United States
Environmental Protection
Agency
Office of Research and
Development
Washington. DC 20460
v>EPA
Guidelines for
Reproductive
Toxicity Risk
Assessment
EPA/600/AP-94/001
February 1994
External Review Draft
Review
Draft
(Do Not
Cite or
Quote)
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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DRAFT EPA/600/AP-94/001
DO NOT QUOTE OR CITE February 1994
External Review Draft
o
Guidelines for Reproductive
Toxicity Risk Assessment
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by
the U.S. Environmental Protection Agency and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its technical accuracy and
policy implications.
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C.
^69 Printed on Recycled Paper
U.S. Environmr.r.- .Action ARency
Region 5, Lit, :,
77 West Jacksc,-. "": •-,,.,-) 10f. r.
Chicago, IL 60604-0^90 ' F'°0f
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DISCLAIMER
This document is an external draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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TABLE OF CONTENTS
List of Tables
Authors, Contributors, and Reviewers
SUPPLEMENTAL INFORMATION 1
A. REGULATORY AUTHORITY 1
B. ENVIRONMENTAL AGENTS AND REPRODUCTIVE TOXICITY 2
C. THE RISK ASSESSMENT PROCESS AND ITS APPLICATION TO
REPRODUCTIVE TOXICITY 5
I. OVERVIEW 9
II. DEFINITIONS AND TERMINOLOGY 12
III. HAZARD IDENTIFICATION/DOSE-RESPONSE EVALUATION OF
REPRODUCTIVE TOXICANTS 13
A. LABORATORY TESTING PROTOCOLS 13
A.I. Introduction 13
A.2. Duration of Dosing 13
A.3. Length of Mating Period 14
A.4. Number of Females Mated to Each Male 15
A. 5. Single- and Multigeneration Reproduction Tests 15
A.6. Alternative Reproductive Tests 18
A.7. Additional Test Protocols That May Provide Reproductive Data 20
B. ENDPOINTS FOR EVALUATING MALE AND FEMALE
REPRODUCTIVE TOXICITY IN TEST SPECIES 23
, B. 1. Introduction 23
B.2. Couple-mediated Endpoints 24
a. Fertility and Pregnancy Outcomes 24
b. Sexual Behavior 33
B.3. Male-specific Endpoints 34
a. Introduction 34
b. Body Weight and Organ Weights 35
c. Histopathologic Evaluations 38
d. Sperm Evaluations 41
e. Endocrine Evaluations 47
f. Biochemical Tests or Markers of Toxicity to the Testes and Other
Male Reproductive Organs 48
g. Paternally-mediated Effects on Offspring 49
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TABLE OF CONTENTS (continued)
B.4. Female-specific Endpoints 49
a. Introduction 49
b. Body Weight, Organ Weight, Organ Morphology, and Histology . 52
c. Oocyte Production 58
d. Alterations in the Female Reproductive Cycle 60
e. Mammary Gland and Lactation 62
f. Developmental and Pubertal Alterations 63
g. Reproductive Senescence 64
C. HUMAN STUDIES 65
C.I. Epidemiologic studies 66
a. General Design Considerations 66
b. Selection of Outcomes for Study 70
c. Reproductive History Studies 74
d. Community Studies and Surveillance Programs 76
e. Identification of Important Exposures for Reproductive Effects . . 77
C.2. Examination of Clusters, Case Reports, or Series 78
D. PHARMACOKINETIC CONSIDERATIONS 79
E. COMPARISONS OF MOLECULAR STRUCTURE 81
F. EVALUATION OF DOSE-RESPONSE RELATIONSHIPS 82
G. CHARACTERIZATION OF THE HEALTH-RELATED DATA BASE . 86
H. DETERMINATION OF THE REFERENCE DOSE OR REFERENCE
CONCENTRATION FOR REPRODUCTIVE TOXICITY 94
I. SUMMARY 97
IV. EXPOSURE ASSESSMENT 97
V. RISK CHARACTERIZATION 102
A. OVERVIEW 102
B. INTEGRATION OF HAZARD IDENTIFICATION/DOSE-RESPONSE AND
EXPOSURE ASSESSMENTS 104
C. DESCRIPTORS OF REPRODUCTIVE RISK 107
C.I. Estimation of the Number/Proportion of Individuals Exposed to Levels
Above the RfD or RfC 107
C.2. Presenting Situation-Specific Exposure Scenarios 107
C.3. Margin of Exposure 108
C.4. Risk Characterization for Highly Exposed Individuals 108
C.5. Risk Characterization for Highly Sensitive or Susceptible Individuals . . 109
D. SUMMARY AND RESEARCH NEEDS 109
VI. REFERENCES Ill
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LIST OF TABLES
1. Couple-mediated Endpoints of Reproductive Toxicity 25
2. Selected Indices That May Be Calculated From Endpoints of Reproductive Toxicity
in Test Species 27
3. Male-Specific Endpoints of Reproductive Toxicity 36
4. Female-Specific Endpoints of Reproductive Toxicity 51
5. Categorization of the Health-Related Data Base Hazard Identification/Dose-Response
Evaluation 89
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AUTHORS AND MANAGERS
This external review draft was prepared by an intra-Agency EPA working group
chaired by Eric Clegg of the Office of Health and Environmental Assessment.
DOCUMENT MANAGER
Eric Clegg
Office of Health and Environmental Assessment
Office of Research and Development
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SUPPLEMENTAL INFORMATION:
A. REGULATORY AUTHORITY
The Environmental Protection Agency is authorized by numerous statutes, including
the Toxic Substances Control Act (TSCA), the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA), the Clean Air Act, the Safe Drinking Water Act, and the Clean
Water Act, to regulate environmental agents that have the potential to adversely affect
human health, including the reproductive system. These statutes are implemented through
offices within the Agency. The Office of Pesticide Programs and the Office of Pollution
Prevention and Toxics within the Agency have issued testing guidelines (U.S.
Environmental Protection Agency, 1982, 1985b) that provide protocols (now under review)
designed to determine the potential of a test substance to produce reproductive (including
developmental) toxicity in laboratory animals. The Organization for Economic Cooperation
and Development (OECD) also has issued testing guidelines for reproduction studies
(Organization for Economic Cooperation and Development, 1993b).
The procedures outlined here in the Guidelines for Reproductive Toxicity Risk
Assessment (hereafter called Guidelines) provide guidance for interpreting, analyzing, and
using the data from studies that follow the above testing guidelines. In addition, the
Guidelines provide information for interpretation of other studies and endpoints (e.g.,
evaluations of epidemiologic data, measures of sperm production, reproductive endocrine
system function, sexual behavior, female reproductive cycle normality) that have not been
required routinely, but may be required subsequently or may be encountered in reviews of
data on particular agents. The Guidelines will promote consistency in the Agency's
assessment of toxic effects on the male and female reproductive systems, including
outcomes of pregnancy and lactation, and inform others of approaches that the Agency will
use in assessing those risks. Guidance is also provided by the Guidelines for
Developmental Toxicity Risk Assessment (U.S. Environmental Protection Agency, 1991)
and the Guidelines for Mutagenicity Risk Assessment (U.S. Environmental Protection
Agency, 1986c). These three guidelines are complementary.
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Reproductive toxicity risk assessments prepared pursuant to these Guidelines will be
used within the requirements and constraints of the applicable statutes to arrive at
regulatory decisions concerning reproductive toxicity. These Guidelines do not change any
statutory or regulatory prescribed standards, such as those specified by Agency or OECD
testing guidelines for the types of data necessary for regulatory action.
The Agency has sponsored or participated in several conferences that addressed
issues related to evaluations of reproductive toxicity data and that provide some of the
scientific basis for these risk assessment guidelines. Numerous publications from these and
other efforts are available that provide background for these Guidelines (U.S.
Environmental Protection Agency, 1982, 1985b; Galbraith et al., 1983; Organization for
Economic Cooperation and Development, 1983; U.S. Congress, 1985, 1988; Kimmel, C.A.
et al., 1986; Francis & Kimmel, 1988; Burger et al., 1989; Sheehan et al., 1989). Also,
numerous resources provide background information on the physiology, biochemistry, and
toxicology of the male and female reproductive systems (Lamb & Foster, 1988; Working,
1989; Russell et al., 1990; Atterwill & Flack, 1992; Scialli & Clegg, 1992; Chapin &
Heindel, 1993; Heindel & Chapin, 1993; Manson & Kang, 1994; Zenick et al., 1994;
Kimmel, G.L. et al., In press). A comprehensive text on reproductive biology has been
published (Knobil & Neill, 1988).
B. ENVIRONMENTAL AGENTS AND REPRODUCTIVE TOXICITY
Disorders of reproduction and hazards to reproductive health have become prominent
public health issues. The perception of risk leads to a corresponding perception that action
is needed to protect people from hazards and prevent disorders of reproduction. Disorders
of reproduction in humans include but are not limited to reduced fertility, impotence,
menstrual disorders, spontaneous abortion, low birth weight and other developmental
(including heritable) defects, premature reproductive senescence and various genetic
diseases affecting the reproductive system and offspring.
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The prevalence of infertility, which is defined clinically as the failure to conceive
after one year of unprotected intercourse, is difficult to estimate. National surveys have
been conducted to obtain demographic information about infertility in the United States
(Mosher & Pratt, 1990). In their 1988 survey, an estimated 4.9 million women aged 15-44
(8.4%) had impaired fertility. The proportion of married couples that was infertile was
7.9%. Of major concern is the report that human sperm concentration has declined from
113 x 106 per ml of semen prior to 1960 to 66 x 10 per ml subsequently (Carlsen et al.,
1992). When combined with a decline in semen volume from 3.4 ml to 2.75 ml, that
indicates a decline in total number of sperm of approximately 50%. Increased incidences of
human male hypospadia, cryptorchidism and testicular cancer have also been indicated over
the last 50 years (Giwercman et al., 1993).
Even though not all infertile couples seek treatment, and infertility is not the only
adverse reproductive effect, it is estimated that Americans spent about $1 billion in 1986 on
medical care to treat infertility alone (U. S. Congress, 1988). With the increased use of
assisted reproduction techniques, that amount has increased substantially since 1986.
Disorders of the male or female reproductive system may also be manifested as
adverse outcomes of pregnancy. For example, it has been estimated that approximately
50% of human conceptuses fail to reach term (Hertig, 1967). Methods that detect
pregnancy as early as 9 days after conception have suggested that 35% of postimplantation
pregnancies end in embryonic or fetal loss (Wilcox et al., 1985). Approximately 3% of
newborn children have one or more significant congenital malformations at birth, and by
the end of the first postnatal year, about 3% more are recognized to have serious
developmental defects (Shepard, 1986). Of these, it is estimated that 20% are of known
genetic transmission, 10% are attributable to known environmental factors, and the
remaining 70% result from unknown causes (Wilson, 1977). Also, approximately 7.4% of
children have low birth weight (i.e., below 2.5 kg) (Selevan, 1981).
Numerous agents have been shown to be reproductive toxicants in male and female
laboratory animals and in humans (Mattison, 1985; Schrag & Dixon, 1985a, b; Waller et
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al., 1985; Lewis, 1991). For example, neonatal or peripubertal exposure to environmental
compounds that possess steroidogenic (e.g., diethylstilbestrol; Steinberger & Lloyd, 1985)
or antisteroidogenic (Schardein et al., 1985) activity affect the onset of puberty and
reproductive function in adulthood. In adult males and females, exposure to agents of
abuse such as cocaine disrupts normal reproductive function in both test species and
humans (Smith, C.G. & Gilbeau, 1985). Numerous chemicals disrupt ovarian cyclicity,
alter ovulation, and impair fertility in experimental animals and humans. These include
agents with steroidogenic activity, certain pesticides and herbicides, and some metals
(Thomas, 1981; Mattison, 1985). In males, estrogenic compounds can be testicular
toxicants in rodents and humans (Colborn et al., 1993). Dibromochloropropane (DBCP)
impairs spermatogenesis in both experimental animals and humans by another mechanism.
These and other examples of toxicant-induced effects on reproductive function have been
reviewed (Katz & Overstreet, 1981; Working, 1988).
Altered reproductive health is often manifested as an adverse effect on the
reproductive success or sexual behavior of the couple even though only one of the pair may
be affected directly. Often, it is difficult to discern which partner has reduced reproductive
capability. For example, exposure of the male to an agent that reduces the number of
normal sperm may result in reduced fertility in the couple, but without further diagnostic
testing the affected partner may not be identified. Also, adverse effects on the reproductive
systems of the two sexes may not be detected until a couple attempts to conceive a child.
For successful reproduction, it is critical that the biologic integrity of the human
reproductive system be maintained. For example, the events in the estrous or menstrual
cycle are closely interrelated; changes in one event in the cycle can alter other events.
Thus, a short or inadequate luteal phase of the menstrual cycle is associated with disorders
in ovarian follicular steroidogenesis, gonadotropin secretion, and endometrial integrity
(Scommegna et al., 1980; Smith, S.K. et al., 1984; Sakai & Hodgen, 1987). Toxicants may
interfere with luteal function by altering hypothalamic or pituitary function and by affecting
ovarian response (La Bella et al., 1973a, b).
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Fertility of the human male is particularly susceptible to agents that reduce the
number or quality of sperm produced. Compared with many other species, human males
produce fewer sperm relative to the number of sperm required for fertility (Amann, 1981;
Working, 1988). As a result, many men are subfertile or infertile (Amann, 1981). The
incidence of infertility in men is considered to increase at sperm concentrations below 20-
40 x 10 sperm per milliliter of ejaculate. As the concentration of sperm drops below that
level, the probability of a pregnancy resulting from a single ejaculation declines. If the
number of normal sperm per ejaculate is sufficiently low, fertilization is unlikely, and an
infertile condition exists. Toxic agents may further decrease production of sperm and
increase risk of impaired fertility.
Chemical or physical agents can affect the female and male reproductive systems at
any time in the life cycle, including susceptible periods in development. The reproductive
system begins to form early in gestation, but structural and functional maturation is not
completed until puberty. Exposure to toxicants early in development can lead to alterations
that may affect reproductive function or performance well after the time of initial exposure.
Adverse effects such as reduced fertility in offspring may appear as delayed consequences
of in utero exposure to toxicants. Effects of toxic agents on other parameters such as
sexual behavior, reproductive cycle normality, or gonadal function can also alter fertility
(Chapman, 1983; Dixon & Hall, 1984; Schrag & Dixon, 1985b; U.S. Congress, 1985).
C. THE RISK ASSESSMENT PROCESS AND ITS APPLICATION TO
REPRODUCTIVE TOXICITY
Risk assessment is the process that defines the potential adverse health consequences
of exposure to a toxic agent. The National Research Council (NRC) of the National
Academy of Sciences defines risk assessment as comprising some or all of the following
components: hazard identification, dose-response assessment, exposure assessment, and risk
characterization (National Research Council, 1983). This approach was derived for
carcinogens, but in general, this process is appropriate for human reproductive risk
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assessment as well. However, several significant factors lead to certain differences in risk
assessment for reproductive effects for which a threshold is assumed. Below a threshold
level of exposure, a chemical is not considered to be a reproductive hazard. Related factors
that are significant include the process of defining an adverse effect, consideration of
reversibility of effects, and consideration of target effects in the presence of other toxic
effects. In practice, hazard identification for reproductive effects usually includes an
evaluation of dose-response relationships, because the determination of a hazard may be
dependent on whether a dose-response relationship exists and, ideally, on whether
information is available on potential human exposures. A reproductive hazard for humans
is defined in terms of the range of effective doses, route of exposure, timing and duration
of exposure, and other relevant factors. For this reason, these Guidelines present hazard
identification and dose-response evaluation together (Sections III.F and III.G.) and include
both in the characterization of the health-related data as sufficient or insufficient to proceed
with a quantitative risk assessment. If data are sufficient for quantitative risk assessment, a
reference dose (RfD), reference concentration (RfC), or margin of exposure (MOE) for
reproductive toxicity can then be derived for comparison with human exposure estimates
(Section III.H.).
As discussed more fully in Section V (Risk Characterization), the components of the
risk assessment process are not considered in isolation. Appreciation of the potential for
human risk comes in part from integration of the hazard identification and dose-response
evaluation with the human exposure estimates in the final risk characterization. The final
assessment of risk depends on full consideration of all of these factors.
Hazard identification/dose-response evaluation involves the evaluation of all
available human and experimental animal data on the effects of exposures as well as the
associated doses, routes, durations, and patterns of exposure to determine if an agent is
likely to cause reproductive toxicity. In addition to reproductive effects, all other
manifestations of toxicity are examined in describing the effects for a given exposure. This
description includes evaluating the relationships between endpoints at a given dose as well
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as the progression of toxicity across doses. Adequacy of the health-related data to be used
further for quantitative risk assessment is characterized then, based on criteria defining a
sufficient data base outlined in these Guidelines (Section III.G.).
The evaluation of dose-response relationships includes the identification of effective
dose levels as well as doses associated with low or no increased incidence of adverse
effects when compared with controls. Ideally, a dose-response relationship would be
established from human epidemiologic data that include the expected levels of exposure.
Such data are seldom available. When the data are limited to test species, the relevance of
the test system to humans must be considered. The Agency typically uses a dose-response
approach in which uncertainty factors are applied to the no-observed-adverse-effect level
(NOAEL), or lowest-observed-adverse-effect level (LOAEL) if a NOAEL is not available,
to extrapolate from experimental animals to humans and to compensate for variability
within the human population. Because of limitations associated with the use of the
NOAEL, the Agency also is evaluating an alternative approach to quantitative dose-
response evaluation, i.e., the benchmark dose (Crump, 1984) (Section III.F.). Uncertainty
factors would be applied also to a benchmark dose. In either case, the value derived is a
RfD or RfC. These Guidelines discuss these approaches (Section III.H.).
The hazard identification/dose-response evaluation concludes with a determination of
the sufficiency of the health-related data base to assess potential human risk (Section
III.G.). This determination reflects the confidence of the risk assessor in the data base with
respect to the evidence for causation and ability to estimate effects at low doses. However,
it is only when the risk assessment process is carried through the remaining phases that the
actual risk to humans can be estimated for the exposure conditions that human populations
may experience. Thus, although an agent may cause an adverse effect in laboratory
animals, the level of potential human exposure should be evaluated before the agent is
considered to have potential risk to the human reproductive system. In the absence of
human exposure information, potential for human toxicity may be assumed from test
species results that show reproductive toxicity.
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Exposure assessment identifies and describes populations exposed or potentially
exposed to an agent, and presents the type, magnitude, frequency, and duration of such
exposures. Those procedures are considered separately in the Guidelines for Exposure
Assessment (U.S. Environmental Protection Agency, 1992). However, unique
considerations for reproductive toxicity exposure assessments are detailed in Section IV.
In risk characterization, the hazard identification/dose-response evaluation and the
exposure assessment are combined to estimate the risk of human reproductive toxicity. As
part of risk characterization, the strengths and weaknesses in each component of the risk
assessment are summarized along with major assumptions, scientific judgments, and to the
extent possible, qualitative descriptions and quantitative estimates of the uncertainties. The
sufficiency of the health-related data is presented with information on dose-response, the
RfD or RfC, and if available, the human exposure estimate. Here the NOAEL (or the
benchmark dose if adopted for Agency use) and the estimated human exposure levels may
be compared in a ratio to provide a margin of exposure (MOE). The considerations for
evaluating the MOE are similar to those used in determining the appropriate size of the
uncertainty factor for calculating the RfD or RfC.
Risk assessment is just one component of the regulatory process. The other
component, risk management, uses the risk characterization along with the directives of the
enabling regulatory legislation and other factors to decide whether to control exposure to
the suspected agent and the level of control. The risk management decisions also consider
socioeconomic, technical, and political factors. Risk management is not discussed directly
in these guidelines.
