects may be
necessary, although not completely justified. For the purposes of
interpretation of animal data for predicting potential human
disease, the dose-effect relationship of the most sensitive effect
has the greatest significance.
Statistical tests should be used as a guide and not as a
substitute for biological judgement. Before statistical
significance can be transformed to toxicological relevance, the
result must make biological sense and the same applies to "non-
significant" results.
Since by using fetus or litter as the statistical unit,
important data may be lost, it is advisable to report effects based
both on the number of litters and on the total number of fetuses
observed (Neubert & Dilmann, 1972). Statistical methods for
combining fetal and litter data for evaluating chemically-induced
effects have been described (Snedecor & Cochran, 1967; Sokal &
Rohlf, 1969; Haseman & Kupper, 1979; Haseman & Hogan, 1975).
6.2.4. Quantitative risk assessment
Since there is no universally-accepted mathematical model for
extrapolating embryotoxic including teratogenic effects below the
observed dose-effect range, safe levels of exposure for human
beings have been estimated by applying a safety factor to the no-
observed-teratogenic-effect level (Lehman et al., 1954; FDA, 1974;
EPA, 1975; NAS, 1977; Faber, 1980; ACGIH, 1981; Khera, 1984).
Usually, large safety factors are used, because it is not possible
to accurately characterize synergistic or antagonistic factors that
may occur in human beings through exposure to a combination of
chemicals in the ambient environment. Khera (in press) compared
the quantitative dose-effect data available in the literature for
embryotoxic effects in human beings and animals. Using crude
estimates, the ratios between the lowest doses of several drugs
reported in literature to have produced adverse effects in human
progeny and the lowest reported embryotoxic doses in various animal
species varied between 1 and 400.
A number of statistical (tolerance) models, such as probit,
logit, and Weibul, which were developed for other toxicological
endpoints, might also be used to describe teratogenic dose-response
relationships. Other mechanistic models such as the multi-hit and
multi-stage models have been specifically developed for the
carcinogenic response (Mantel & Schreiderman, 1975; Chaud & Hoel,
1974; Brown, 1980; Krewski & Van Ryzin, 1981). The tolerance
models were developed on the basis of the notion that each
individual in the population possesses a tolerance to the test
compound. If dose D does not exceed the tolerance for an
individual, then there will be no effect on that individual; while
a dose exceeding the tolerance will result in a positive response.
The probability that the individual selected at random will respond
at a dose D is given by,
P = f (D) = Pr [tolerance < D]
The probit model is obtained when the tolerance is considered
as a random variable, and follows a log normal distribution. Logit
and Weibul models are obtained when the tolerance is assumed to
follow logistic or extreme value distribution, respectively. The
logit, like the probit model, has an S-shaped dose-response curve
that is symmetric about the 50% response point. The logit curve
approaches the zero response more slowly than the probit curve,
leading to a higher response rate for a given dose in the low-dose
region (Brown, 1980).
All these statistical (tolerance) models and the mechanistic
models will probably fit teratological dose-response data in the
observed range reasonably closely; but would be expected to diverge
substantially in the low-dose region. Since the risk estimates
obtained at low doses from both the statistical (tolerance) models
and the mechanistic models are expected to cover a wide spectrum at
low doses, they could be used to define a range of risk estimates
at a given exposure level. The primary reason for believing that
such a risk range is plausible is based on the hypothesis that most
biological effects have a dose-response curve in the low-dose range
that is concave upwards. If this is also true for teratogens, then
the above models would establish a broad risk range for a given low
dose exposure. However, low-dose extrapolation for teratogens is
largely an uninvestigated field. Only with knowledge of the
mechanisms of action, can an appropriate extrapolation model be
selected for better describing risk at low doses for embryotoxic
agents including teratogens.
Probit models have been proposed as an extrapolation model to
determine safe human exposure levels (Mantel & Bryan, 1961; Weil,
1972). Probit models have also been used to compare the relative
teratogenicity of different chemicals in a specific animal species
(Fabro et al., 1981) and to fit experimental data in an observed
dose-effect range (Neubert et al., 1973).
Biddle (1978) studied the potential of cortisone and 6-
aminonicotinamide to induce cleft palate in a variety of inbred and
hybrid mouse strains using a probit model. For both compounds, a
separate family of parallel dose-effect curves was derived from the
experimental data generated by studying the different genotypes.