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I. OVERVIEW
These Guidelines for Reproductive Toxicity Risk Assessment (hereafter called
Guidelines) describe the procedures that the U.S. Environmental Protection Agency (EPA;
Agency) will follow in using existing data to evaluate the potential toxicity of
environmental agents to the human male and female reproductive systems and to outcomes
of pregnancy. These Guidelines focus on reproductive function as it relates to sexual
behavior, fertility, pregnancy outcomes, and lactating ability, and the processes that can
affect those functions directly. Included are effects on gametogenesis and gamete
maturation and function, the reproductive organs, and the components of the endocrine
system that directly support those functions. These Guidelines concentrate on the integrity
of the male and female reproductive systems as required to ensure successful procreation.
They also emphasize the importance of maintaining the integrity of the reproductive system
for overall physical and psychologic health. The Guidelines for Developmental Toxicity
Risk Assessment (U.S. Environmental Protection Agency, 1991) focus on effects of agents
on development specifically, and should be used as a companion to these Guidelines.
In evaluating reproductive effects, it is important to consider the presence, and where
possible, the contribution of other manifestations of toxicity such as developmental toxicity,
mutagenicity, or carcinogenicity. The reproductive process is such that these areas overlap,
and all should be considered in reproductive risk assessments.
Although the endpoints discussed in these Guidelines can detect impairment to
components of the reproductive process, they may not discriminate effectively between
nonmutagenic (e.g., cytotoxic) and mutagenic mechanisms. Examples of endpoints affected
by either type of mechanism are sperm head morphology and preimplantation loss. If the
effects seen may result from mutagenic events, then there is the potential for transmissible
genetic damage. In such cases, the Guidelines for Mutagenicity Risk Assessment (U.S.
Environmental Protection Agency, 1986c) should be consulted in conjunction with this
document. The Guidelines for Cancer Risk Assessment (currently under review) (U.S.
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Environmental Protection Agency, 1986a) should be consulted if reproductive system or
developmentally-induced cancer is detected.
For assessment of risk to the human reproductive systems, the most appropriate data
are those derived from human studies. In the absence of adequate human data, our
understanding of the mechanisms controlling reproduction supports the use of data from
experimental animal studies to estimate the risk of reproductive effects in humans.
However, some information needed for extrapolation of data from experimental animal
studies to humans is not generally available. Therefore, to bridge these gaps in information,
a number of assumptions are made. These assumptions should not preclude inquiry into the
relevance of the data to potential human risk. These assumptions provide the inferential
basis for the approaches to risk assessment in these Guidelines. Each assumption should be
evaluated along with other relevant information in making a final judgement as to human
risk for each agent, and that information summarized in the risk characterization.
First, an agent that produces an adverse reproductive effect in experimental animal
studies is assumed to pose a potential reproductive hazard to humans. This assumption is
based on comparisons of data for known human reproductive toxicants (Thomas, 1981;
Nisbet & Karch, 1983; Kimmel, C.A. et al., 1984, 1990; Hemminki & Vineis, 1985;
Meistrich, 1986; Working, 1988). In general, the experimental animal data indicated
adverse reproductive effects that are also seen in humans.
Because similar mechanisms can be identified in the male and female of many
mammalian species, effects of xenobiotics on male and female reproductive processes are
assumed generally to be similar across species unless demonstrated otherwise. However,
for effects on pregnancy outcomes, it is assumed that the effects seen in experimental
animal studies are not necessarily the same as those produced in humans. This latter
assumption is made because every species may not react in the same way because of
species-specific differences in timing of exposure relative to critical periods of
development, pharmacokinetics (including metabolism), developmental patterns,
placentation, or mechanisms of action.
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When sufficient data are available (e.g., pharmacokinetic), the most appropriate
species should be used to estimate human risk. In the absence of such data, it is assumed
that the most sensitive species is most appropriate because, for the majority of known
human reproductive toxicants, humans appear to be as or more sensitive than the most
sensitive animal species tested (Nisbet & Karch, 1983; Kimmel, C.A. et al., 1984, 1990;
Hemminki & Vineis, 1985; Meistrich, 1986; Working, 1988), based on data from studies
that determined dose on a body weight or air concentration basis.
In the absence of specific information to the contrary, it is assumed that a chemical
that acts as a reproductive toxicant in one sex may also adversely affect reproductive
function in the other sex. This assumption for reproductive risk assessment is based on
three considerations: (1) For most agents, the nature of the testing and the data available are
limited, reducing confidence that the potential for toxicity to both sexes and their offspring
has been examined equally; (2) Exposures of either males or females have resulted in
developmental toxicity, although studies of developmental effects resulting from male
exposures have been limited (Davis et al., 1992); and (3) Many of the mechanisms
controlling important aspects of reproductive system function are similar in females and
males, and could therefore be susceptible to the same agents. Information that could negate
this assumption would demonstrate (1) that a mechanistic difference existed between the
sexes that would preclude toxic action on the other sex or (2) that, on the basis of sufficient
testing, an agent did not produce an adverse reproductive effect when administered to the
other sex.
In general, a threshold is assumed for the dose-response curve for reproductive
toxicity. This is based on the known capacity of cells, tissues, and organs of the
reproductive systems and the developing organism to compensate for or to repair a certain
amount of damage. Furthermore, multiple insults at the molecular or cellular level may be
required to produce an adverse effect. Therefore, although the levels of exposure at which
different individuals react adversely to an agent may differ, each individual should have an
exposure level below which no increased risk exists.
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II. DEFINITIONS AND TERMINOLOGY
The following terms are defined according to their usage in this document:
Reproductive toxicity - The occurrence of adverse effects on the reproductive systems that
may result from exposure to environmental agents. The toxicity may be expressed as
alterations to the female or male reproductive organs, the related endocrine system, or
pregnancy outcomes. The manifestation of such toxicity may include, but not be limited to,
adverse effects on onset of puberty, gamete production and transport, reproductive cycle
normality, sexual behavior, fertility, gestation, parturition, lactation, pregnancy outcomes,
premature reproductive senescence, or modifications in other functions that are dependent
on the integrity of the reproductive systems.
Fertility - The ability to conceive and to produce offspring within a defined period of time.
For litter-bearing species, the number of offspring per litter is also a measure of fertility.
Fertile - Having a level of fertility that is within or exceeds the normal range for that
species.
Infertile - Lacking fertility for a specified period. The infertile condition may be
temporary; permanent infertility is termed sterility.
Subfertile - Having a level of fertility that is below the normal range for that species but
not infertile.
Developmental toxicity - The occurrence of adverse effects on the developing organism that
may result from exposure prior to conception (either parent), during prenatal development,
or postnatally to the time of sexual maturation. Adverse developmental effects may be
detected at any point in the life span of the organism. The major manifestations of
developmental toxicity include (1) death of the developing organism, (2) structural
abnormality, (3) altered growth, and (4) functional deficiency (U.S. Environmental
Protection Agency, 1991).
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III. HAZARD IDENTIFICATION/DOSE-RESPONSE EVALUATION OF
REPRODUCTIVE TOXICANTS
This section presents the traditional testing protocols for rodents and endpoints used
to evaluate male and female reproductive toxicity along with evaluation of their strengths
and limitations. Because many endpoints are common to multiple protocols, endpoints are
considered separately from the discussion of the overall protocol structures.
III.A. LABORATORY TESTING PROTOCOLS
III.A. 1. Introduction
Testing protocols describe the procedures to be used to provide data for risk
assessments. The quality and usefulness of those data are dependent on the design and
conduct of the tests, including endpoint selection and resolving power. A single protocol is
unlikely to provide all of the information that would be optimal for conducting a
comprehensive risk assessment. For example, the test design to study reversibility of
adverse effects or mechanism of toxic action may be different from that needed to
determine time of onset of an effect or for calculation of a safe level for repeated exposure
over a long term. Ideally, results from several different types of tests should be available
when performing a risk assessment. Typically, only limited data are available. Under those
conditions, the limited data should be used to the extent possible to assess risk.
An integral part of the hazard identification and dose-response process includes
evaluation of the protocols from which data are available and the quality of the resulting
data. In this section, design factors that are of particular importance in reproductive
toxicity testing are discussed. Then, standardized protocols that may provide useful data for
reproductive risk assessments are described.
III.A.2. Duration of Dosing
To evaluate adequately the potential effects of an agent on the reproductive systems,
a prolonged treatment period is needed. For example, damage to spermatogonial stem cells
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will not appear in samples from the cauda epididymis or in ejaculates for 8 to 14 weeks,
depending on the test species. With some chemical agents that bioaccumulate, the full
impact on a given cell type could be further delayed, as could the impact on functional
endpoints such as fertility. In such situations, adequacy of the dosing duration is a critical
factor in the risk assessment.
Conversely, adaptation may occur that allows tolerance to levels of a chemical that
initially caused an effect that could be considered adverse. An example is interference with
ovulation by chlordimeform (Goldman et al., 1991); an effect for which a compensatory
mechanism is available. Thus, with continued dosing, the compensatory mechanism can be
activated so that the initial adverse effect is masked.
In these situations, knowledge of the relevant pharmacokinetic and
pharmacodynamic data can facilitate selection of dose levels and treatment duration (see
also section on Exposure Assessment). Equally important is proper timing of examination
of treated animals relative to initiation and termination of exposure to the agent.
III.A.3. Length of Mating Period
Traditionally, pairs of rats or mice are allowed to cohabit for periods ranging from
several days to 3 weeks. Given a 4- or 5-day estrous cycle, each female that is cycling
normally should be in estrus four or five times during a 21-day mating period. Therefore,
information on the interval or the number of cycles needed to achieve pregnancy may
provide evidence of reduced fertility that is not available from fertility data. Additionally,
during each period of behavioral estrus, the male has the opportunity to copulate a number
of times, resulting in delivery of many more sperm than are required for fertilization.
When an unlimited number of matings is allowed in fertility testing, a large effect on sperm
production is necessary before an effect on fertility can be detected.
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III.A.4. Number of Females Mated to Each Male
Current EPA test guidelines prepared pursuant to FIFRA and TSCA specify the use
of 20 males and enough females to produce at least 20 pregnancies for each dose group in
each generation in the multigeneration reproduction test (U.S. Environmental Protection
Agency, 1982, 1985b). However, in some tests that were not designed to conform to EPA
test guidelines (Organization for Economic Cooperation and Development, 1983), 20
pregnancies may have been achieved by mating two females with each male and using
fewer than 20 males per treatment group. In such cases, the statistical treatment of the data
should be examined carefully. With multiple females mated to each male, the degree of
independence of the observations for each female may not be known. In that situation,
when the cause of the adverse effect cannot be assigned with confidence to only one sex,
dependence should be assumed and the male used as the experimental unit in statistical
analyses. Using fewer males as the experimental unit reduces ability to detect an effect.
III.A.5. Single- and Multigeneration Reproduction Tests
Reproductive toxicity studies in laboratory animals generally involve continuous
exposure to a test substance for one or more generations. The objective is to detect effects
on the integrated reproductive process as well as to study effects on the individual
reproductive organs. Test guidelines for the conduct of single- and multigeneration repro-
duction protocols have been published by the Agency pursuant to FIFRA and TSCA and by
OECD (U.S. Environmental Protection Agency, 1982, 1985b; Galbraith et al., 1983;
Organization for Economic Cooperation and Development, 1983).
The single-generation reproduction test evaluates effects of subchronic exposure of
peripubertal and adult animals. In the multigeneration reproduction protocol, Fj and ^2
offspring are exposed continuously in utero from conception until birth and during the
preweaning period. This allows detection of effects that occur from exposures throughout
development, including the peripubertal and young adult phases. Because the parental and
subsequent filial generations have different exposure histories, reproductive effects seen in
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any particular generation are not necessarily comparable with those of another generation.
Also, successive litters from the same parents cannot be considered as replicates because of
factors such as continuing exposure of the parents, increased parental age, sexual
experience, and parity of the females.
In a single- or multigeneration reproduction test, rats are used most often. In a
typical reproduction test, dosing is initiated at 5 to 8 weeks of age and continued for 8 to
10 weeks prior to mating to allow effects on gametogenesis to be expressed in ejaculates.
Three dose levels plus one or more control groups are usually included. Enough males and
females are mated to ensure 20 pregnancies per dose group for each generation. Animals
producing the first generation of offspring should be considered the parental (P) generation,
and all subsequent generations should be designated filial generations (e.g., Fj, F2). Only
the P generation is mated in a single-generation test, while both the P and Fj generations
are mated in a two-generation reproduction test.
In the P generation, both females and males are treated prior to and during mating,
with treatment usually beginning around puberty. Cohabitation is allowed for up to 3
weeks, during which the females are monitored for evidence of mating. Females continue
to be exposed during gestation and lactation.
In the two-generation reproduction test, randomly selected Fj offspring continue to
be exposed after weaning (day 21) and then are mated at 11 to 13 weeks of age. Treatment
of mated Fj females is continued throughout gestation and lactation. More than one litter
may be produced from either P or Fj animals. Depending on the route of exposure of
lactating females, it is important to consider that offspring may be exposed additionally to a
chemical by ingestion of maternal feed or water (diet or drinking water studies), by licking
of exposed fur (inhalation study), by contact with treated skin (dermal study), or by
coprophagia.
In single- and multigeneration reproduction tests, reproductive endpoints evaluated in
P and F generations usually include visual examination of the reproductive organs. Weights
and histopathology of the testes, epididymides, and accessory sex glands may be available
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from males, and histopathology of the vagina, uterus, cervix, ovaries, and mammary glands
from females. Uterine and ovarian weights are also often available. Male and female
mating and fertility indices (Section III.B.2.a.) are usually presented. In addition, litters
(and often individual pups) are weighed at birth and examined for number of live and dead
offspring, gender, gross abnormalities, and growth and survival to weaning. Maturation and
behavioral testing may also be performed on the pups.
If effects on fertility or pregnancy outcome are the only adverse effects observed in
a study using one of these protocols, the contributions of male- and female-specific effects
often cannot be distinguished. If testicular histopathology or sperm evaluations have been
included, it may be possible to characterize a male-specific effect. Similarly, ovarian and
reproductive tract histology or changes in estrous cycle normality may be indicative of
female-specific effects. However, identification of effects in one sex does not exclude the
possibility that both sexes may have been affected adversely. Data from matings of treated
males with untreated females and vice versa (crossover matings) are necessary to separate
sex-specific effects.
An EPA workshop has considered the relative merits of one- versus two-generation
reproductive effects studies (Francis & Kimmel, 1988). The participants concluded that a
one-generation study is insufficient to identify all potential reproductive toxicants, because
it would exclude detection of effects caused by prenatal and postnatal exposures (including
the prepubertal period) as well as effects on germ cells that could be transmitted to and
expressed in the next generation. A one-generation test might also miss adverse effects
with delayed or latent onset because of the shorter duration of exposure for the P
generation. Therefore, a comprehensive reproductive risk assessment should include results
from a two-generation test. A further recommendation from that workshop was to include
sperm analyses and estrous cycle normality as endpoints in reproductive effects studies.
In studies where parental and offspring generations are evaluated, there are
additional risk assessment issues regarding the relationships of reproductive outcomes
across generations. Increasing vulnerability of subsequent generations is often, but not
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always, observed. Qualitative predictions of increased risk of the filial generations could be
strengthened by knowledge of the reproductive effects in the adult, the likelihood of
bioaccumulation of the agent, and the potential for increased sensitivity resulting from
exposure during critical periods of development (Gray, 1991).
Occasionally, the severity of effects may be static or decrease with succeeding
generations. When a decrease occurs, one explanation may be that the animals in the Fj
and ?2 generations represent "survivors" who are (or become) more resistant to the agent
than the average of the P generation. If such selection exists, then subsequent filial
generations may show a reduced toxic response. Thus, significant adverse effects in any
generation may be cause for concern regardless of results in other generations unless
inconsistencies in the data indicate otherwise.
III.A.6. Alternative Reproductive Tests
A number of alternative test designs have appeared in the literature (Lamb, 1985;
Lamb & Chapin, 1985; Gray et al., 1988, 1989, 1990; Morrissey et al., 1989). Although
not necessarily viewed as replacements for the standard two-generation reproduction tests,
data from these protocols may be used on a case-by-case basis depending on what is known
about the test agent in question. When mutually agreed on by the testing organization and
the Agency, such alternative protocols may offer an expanded array of endpoints and
increased flexibility (Francis & Kimmel, 1988).
A continuous breeding protocol, Fertility (or Reproductive) Assessment by
Continuous Breeding (FACE or RACE), has been developed by the National Toxicology
Program (Lamb & Chapin, 1985; Morrissey et al., 1989; Gulati et al., 1991). As originally
described, this protocol was a one-generation test. However, dosing can be extended into
the F! generation to make it compatible with the EPA workshop recommendations for a
two-generation design (Francis & Kimmel, 1988). The RACB protocol is being used with
both mice and rats. A distinctive feature of this protocol is the continuous cohabitation of
male-female pairs (in the P generation) for 14 weeks. Up to five litters can be produced
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with the pups removed soon after birth. This protocol provides information on changes in
the spacing, number, and size of litters over the 14 week dosing interval. Treatment (three
dose levels plus controls) is initiated in postpubertal males and females (11 weeks of age)
seven days before cohabitation and continues throughout the test. Offspring that are
removed from the dam soon after birth are counted and examined for viability, litter and/or
pup weight, sex, and external abnormalities. The last litter may remain with the dam until
weaning to study the effects of in utero as well as perinatal and postnatal exposures. If
effects on fertility are observed in the P or F generations, additional reproductive
evaluations may be conducted, including fertility studies and crossover matings to define
the affected gender and site of toxicity.
The sequential production of litters from the same adults allows observation of the
timing of onset of an adverse effect on fertility. In addition, it may improve ability to
detect subfertility due to the potential to produce larger numbers of pregnancies and litters
than in a standard single- or multigeneration reproduction study. With continuous
treatment, a cumulative effect could increase the incidence or extent of expression with
subsequent litters. However, unless offspring are allowed to grow and reproduce (as they
are in the more recent version of the RACB protocol) (Gulati et al., 1991), little or no
information will be available on postnatal development or reproductive capability of a
second generation.
Sperm measures (including sperm number, morphology, and motility) and vaginal
smear cytology to detect changes in estrous cyclicity have been added to the RACB
protocol at the end of the test period (although not at all dose levels) and their utility has
been examined using model compounds in the mouse (Morrissey et al., 1989).
Another test method under development combines the use of multiple endpoints in
both sexes of rats with initiation of treatment at weaning (Gray et al., 1988). Thus,
morphologic and physiologic changes associated with puberty are included as endpoints.
Both P sexes are treated (at least three dose levels plus controls) continuously through
breeding, pregnancy, and lactation. The Fj generation is mated in a continuous breeding
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protocol. Vaginal smears are recorded daily throughout the test period to evaluate estrous
cycle normality and confirm breeding and pregnancy (or pseudopregnancy). Pregnancy
outcome is monitored in both the P and Fj generations at all doses, and terminal studies on
both generations include comprehensive assessment of sperm measures (numbers,
morphology, motility) as well as organ weights, histopathology, and the serum and tissue
levels of appropriate reproductive hormones. As with the RACB, crossover mating studies
may be conducted to identify the affected sex as warranted. This protocol combines the
advantages of a continuous breeding design with acquisition of sex-specific multiple
endpoint data at all doses. In addition, identification of pubertal effects makes this protocol
particularly useful for detecting compounds with hormone-mediated actions such as
environmental estrogens.
III.A.7. Additional Test Protocols That May Provide Reproductive Data
Several shorter-term reproductive toxicity screening tests have been developed.
Among those are the Reproductive/Developmental Toxicity Screening Test, which is part of
the OECD's Screening Information Data Set (SIDS) protocol (Scala et al., 1992; Tanaka et
al., 1992; Organization for Economic Cooperation and Development, 1993a), a draft
protocol from the International Conference on Harmonization (Manson, 1994), and the
National Toxicology Program's Short-Term Reproductive and Developmental Toxicity
Screen (Harris et al., 1992). These protocols have been developed for setting priorities for
further testing and should not be considered sufficient by themselves to establish regulatory
exposure levels. Their limited exposure periods do not allow assessment of certain aspects
of the reproductive process, such as developmentally-induced effects on the reproductive
systems of offspring, that are covered by the multi-generation reproduction protocols.
The dominant lethal test was designed to detect mutagenic effects in the male
spermatogenic process that are lethal to the offspring. A review of this test has been
published as part of the EPA's Gene-Tox program (Green et al., 1985). Dominant lethal
protocols may use acute dosing (1 to 5 days) followed by serial matings with one or two
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females per male per week for the duration of the spermatogenic process. An alternative
protocol may use subchronic dosing for the duration of the spermatogenic process followed
by mating. Females are monitored for evidence of mating, killed at approximately
midgestation, and examined for incidence of pre- and postimplantation loss (see Section
III.B.2. for discussions of these endpoints).
Pre- or postimplantation loss in the dominant lethal test is often considered evidence
that the agent has induced mutagenic damage to the male germ cell (U.S. Environmental
Protection Agency, 1986c). A genotoxic basis for a substantial portion of postimplantation
loss is accepted widely. However, methods used to assess preimplantation loss do not
distinguish between contributions of mutagenic events that cause embryo death and
nonmutagenic factors that result in failure of fertilization or early embryo mortality (e.g.,
inadequate number of normal sperm, failure in sperm transport or ovum penetration).
Similar effects (fertilization failure, early embryo death) could also be produced indirectly
by effects that delay the timing of fertilization relative to time of ovulation. Such
distinctions are important because cytotoxic effects on gametogenic cells do not imply the
potential for transmittable genetic damage that is associated with mutagenic events. The
interpretation of an increase in preimplantation loss may require additional data on the
agent's mutagenic and gametotoxic potential if genotoxicity is to be factored into the risk
assessment. Regardless, significant effects may be observed in a dominant lethal test that
are considered reproductive in nature.
An acute exposure protocol, combined with serial mating, may allow identification
of the spermatogenic cell types that are affected by treatment. However, acute dosing may
not produce adverse effects at levels as low as with subchronic dosing because of factors
such as bioaccumulation. Conversely, if tolerance to an agent is developed with longer
exposure, an effect may be observed after acute dosing that is not detected after longer term
dosing.
Subchronic toxicitv tests may have been conducted before a detailed reproduction
study is initiated. In the subchronic toxicity test with rats, exposure usually begins at six to
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eight weeks of age and is continued for 90 days (U.S. Environmental Protection Agency,
1982, 1985b). Initiation of exposure at eight weeks of age (compared with six) and
exposure for approximately 90 days allows the animals to reach a more mature stage of
sexual development and assures an adequate length of dosing for observation of effects on
the reproductive organs with most agents. The route of administration is often oral or by
gavage but may be dermal or by inhalation. Animals are monitored for clinical signs
throughout the test and are necropsied at the end of dosing.
The endpoints that are usually evaluated for the male reproductive system include
visual examination of the reproductive organs, plus weights and histopathology for the
testes, epididymides, and accessory sex glands. For the female, endpoints may include
visual examination of the reproductive organs, uterine and ovarian weights, and
histopathology of the vagina, uterus, cervix, ovaries, and mammary glands.
This test may be useful to identify an agent as a potential reproductive hazard, but
usually does not provide information about the integrated function of the reproductive
systems (sexual behavior, fertility and pregnancy outcomes), nor does it include effects of
the agent on immature animals.
Chronic toxicity tests provide an opportunity to evaluate toxic effects of long-term
exposures. Oral, inhalation or dermal exposure is initiated soon after weaning and is
usually continued for 12 to 24 months. Because of the extended treatment period, interim
sacrifices may be available to provide useful information regarding the onset and sequence
of toxicity. In males, the reproductive organs are examined visually, testes are weighed,
and histopathologic examination is done on the testes and accessory sex glands. In females,
the reproductive organs are examined visually, uterine and ovarian weights may be
obtained, and histopathologic evaluation of the reproductive organs is done. The incidence
of abnormalities is often increased in the reproductive tracts of aged control animals.
Therefore, findings should be interpreted carefully.
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III.B. ENDPOINTS FOR EVALUATING MALE AND FEMALE REPRODUCTIVE
TOXICITY IN TEST SPECIES
III.B. 1. Introduction
The following discussion emphasizes endpoints that measure characteristics that are
necessary for successful sexual performance and procreation. Other areas that are related
less directly to reproduction are beyond the scope of these Guidelines. For example,
adverse health effects that may result from toxicity to the reproductive organs (e.g.,
osteoporosis or altered immune function), although important, are not included.
In these Guidelines, the endpoints of reproductive toxicity are separated into three
categories: couple-mediated, female-specific, and male-specific. Couple-mediated endpoints
are those in which both sexes have a contributing role. Thus, an effect on either sex or
both sexes may result in an effect on that endpoint.
The discussions of endpoints and the factors influencing results that are presented in
this section are directed to evaluation and interpretation of results with test species. Many
of those endpoints require invasive techniques that preclude routine use with humans.
However, in some instances (e.g., Tables 3 and 4), related endpoints that can be used with
humans are identified. Information that is specific for evaluation of effects on humans is
presented in Section III.C.
Although statistical analyses are important in determining the effects of a particular
agent, the biological significance of data is most important. It is important to be aware that
when many endpoints are investigated, statistically significant differences may occur by
chance. On the other hand, apparent trends with dose may be biologically relevant even
though pair-wise comparisons do not indicate a statistically significant effect. In each
section, endpoints are identified in which significant changes may be considered adverse.
However, concordance of results and known biology should be considered in interpreting
all results. Results should be evaluated on a case-by-case basis with all of the evidence
considered. Scientific judgment should be used extensively. All effects that may be
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considered as adverse are appropriate for use in establishing a NOAEL, LOAEL, or
benchmark dose.
III.B.2. Couple-mediated Endpoints
Data on fertility potential and associated reproductive outcomes provide the most
comprehensive and direct insight into reproductive capability. As noted previously, most
protocols specify cohabitation of exposed males with exposed females. This complicates
the resolution of gender-specific influences. Conclusions may need to be restricted to
noting that the "couple" is at reproductive risk when one or both parents are potentially
exposed.
III.B.2.a. Fertility and Pregnancy Outcomes
Breeding studies with test species are a major source of data on reproductive
toxicants. Evaluations of fertility and pregnancy outcomes provide measures of the
functional consequences of reproductive injury. Measures of fertility and pregnancy
outcome that are often obtained from multigeneration reproduction studies are presented in
Table 1. Many endpoints that are pertinent for developmental toxicity are also listed and
discussed in the Agency Guidelines for Developmental Toxicity Risk Assessment (U.S.
Environmental Protection Agency, 1991). Also included in Table 1 are measures that may
be obtained from other types of studies (e.g., single-generation reproduction studies,
developmental toxicity studies, dominant lethal studies) in which offspring are not retained
to evaluate subsequent reproductive performance. Significant detrimental effects on any of
those endpoints should be considered adverse. Whether effects are on the female
reproductive system or directly on the embryo or fetus is often not distinguishable, but the
distinction may not be important because all of these effects should be cause for concern.
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TABLE 1
Couple-mediated Endpoints of Reproductive Toxicity
Multigeneration studies Other reproductive endpoints
Mating rate, time to mating Ovulation rate
(Time to pregnancy*) Fertilization rate
Pregnancy rate* Preimplantation loss
Delivery rate* Implantation number
Gestation length* Postimplantation loss*
Litter size (total and live) Internal malformations
Number of live and dead offspring and variations*
(Fetal death rate*) Postnatal structural and
Offspring gender* functional development*
Birth weight*
Postnatal weights*
Offspring survival*
External malformations
and variations*
Offspring reproduction*
* Endpoints that can be obtained with humans
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Some of the endpoints identified above are used to calculate ratios or indices
(National Research Council, 1977; Collins, 1978; Schwetz et al., 1980; U.S. Environmental
Protection Agency, 1982, 1985b; Dixon & Hall, 1984; Lamb et al., 1985; Thomas, 1991).
While the presentation of such indices is not discouraged, the measurements used to
calculate those indices should also be available for evaluation. Definitions of some of these
indices in published literature vary substantially. Also, the calculation of an index may be
influenced by the test design. Therefore, it is important that the methods used to calculate
indices be specified. Some commonly reported indices are in Table 2.
Mating rate may be reported for the mated pairs, males only or females only.
Evidence of mating may be direct observation of copulation, observation of copulatory
plugs, or observation of sperm in the vaginal fluid (vaginal lavage). The mating rate may
be influenced by the number of estrous cycles allowed or required for pregnancy to occur.
The most meaningful measure is derived from the occurrence of mating during the first
estrous cycle after initiation of cohabitation. Evidence of mating does not necessarily mean
successful impregnation.
A useful indicator of impaired reproductive function may be the length of time
required for each pair to mate (time to mating). An increased interval between initiation of
cohabitation and evidence of mating suggests abnormal estrous cyclicity in the female or
impaired sexual behavior in one or both partners.
The time to mating for normal pairs (rat or mouse) could vary by 3 or 4 days
depending on the stage of the estrous cycle at which they were paired. If the stage of the
estrous cycle at time of cohabitation is known, the component of the variance due to
variation in stage at cohabitation can be removed in the statistical analysis.
Data on fertilization rate, the proportion of available ova that were fertilized, are
seldom available because the measurement requires necropsy very early in gestation.
Pregnancy rate is the proportion of mated pairs that have produced at least one pregnancy
within a fixed period where pregnancy is determined by the earliest available evidence that
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TABLE 2
Selected Indices That May Be Calculated From
Endpoints of Reproductive Toxicity in Test Species
MATING INDEX
Number of males or females mating X 100
Number of males or females cohabited
Note: Mating is used to indicate that evidence of copulation (observation or other
evidence of ejaculation such as vaginal plug or sperm in vaginal smear) was obtained.
FERTILITY INDEX
Number of cohabited females becoming pregnant X 100
Number of nonpregnant couples cohabited
Note: Because both sexes are often exposed to an agent, distinction between sexes
is often not possible. If responsibility for an effect can be clearly assigned to one sex (as
when treated animals are mated with controls), then a female or male fertility index could
be useful.
GESTATION INDEX
Number of females delivering live young X 100
Number of females with evidence of pregnancy
LIVE BIRTH INDEX
Number of live offspring X 100
Number of offspring delivered
SEX RATIO
Number of male offspring
Number of female offspring
4-DAY SURVIVAL INDEX (VIABILITY INDEX)
Number of live offspring at lactation day 4 X 100
Number of live offspring delivered
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TABLE 2 (continued)
Note: This definition assumes that no standardization of litter size is done until after the
day 4 determination is completed.
LACTATION INDEX (WEANING INDEX)
Number of live offspring at day 21 X 100
Number of live offspring born
Note: If litters were standardized to equalize numbers of offspring per litter, number of
offspring after standardization should be used instead of number born alive. When no
standardization is done, measure is called weaning index. When standardization is done,
measure is called lactation index.
PREWEANING INDEX
Number of live offspring born -
Number of offspring weaned X 100
Number of live offspring born
Note: If litters were standardized to equalize numbers of offspring per litter, then number
of offspring remaining after standardization should be used instead of number born.
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fertilization has occurred. Generally, a more meaningful measure of fertility results when
the mating opportunity was limited to one mating couple and to one estrous cycle (see
Sections III.A.3. and III.A.4.).
The timing and integrity of gamete and zygote transport are important to fertilization
and embryo survival and are quite susceptible to chemical perturbation. Disruption of the
processes that contribute to a reduction in fertilization rate and early embryo loss are
usually identified simply as preimplantation loss. Additional studies using direct
assessments of fertilized ova and early embryos would be necessary to identify the cause of
increased preimplantation loss (Cummings & Perreault, 1990). Preimplantation loss
(described below) occurs in untreated as well as treated rodents and contributes to the
variation in litter size.
After mating, uterine and oviductal contractions are critical in the transport of
spermatozoa from the vagina. In rodents, sufficient stimulation during mating is necessary
for initiation of those contractions. Thus, impaired mating behavior may affect sperm
transport and fertilization rate. Exposure of the female to estrogenic compounds is known
to alter gamete transport. In women, low doses of exogenous estrogens may accelerate
ovum transport to a detrimental extent, whereas high doses of estrogens or progestins delay
transport and increase the incidence of ectopic pregnancies.
Mammalian ova are surrounded by investments that the sperm must penetrate before
fusing with ova. Chemicals may block fertilization by preventing this passage. Other
agents may impair fusion of the sperm with the ovum plasma membrane, transformations of
the sperm or ovum chromatin into the male and female pronuclei, fusion of the pronuclei,
or the subsequent cleavage divisions. Carbendazim, an inhibitor of microtubule synthesis,
is an example of a chemical that can interfere with fertilization (Perreault et al., 1992). The
early zygote is also susceptible to detrimental effects of mutagens such as ethylene oxide
(Generoso et al., 1987).
Fertility assessments in test animals have limited sensitivity as measures of
reproductive injury. Therefore, results demonstrating no treatment-related effect on fertility
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may be given less weight than other endpoints that are more sensitive. Unlike humans,
normal males of most test species produce sperm in numbers that greatly exceed the
minimum requirements for fertility, particularly as evaluated in protocols that allow
multiple matings (Amann, 1981; Working, 1988). In some strains of rats and mice,
production of normal sperm can be reduced by up to 90% or more without compromising
fertility (Aafjes et al., 1980; Meistrich, 1982; Robaire et al., 1984; Working, 1988).
However, less severe reductions can cause reduced fertility in human males who appear to
function closer to the threshold for the number of normal sperm needed to ensure full
reproductive competence (see Supplemental Information). This difference between test
species and humans means that negative results with test species in a study that was limited
to endpoints that examined only fertility and pregnancy outcomes would provide
insufficient information to conclude that the test agent poses no reproductive hazard in
humans. It is unclear whether a similar consideration is applicable for females for some
mechanisms of toxicity.
The limited sensitivity of fertility measures in rodents also suggests that a NOAEL,
LOAEL, or benchmark dose (see Section III.H.) based on fertility may not reflect
completely the extent of the toxic effect. In such instances, data from additional
reproductive endpoints might indicate that an adverse effect could occur at a lower dose
level. In the absence of such data, the margin of exposure or uncertainty factor applied to
the NOAEL, LOAEL, or benchmark dose may need to be adjusted to reflect the additional
uncertainty (see Section III.H.).
Both the blastocyst and the uterus must be ready for implantation, and their
synchronous development is critical (Cummings & Perreault, 1990). The preparation of the
uterine endometrium for implantation is under the control of sequential estrogen and
progesterone stimulation. Treatments that alter the internal hormonal environment, inhibit
protein synthesis, or inhibit mitosis or cell differentiation can block implantation and cause
embryo death.
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Gestation length can be determined in test animals from data on day of mating
(observation of vaginal plug or sperm-positive vaginal lavage) and day of parturition.
Significant shortening of gestation can lead to adverse outcomes of pregnancy such as
decreased birth weight and offspring survival. Significantly longer gestation may be caused
by failure of the normal mechanism for parturition and may result in death or impairment
of offspring if dystocia (difficulty in parturition) occurs. Dystocia also constitutes a
maternal health threat. Lengthened gestation may result in higher birth weight; an effect
that could mask a slower growth rate in utero because of exposure to a toxic agent.
Comparison of offspring weights based on conceptional age may allow insight, although
this comparison is complicated by generally faster growth rates postnatally than in utero.
Litter size is the number of offspring delivered and is measured at or soon after
birth. Unless this observation is made soon after parturition, the number of offspring
observed may be less than the actual number delivered because of cannibalism by the dam.
Litter size is affected by the number of ova available for fertilization (ovulation rate),
fertilization rate, implantation rate, and the proportion of the implanted embryos that
survives to parturition. Litter size may include dead as well as live offspring, therefore data
on the numbers of live and dead offspring should be available also.
When pregnant animals are examined by necropsy in mid- to late gestation,
pregnancy status, including pre- and postimplantation losses can be determined.
Preimplantation loss is the number of corpora lutea minus number of implantation
sites/number of corpora lutea. Postimplantation loss is the (number of implantation sites
minus number of live pups)/number of implantation sites.
Offspring gender is determined by the male through fertilization of an ovum by a Y-
or an X-chromosome-bearing sperm. Therefore, selective impairment in the production,
transport, or fertilizing ability of either of these sperm types can produce an alteration in the
sex ratio. An agent may also induce selective loss of male or female fetuses. Although not
examined routinely, these factors provide the most likely explanations for alterations in the
sex ratio.
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Birth weight should be measured on the day of parturition. Often data from
individual pups as well as the entire litter (litter weight) are provided. Birth weights are
influenced by intrauterine growth rates, litter size, and gestation length. Growth rate in
utero is influenced by the normality of the fetus, the maternal environment, and gender,
with females tending to be smaller than males. Individual pups tend to be smaller in larger
litters than individual pups in smaller litters. Thus, reduced birth weights that can be
attributed to large litter size should not be considered an adverse effect unless the increased
litter size is treatment related and the subsequent ability of the offspring to survive or
develop is compromised. When litter weights only are reported, the increased numbers of
offspring and the lower weights of the individuals tend to offset each other. When prenatal
or postnatal growth is impaired by an acute exposure, compensatory growth after cessation
of dosing could obscure the earlier effect.
Postnatal weights are dependent on birth weight, sex and normality of the individual,
as well as the litter size, lactational ability of the dam, and suckling ability of the offspring.
With large litters, small or weak offspring may not compete successfully for milk and show
impaired growth. Because it is not possible usually to determine whether the effect was
due solely to the increased litter size, growth retardation or decreased survival rate should
be considered adverse in the absence of information to the contrary. Also, offspring
weights may appear normal in very small litters and should be considered carefully in
relation to controls.
Offspring survival is dependent on the same factors as postnatal weight, although
more severe effects are necessary usually to affect survival. All weight and survival
endpoints can be affected by toxicity of an agent, either by direct effects on the offspring or
indirectly through effects on the ability of the dam to support the offspring.
Measures of malformations and variations, as well as postnatal structural and
functional development, are presented in the Guidelines for Developmental Toxicity Risk
Assessment (U.S. Environmental Protection Agency, 1991). That document should be
consulted for additional information on those parameters.
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III.B.2.b. Sexual Behavior
Sexual behavior reflects complex neural, endocrine and reproductive organ
interactions and is therefore susceptible to disruption by a variety of toxic agents, diseases,
and pathologic conditions. Interference with sexual behavior in either sex by environmental
agents represents a potentially significant human reproductive problem. Most human
information comes from from clinical reports in which the detection of exposure-effect
associations is unlikely. Data on sexual behavior are usually not available from studies of
human populations that were exposed occupationally or environmentally to potentially toxic
agents, nor are such data obtained routinely in studies of environmental agents with test
species.