Parallel dose-effect curves were assumed to suggest a common
mechanism of teratogenicity. Biddle concluded that when dose-effect
curves for the different strains have the same slope, the ratios of
estimated ED50s provide the most reasonable measure of relative
strain sensitivity. Hogan & Hoel (1982) pointed out the
limitations of this approach. Their main criticism was that,
because approximately parallel dose-response curves for different
strains of mice were restricted to the experimental dose range, it
was not possible to know whether they would persist in the low-dose
region, near the origin of the curve.
An attempt to ascertain whether a "threshold" exists in
prenatal toxicity and whether embryotoxic agents may be grouped in
different categories in this respect was made by Jusko (1972).
Using a mathematical model and a graphing procedure, he suggested
that embryotoxic substances might be grouped in two mechanists
categories: agents that show a "threshold" and others that do not,
with implications for low-dose extrapolation. Although the method
suffers from the common difficulty of having to project data in the
low-dose range, the approach seems worthy of consideration with
appropriate experimental data.
In any attempt to extrapolate animal data, the possible
contribution of maternal toxicity to the toxic effects in the fetus
must be considered, because most chemicals could damage the fetus
by this mechanism, if exposure were high enough (Martson &
Shepelskaja, 1982, 1983). In recent studies, attempts have been
made to quantify the toxic effects on the mother and the embryo and
to associate dose-response relationships of the various parameters.
From the data, it is possible to calculate indices, derived by
comparing doses for the maternal toxic responses with the dose
producing embryotoxic effects, including teratogenic effects
(Murphy, 1965; Chaube & Murphy, 1968; Johnson, 1981; Vergieva,
1981, 1982; Bass et al., 1982; Fabro et al., 1981, 1982; Platzek et
al., 1982). In some situations, these indices are being used to
classify the relative hazard of environmental agents such as
pesticides (Kalojanova, 1982) and to estimate safe levels for water
pollutants (Vitvitskaja et al., 1980; Korolev & Agareva, 1983).
6.3. Interpretation of Human Data for Risk Assessment
Human data are the most reliable basis for estimating risks of
human disease. These data, however, are difficult to obtain and
may be of limited value for risk assessment because of confounding
variables common to all epidemiology studies, including
difficulties in exposure estimation, high background rates of birth
defects in the human population, and wide variation in diagnosing
and reporting defects. These factors were discussed in detail in
section 3.1 (Human studies). For the purposes of quantitative risk
extrapolation, the model that best fits the human dose-effect data
in the observed range should be used and extrapolated to lower
doses, if possible, on the basis of plausible mechanisms of action.
Even negative human data can be useful for comparison with dose-
effect relationships and risk levels estimated from animal data, in
determining an upper level for possible human risk.
7. CONCLUSIONS
In concluding this report, it is worthwhile to return to the
questions posed in the introduction to the document:
(a) What is the value of currently used toxicity tests for
predicting human risk?
(b) What are the difficulties involved in applying these test
results to estimate human health risks and hazards?
(c) Can better testing strategies be developed?
1. Animal tests are valuable in identifying chemicals that
can alter morphological and functional development. Extrapolation
from such data to man is the principal method of predicting health
risks. Prenatally-induced toxicity is identified in animals and
its transposition to man depends on essential elements in the
experimental design. The species (and strain) of the test animal
must be carefully selected, bearing in mind availability,
fecundity, and the incidence of spontaneous malformations or
variations. A knowledge of toxicokinetic variables, homeostatic
mechanism, and ability to adapt to a chemical insult, relative to
man, greatly aid in extrapolating laboratory data to man. If a
study is properly executed, it is possible to compare fetal and
maternal levels of toxicity.
2. A number of difficulties are involved in applying laboratory
animal test data to estimate human risks.They become all the more
pronounced, when attempting a quantitative risk assessment. Animal
studies usually involve healthy animals; food and water and other
environmental factors are controlled; adequate group sizes are
possible; and exposure is usually to a single agent. The offspring
can be carefully examined at any time. In contrast, an exposed
human population varies in genetic constitution, health, diet, and
physical environment. In addition, the subjects under study may
elect to smoke, consume alcoholic beverages, or take drugs.