In the absence of human data, the perturbation of sexual behavior in test species
may suggest the potential for similar effects on humans. Consistent with this position are
data showing that central nervous system effects can disrupt sexual behavior in both test
species and humans (Rubin & Henson, 1979; Waller et al., 1985). Although the functional
components of sexual performance can be quantified in most test species, no direct
evaluation of this behavior is done in most breeding studies. Rather, copulatory plugs or
sperm-positive vaginal lavages are taken as evidence of sexual receptivity and successful
mating. However, these markers do not demonstrate whether male performance resulted in
adequate sexual stimulation of the female. Failure of the male to provide adequate
stimulation to the female may impair sperm transport in the genital tract of female rats,
thereby reducing the probability of successful impregnation (Adler & Toner, 1986). Such a
"mating" failure would be reflected in the calculated fertility index as reduced fertility and
could be attributed erroneously to an effect on the spermatogenic process in the male or on
fertility of the female.
In the rat, a direct measure of female sexual receptivity is the occurrence of lordosis.
Sexual receptivity of the female rat is normally cyclic, with receptivity commencing during
the late evening of vaginal proestrus. Agents that interfere with normal estrous cyclicity
also could cause absence of or abnormal sexual behavior. In the male, measures include
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latency periods to first mount, mount with intromission, and first ejaculation, number of
mounts with intromission to ejaculation, and the post-ejaculatory interval (Beach, 1979).
Direct evaluation of sexual behavior is not warranted for all agents being tested for
reproductive toxicity. Some likely candidates may be agents reported to exert neurotoxic
effects. Chemicals possessing or suspected to possess androgenic or estrogenic properties
(or antagonistic properties) also merit consideration as potentially causing adverse effects on
sexual behavior concomitant with effects on the reproductive organs. Effects on sexual
behavior (within the limited definition of these Guidelines) should be considered as adverse
reproductive effects.
III.B.3. Male-specific Endpoints
III.B.3.a. Introduction
The following sections (III.B.3. and III.B.4.) describe various male-specific and
female-specific endpoints of reproductive toxicity that can be obtained. Included are
endpoints for which data are obtained routinely by the Agency and other endpoints for
which data may be encountered in the review of chemicals. Guidance is presented for
interpretation of results involving these endpoints and their use in risk assessment. Effects
are identified that should be considered as adverse reproductive effects if significantly
different from controls. Because of substantial overlap between the sexes in discussion of
effects on the reproductive systems during development, that topic is presented for both
sexes in Section III.BAf, Developmental and Pubertal Alterations.
The Agency may obtain data on the potential male reproductive toxicity of an agent
from many sources including, but not limited to, studies done according to Agency test
guidelines. These may include acute, subchronic, and chronic testing and reproduction and
fertility studies. Male-specific endpoints that may be encountered in such studies are
identified in Table 3.
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III.BJ.b. Body Weight and Organ Weights
Monitoring body weight during treatment provides an index of the general health
status of the animals, and such information may be important for the interpretation of
reproductive effects (see also Section III.B.2.). Depression in body weight or reduction in
weight gain may reflect a variety of responses, including rejection of chemical-adulterated
food or water because of reduced palatability, treatment-induced anorexia, or systemic
toxicity. Less than severe reductions in adult body weight may have little effect on the
male reproductive organs or on male reproductive function (Chapin et al., 1993a, b).
When a meaningful, biologic relationship between a body weight decline and a significant
effect on the male reproductive system is not apparent, it is not appropriate to dismiss
significant alteration of the male reproductive system as secondary to the occurrence of
non-reproductive toxicity. Unless additional data provide the needed clarification, alteration
in a reproductive measure that would otherwise be considered adverse should still be
considered as an adverse male reproductive effect in the presence of mild to moderate body
weight changes. In the presence of severe body weight depression, it should be noted that
an adverse effect on a reproductive endpoint occurred but that the effect may have resulted
from another, non-reproductive effect. Regardless, adverse effects would have been
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TABLE 3
Male-Specific Endpoints of Reproductive Toxicity
Organ weights Testes, epididymides, seminal vesicles,
prostate, pituitary
Visual examination & Testes, epididymides, seminal vesicles,
histopathology prostate, pituitary
Sperm evaluation* Sperm number (count) and quality (morphology,
motility)
Hormone levels* Luteinizing hormone, follicle stimulating
hormone, testosterone, estrogen, prolactin
Developmental Testis descent*, preputial separation, sperm production*,
ano-genital distance, normality of external genitalia*
* Reproductive endpoints that can be obtained or estimated relatively
noninvasively with humans
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observed in that situation and a risk assessment should be pursued if sufficient data are
available.
The male reproductive organs for which weights may be useful for reproductive risk
assessment include the testes, epididymides, pituitary gland, seminal vesicles (with
coagulating glands), and prostate. Organ weight data may be presented as both absolute
weights and as relative weights (i.e., organ weight to body weight ratios). Organ weight
data may also be reported relative to brain weight since, subsequent to development, the
weight of the brain remains quite stable (Stevens & Gallo, 1989). Evaluation of data on
absolute organ weights is important, because a decrease in a reproductive organ weight may
occur that was not necessarily related to a reduction in body weight gain. The organ
weight-to-body weight ratio may show no significant difference if both body weight and
organ weight change in the same direction, masking a potential organ weight effect.
Normal testis weight varies only modestly within a given test species (Schwetz et
al., 1980; Blazak et al., 1985). This relatively low interanimal variability suggests that
testis weight should be a precise indicator of gonadal injury. However, damage to the
testes may be detected as a weight change only at doses higher than those required to
produce significant effects in other measures of gonadal status (Berndtson, 1977; Foote et
al., 1986). This contradiction may arise from several factors, including a delay before cell
deaths are reflected in a weight decrease (due to edema and inflammation, cellular
infiltration) or Leydig cell hyperplasia. Blockage of the efferent ducts by cells sloughed
from the germinal epithelium or the efferent ducts themselves can lead to an increase in
testis weight due to fluid accumulation (Hess et al., 1991; Nakai et al., 1993), an effect that
could offset the effect of depletion of the germinal epithelium on testis weight. Thus, testis
weight measurements do not indicate the nature of an effect, but a significant increase or
decrease is indicative of an adverse effect.
Pituitary gland weight can provide valuable insight into the reproductive status of
the animal. However, the pituitary contains cell types that are responsible for the regulation
of a variety of physiologic functions including some that are separate from reproduction.
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Thus, changes in pituitary weight may not necessarily reflect reproductive impairment. If
weight changes are observed, gonadotroph-specific histopathologic evaluations may be
useful in identifying the affected cell types. This information may then be used to judge
whether the observed effect on the pituitary is related to reproductive system function and
therefore an adverse reproductive effect. Prostate and seminal vesicle weights are
androgen-dependent and may reflect changes in the animal's endocrine status or testicular
function. Separation of the seminal vesicles and coagulating gland (dorsal prostate) is dif-
ficult in rodents. However, the seminal vesicle and prostate can be separated and results
may be reported for these glands separately or together. Because the seminal vesicles and
prostate may respond differently to an agent (endocrine dependency and developmental
susceptibility differ), more information may be gained if the weights were examined
separately.
Significant changes in absolute or relative male reproductive organ weights may
constitute an adverse reproductive effect. Such changes also may provide a basis for
obtaining additional information on the reproductive toxicity of that agent. However,
significant changes in other important endpoints that are related to reproductive function
may not be reflected in organ weight data. Therefore, lack of an organ weight effect
should not be used to negate significant changes in other endpoints that may be more
sensitive.
III.B.3.C. Histopathologic Evaluations
Histopathologic evaluations of test animals have a prominent role in male
reproductive risk assessment. Organs that are often evaluated include the testes,
epididymides, prostate, seminal vesicles (often including coagulating glands), and pituitary.
Tissues from lower dose exposures are often not examined histologically if the high dose
produced no difference from controls. Histologic evaluations can be especially useful by 1)
providing a relatively sensitive indicator of damage; 2) providing information on toxicity
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from a variety of protocols; 3) with short-term dosing, providing information on site
(including target cells) and extent of toxicity; and 4) indicating the potential for recovery.
The quality of the information presented from histologic analyses of spermatogenesis
is improved by proper fixation and embedding of testicular tissue. With adequately
prepared tissue (Chapin, 1988; Russell et al., 1990; Hess & Moore, 1993), a description of
the nature and background level of lesions in control tissue, whether preparation induced or
otherwise, can facilitate interpreting the nature and extent of the lesions observed in tissues
obtained from exposed animals. Many histopathologic evaluations of the testis only detect
lesions if the germinal epithelium is severely depleted, degenerating cells are obvious, or
sloughed cells are present in the tubule lumen. More subtle lesions that can significantly
affect the number of sperm being released normally into the tubule lumen may not be
detected when less adequate methods of tissue preparation are used. Also, familiarity with
the detailed morphology of the testis and the kinetics of spermatogenesis of each test
species can assist in the identification of less obvious lesions that may accompany lower
dose exposures or lesions that result from short-term exposure (Russell et al., 1990).
Several approaches for qualitative or quantitative assessment of testicular tissue are
available that can assist in the identification of less obvious lesions that may accompany
lower-dose exposures, including use of the technique of "staging." A text has been
prepared (Russell et al., 1990) that provides extensive information on tissue preparation,
examination, and interpretation of observations for normal and high resolution histology of
the germinal epithelium of rats, mice, and dogs. Included is guidance for identification and
quantification of the various cell types and associations for each stage of the spermatogenic
cycle. Also, a decision-tree scheme for staging with the rat has been published (Hess,
1990).
The basic morphology of other male reproductive organs (e.g., epididymides,
accessory sex glands, and pituitary) has been described as well as the histopathologic altera-
tions that may accompany certain disease states (Fawcett, 1986; Jones et al., 1987;
Haschek & Rousseaux, 1991). Compared with the testes, less is known about structural
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changes in these tissues that are associated with exposure to toxic agents. With the
epididymides and accessory sex glands, histologic evaluation is usually limited to the height
and possibly the integrity of the secretory epithelium. Presence of debris and sloughed cells
in the epididymal lumen are valuable indicators of damage to the germinal epithelium or
the excurrent ducts. Information from examinations of the pituitary should include
evaluation of the morphology of the cell types that produce the gonadotropins and prolactin.
The degree to which histopathologic effects are quantified is usually limited to
classifying animals, within dose groups, as either affected or not affected by qualitative
criteria. Little effort has been made to quantify the extent of injury, and procedures for
such classifications are not applied uniformly (Linder et al., 1990). Evaluation procedures
would be facilitated by adoption of more uniform approaches for quantifying the extent of
histopathologic damage per individual. In the absence of a standardized quantification
system, the evaluation of histopathologic data would be facilitated by the presentation of the
evaluation criteria and the manner in which the level of lesions in exposed individuals was
judged to be in excess of controls.
If properly obtained (i.e., proper preparation and analysis of tissue), data from
histopathologic evaluations may provide a relatively sensitive tool that is useful for
detection of low-dose effects. This approach may also provide insight into sites and
mechanisms of action for the agent on that reproductive organ. When similar targets or
mechanisms exist in humans, the basis for interspecies extrapolation is strengthened.
Depending on the experimental design, information can also be obtained that may allow
prediction of the eventual extent of injury and degree of recovery in that species and
humans (Russell, 1983).
Significant histopathologic damage in excess of the level seen in control tissue of
any of the male reproductive organs should be considered an adverse reproductive effect.
Significant histopathologic damage in the pituitary should be considered as an adverse
effect but should be shown to involve cells that control gonadotropin or prolactin
production to be called a reproductive effect. Although thorough histopathologic evalu-
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ations that fail to reveal any treatment-related effects may be quite convincing,
consideration should be given to the possible presence of other testicular or epididymal
effects that are not detected histologically (e.g., genetic damage to the germ cell, decreased
sperm motility), but may affect reproductive function.
III.B.3.d. Sperm Evaluations
The parameters that are important for sperm evaluations are sperm number, sperm
morphology, and sperm motility. Data on those parameters allow more adequate estimation
of the number of "normal" sperm; a parameter that is likely to be more informative than
sperm number alone. Although effects on sperm production can be reflected in other
measures such as spermatid count or cauda epididymal weight, no surrogate measures are
adequate to reflect effects on sperm morphology or motility. Similar data can be obtained
noninvasively from human ejaculates, enhancing the ability to confirm effects seen in test
species or to detect effects directly in humans. Brief descriptions of these measures are
provided below, followed by discussion of use of the various sperm measures in male
reproductive risk assessment.
Sperm number
Measures of sperm concentration (count) have been the most frequently reported
semen variable in the literature on humans (Wyrobek et al., 1983a). Sperm number or
sperm concentration from test species may be derived from ejaculated, epididymal, or
testicular samples. Of the common test species, ejaculates can only be obtained readily
from rabbits or dogs. Ejaculates can be recovered from the reproductive tracts of mated
females of other species (Zenick et al., 1984). Measures of human sperm production are
usually derived from ejaculates, but could also be obtained from spermatid counts or
quantitative histology using testicular biopsy tissue samples. With ejaculates, both sperm
concentration (number of sperm/ml of ejaculate) and total sperm per ejaculate (sperm
concentration x volume) should be evaluated.
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Ejaculated sperm number from any species is influenced by several variables,
including the length of abstinence and the ability to obtain the entire ejaculate. Intra- and
interindividual variabilities are often high, but are reduced somewhat if ejaculates were
collected at regular intervals from the same male (Williams et al., 1990). Such a
longitudinal study design has improved detection sensitivity and thus requires a smaller
number of subjects (Wyrobek et al., 1984). In addition, if a pre-exposure baseline is
obtained for each male (test animal or human studies), then changes during exposure or
recovery can be better defined.
Epididymal sperm evaluations with test species usually use sperm from only the
cauda portion of the epididymis. It has been customary to express the sperm count in
relation to the weight of the cauda epididymis. However, because sperm contribute to
epididymal weight, expression of the data as a ratio may actually mask declines in sperm
number. The inclusion of data on absolute sperm counts can improve resolution. As is
true for ejaculated sperm counts, epididymal sperm counts are influenced directly by level
of sexual activity (Amann, 1981; Hurtt & Zenick, 1986).
Sperm production data may be derived from counts of the distinctive elongated
spermatid nuclei that remain after homogenization of testes in a detergent-containing
medium (Amann, 1981; Meistrich, 1982; Cassidy et al., 1983; Blazak et al., 1993). The
elongated spermatid counts are a measure of sperm production from the stem cells and their
ensuing survival through spermatocytogenesis and spermiogenesis (Meistrich, 1982;
Meistrich & van Beek, 1993). If evaluation was conducted when the effect of a lesion
would be reflected adequately in the spermatid count, spermatid count may serve as a
substitute for quantitative histologic analysis of sperm production (Russell et al., 1990).
However, spermatid counts may be misleading when duration of exposure is shorter than
the time required for a lesion to be fully expressed in spermatid count. Also, spermatid
counts reported from some laboratories have large coefficients of variation that may reduce
the usefulness of that measure.
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The ability to detect a decrease in testicular sperm production may be enhanced if
spermatid counts are available. However, spermatid enumerations only reflect the integrity
of spermatogenic processes within the testes. Posttesticular effects or toxicity expressed as
alterations in motility, morphology, viability, fragility, and other properties of sperm can be
determined only from epididymal or ejaculated samples.
Sperm morphology
Sperm morphology refers to structural aspects of sperm and can be evaluated in
cauda epididymal, vas deferens, or ejaculated samples. A thorough morphologic evaluation
identifies abnormalities in the sperm head and flagellum. Because of the suggested
correlation between an agent's mutagenicity and its ability to induce abnormal sperm, sperm
head morphology has been a frequently reported sperm variable in toxicologic studies on
test species (Wyrobek et al., 1983b). The tendency has been to conclude that increased
incidence of sperm head malformations reflects germ cell mutagenicity. However, not
every mutagen induces sperm head abnormalities, and other nonmutagenic chemicals may
alter sperm head morphology. For example, microtubule poisons may cause increases in
abnormal sperm head incidence, presumably by interfering with spermiogenesis; a
microtubule-dependent process (Russell et al., 1981). Sperm morphology may be altered
also due to degeneration subsequent to cell death.
An increase in abnormal sperm morphology has been considered evidence that the
agent has gained access to the germ cells (U.S. Environmental Protection Agency, 1986c).
Exposure of males to toxic agents may lead to sperm abnormalities in their progeny
(Wyrobek & Bruce, 1978; Hugenholtz & Bruce, 1983). However, transmissible germ-cell
mutations might exist in the absence of any warning morphologic indicator such as
abnormal sperm. The relationships between these morphologic alterations and other
karyotypic changes remains uncertain (de Boer et al., 1976).
The traditional approach to characterizing morphology in toxicologic testing has
relied on subjective categorization of sperm head shape from examination of stained slides
at the light microscopic level (Filler, 1993). Such an approach may be adequate for mice
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and rats with their distinctly angular head shapes. However, the observable heterogeneity
of structure in human sperm and in nonrodent species makes it difficult for the
morphologist to define clearly the limits of normality. More systematic, quantitative, and
automated approaches have been offered that can be used with humans and test species
(Katz et al., 1982; Wyrobek et al., 1984). Data that identify the types of abnormalities
observed and the frequencies of their occurrences are preferred to estimation of overall
proportion of abnormal sperm. Objective, quantitative approaches that are done properly
should result in a higher level of confidence than more subjective measures.
Sperm morphology profiles are relatively stable and characteristic of a normal
individual (and a strain within a species) over time. Sperm morphology is one of the least
variable sperm measures in normal individuals, which may enhance its use in the detection
of spermatotoxic events (Zenick & Clegg, 1986). However, the reproductive implications
of the various types of abnormal sperm morphology need to be delineated more fully. The
majority of studies in test species and humans have suggested that abnormally shaped
sperm may not reach the oviduct or participate in fertilization (Nestor & Handel, 1984;
Redi et al., 1984). The implication is that the greater the number of abnormal sperm in the
ejaculate, the greater the probability of reduced fertility.
Sperm motility
The biochemical environments in the testes and epididymides are highly regulated to
assure the proper development and maturation of the sperm and the acquisition of critical
functional characteristics, i.e., progressive motility and the potential to fertilize. With
chemical exposures, perturbation of this balance may occur, producing alterations in sperm
properties such as motility. Chemicals (e.g., epichlorohydrin) have been identified that
selectively affect sperm motility and also reduce fertility. Studies have examined rat sperm
motility as a reproductive endpoint (Toth et al., 1989b, 1991), and sperm motility
assessments are an integral part of some reproductive toxicity tests.
Motility estimates may be obtained on ejaculated, vas deferens, or cauda epididymal
samples. Standardized methods are needed because motility is influenced by a number of
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experimental variables, including abstinence interval, the elapsed time and temperature
history of the sample, dilution, diluent, or sample chamber (Cassidy et al., 1983).
Historically, motility has been measured using subjective, microscopic evaluations.
Estimates of percent motile sperm can be made, and a scaling system used to describe the
quality of motility (i.e., the degree to which sperm show progressive, linear motility). More
quantitative approaches have been taken in test species (Linder et al., 1986; Toth et al.,
1989a; Slott et al., 1990) and humans (Boyers et al., 1989), including enumeration of motile
and immptile sperm from videotapes. Videotaping has the advantage that a record can be
retained.
Computer-assisted methods for evaluation of sperm motility allow measurement of
an extensive array of motion parameters. Included may be measures such as linear
velocity, curvilinear velocity, lateral sperm head displacement, and linearity of motion.
This technology has been applied in a limited number of toxicologic studies using test
species (Working & Hurtt, 1987; Toth et al., 1989a; Slott et al., 1990, 1991). The ability of
some of these measures to predict a potential effect on ability of sperm to fertilize has not
been established. Therefore, judgments concerning adversity should consider the status of
knowledge on those measures. Significant reductions in proportion of sperm that are
progressively motile should be considered adverse. Reductions in velocity or pattern of
motility may indicate a need for further testing. For example, a reduction in velocity could
indicate potential for reduced ability of the sperm to survive or fertilize. Efforts are in
progess to validate and standardize these automated techniques for application in
reproductive toxicity studies and to determine the relationships between these motility
endpoints and fertility.
In vitro tests of reproductive function
Numerous in vitro tests are available that can measure effects on different aspects of
the reproductive systems of males and females. These include in vitro fertilization, whole
organ (e.g., testis, ovary) perfusion, culture of cell populations, and incubations of
subcellular fractions or cytosol from specific cell types. Tests of sperm properties and
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function include sperm-cervical mucus penetration, in vitro sperm capacitation, in vitro
fertilization using zona pellucida-free hamster ova and the hemizona penetration assay
(Overstreet, 1984; Franken et al., 1990).
The diagnostic information obtained from such tests may help to identify potential
effects on the reproductive systems. However, each test bypasses essential components of
the intact animal system and therefore, by itself, is not capable of predicting exposure levels
that would result in toxicity in intact animals. While it is desirable to replace whole animal
testing to the extent possible with in vitro tests, the use of such tests currently is to screen
for toxicity potential and to study mechanisms of action and metabolism (Perreault, 1989;
Holloway et al., 1990a, b).
Use of sperm evaluations in risk assessment
The relationships between the various endpoints that measure effects on
spermatogenesis or sperm maturation in humans and test species have not been evaluated
adequately. Thus, how toxicity that is reflected in one such measure may influence other
measures is not always clear. The quantitative relationships between these measures and
fertility also are not well characterized for any species. Certain qualitative and quantitative
standards must be met to ensure full fertility, but the lower limits of these standards have
not been delineated adequately. For instance, the distributions of sperm counts for fertile
and infertile men overlap, with the mean for fertile men being higher (Meistrich & Brown,
1983). However, observations with farm species (cattle, sheep, swine) have shown more
clearly that reductions in the parameters of sperm quality become important when either the
total number of sperm or the number of apparently normal sperm is reduced below a certain
level. Additional research is needed to quantify the biologic consequences of reductions in
sperm number and quality in the laboratory animal species and humans.
Human male fertility is generally lower than that of test species and may be more
susceptible to damage from toxicants (see Supplemental Information). Therefore, the
conservative view should be taken that, within the limits indicated in the sections on those
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parameters, significant changes in measures of sperm count, morphology or motility as well
as number of normal sperm should be considered adverse effects.
III.B.S.e. Endocrine Evaluations
Measurement of the reproductive hormones in males offers useful supplemental
information in assessing potential reproductive toxicants for test species (Sever & Hessol,
1984; Heywood & James, 1985; National Research Council, 1989). However, such
measurements have increased importance with humans where invasiveness of approaches
must be limited. The reproductive hormones measured often are luteinizing hormone (LH),
follicle stimulating hormone (FSH), and testosterone. Other useful measures that may be
available include prolactin, inhibin, and androgen binding protein levels. In addition,
challenge tests with exogenous agents (e.g., gonadotropin releasing hormone, LH, or human
chorionic gonadotropin) may provide insight into the functional responsiveness of the
pituitary or Leydig cells.
Toxic agents can alter endocrine function by affecting any part of the hypothalamic-
pituitary-gonadal axis. If a compound affects the hypothalamus or pituitary, then serum LH
and FSH may be decreased, leading to decreased testosterone levels. On the other hand,
severe interference with Sertoli cell function or spermatogenesis would be expected to
elevate serum FSH levels.
A toxicant having antiandrogenic activity might cause endocrine and morphologic
changes that differ from those described previously. In adult male rats, exposure to an
antiandrogen might elevate serum LH and testosterone. Testis weight might be unaffected,
while the weight and size of the accessory sex glands may be altered. The profile
presented by specific antiandrogens can differ markedly because of differences in tissue
specificity and receptor kinetics.
Interpretation of endocrine effects is facilitated if information is available on a
battery of hormones. However, in evaluating such data, it is important to consider that
serum hormones such as FSH, LH, prolactin, and androgens exhibit cyclic variations within
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a 24-hour period (Fink, 1988). Thus, the time of sampling should be controlled rigorously
to avoid excessive variability. Sequential sampling can allow detection of treatment-related
changes in circadian and pulsatile rhythms.
In the absence of endocrine data, significant effects on pituitary or accessory sex
gland weights or histopathology or on Leydig cell histopathology may suggest disruption of
the endocrine system. In those instances, additional testing for endocrine effects may be
indicated. Significant alterations in circulating levels of testosterone, LH, or FSH may be
indicative of existing pituitary or gonadal injury. When significant alterations from control
levels are observed in those hormones, the changes should be considered cause for concern
because they are likely to affect, or occur in concert with, alterations in spermatogenesis,
sperm maturation, mating ability, or fertility. Such effects, if compatible with other
available information, may be considered adverse and may be used to establish a NOAEL
or LOAEL. Furthermore, endocrine data may facilitate identification of sites or
mechanisms of toxicant action, especially when obtained after short term exposures.
III.B.S.f. Biochemical Tests or Markers of Toxicity to the Testes and Other Male
Reproductive Organs
Numerous biomarkers and biochemical tests exist that are related less directly to
integrated reproductive function than the endpoints discussed previously in this section.
Currently, the value of such tests as endpoints of reproductive toxicity remains to be
demonstrated, although a number of potential chemical markers are available (National
Research Council, 1989; Kimmel, G.L. et al., In press). However, the results of such
measurements may suggest the presence of an effect that should be investigated further.
Another valuable role for these tests may be in delineating the target or mechanism of
action for a given agent. Such data may be of use in the design of subsequent tests,
interspecies extrapolation, and in estimating the potential for reversibility (Scialli & Clegg,
1992).
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III.B.3.g. Paternally-mediated Effects on Offspring
The concept is well accepted that exposure of a female to toxic chemicals during
gestation or lactation may produce death, structural abnormalities, growth alteration, or
postnatal functional deficits in her offspring. Sufficient data now exist with a variety of
agents to conclude that male-only exposure also can produce deleterious effects in offspring
(Hood, 1989; Nagao & Fujikawa, 1990; Davis et al., 1992).
These effects may be the result of direct damage to the sperm. However,
xenobiotics present in seminal plasma or bound to the fertilizing sperm could be introduced
into the female genital tract, or even the oocyte directly, and might also interfere with fer-
tilization or early development. With humans, the possibility also exists that a parent could
transport the toxic agent from the work environment to the home (e.g., on work clothes),
exposing other adults or children. Further work is needed to clarify the extent to which
paternal exposures may be associated with adverse effects on offspring. Regardless, if an
agent is identified in test species as causing a paternally- mediated adverse effect on
offspring, the effect should be considered an adverse reproductive effect.
III.B.4. Female-specific Endpoints
III.BAa. Introduction
The reproductive life cycle of the female may be divided into phases that include
fetal, prepubertal, cycling adult, pregnant, lactating, and reproductively senescent. Detailed
descriptions of all phases are available (Knobil & Neill, 1988). It is important to detect
adverse effects occurring in any of these stages. Traditionally, the endpoints that have been
used have emphasized ability to become pregnant, pregnancy outcome, and offspring
survival and development. Although reproductive organ weights may be obtained and these
organs examined histologically in test species, these measures do not necessarily detect
abnormalities in dynamic processes such as estrous cyclicity or follicular atresia unless
degradation is severe. Similarly, toxic effects on onset of puberty have not been examined,
nor have the long-term consequences of exposure on reproductive senescence. Thus, the
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amount of information obtained routinely to detect toxic effects on the female reproductive
system is limited.
The consequences of impairment in the nonpregnant female reproductive system are
equally important, and endpoints to detect adverse effects on the nonpregnant reproductive
system, when available, can be useful in evaluating reproductive toxicity. Such measures
may also provide additional interrelated endpoints and information on mechanism of action.
Alterations in the nonpregnant female reproductive system have been observed at
dose levels below those that result in reduced fertility or produce other overt effects on
pregnancy or pregnancy outcomes (Le Vier & Jankowiak, 1972; Barsotti et al., 1979;
Sonawane & Yaffe, 1983; Cummings & Gray, 1987). In contrast to the male reproductive
system, the status of the normal female system fluctuates in adults. Thus, in nonpregnant
animals (including humans), the ovarian structures and other reproductive organs change
throughout the estrous or menstrual cycle. Although not cyclic, normal changes also
accompany the progression of pregnancy, lactation, and return to cyclicity during or after
lactation. These normal fluctuations may affect the endpoints used for evaluation.
Therefore, knowledge of the reproductive status of the female at necropsy, including the
stage of the estrous cycle, can facilitate detection and interpretation of effects with
endpoints such as uterine weight and histopathology of the ovary and uterus. Necropsy of
all test animals at the same stage of the estrous cycle can reduce the variance of test results
with such measures.
A variety of measures to evaluate the integrity of the female reproductive system has
been used in toxicity studies. With appropriate measures, a comprehensive evaluation of
the reproductive process can be achieved, including identification of target organs and
possible elucidation of the mechanisms involved in the toxicant's effect. Areas that may be
examined in evaluations of the female reproductive system are listed in Table 4.
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TABLE 4
Female-Specific Endpoints of Reproductive Toxicity
Body weight
Organ weights Ovary, uterus, vagina, pituitary
Visual examination & Ovary, uterus, vagina, pituitary, oviduct,
histopathology mammary gland
Estrous (menstrual*) Vaginal smear cytology
cycle normality
Hormone levels* LH, FSH, estrogen, progesterone, prolactin
Lactation* Offspring growth
Development Normality of external genitalia*, vaginal opening,
vaginal smear cytology, onset of estrus behavior
(menstruation*)
Senescence Vaginal smear cytology, ovarian histology
(menopause*)
* Endpoints that can be obtained relatively noninvasively with humans
The female reproductive system is also primarily controlled by the endocrine system.
This control is accomplished through complex interactions involving the central nervous
system (e.g. hypothalamus), pituitary, ovaries, the reproductive tract, and the secondary
sexual organs. Other non-gonadotrophic components of the endocrine system may also
modulate reproductive system function. Because it is difficult to measure certain important
aspects of female reproductive function (e.g., increased rate of follicular atresia, ovulation
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failure), assessment of the endocrine status may provide needed insight that is not otherwise
available.
To understand the significance of effects on the reproductive endpoints, it is critical
that the relationships between the various reproductive hormones and the female
reproductive organs be understood. Although certain effects may be identified routinely as
adverse, all of the results should be considered in the context of the known biology.
The format for presentation of the female reproductive endpoints is altered from that
used for the male to allow examination of events that are linked and that fluctuate with the
changing endocrine status. Particularly, the organ weight, gross morphology, and histology
are combined for each organ. Endpoints and endocrine factors for the individual female
reproductive organs are discussed, with emphasis on the nonpregnant animal. This is
followed by examination of measures of cyclicity and their interpretation. Then,
considerations relevant to prepubertal, pregnant, lactating, and aging females are presented.
III.BAb. Body Weight, Organ Weight, Organ Morphology, and Histology
III.BAb. 1. Body weight
Toxicologists are often concerned about how a change in body weight may affect
reproductive function. In females, an important consideration is that weights may fluctuate
normally with the physiologic state of the animal because estrogen and progesterone are
known to influence food intake and energy expenditure to an important extent (Wang,
1923; Wade, 1972). Water retention and fat deposition rates are also affected (Gelletti &
Klopper, 1964; Hervey & Hervey, 1967). Food consumption is elevated during pregnancy,
in part because of the elevated serum progesterone level. One of the most sensitive
indicators of a compound with estrogenic action in the female rat is a reduction in food
intake and body weight. Also, growth retardation induced by effects on extragonadal
hormones (e.g., thyroid or growth hormone) can cause a delay in pubertal development, and
induce acyclicity and infertility. Because of these endocrine-related fluctuations, the
weights of the reproductive organs are poorly correlated with body weight, except in
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extreme cases. Thus, actual organ weight data, rather than organ to body weight ratios,
should be reported and evaluated for the female reproductive system.
Chapin et al. (1993a, b) have studied the influence of food restriction on female
Sprague-Dawley rats and Swiss CD-I mice when body weights were 90, 80 or 70% of
controls. Female rats were resistant to effects on reproductive function at 80% of control
weight whereas mice showed adverse effects at 80%. These results indicate that differences
exist between species (and probably between strains) in the response of the female rodent
reproductive system to reduced food intake or body weight reduction.
III.B.4.b.2. Ovary
The ovary serves a number of functions that are critical to reproductive activity,
including production and ovulation of oocytes. Estrogen is produced by developing
follicles and progesterone is produced by corpora lutea that are formed after ovulation.
Ovarian weight
Significant increases or decreases in ovarian weight compared with controls should be
considered an indication of female reproductive toxicity. Although ovarian function shifts
throughout the estrous cycle, ovarian weight in the normal rat does not show significant
fluctuations. Still, oocyte and follicle depletion, persistent polycystic ovaries, inhibition of
corpus luteum formation, luteal cyst development, reproductive aging, and altered
hypothalamic-pituitary function may all be associated with changes in ovarian weight.
Therefore, it is important that ovarian gross morphology and histology also be examined to
allow correlation of alterations in those parameters with changes in ovarian weight.
However, not all adverse histologic alterations in the ovary are concurrent with changes in
ovarian weight. Therefore, a lack of effect on organ weights does not preclude the need for
histologic evaluation.
Histopathology
Histologic evaluation of the three major compartments of the ovary (i.e., follicular,
luteal, and interstitial) plus the epithelial capsule and ovarian stroma may indicate ovarian
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toxicity. A number of pathologic conditions can be detected by ovarian histology (Kurman
& Norris, 1978; Langley & Fox, 1987). Methods are available to quantify the number of
follicles and their stages of maturation (Plowchalk et al., 1993). These techniques may be
useful when a compound depletes the pool of primordial follicles or alters their subseqent
development and recruitment during the events leading to ovulation.
Adverse effects
Significant changes in the ovaries in any of the following effects should be
considered adverse:
* Increase or decrease in ovarian weight
* Increased incidence of follicular atresia
* Decreased number of primary follicles
* Decreased number or lifespan of corpora lutea
* Evidence of abnormal folliculogenesis or luteinization,
including cystic follicles, luteinized follicles, and failure of ovulation
* Evidence of altered puberty or premature reproductive
senescence
III.B.4.b.3. Uterus
Uterine weight
An alteration in the weight of the uterus may be considered an indication of female
reproductive organ toxicity. Compounds that inhibit cyclicity can dramatically reduce the
weight of the uterus so that it appears atrophic and small. However, uterine weight
fluctuates 3 to 4 fold throughout the estrous cycle, peaking at proestrus when, in response
to increased estrogen secretion, the uterus is fluid filled and distended. This increase in
uterine weight has been used as a basis for comparing relative potency of estrogenic
compounds in bioassays (Kupfer, 1987). As a result of the wide fluctuation because of the
influence of estrogenic compounds, uterine weights taken from cycling animals have a high
variance, and large compound-related effects are required to demonstate a significant effect
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unless interpreted relative to that animal's estrous cycle stage. A number of environmental
compounds (e.g., pesticides such as methoxychlor and chlordecone, mycotoxins,
polychlorinated biphenyls, and phytoestrogens) possess varying degrees of estrogenic
activity and have the potential to stimulate the female reproductive tract (Barlow &
Sullivan, 1982; Bulger & Kupfer, 1985; Hughes, 1988).
When pregnant or postpartum animals are examined, the numbers of implantation
sites or implantation scars should be counted. This information, along with corpus luteum
counts, can be used to calculate pre- and postimplantation losses.
Histopathology
The histologic appearance of the normal uterus fluctuates with stage of the estrous
cycle and pregnancy. The uterine endometrium is sensitive to influences of estrogens and
progestogens (Warren et al., 1967), potentially leading to hypertrophy and hyperplasia.
Conversely, interference with ovarian activity results in endometrial hypoplasia and atrophy.
Effects induced during development may delay or prevent puberty, resulting in persistence
of infantile genitalia.
Adverse effects
Effects on the uterus that may be considered adverse include significant dose-related
alteration of weight, as well as gross anatomic or histologic abnormalities. In particular,
any of the following effects should be considered as adverse.
* Infantile or malformed uterus or cervix
* Decreased or increased uterine weight
* Endometrial hyperplasia, hypoplasia, or aplasia
* Decreased number of implantation sites
III.BAbA Oviducts
Typically, the oviducts are not weighed or examined histologically in tests for
reproductive toxicity. However, information from visual and histologic examinations is of
value to detect morphologic anomalies. Descriptions of pathologic effects within the
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oviducts of animals other than humans are not common. Hypoplasia of otherwise
well-formed oviducts results most commonly from a lack of estrogen stimulation, and for
this reason, this condition may not be recognized until after puberty. Hyperplasia of the
oviductal epithelium results from prolonged estrogenic stimulation. Anomalies induced
during development have also been described, including agenesis, segmental aplasia, and
hypoplasia.
Anatomic anomalies in the oviduct occurring in excess of control incidence should
be considered as adverse effects. Hypoplasia or hyperplasia of the oviductal epithelium
may be considered as an adverse effect, particularly if that result is consistent with
observations in the uterine histology.
III.BAb.5. Vagina and external genitalia
Vaginal weight
Vaginal weight changes should parallel those seen in the uterus during the estrous
cycle, although the magnitude of the changes is smaller.
Histopathology
In rodents, cytologic changes in the vaginal epithelium (vaginal smear) may be used
to identify the different stages of the estrous cycle (see Section III.BAd.). The vaginal
smear pattern may be useful to identify conditions that would delay or preclude fertility, or
affect sexual behavior. Other histologic alterations that may be observed include aplasia,
hypoplasia, and hyperplasia of the vaginal epithelial cell lining.
Developmental effects
Developmental abnormalities, either genetic or related to prenatal exposure to
compounds that disrupt the endocrine balance, include agenesis, hypoplasia, and dysgenesis.
Hypoplasia of the vagina may be concomitant with hyperplasia of the external genitalia and
can be induced by gonadal or adrenal steroid exposure. In rodents, malpositioning of the
vaginal and urethral ducts is common in steroid-treated females. Such developmentally
induced lesions are irreversible.
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The sex ratio observed at birth may be affected by developmental exposure to
androgens. In cases of incomplete sex reversal because of such exposures, female rodents
may appear more male-like and have an increased anogenital distance.
At puberty, the opening of the vaginal orifice normally provides a simple and useful
developmental marker. However, estrogenic or antiestrogenic chemicals can act directly on
the vaginal epithelium and alter the age at which vaginal patency is lost without truly
affecting puberty (see Section III.BAf).
Adverse effects
Significant effects on the vagina that may be considered adverse include the
following:
* Increases or decreases in weight
* Infantile or malformed vagina or vulva, including masculinized vulva
or increased anogenital distance
* Vaginal hypoplasia or aplasia
* Altered timing of vaginal opening
* Abnormal vaginal smear cytology pattern
III.B.4.b.6. Pituitary
Pituitary weight
Alterations in weight of the pituitary gland may be considered an adverse effect.
The discussion on pituitary weight and histology for males (see Section III.B.3.) is pertinent
also for females. Pituitary weight increases normally with age, as well as during pregnancy
and lactation. Changes in pituitary weight can occur also as a consequence of chemical
stimulation. Increased pituitary weight often precedes tumor formation, particularly in
response to treatment with estrogenic compounds. Increased pituitary size associated with
estrogen treatment may be accompanied by hyperprolactinemia and constant vaginal estrus.
Decreased pituitary weight is less common but may result from decreased estrogenic
stimulation (Cooper et al., 1989).