Human studies are perhaps the weakest link in the chain of
events, necessary to develop and validate means of predicting human
risk to a toxic chemical. In epidemiology studies, it is rarely
possible to identify the ideal test population and its control; the
numbers of individuals in the test group are usually small and the
incidence of the effect low and difficult to detect. Toxic effects
may be apparent at birth or manifest later during postnatal life.
Therefore, few environmental chemicals have been shown to be toxic
to the human fetus. However, when collecting human epidemiological
data, data on accidental exposure and other evidence of human
hazards is emphasized. Toxicokinetic data can sometimes be
obtained on human beings occupationally or accidentally exposed to
chemicals. Quantitative data on human exposures are essential,
both in conducting human studies to establish dose-response
relationships and for the estimation of risks to sub-populations
exposed to various levels of environmental agents.
3. There is no question that better testing strategies could be
developed in both laboratory animals and human studies. Topics that
deserve emphasis include comparative toxicokinetic studies to
enable interspecies comparison of test results and extrapolation to
man; a better understanding of the genetic, molecular, and cellular
processes that are the targets for toxic chemicals, which disturb
them and cause dysfunction; short-term tests in which enzymes,
cells, organs, and embryos in culture are used as well as sub-
mammalian species; and studies on effects of prenatal chemical
exposure on neurobehavioural development and on cancer incidence.
4. Though data on the molecular biology of development and
morphogenetics is also increasing, information and understanding of
crucial development processes is still rudimentary. Nevertheless,
available data seem to suggest that the biological mechanisms
underlying normal development are similar and that interspecies
differences are often only temporal in nature. The disciplines of
genetics, and molecular and cellular biology provide the
experimental and clinical scientist with tools to detect altered
genes or genetic regulation. A molecular explanation should be
sought for every endpoint induced by chemicals in experimental
animals.
5. Considerable scientific activity is concerned with the
development of short-term tests.They are useful research tools for
investigating the processes underlying normal and abnormal
development. However, the Task Group was of the opinion that, with
the present state of knowledge, short-term tests cannot replace
whole animal-testing procedures for assessing risk to human
subjects. Short-term tests should be further developed to provide
information to support data obtained in whole experimental animal
studies, to provide a basis for selecting chemicals for further
testing, and to develop models for possible correlation with
results from whole animal tests.
6. Morphological defects at birth represent only a part of the
effects produced by prenatal toxicity. The process of development
during its various stages is also susceptible to chemically-induced
adverse effects, manifested during postnatal life.These could
involve every tissue and organ system and function, such as the
immune system, reproductive capacity, neurobehavioural function.
Transplacental carcinogenesis belongs to this category.
Although the importance of the potential effects of prenatal
exposure to chemicals on postnatal development is acknowledged,
there are, at present, no generally agreed testing strategies for
the evaluation of the postnatal manifestation of such effects.
However, it may be worth investigating the relative adult versus
fetal sensitivity to chemicals to which many pregnant women have
been, or are, unavoidably exposed. As a primary task, compounds
that are suspected of being human carcinogens (IARC, 1982), should
be tested for transplacental carcinogenicity.
7. For qualitative risk assessment,results from human studies,
backed up by animal studies, provide the strongest evidence that a
chemical is toxic to the developing progeny, if the mother is
exposed during pregnancy. In the absence of human studies,
positive results in animal studies provide evidence that the agent
is potentially toxic for the developing fetus, although the
importance of carefully evaluating each individual study and all
related biomedical evidence in completing the qualitative risk
assessment is essential. Categorizing of embryotoxic effects into
specific fetal effects and non-specific effects is useful for
establishing safety guidance.
8. At present, the statistical (tolerance) and mechanistic
extrapolation modelsthat have been designed for extrapolating
high dose-effect data to lower doses for cancer and other
toxicological endpoints, remain largely uninvestigated for
teratogens. Use of these models for teratogenic extrapolation, as
a general matter, can only establish broad risk ranges associated
with low-dose levels of exposure, unless there is some indication
of the mode of action and some rational basis for extrapolating
from the test species to man.