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Histopathologv
In histologic evaluations with rats and mice, the relative size of cell types in the
anterior pituitary (acidophils and basophils) has been reported to vary with the stages of the
reproductive cycle and in pregnancy (Holmes & Ball, 1974). Therefore, the relationship of
morphologic pattern to estrous or menstrual cycle stage or pregnancy status should be
considered in interpreting histologic observations on the female pituitary.
Adverse effects
A significant increase or decrease in pituitary weight should be considered an
adverse effect. Significant histopathologic damage in the pituitary should be considered an
adverse effect, but should be shown to involve cells that control gonadotropin or prolactin
production to be called a reproductive effect.
III.B.4.C Oocyte Production
III.B.4.C.1. Folliculogenesis
In normal females, all of the follicles (and the resident oocytes) are present at or
soon after birth. The large majority of these follicles undergo atresia and are not ovulated.
Therefore, the ovaries from control animals have a background rate of follicular atresia that
must be distinguished from an increased rate resulting from exposure to toxicants (Smith,
B.J. et al., 1991; Heindel & Chapin, 1993). If the population of follicles is depleted, it
cannot be replaced and the female will be rendered infertile. In humans, depletion of
oocytes can lead to premature menopause.
In rodents, lead, mercury, cadmium, and polyaromatic hydrocarbons have all been
implicated in the arrest of follicular growth at various stages of the life cycle (Mattison &
Thomford, 1989). Susceptibility to oocyte toxicity varies considerably between species
(Mattison & Thorgeirsson, 1978).
Environmental toxicants that affect gonadotropin-mediated steroidogenesis or
follicular maturation can prolong the follicular phase of the estrous or menstrual cycle and
cause atresia of follicles that would otherwise ovulate. Estrogenic as well as antiestrogenic
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agents can produce that effect. Also, normal follicular maturation is essential for normal
formation and function of the corpus luteum formed after ovulation (McNatty, 1979).
IH.B.4.C.2. Ovulation
Chemicals can delay or block ovulation by disrupting the ovulatory surge of LH or
by interfering with the ability of the maturing follicle to respond to that gonadotropic
signal. Examples for rats are the pesticides chlordimeform and amitraz (Goldman et al.,
1990) and compounds that interfere with normal central norepinephrine receptor stimulation
(Drouva et al., 1982). Compounds that increase central opioid receptor stimulation also
decrease serum LH and inhibit ovulation in monkeys and rats (Pang et al., 1977; Smith,
C.G., 1983). Delayed ovulation can alter oocyte viability and cause trisomy and polyploidy
in the conceptus (Fugo & Butcher, 1966; Butcher & Fugo, 1967; Butcher et al., 1969,
1975; Na et al., 1985).
III.B.4.C.3. Corpus luteum
The corpus luteum arises from the ruptured follicle and secretes progesterone, which
has an important role in the estrous or menstrual cycle. It also serves as the principal source
of progesterone required for the maintenance of early pregnancy in the human (Csapo &
Pulkkinen, 1978). Therefore, establishment and maintainance of normal corpora lutea are
essential to normal reproductive function. However, with the exception of histopathologic
evaluations that may establish only their presence or absence, these structures are not
evaluated in routine testing. Additional research is needed to determine the importance of
incorporating endpoints that examine direct effects on luteal function in routine toxicologic
testing.
Adverse effects
Increased rates of follicular atresia and oocyte toxicity can lead to premature
menopause. Altered follicular development, ovulation failure, or altered corpus luteum
formation and function can result in disruption of cyclicity, reduced fertility, and, in non-
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primates, interference with normal sexual behavior. Therefore, significant increases in rate
of follicular atresia, evidence of oocyte toxicity, interference with ovulation, or altered
corpus luteum formation or function should be considered adverse effects.
III.BAd. Alterations in the Female Reproductive Cycle
The pattern of events in the estrous cycle provides a useful indicator of the
normality of reproductive neuroendocrine and ovarian function in the nonpregnant female.
It also provides a means to interpret hormonal, histologic, and morphologic measurements
relative to stage of the cycle, and can be useful to monitor the status of mated females.
Estrous cycle normality can be monitored in the rat and mouse by observing the changes in
the vaginal smear cytology (Long & Evans, 1922; Cooper et al., 1993). To be most useful
with cycling females, vaginal smear cytology should be examined daily for at least three
normal estrous cycles prior to treatment, after onset of treatment, and before necropsy
(Kimmel, G.L. et al., In press).
Daily vaginal smear data from rodents can provide useful information on (1) cycle
length, (2) occurrence or persistence of estrus, (3) duration or persistence of diestrus, (4)
incidence of spontaneous pseudopregnancy, (5) distinguishing pregnancy from
pseudopregnancy (based on the number of days smear remains leukocytic), and (6)
indications of fetal death and resorption by the presence of blood in the smear after day 12
of gestation. The technique also can detect onset of reproductive senescence in rodents
(LeFevre & McClintock, 1988). It is useful further to detect the presence of sperm in the
vagina as an indication of mating.
In nonpregnant females, repetitive occurence of the four stages of the estrous cycle
at regular, normal intervals suggests that neuroendocrine control of the cycle and ovarian
responses to that control are normal. Occasionally, even normal, control animals show an
irregular cycle. However, a significant alteration compared with controls in the interval
between occurrence of estrus smears for a treatment group is cause for concern. Generally,
the cycle will be lengthened or terminated. Lengthening of the cycle may be a result of
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increased duration of either the estrus or diestrus phase. Knowing the affected phase can
provide direction for further investigation.
The persistence of regular vaginal cycles after treatment does not necessarily indicate
that ovulation occurred, because luteal tissue may form in follicles that have not ruptured.
This effect has been observed after treatment with anti-inflammatory agents (Walker et al.,
1988). However, that effect should be reflected in reduced fertility. Conversely, subtle
alterations of cyclicity can occur at doses below those that alter fertility (Gray et al., 1989).
Irregular cycles may reflect impaired ovulation. Extended vaginal estrus usually
indicates that the female cannot spontaneously achieve the ovulatory surge of LH (Huang &
Meites, 1975). A number of compounds have been shown to alter the characteristics of the
LH surge including anesthetics (Nembutal), neurotransmitter receptor binding agents
(Drouva et al., 1982), and the pesticides chlordimeform and lindane (Cooper et al., 1989;
Morris et al., 1990). The female in constant estrus may be sexually receptive and ovulation
may be induced by mating (Brown-Grant et al., 1973; Smith, E.R. & Davidson, 1974), but
the fertility of such matings has not been evaluated thoroughly. Significant delays in
ovulation can result in increased embryonic abnormalities and loss (Fugo & Butcher,
1966).
Persistent diestrus indicates cessation of follicular development and ovulation and
thus infertility. Prolonged vaginal diestrus, or anestrus, may be indicative of agents (e.g.,
polyaromatic hydrocarbons) that interfere with follicular development or deplete the pool of
primordial follicles (Mattison & Nightingale, 1980). The ovaries of the anestrus female
are atrophic, with few primary follicles and an unstimulated uterus (Huang & Meites,
1975). Serum estradiol and progesterone are minimal.
Adverse effects
Significant evidence that the estrous cycle (or menstrual cycle in primates) has been
disrupted should be considered an adverse effect. Included should be evidence of abnormal
cycle length or pattern, ovulation failure, or abnormal menstruation.
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III.B.4.e. Mammary Gland and Lactation
The mammary glands of normal adults change dramatically during the period around
parturition because of the sequential effects of a number of gonadal and extragonadal
hormones. Milk letdown is dependent on the suckling stimulus and the release of oxytocin
from the posterior pituitary. Thus, mammary tissue is highly endocrine-dependent for
development and function.
Mammary gland size, milk production and release, and histology can be affected
adversely by toxic agents. Reduced growth of young could be caused by reduced milk
availability, by ingestion of a toxic agent secreted into the milk, or by other factors
unrelated to lactational ability (e.g., reduced growth could be caused by poor suckling
ability). Perinatal exposure to steroid hormones and other chemicals can alter mammary
gland morphology and tumor potential in adulthood. Because of the tendency for
mobilization of lipids from adipose tissue and secretion of those lipids into milk by
lactating females, milk may contain lipophilic agents at concentrations equal to or higher
than those present in the blood or organs of the dam. Thus, suckling offspring may be
exposed to elevated levels of such toxicants.
During lactation, the mammary glands can be dissected and weighed only with
difficulty. This provides a measure of milk production by the dam. A simple estimate of
milk production may be obtained by measuring litter weights taken after one or two hours
of nursing by milk-deprived pups (6 hours). Milk from the stomachs of pups can also be
weighed. Cleared and stained whole mounts of the mammary gland can be prepared at
necropsy for histologic examination. In addition, the DNA, RNA, and lipid content of the
mammary gland and the composition of the milk have been measured following toxicant
administration as indicators of toxicity to this target organ.
Significant reductions in milk production or negative effects on milk quality,
whether measured directly or reflected in impaired development of young, should be
considered adverse reproductive effects.
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III.BAf. Developmental and Pubertal Alterations
Developmental effects
Alterations of reproductive differentiation and development can result in infertility,
functional and morphologic alterations of the reproductive system, and cancer (Steinberger
& Lloyd, 1985; Gray, 1991). Prenatal and postnatal exposure to toxicants can produce
changes that may not be predicted from effects seen in adults, and those effects are often
irreversible. Adverse developmental outcomes in either sex can result from exposure to
toxicants in utero, through contact with exposed dams, or in milk. Dosing of dams during
lactation also can result in developmental toxicity through impaired nursing capability of
the dams.
Effects observed following exposure to agents in rodents include alterations in the
genitalia (including ano-genital distance), impaired sexual behavior, delay or acceleration of
the onset of puberty, and reduced fertility. Many of these effects have been detected in
human females and males exposed prenatally to diethylstilbestrol (DBS), other estrogens,
progestins, and androgens. Accelerated reproductive aging and tumors of the reproductive
tract have been observed in laboratory animal and human females. Generally, the type of
effect seen may differ depending on the stage at which the exposure occurred. Effects may
include anomalies in sexual behavior or ability to produce normal gametes that are not
observed until after puberty. Hepatic enzyme systems for steroid metabolism that are
imprinted during development may be altered in males. Testis descent from the abdominal
cavity into the scrotum may be delayed or may not occur. Other agents, such as busulfan,
that do not have endocrine activity may act via different mechanisms during critical periods
of development to cause similar effects.
Effects on puberty
In female rats and mice, the age at vaginal opening is the most commonly measured
marker of puberty. This event results from increases in the levels of estradiol in the blood.
The ages and weights of females at the first cornified (estrus) vaginal smear, the first
diestrus smear, and the onset of vaginal cycles have also been used as endpoints for onset
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of puberty. In males, preputial separation or appearance of sperm in expressed urine or
ejaculates can serve as markers of puberty. Body weight at puberty may provide a means
to separate specific delays in puberty from those that are related to general delays in
development. Agents may differentially affect the endpoints related to puberty onset, so it
is useful to have information on more than one marker.
Puberty can be accelerated or delayed by exogenous agents, and both types of effects
may be adverse. For example, an acceleration of vaginal opening may be associated with a
delay in the onset of cyclicity, infertility, and with accelerated reproductive aging (Gorski,
1979). Delays in pubertal development in rodents are usually related to delayed maturation
or inhibition of function of the hypothalamic-pituitary axis. Adverse reproductive outcomes
have been reported in rodents when puberty is altered by a week or more, but the biologic
relevance of a change in these measures of a day or two is unknown (Gray, 1991).
Adverse effects
Effects induced or observed during the perinatal period should be judged using
guidance from the Guidelines for Assessment of Developmental Risk (U.S. Environmental
Protection Agency, 1991) as well as from these Guidelines. Significant effects on age at
puberty, either early and delayed, should be considered adverse as should malformations of
the internal or external genitalia. Included as adverse effects for females should be effects
on age at vaginal opening, onset of cyclic vaginal smears, onset of menstruation, or onset of
an endocrine or behavioral pattern consistent with estrous or menstrual cyclicity. Included
as adverse effects for males should be delay or failure of testis descent, as well as delays in
age at preputial separation or appearance of sperm in expressed urine or ejaculates.
III.BAg. Reproductive Senescence
With advancing age, there is a loss of the regular ovarian cycles and associated normal
cyclical changes in the uterine and vaginal epithelium that are typical of the young-adult
female rat (Cooper & Walker, 1979). Although the mechanisms responsible for this loss of
cycling are not thoroughly understood, age-dependent changes within the hypothalamic-
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pituitary control of ovulation are impaired (Cooper et al., 1980; Finch et al., 1984).
Cumulative exposure to estrogen secreted by the ovary may play a role, as treatment with
estrogens during adulthood can accelerate the age-related loss of ovarian function (Drawer
& Finch, 1983). In contrast, the principal cause of the loss of ovarian cycling in humans
appears to be the depletion of oocytes (Mattison, 1985).
Prenatal or postnatal treatment of females with estrogens or estrogenic pesticides can
also cause impaired ovulation and sterility (Gorski, 1979). These observations imply that
alterations in ovarian function may not be noticeable immediately after treatment but may
become evident at puberty or influence the age at which reproductive senescence occurs.
Adverse effects
Significant effects on measures showing a decrease in the age of onset of
reproductive senescence in females should be considered adverse. Included as adverse
effects should be cessation of normal cycling measured by vaginal smear cytology, ovarian
histopathology, or an endocrine pattern that is consistent with this interpretation.
III.C. HUMAN STUDIES
In principle, human data are preferred for risk assessment. At this time,
reproductive data for humans are available for only a limited number of toxicants. As the
field develops further, expanding both the endpoints available for study and agents covered,
risk assessments will more frequently incorporate human data. The following describes the
methods of generation and evaluation of human data and the weight human data should be
given in risk assessments.
"Human studies" include both epidemiologic studies and other reports of individual
cases or clusters of events. Greatest weight should be given to carefully designed
epidemiologic studies with more precise measures of exposure, because they can best
evaluate exposure-response relationships. Epidemiologic studies in which exposure is
presumed, based on occupational title or residence (e.g., some case-referent and all ecologic
studies), may contribute data to qualitative risk assessments, but are of limited use for
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quantitative risk assessments because of the generally broad categorical groupings. Reports
of individual cases or clusters of events may generate hypotheses of exposure-outcome
associations, but require further confirmation with well-designed epidemiologic or
laboratory studies. These reports of cases or clusters may support associations suggested by
other human or animal data, but cannot stand by themselves in risk assessments. Risk
assessors should seek the assistance of professionals trained in epidemiology when
conducting a detailed analysis.
III.C.I. Epidemiologic Studies
Good epidemiologic studies provide the most relevant information for assessing
human risk. As there are many different designs for epidemiologic studies, simple rules for
their evaluation do not exist.
III.C.I.a. General Design Considerations
The factors that enhance a study and thus increase its usefulness for risk assessment
have been noted in a number of publications (Selevan, 1980; Bloom, 1981; Hatch & Kline,
1981; Wilcox, 1983; Sever & Hessol, 1984; Axelson, 1985; Tilley et al., 1985; Kimmel,
C.A. et al., 1986). Some of the more prominent factors are discussed below.
The power of the study
The power, or ability of a study to detect a true effect, is dependent on the size of
the study group, the frequency of the outcome in the general population, and the level of
excess risk to be identified. In a cohort study, groups are defined by exposure, and their
health outcomes examined. Common outcomes, such as recognized fetal loss, require
hundreds of pregnancies to have a high probability of detecting a modest increase in risk
(e.g., 133 participants in both exposed and unexposed groups to detect a twofold increase;
alpha = 0.05, power = 80%), while less common outcomes, such as the total of all
malformations recognized at birth, require thousands of pregnancies to have the same
probability (e.g., more than 1,200 pregnancies in both exposed and unexposed groups)
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(Bloom, 1981; Selevan, 1981, 1985; Sever & Hessol, 1984; Stein, Z. et al., 1985; Kimmel,
C.A. et al., 1986). Semen evaluation may require fewer subjects depending on the sperm
parameters evaluated, especially when each man is used as his own control (Wyrobek,
1982, 1984). In case-referent studies, groups are defined by health status and prior
exposures are examined. Study sizes are dependent upon the frequency of exposure within
the source population. The confidence one has in the results of a study with negative
findings is related directly to the power of the study to detect meaningful differences in the
endpoints.
Power may be enhanced by combining populations from several studies using a
meta-analysis (Greenland, 1987). The combined analysis would increase confidence in the
absence of risk for agents with negative findings. However, care must be exercised in the
combination of potentially dissimilar study groups.
A posteriori determination of power of the actual study is useful in evaluating
negative findings. Negative findings in a study of low power would be given considerably
less weight than either a positive study or a negative study with high power. Positive
findings from very small studies are open to question because of the instability of the risk
estimates and the highly selected nature of the population.
Potential bias in data collection
Bias may result from the way the study group is selected or information collected
(Rothman, 1986). Selection bias may occur when an individual's willingness to participate
varies with certain characteristics relating to the exposure status or health status of that
individual. In addition, selection bias may operate in the identification of subjects for
study. For example, in studies of embryonic loss, use of hospital records to identify
embryonic or early fetal loss will under-ascertain events, because women are not always
hospitalized for these outcomes. More weight would be given in a risk assessment to a
study in which a more complete list of pregnancies is obtained by, for example, collecting
biologic data [e.g., human chorionic gonadotropin (hCG) measurements] of pregnancy
status from study members. These studies may also be affected by bias. The
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representativeness of these data may be affected by selection factors related to the
willingness of different groups of women to continue participation over the total length of
the study. Interview data result in more complete ascertainment; however this strategy
carries with it the potential for recall bias, discussed in further detail below. A second
example of different levels of ascertainment of events is the use of hospital records to study
congenital malformations. Hospital records contain more complete data on malformations
than do birth certificates (Mackeprang et al., 1972). Thus a study using hospital records to
identify congenital malformations would be given more emphasis in a risk assessment than
one using birth certificates. Studies of working women present the potential for additional
bias because some factors that influence employment status may also affect reproductive
endpoints. For example, because of child-care responsibilities, women may terminate
employment, as might women with a history of reproductive problems who wish to have
children and are concerned about workplace exposures (Joffe, 1985). Thus, retrospective
studies of female exposure that do not include terminated women workers may be of
limited use in risk assessment because the level of risk for these outcomes is likely to be
overestimated (Lemasters & Pinney, 1989).
Information bias may result from misclassification of characteristics of individuals or
events identified for study. Recall bias, one type of information bias, may occur when
respondents with specific exposures or outcomes recall information differently than those
without the exposures or outcomes. Interview bias may result when the interviewer knows
a priori the category of exposure (for cohort studies) or outcome (for case-referent studies)
in which the respondent belongs. Use of highly structured questionnaires and/or "blinding"
of the interviewer reduces the likelihood of such bias. Studies with lower likelihood of
such bias should carry more weight in a risk assessment.
When data are collected by interview or questionnaire, the appropriate respondent
depends on the type of data or study. For example, a comparison of husband-wife
interviews on reproduction found the wives' responses to questions on pregnancy-related
events to be more complete and valid than those of the husbands (Selevan, 1980; Selevan et
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al., 1982). Studies based on interview data from the appropriate respondents would carry
more weight than those from proxy respondents (e.g., the specific individual when
examining exposure history and the woman or both partners when examining pregnancy
history). Data on male workers' exposures and factors relating to semen quality (e.g., fever
within the past 2 to 3 months) should be obtained from the workers themselves.
Data from any source may be prone to errors or bias. All types of bias are difficult
to assess; however, validation with an independent data source (e.g., vital or hospital
records), or use of biomarkers of exposure or outcome, where possible, may indicate the
degree of bias present and increase confidence in the results of the study. Those studies
with a low probability of biased data should carry more weight (Axelson, 1985; Stein, A. &
Hatch, 1987).
Differential misclassification (i.e., when certain subgroups are more likely to have
misclassified data than others) may either raise or lower the risk estimate. Nondifferential
misclassification will bias the results toward a finding of "no effect" (Rothman, 1986).
Collection of data on other risk factors, effect modifiers, and confounders
Risk factors for reproductive toxicity include such characteristics as age, smoking,
alcohol consumption, drug use, and past reproductive history. Additionally, occupational
and environmental exposures may be risk factors for these effects. Known and potential
risk factors should be examined to identify those that may be confounders or effect
modifiers. An effect modifier is a factor that produces different exposure-response
relationships at different levels of that factor. For example, age would be an effect
modifier if the risk associated with a given exposure increased with the individual's age. A
confounder is a variable that is a risk factor for the disease under study and is associated
with the exposure under study, but is not a consequence of the exposure. A confounder
may distort both the magnitude and direction of the measure of association between the
exposure of interest and the outcome. For example, smoking might be a confounder in a
study of the association of socioeconomic status and fertility because smoking may be
associated with both.
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Both effect modifiers and confounders need to be controlled in the study design
and/or analysis to improve the estimate of the effects of exposure (Kleinbaum et al., 1982).
A more in-depth discussion may be found elsewhere (Epidemiology Workgroup for the
Interagency Regulatory Liaison Group, 1981; Kleinbaum et al., 1982; Rothman, 1986). The
statistical techniques used to control for these factors require careful consideration in their
application and interpretation (Kleinbaum et al., 1982; Rothman, 1986). Studies that fail to
account for these important factors should be given less weight in a risk assessment.
Statistical factors
As in studies of test animals, pregnancies experienced by the same woman are not
fully independent events (Kissling, 1981; Selevan, 1985). Women who have had fetal loss
are reported to be more likely to have subsequent losses (Leridon, 1977). In test animal
studies, the litter is generally used as the unit of measure to deal with nonindependence of
events. In studies of humans, pregnancies are sequential, making analysis considering
nonindependence of events difficult (Epidemiology Workgroup for the Interagency
Regulatory Liaison Group, 1981; Kissling, 1981; Selevan, 1981). If more than one
pregnancy per woman is included, as is often necessary with small study groups, the use of
nonindependent observations overestimates the true size of the groups being compared, thus
artificially increasing the probability of reaching statistical significance (Stiratelli et al.,
1984). Biased estimates of risk might also result, if family size confounds the relationship
between exposure and outcome. Some approaches to deal with these issues have been
suggested (Kissling, 1981; Stiratelli et al., 1984; Selevan, 1985). At this time, a generally
accepted solution to this problem has not been developed.
Ill.C.l.b. Selection of Outcomes for Study
As already discussed, a number of endpoints can be considered in the evaluation of
adverse reproductive effects. However, some of the outcomes are not easily observed in
humans, such as early embryonic loss, reproductive capacity of the offspring, and invasive
evaluations of reproductive function (e.g., testicular biopsies). Currently, the most feasible
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endpoints for epidemiologic studies are (1) indirect measures of fertility/infertility; (2)
reproductive history studies of some pregnancy outcomes (e.g., embryonic/fetal loss, birth
weight, sex ratio, congenital malformations, postnatal function, and neonatal growth and
survival); (3) semen evaluations; and (4) menstrual history. Factors requiring control in the
design or analysis (such as effect modifiers and confounders) may vary depending on the
specific outcomes selected for study.
The reproductive outcomes available for epidemiologic examination are limited by a
number of factors, including the relative magnitude of the exposure, the size and
demographic characteristics of the population, and the ability to observe the reproductive
outcome in humans. Improved methods for identifying some outcomes such as embryonic
loss using more sensitive hCG assays may change the spectrum of outcomes available for
study (Wilcox et al., 1985; Sweeney et al., 1988). Other endpoints require invasive
techniques to obtain samples (e.g., histopathology) or have high intra- or interindividual
variability (e.g., serum hormone levels, sperm count).
Demographic characteristics of the population, such as marital status, age
distribution, education, socioeconomic status (SES), and prior reproductive history are
associated with the probability of whether couples will attempt to have children.
Differences in birth control practices would also affect the number of outcomes available
for study.
In addition to the above-mentioned factors, reproductive endpoints may be
envisioned as effects recognized at various points in a continuum, starting before
conception and continuing through death of the offspring. Thus, a malformed stillbirth
would not be included in a study of defects observed at live birth, even though the etiology
could be identical (Bloom, 1981). A shift in the patterns of outcomes could result from
differences in timing or in level of exposure (Selevan & Lemasters, 1987).
The following section discusses various human male and female reproductive
endpoints. These are followed by a discussion of reproductive history studies.
Male Endpoints - Semen Evaluations
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The use of semen analysis was discussed in Section III.B.S.d. Most epidemiologic
studies of semen characteristics have been conducted in occupational groups and patients
receiving drug therapy. Obtaining specimens with a high level of participation in the
workforce has been difficult,'because social and cultural attitudes concerning sex and
reproduction may affect cooperation of the study groups. Increased participation may occur
in men who are planning to have children or who are concerned either about existing
reproductive problems or about possible ill effects of their exposures. Unless controlled,
such biased participation may yield unrepresentative estimates of risk associated with
exposure, resulting in data that are less useful for risk assessment. Response rates are
typically less than 70% in such studies and may be even lower in the comparison group
(Egnatz et al., 1980; Lipshultz et al., 1980; Milby & Whorton, 1980; Lantz et al., 1981;
Meyer, 1981; Milby et al., 1981; Rosenberg et al., 1985). Some of the low response rates
may be caused by inclusion of vasectomized men in the total population, although this
could vary widely by population (Milby & Whorton, 1980). Participation in the
comparison group may be biased toward those with pre-existing reproductive problems.
The response rate may be improved substantially with proper education and payment of
subjects (Ratcliffe et al., 1986, 1987).
Several factors may influence the semen evaluation, including the period of
abstinence preceding collection of the sample, health status, and social habits (e.g., alcohol,
drugs, smoking). Data on these factors may be collected by interview, subject to the
limitations described for pregnancy outcome studies.
Such studies have also included an evaluation of endocrine status of exposed males.
These evaluations include determination of hormone levels in the blood and urine.
Female Endpoints
Reproductive effects may result from a variety of exposures. For example,
environmental exposures may result in oocyte toxicity, in which a loss of primary oocytes
irreversibly affects the woman's fertility. The exposures of importance may occur during
the prenatal period, and beyond. Oocyte depletion is difficult to examine directly in women
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because of the invasiveness of the tests required; however, it can be studied indirectly
through evaluation of the age at reproductive senescence (menopause) (Everson et al.,
1986).
Numerous diagnostic methods have been developed to evaluate female reproductive
dysfunction. Although these methods have rarely been used for occupational or
environmental toxicologic evaluations, they may be helpful in defining biologic parameters
and the mechanisms related to female reproductive toxicity. If clinical observations are
able to link exposures to the reproductive effect of concern, these data may aid the
assessment of adverse female reproductive toxicity. The following clinical observations
include endpoints that may be reported in case reports or epidemiologic research studies.
Reproductive dysfunction can be studied by the evaluation of irregularities of
menstrual cycles. However, menstrual cyclicity is affected by many parameters such as
age, nutritional status, stress, certain drugs, and the use of contraceptive measures that alter
endocrine feedback. Vaginal bleeding at menstruation is a reflection of withdrawal of
steroidogenic support, particularly progesterone. Vaginal bleeding can occur at midcycle, in
early miscarriage, after withdrawal of contraceptive steroids, or after an inadequate luteal
phase. The length of the menstrual cycle, particularly the follicular phase, can vary
between individuals and may make it difficult to determine significant effects in populations
of women (Burch et al., 1967; Treloar et al., 1967). However, menstrual dysfunction data
have been used to examine adverse reproductive effects in women exposed to styrene in the
workplace (Lemasters et al., 1985).
Vaginal cytology may provide information on the functional state of reproductive
cycles. Cytologic evaluations, along with the evaluation of changes in cervical mucus
viscosity, can be used to estimate the occurrence of ovulation and determine different stages
of the reproductive cycle.
The endocrine status of a woman can be evaluated by the measurement of hormones in
blood and urine. Progesterone can also be measured in saliva. Because the female
reproductive endocrine milieu is changing in a cyclic pattern, single sample analysis does
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not provide adequate information for evaluating alterations in the reproductive function.
Still, a single sample for progesterone determination some 7 to 9 days after the estimated
midcycle surge of gonadotropins in a regularly cycling woman may provide suggestive
evidence for the presence of a functioning corpus luteum and prior follicular maturation and
ovulation. Clearly clinically abnormal levels of gonadotropins, steroids, or other
biochemical parameters may be detected from a single sample. Preferably, multiple
samples could be collected and observed in conjunction with events in the menstrual cycle.
Ovulation can be estimated by the biphasic shift in basal body temperature. Ovulation
can also be detected by serial measurement of hormones in the blood or urine and analysis
of estradiol and gonadotropins at midcycle. After ovulation, luteal phase function can be
assessed by analysis of progesterone secretion and by evaluation of endometrial histology.
Tubal patency is an important endpoint that can be observed in clinical evaluations of
reproductive function (Forsberg, 1981). These latter evaluations are less likely to be
present in epidemiologic studies or surveillance programs because of the invasiveness of the
procedures.
III.C.l.c. Reproductive History Studies
Measures of fertility
Infertility or subfertility may be thought of as a nonevent: a couple is unable to
have children within a specific time frame. Therefore, the epidemiologic measurement of
reduced fertility is typically indirect and is accomplished by comparing birth rates or time
intervals between births or pregnancies. In these evaluations, the couple's joint ability to
procreate is estimated. One method, the Standardized Birth Ratio (SBR; also referred to as
the Standardized Fertility Ratio), compares the number of births observed to those expected
based on the person-years of observation stratified by factors such as time period, age, race,
marital status, parity, and contraceptive use (Wong et al., 1979; Levine et al., 1980, 1981,
1983; Levine, 1983; Starr et al., 1986). The SBR is analogous to the Standardized
Mortality Ratio (SMR), a measure frequently used in studies of occupational cohorts and
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has similar limitations in interpretation (Gaffey, 1976; McMichael, 1976; Tsai & Wen,
1986).
Analysis of the time between recognized pregnancies or live births has been
suggested as another indirect measure of fertility (Dobbins et al., 1978; Baird & Wilcox,
1985; Baird et al., 1986; Weinberg & Gladen, 1986). Because the time between births
increases with increasing parity (Leridon, 1977), comparisons within birth order (parity) are
more appropriate. A statistical method (Cox regression) can stratify by birth or pregnancy
order to help control for nonindependence of these events in the same woman or couple.
Fertility may also be affected by alterations in sexual behavior. However, data
linking toxic exposures to these alterations in humans are limited and are not easily
obtained in epidemiology studies (see Section IILC.l.e.).
Pregnancy outcomes
Pregnancy outcomes examined in human studies of parental exposures may include
embryo or fetal loss, congenital malformations, birth weight effects, sex ratio at birth, and
possibly postnatal effects (e.g., physical growth and development, organ or system function,
and behavioral effects of exposure). Postnatal effects are discussed in more detail in the
Guidelines for Developmental Toxicity Risk Assessment (U.S. Environmental Protection
Agency, 1991). As mentioned above, epidemiologic studies that focus on only one type of
pregnancy outcome may miss a true effect of exposure. Studies that examine multiple
endpoints could yield more information, but results may be more difficult to interpret.
Evidence of a dose-response relationship is usually an important criterion in the
assessment of exposure to a potentially toxic agent. However, traditional dose-response
relationships may not always be observed for some endpoints (Wilson, 1973). For
example, with increasing dose, a pregnancy might end in embryo or fetal loss, rather than a
live birth with malformations. A shift in the patterns of outcomes could result from
differences either in level of exposure or in timing (Wilson, 1973; Selevan & Lemasters,
1987) (for a more detailed description, see Section IILC.l.e.). Therefore, a risk assessment
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should, when possible, attempt to look at the relationship of different reproductive
endpoints and patterns of exposure.
In addition to the above effects, genetic damage to germ cells may potentially result
from exposures to the reproductive system. Outcomes resulting from germ cell mutations
could include reduced probability of fertilization and increased probability of embryo or
fetal loss and postnatal developmental effects. Based on studies with test species, critical
exposures are to germ cells or early zygotes. Germ cell mutagenicity could be expressed
also as genetic diseases in future generations. Unfortunately, these studies are difficult to
conduct in human populations because of the long time between exposure and outcome.
For more information, refer to the Guidelines for Mutagenicity Risk Assessment (U.S.
Environmental Protection Agency, 1986c).
Ill.C.l.d. Community Studies and Surveillance Programs
Epidemiologic studies may also be based on broad populations such as a community,
a nationwide probability sample, or surveillance programs (such as birth defects registries).
Other studies have examined environmental exposures such as toxicants in the water system
and adverse pregnancy outcome (Deane et al., 1989; Swan et al., 1989). Unfortunately, in
these studies, maternally-mediated effects may be difficult to distinguish from paternally-
mediated effects. In addition, the presumably lower exposure levels (compared with
industrial settings) may require very large groups for study. A number of case-referent
studies have examined the relationship between broad classes of parental occupation in
certain communities or countries and embryo/fetal loss (Silverman et al., 1985), birth
defects (Hemminki et al., 1980; Kwa & Fine, 1980; Papier, 1985), and childhood cancer
(Fabia & Thuy, 1974; Hakulinen et al., 1976; Kwa & Fine, 1980; Zack et al., 1980;
Hemminki et al., 1981; Peters et al., 1981). In these reports, jobs are typically classified
into broad categories based on the probability of exposure to certain classes or levels of
exposure (e.g., Kwa & Fine, 1980). Such studies are most helpful in the identification of
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topics for additional study. However, because of the broad groupings of types or levels of
exposure, such studies are not typically useful for risk assessment of a particular agent.
Surveillance programs may also exist in occupational settings. In this case,
reproductive histories or semen evaluations could be followed to monitor reproductive
effects of exposures. Both could yield very useful data for risk assessment; however, a
semen evaluation or other clinical program would be costly to maintain, and there are
numerous impediments to the collection of reliable and valid information in the workplace.
In addition to these concerns, impediments include potentially low employee participation
rates, employee sensitivities, and confidentiality requirements.
III.C.I.e. Identification of Important Exposures for Reproductive Effects
For all examinations of the relationship between reproductive effects and potentially
toxic exposures, the identification of the appropriate exposure is crucial. Preconceptional
exposures to either parent and in utero exposures have been associated with the more
commonly examined outcomes (e.g., fetal loss, malformations, low birth weight, and
measures of infertility). These exposures, plus postnatal exposure from breast milk, food,
and the environment, may also be associated with postnatal developmental effects (e.g.,
changes in behavioral and cognitive function or growth).
General environmental exposures are typically lower than in industrial or agricultural
settings. However, this relationship may change as exposures are reduced in workplaces
and as more is learned about environmental exposures (e.g. indoor air exposures, pesticides
usage). Larger populations are necessary in settings with lower exposures (Lemasters &
Selevan, 1984). Other factors affect the identification of reproductive or developmental
events with various levels of exposure. Exposed individuals may move in and out of areas
with differing levels and types of exposures, affecting the number of exposed and
comparison events for study.
Data on exposure from human studies are frequently qualitative, such as employment
or residence histories. More quantitative data may be difficult to obtain because of the
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nature of certain study designs (e.g. retrospective studies) and historical limitations in
exposure measurements. Many developmental outcomes result from exposures during
certain critical times. The appropriate exposure classification depends on the outcomes
studied, the biologic mechanism affected by exposure, and the biologic half-life of the
agent. The half-life, in combination with the patterns of exposure (e.g. continuous or
intermittent) affect the individual's body burden and consequently the "true" dose during
the critical period. The probability of misclassification of exposure status may affect the
ability to recognize a true effect in a study (Smith, P.E., 1939; Leridon, 1977; Collins,
1978; Scommegna et al., 1980; Deane et al., 1989; Swan et al., 1989). As more
prospective studies are done, better estimates of exposure will be developed.
III.C.2. Examination of Clusters, Case Reports, or Series
The identification of cases or clusters of adverse reproductive effects is generally
limited to those identified by the individuals involved or clinically by their physicians. The
likelihood of identification varies with the gender of the exposed person. Identification of
infertility in either gender is difficult. This might be thought of as identification of a
nonevent (e.g., lack of pregnancies or children), and thus is much harder to recognize than
are some developmental effects, including malformations, resulting from in utero exposure.
The identification of cases or clusters of adverse male reproductive outcomes may be
limited because of cultural norms that may inhibit the reporting of impaired fertility in men.
Identification is also limited by the decreased likelihood of recognizing adverse pregnancy
outcomes as a result of paternal exposure rather than maternal exposure. Thus far, only one
human male reproductive toxicant, dibromochloropropane (DBCP), has been identified after
observation of a cluster of male infertility through an atypically high level of
communication among the workers' wives (Whorton et al., 1977, 1979; Biava et al., 1978;
Whorton & Milby, 1980).
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Adverse effects identified in females have, thus far, been limited to adverse
pregnancy outcomes such as fetal loss and congenital malformations. Identification of other
effects, such as infertility or menstrual disorders, may be difficult, as noted above.
Case reports may have importance in the recognition of reproductive toxicants.
However, they are probably of greatest use in suggesting topics for further investigation.
Reports of clusters and case reports/series are best used in risk assessment in conjunction
with strong laboratory data to suggest that effects observed in test animals also occur in
humans.
III.D. PHARMACOKINETIC CONSIDERATIONS
Extrapolation of toxicity data between species can be aided considerably by the
availability of data on the pharmacokinetics of a particular agent in the species tested and,
when available, in humans. Information on absorption, half-life, steady-state or peak
plasma concentrations, placental metabolism and transfer, comparative metabolism, and
concentrations of the parent compound and metabolites in target organs may be useful in
predicting risk for reproductive toxicity. Such data may also be helpful in defining the
dose-response curve, developing a more accurate comparison of species sensitivity,
including that of humans (Wilson et al., 1975, 1977), determining dosimetry at target sites,
and comparing pharmacokinetic profiles for various dosing regimens or routes of exposure.
EPA's Office of Prevention, Pesticides, and Toxic Substances has published protocols for
metabolism studies that may be adapted to provide information useful in reproductive
toxicity risk assessment for a suspect toxicant. Pharmacokinetic studies in reproductive
toxicology are most useful if the data are obtained with animals that are at the same
reproductive status and stage of life (e.g., pregnant, nonpregnant, embryo or fetus, neonate,
prepubertal, adult) at which reproductive insults are expected to occur in humans.
Specific guidance regarding both the development and application of
pharmacokinetic data was agreed on by the participants of the Workshop on Dermal
Developmental Toxicity Studies (Kimmel, C.A. & Francis, 1990). This guidance is also
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applicable to non-dermal reproductive toxicity studies. Participants of the Workshop
concluded that absorption data are needed both when a dermal study does or does not show
effects. The results of a dermal study showing no effects and without blood level data are
potentially misleading and are inadequate for risk assessment, especially if interpreted as a
"negative" study. In studies where adverse effects are detected, regardless of the route of
exposure, pharmacokinetic data can be used to establish the internal dose in maternal and
paternal animals for risk extrapolation purposes.
The existence of a Sertoli cell barrier (formerly called the blood-testis barrier) in the
seminiferous tubules may influence the pharmacokinetics of a potential testicular toxicant
by restricting access of compounds to the adluminal compartment of seminiferous tubules.