8. RECOMMENDATIONS FOR FUTURE ACTIVITIES
The Task Group was of the opinion that, though progress has
been made in the evaluation of risks for progeny following prenatal
exposure to chemicals, very little information on the mechanisms by
which adverse effects are produced has been forthcoming. The Group
believed that the following recommendations, if carried out, would
enhance the predictive value of the present approaches:
1. In the areas of genetics, and cellular and molecular biology, a
number of new techniques have been developed. These involve:
cell fusion, cellular migration, interaction and pattern
formation, monoclonal antibodies, embryo transfer, whole
chromosome and/or gene transfer, production and use of chimeric
animals, gene isolation, in vitro mutagenesis, and general
recombinant-DNA methods. Such techniques should be applied to
determine the underlying mechanisms in normal and abnormal
developmentwith a view to understanding and controlling
chemically-induced congenital abnormalities.
2. The following improvements in the design and conduct of human
studies are recommended:
(a) Collaborative international epidemiological
studies, using standard techniques and quantitative
assessments of exposure, should be performed. The
information will be useful in the study of the
responses of different sub-populations to particular
chemicals. These studies can also provide
dose-response data to validate animal models for
human risk assessment.
(b) Registries monitoring congenital malformations
and pediatric tumours should be expanded to increase
the number of participating countries. Data on the
frequency and geographical distribution of
malformations and infant neoplasms should be
published regularly.
(c) Monitoring of occupational health should include
complete reproductive and exposure data of workers.
Special risk groups should be studied for pregnancy
outcomes. Pregnancy wastage, including spontaneous
abortions, intrauterine deaths, and the more subtle
effects that occur prenatally or develop postnatally,
must also be considered.
(d) Relationships, if any, between structural defects
and biochemical and/or functional defects, and fetal
loss should be investigated.
(e) Methods should be established to diagnose
so-called "minor abnormalities" in the general
population and in risk groups.
3. In order to make the animal embryotoxicity studiesmore
meaningful for extrapolation to human safety, the following
suggestions are made:
(a) It has been suggested that 3.5% of all human
malformations are associated with the ill health and
disease conditions of the mother (Kalter & Warkany,
1983). However, in animal studies, the role of
maternal toxicity in the induction of fetal anomalies
and embryotoxicity needs clarification. Such a
clarification would be extremely helpful in the
embryo/fetal evaluation of chemicals and
extrapolation of human safety from animal data.
(b) The role of maternal, placental, and fetal
factors in activating or detoxifying environmental
chemicals in different animal species and man should
be clarified.
(c) The significance of different kinds of
experimentally-induced gross structural abnormalities
for a human risk assessment must be determined by
well-designed studies.
(d) Studies should be designed with a view to
defining target cells or receptors in the fetus that
are specific for teratogens.
(e) Since human beings are usually exposed to a
variety of chemicals, studies on the treatment of
pregnant animals with a combination of two or more
chemicals are suggested.
(f) In view of increasing knowledge, the design and
interpretation of animal studies should be
periodically appraised by an international group of
experts.
4. Attention must be directed to the entire spectrum of
developmental toxicity including postnatal manifestations in
human and animal studies. Test methods must be developed to
monitor neonates and young children of mothers exposed to
chemicals during pregnancy, for functional abnormalities. This
should include evaluation of neurobehavioural effects, the
immune and endocrine systems, and of xenobiotic metabolism,
preferably with non-invasive methods. The background data for
these functions must be established.
5. Additional effort is needed to evaluate neurobehavioural tests
for routine use in developmental toxicology. Attention should
be given to:
(a) selection of chemicals that can be used as
positive controls;
(b) identification of the most suitable tests and
periods during postnatal development;
(c) development of procedures for the early detection
of latent neurotoxic effects; and
(d) better understanding of basic mechanisms
underlying neurobehavioural effects.
6. The following research needs were recognized in the area of
transplacental carcinogenicity:
(a) Fetal exposure to carcinogens in animal studies
has been reported to increase susceptibility to
tumour induction following further exposure,
postnatally, to various carcinogens or promoting
agents (section 4.4). Verification of these results
is needed.
(b) Results of a few animal studies have suggested
that chemical exposure of the parent generation can
be followed by an increased incidence of tumours in
subsequent generations (section 4.4). This
phenomenon should be explored further.
(c) Animal models with a comparatively long pregnancy
should be investigated to estimate the susceptibility
of the embryo/fetus to transplacental carcinogens at
various stages of development.
7. (a) Short-term testsshould be further developed to
provide information on embryotoxic mechanisms and to
determine their use for selecting chemicals for
further animal testing;
(b) Short-term tests that show promise for chemical
selection should be validated using the principles
provided in section 5. An international meeting to
discuss validation methods is needed. The
sensitivity and susceptibility of any technique
considered for use in short-term selection tests must
be established taking into account results from in
vivostudies.
8. (a) Toxicokinetic modelsshould be developed in order
to compare toxic effects among species, based on the
amount of chemical present in the maternal blood,
various tissues, or even at receptor sites of the dam
or embryo. These data are needed because a
satisfactory risk assessment for human beings can
never be based solely on the doses administered to
animals and the human exposure levels. Toxicokinetics,
and homeostatic and adaptive factors largely account for
differences in response. Finally, these data are needed to
establish a better understanding of the mechanisms of
action as a basis for qualitative risk extrapolation.
(b) Research is needed to (i) generate basic
biological information that will provide a more
rational scientific basis for the proper application
of safety factors for estimating "safe levels" of
exposure; and (ii) develop the biological basis for
selecting appropriate mathematical models. These
methods are germane to extrapolation from high doses
in animal studies to low exposure levels in human
beings, in order to estimate the magnitude of effect
in human beings.
9. Priority for testingshould be high for chemicals: (i)
that show an increased potential for altering development by
affecting genetic, molecular, or cellular processes; (ii) that
show structural similarities to known human teratogens; and
(iii) to which women of child-bearing age will probably be
exposed.
9. EXPLANATION OF TERMS USED IN THE DOCUMENT
This section explains the meanings of certain terms as used in
this document. These meanings are not necessarily valid for other
purposes.
Conceptus
All products of conception derived from and including the
fertilized ovum at any time during pregnancy, including the embryo
or fetus and embryonic membranes.
Congenital malformation
A permanent structural abnormality present at birth.
Critical period
A particular developmental phase during which a morphogenetic
system is especially vulnerable (e.g., neurulation, fusion of the
facial swellings, elevation of palatal shelves).
Developmental toxicity
Any adverse effect induced prior to attainment of adult life.
This is a new term and includes effects induced or manifested in
the embryonic or fetal period and those induced or manifested
postnatally (before sexual maturity).
Embryo/fetotoxicity
Any toxic effect on the conceptus resulting from prenatal
exposure, including structural or functional abnormalities or
postnatal manifestation of such effects.
Embryonic period
The period from conception to the end of major organogenesis.
Generally, the organ systems are identifiable at the end of this
period.
Fetal period
The period from the end of embryogenesis to the completion of
pregnancy.
Poor pregnancy outcome
A term used to describe the failure of a pregnancy to produce
viable, biologically normal offspring. It covers all adverse
effects: fetal death (including abortion), intra-uterine growth
retardation and abnormal development.
Pregnancy dating and conceptional age
Throughout the text, Day 0 is considered to be the first day of
pregnancy. In human beings, gestational age (date from first day
of last menstrual period) and conceptional age (date from
conception) may be used. In this document, conceptional age has
been adopted.
Prenatally-induced abnormality
Any structural, functional, or biochemical deviation from the
norm, initiated prenatally, that can be detected during prenatal or
postnatal life.
Sensitive period
A developmental phase during which differentiating cells become
susceptible to a given toxic agent. The period may not be related
to critical morphogenetic periods, but may be related to the
appearance of specific receptors (e.g., for glucocorticoids).
Teratogena
An agent which when administered prenatally induces permanent
abnormalities in structure.
Teratogenicitya
A type of embryo/fetotoxicity that is restricted to permanent
structural abnormalities produced by prenatal exposure to toxic
agents.
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a "Teratogen" and "Teratogenicity" as agreed by the Task Group
are used here in the traditional sense derived from Greek word
"teratas". They refer to a particular case of embryotoxic effect
demonstrated by structural malformation detectable by the present
routinely used methods (Section 3.2). The Group recognized that
these meanings differ from those used by other bodies.
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ACGIH (1981) American Conference on Governmental Industrial
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and the work room environment with intended changes for 1981,
Cincinnati, Ohio, ACGIH (Publication Office).
ADAMS, J. & BUELKE-SAM, J. (1981) Behavioral assessment of
the postnatal animal: testing and methods development. In:
Kimmel, C. A. & Buelke-Sam, J., ed. Developmental toxicology,
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