The Sertoli cell barrier is formed by tight junctions between Sertoli cells and divides the
seminiferous epithelium into basal and adluminal compartments (Russell et al., 1990). The
basal compartment contains the spermatogonia and primary spermatocytes to the
preleptotene stage, whereas more advanced germ cells are located on the adluminal side.
This selectively permeable barrier is most effective in limiting the access of large,
hydrophilic molecules in the intertubular lymph to cells on the adluminal side. An
analogous barrier in the ovary has not been found, although the zona pellucida and
granulosa cells may modulate access of chemicals to oocytes.
The reproductive organs appear to have a wide range of metabolic capabilities
directed at both steroid and xenobiotic metabolism. However, there are substantial
differences between compartments within the organs in types and levels of enzyme
activities (Mukhtar et al., 1978). Recognition of these differences can be important in
understanding the potential of agents to have specific toxic effects.
Most pharmacokinetic studies have incompletely characterized the distribution of
toxic agents and their subsequent metabolic fate within the reproductive organs.
Generalizations based on hepatic metabolism are not necessarily adequate to predict the fate
of the agent in the testis, ovary, placenta, or conceptus. For example, the metabolic profile
for a given agent may differ in the male between the liver and the testis and between the
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maternal liver and placenta. Detailed interspecies comparisons of the metabolic capabilities
of the testis, ovary, placenta, and conceptus also have not been conducted. For some
xenobiotics, significant differences in metabolism have been identified between males and
females. This is, in part, attributable to organizational effects of the gonadal steroids in the
developing liver (Gustafsson et al., 1980; Skett, 1988). Also, in adults, the sex steroids
have been shown to affect the activity of a number of enzymes involved in the metabolism
of administered compounds. Thus, the blood levels of a toxicant, as well as the final
concentration in the target tissue, may differ significantly between sexes. If data are to be
used effectively in interspecies comparisons and extrapolations for these target systems,
more attention should be directed to the pharmacokinetic properties of chemicals in the
reproductive organs and in other organs that are affected by reproductive hormones.
III.E. COMPARISONS OF MOLECULAR STRUCTURE
Comparisons of the chemical or physical properties of an agent with those of known
reproductive toxicants may provide some indication of a potential for reproductive toxicity.
Such information may be helpful in setting priorities for testing of agents or for evaluation
of potential toxicity when only minimal data are available. Structure-activity relationships
have not been well studied in reproductive toxicology, although data are available that
suggest structure-activity relationships for certain classes of chemicals (e.g., glycol ethers,
some estrogens, androgens, other steroids, retinoids, phthalate esters, short-chain
halogenated hydrocarbon pesticides, metals). The literature has been reviewed and a set of
classifications offered relating structure to reported male reproductive activity (Bernstein,
1984). Although limited in scope and in need of validation, such schemes do provide
hypotheses that can be tested.
In spite of the limited information available on structure-activity relationships in this
field, under certain circumstances (e.g., in the case of new chemicals), this is one of several
procedures used to evaluate the potential for toxicity when little or no other data are
available.
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III.F. EVALUATION OF DOSE-RESPONSE RELATIONSHIPS
The evaluation of dose-response relationships for reproductive toxicity includes the
evaluation of data from both human and laboratory animal studies. When adequate dose-
response data are available in humans and with a sufficient range of exposure, dose-
response relationships in humans may be examined. Because data on human dose-response
relationships are available infrequently, the dose-response evaluation is usually based on the
assessment of data from tests performed in laboratory animals.
The dose-response relationships for individual endpoints, as well as the combination
of endpoints, must be examined in data interpretation.
Dose-response evaluations should consider the effects that competing risks between
different endpoints may have on outcomes observed at different exposure levels. For
example, a toxicant may interfere with cell function in such a manner that, at a low dose
level, an increase in abnormal sperm morphology is observed. At higher doses cell death
may occur, leading to a decrease in sperm counts and a possible decrease in proportion of
abnormal sperm.
When data on several species are available, the selection of the data for the dose-
response evaluation is based ideally on the response of the species most relevant to humans
(e.g., comparable physiologic, pharmacologic, pharmacokinetic, and pharmacodynamic
processes), the adequacy of dosing, the appropriateness of the route of administration, and
the endpoints selected. However, availability of information on many of those components
is usually very limited. For dose-response assessment, no single laboratory animal species
can be considered the best in all situations for predicting reproductive toxicologic risk to
humans. However, in some cases, such as in the assessment of physiologic parameters
related to menstrual disorders, higher nonhuman primates are considered generally similar
to the human. In the absence of a clearly most relevant species, data from the most
sensitive species (i.e., the species showing a toxic effect at the lowest administered dose)
are used, because humans are assumed to be as sensitive generally as the most sensitive
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animal species tested (Nisbet & Karch, 1983; Kimmel, C.A. et al., 1984, 1990; Hemminki
& Vineis, 1985; Meistrich, 1986; Working, 1988).
The evaluation of dose-response relationships includes the identification of effective
dose levels as well as doses that are associated with low or no increased incidence of
adverse effects compared with controls. Much of the focus is on the identification of the
critical effect(s) (i.e., the adverse effect occurring at the lowest dose level) and the LOAEL
and NOAEL associated with the effect(s). The NOAEL is the highest dose at which there
is no significant increase in the frequency of an adverse effect in any manifestation of
reproductive toxicity compared with the appropriate control group in a data base having
sufficient evidence for use in a risk assessment (see Section III.H. below). The LOAEL is
the lowest dose at which there is a significant increase in the frequency of adverse
reproductive effects compared with the appropriate control group in a data base having
sufficient evidence. An effect, whose incidence is statistically significant at a higher
exposure level, may be considered exposure-related if a biologically consistent trend is seen
at a lower level in which the observed difference hi incidence from the concurrent control
group may not reach statistical significance. Although a threshold is assumed for
reproductive effects, the existence of a NOAEL hi an experimental animal study does not
prove or disprove the existence or level of a biologic threshold; it only defines the highest
level of exposure under the conditions of the study that is not associated with a significant
increase in an adverse effect.
Several limitations in the use of the NOAEL have been described (Gaylor, 1983,
1989; Crump, 1984; Kimmel, C.A. & Gaylor, 1988; Brown & Erdreich, 1989; Kimmel,
C.A., 1990): 1) Use of the NOAEL focuses only on the dose that is the NOAEL and does
not incorporate information on the slope of the dose-response curve or the variability in the
data; 2) Because data variability is not taken into account (i.e., confidence limits are not
used), the NOAEL will likely be higher with decreasing sample size or poor study conduct,
either of which are usually associated with increasing variability in the data; 3) The
NOAEL is limited to one of the experimental doses; 4) The number and spacing of doses
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in a study can influence the dose that is chosen for the NOAEL; and 5) Because the
NOAEL is defined as a dose that does not produce an observable change in adverse
responses from control levels and is dependent on the power of the study, theoretically the
risk associated with it may fall anywhere between zero and an incidence just below that
detectable from control levels (usually in the range of 7 to 10% for quantal data). The
upper confidence limit on developmental risk at the NOAEL has been estimated for several
data sets to be 2 to 6% (Crump, 1984; Gaylor, 1989); similar evaluations have not been
conducted on data for other reproductive effects.
Because of the limitations associated with the use of the NOAEL (Kimmel, C.A. &
Gaylor, 1988; Gaylor, 1989; Kimmel, C.A., 1990), the Agency is evaluating the use of an
additional approach for more quantitative dose-response evaluation when sufficient data are
available, i.e., the benchmark dose (Crump, 1984). Calculation and use of the benchmark
dose are described in the Guidelines for Developmental Toxicity Risk Assessment (U.S.
Environmental Protection Agency, 1991). The benchmark dose is based on a model-
derived estimate of a particular incidence level, such as a 5 or 10% incidence. More
specifically, the benchmark dose is derived by modeling the data in the observed range,
selecting an incidence level within or near the observed range (e.g., the effective dose to
produce a 10% increased incidence of response, the ED^Q), and determining the upper
confidence limit on the model. The upper confidence value corresponding to, for example,
a 10% excess in response is used to derive the benchmark dose that is the lower confidence
limit on dose for that level of excess response, in this case, the LEDjQ.
With the benchmark dose approach, an LEDjQ should be calculated for each effect
of an agent for which there is a database with sufficient evidence to conduct a risk
assessment. In some cases, the data may be sufficient to also estimate the EDQ5 or EDQ1
which may be closer to a true no-effect dose. A level between the ED0j and the EDjQ is
usually the lowest level of risk that can be estimated adequately for binomial endpoints
from standard developmental toxicity studies.
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Various mathematical approaches have been proposed for deriving a benchmark dose
in modeling developmental toxicity data (Crump, 1984; Rai & Van Ryzin, 1985; Kimmel,
C.A. & Gaylor, 1988; Chen & Kodell, 1989; Faustman et al., 1989; Kodell et al., 1991). A
benchmark dose approach has been applied to male reproductive effects of
dibromochloropropane (Pease et al., 1991). Such models may be used to calculate the
benchmark dose, and choice of the model may not be critical since estimation is within the
observed dose range. Because the model is only used to fit the observed data, the
assumptions about the existence of a threshold do not affect choice of model. Thus, any
model that fits the empirical data well is likely to provide a reasonable estimate of the
benchmark dose, although if there is some biologic reason to incorporate particular factors
in the model (e.g., intralitter correlation; sex-specific dosing), these should be included to
account as much as possible for variability in the data. The Agency is exploring the
application of several models to data sets for calculating the benchmark dose, as well as to
determine the minimum data set that can be modeled and how to apply this approach to
continuous data. In addition, information from these studies will be used to develop
guidance for application of the benchmark dose approach to the calculation of the RfD or
RfC since the Agency has limited experience with this approach.
Generally, in studies that do not evaluate reproductive toxicity, only adult male and
nonpregnant females are examined. Therefore, the possibility that pregnant females may be
more sensitive to the agent is not tested. In studies in which reproductive toxicity has been
evaluated, the NOAEL, LOAEL, or benchmark dose should be identified for both
reproductive and other forms of systemic toxicity. The NOAEL, LOAEL, or benchmark
dose for systemic toxicity in the reproductive study should be compared with the
corresponding values from other adult toxicity data to determine if the pregnant or lactating
female may be more sensitive to an agent based on results from that study or compared
with results for adult males or nonpregnant females in other toxicity studies.
In addition to identification of the NOAEL, LOAEL, or benchmark dose, the dose-
response evaluation defines the range of doses that is effective in producing reproductive
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and other forms of systemic toxicity for a given agent, the route of exposure, timing and
duration of exposure, species specificity of effects, and any pharmacokinetic or other
considerations that might influence the comparison with human exposure scenarios. This
information should always accompany the characterization of the health-related data base
(discussed in the next section).
For developmental toxic effects, an assumption is made that a single exposure at a
critical time in development may produce an adverse developmental effect (U.S.
Environmental Protection Agency, 1991). Therefore, the daily dose is usually not adjusted
for duration of exposure with developmental toxicity unless appropriate pharmacokinetic
data are available. However, for other reproductive effects, daily dose may be adjusted for
duration of exposure. The Agency is planning to review these stances to determine the
most appropriate approach for the future.
III.G. CHARACTERIZATION OF THE HEALTH-RELATED DATA BASE
This section describes evaluation of the health-related data base on a particular
chemical and provides criteria for judging the potential for that chemical to produce
reproductive toxicity under the exposure conditions inherent in the data base. This
determination provides the basis for judging whether the available data are sufficient to
proceed with a quantitative risk assessment. Characterizing the available evidence in this
way clarifies the strengths and uncertainties in a particular data base. It does not address
the level of concern, nor does it completely address determining relevancy of available data
for estimating human risk. Both level of concern and revelancy are discussed as part of the
final characterization of risk because they depend on information concerning potential
human exposure.
A complex interrelationship exists among study design, statistical analysis, and
biologic significance of the data. Thus, substantial scientific judgment, based on experience
with reproductive toxicity data and with the principles of study design and statistical
analysis, may be required to adequately evaluate the data base. In some cases, a data base
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may contain conflicting data. In these instances, the risk assessor must consider each
study's strengths and weaknesses within the context of the overall data base to characterize
the evidence for assessing the potential hazard for reproductive toxicity. Scientific
judgment is always necessary, and in many cases, interaction with scientists in specific
disciplines (e.g., reproductive toxicology, developmental toxicology, epidemiology, genetic
toxicology, statistics) is recommended.
A scheme for judging the available evidence on the reproductive toxicity of a
particular agent is presented below (Table 5). The scheme contains two broad categories,
"Sufficient" and "Insufficient," which are defined in Table 5. Data from all available
studies, whether or not indicative of potential concern, are evaluated and used to judge
whether available evidence allows a hazard assessment for reproductive toxicity. The
primary considerations are the human data, if available, and the experimental animal data.
The judgment of whether data are sufficient or insufficient should consider a variety of
parameters that contribute to the overall quality of the data, such as the power of the studies
(e.g., number of animals and variation in the data), the number and types of endpoints
examined, replication of effects, relevance of route and timing of exposure for both human
and experimental animal studies, and the appropriateness of the dose selection in
experimental animal studies. In addition, pharmacokinetic data and structure-activity
considerations, data from other toxicity studies, as well as other factors that may affect the
overall decision about the evidence, should be taken into account.
In general, the characterization is based on criteria defined by these Guidelines as
the minimum evidence necessary to complete a hazard identification/dose-response
evaluation. Establishing the minimum human evidence to do a hazard identification/dose-
response evaluation is often difficult because there are often considerable variations in study
designs and study group selection. The body of data should contain convincing evidence as
described in the "Sufficient Human Evidence" category. Because the human data necessary
to judge whether or not a causal relationship exists are generally limited, few agents can be
classified in this category. Agents that have been tested in laboratory animals according to
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EPA's current two-generation reproductive effects test guidelines (U.S. Environmental
Protection Agency, 1982, 1985b), but not limited to such designs (e.g., a continuous
breeding study with two generations), generally would be included in the "Sufficient
Experimental Animal Evidence/Limited Human Data" category. There are occasions in
which more limited data regarding the potential reproductive toxicity of an agent (e.g., a
one-generation reproductive effects study, a standard subchronic or chronic toxicity study in
which the reproductive organs were well examined) are available. If reproductive toxicity
is observed in these limited studies, the data may be used to the extent possible to reach a
decision regarding hazard to the reproductive system. In cases in which only such limited
data are available, it would be appropriate to adjust the uncertainty factor to reflect the
attendant increased uncertainty regarding the use of these data until more definitive data are
developed. Identification of the increased uncertainty and justification for the adjustment of
the uncertainty factor should be stated clearly.
Because it is more difficult both biologically and statistically to support a finding of
no apparent hazard, more data are generally required to support this conclusion than a
finding for a potential hazard. For example, to judge that a hazard for reproductive toxicity
could exist for a given agent, the minimum evidence could be data from a single
appropriate, well-executed study in a single test species that demonstrates an adverse
reproductive effect, or suggestive evidence from adequately conducted clinical or
epidemiologic studies. As in all situations, it is important that the results be biologically
consistent. On the other hand, to judge that an agent is unlikely to pose a hazard for
reproductive toxicity, the minimum evidence would include data on an array of endpoints
and from more than one study that showed no reproductive effects at doses that were
otherwise minimally toxic to the adult animal. In addition, there may be human data from
appropriate studies that are supportive of no apparent hazard. In the event that a substantial
data base exists for a given chemical, but no single study meets current test guidelines, the
risk assessor should use scientific judgment to determine whether the composite data base
may be viewed as meeting the "Sufficient" criteria.
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TABLE 5
CATEGORIZATION OF THE HEALTH-RELATED DATA BASE
HAZARD IDENTIFICATION/DOSE-RESPONSE EVALUATION
SUFFICIENT EVIDENCE
The Sufficient Evidence category includes data that collectively provide enough
information to judge whether or not a reproductive hazard could exist within the context of
dose, duration, timing and route of exposure. This category includes both human and
experimental animal evidence.
Sufficient Human Evidence: This category includes data from epidemiologic studies (e.g.,
case control and cohort) that provide convincing evidence for the scientific community to
judge that a causal relationship is or is not supported. A case series in conjunction with
strong supporting evidence may also be used. Supporting test animal data may or may not
be available.
Sufficient Experimental Animal Evidence/Limited Human Data: This category includes
data from experimental animal studies and/or limited human data that provide convincing
evidence for the scientific community to judge if the potential for reproductive toxicity
exists. Generally, agents that have been tested according to EPA's current two-generation
reproductive effects test guidelines (but not limited to such designs) would be included in
this category. The minimum evidence necessary to judge that a potential hazard exists
would be data demonstrating an adverse reproductive effect in a single appropriate, well-
executed study in a single test species. The minimum evidence needed to judge that a
potential hazard does not exist would include data on an adequate array of endpoints and
from more than one study that showed no adverse reproductive effects at doses that were
minimally toxic to the adult animal.
INSUFFICIENT EVIDENCE
This category includes situations for which there is less than the minimum sufficient
evidence necessary for assessing the potential for reproductive toxicity, such as when no
data are available on reproductive toxicity, as well as for data bases from studies in test
animals or humans that have a limited study design (e.g., small numbers of animals or
human subjects, inappropriate dose selection or exposure information, other uncontrolled
factors), data from studies that examined only a limited number of endpoints and reported
no adverse reproductive effects, or data bases that were limited to information on structure-
activity relationships, short-term tests, pharmacokinetic data, or metabolic
precursors.attendant increased uncertainty regarding the use of these data until more
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TABLE 5 (Continued)
definitive data are developed. Identification of the increased uncertainty and justification
for the adjustment of the uncertainty factor should be stated clearly.
Because it is more difficult both biologically and statistically to support a finding of
no apparent hazard, more data are generally required to support this conclusion than a
finding for a potential hazard. For example, to judge that a hazard for reproductive toxicity
could exist for a given agent, the minimum evidence could be data from a single
appropriate, well-executed study in a single test species that demonstrates an adverse
reproductive effect, or suggestive evidence from adequately conducted clinical or
epidemiologic studies. As in all situations, it is important that the results be biologically
consistent. On the other hand, to judge that an agent is unlikely to pose a hazard for
reproductive toxicity, the minimum evidence would include data on an array of endpoints
and from more than one study that showed no reproductive effects at doses that were
otherwise minimally toxic to the adult animal. In addition, there may be human data from
appropriate studies that are supportive of no apparent hazard. In the event that a
substantial data base exists for a given chemical, but no single study meets current test
guidelines, the risk assessor should use scientific judgment to determine whether the
composite data base may be viewed as meeting the "Sufficient" criteria.
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Some important considerations in determining the confidence in the health data base
are as follows:
* Data of equivalent quality from human exposures are given more weight than
data from exposures of test species.
* Although a single study of high quality could be sufficient to achieve a
relatively high level of confidence, replication increases the
confidence that may be placed in such results.
* Data are available from one or more in vivo studies of acceptable quality
with humans or other mammalian species that are believed to be predictive of
human responses.
* Data exhibit a dose-response relationship.
* Results are statistically significant and biologically plausible.
* When multiple studies are available, results are reproducible.
* When multiple studies are available, the lines of evidence from independent
study types are reinforcing.
* Sufficient information is available to reconcile discordant data.
* Route, level, duration, and frequency of exposure are appropriate.
* An adequate array of endpoints has been examined.
* The power and statistical treatment of the studies are appropriate.
Any statistically significant deviation from baseline levels for an in vivo effect
warrants closer examination. To determine whether such a deviation constitutes an adverse
effect requires an understanding of its role within a complex system and the determination
of whether a "true effect" has been observed. Application of the above criteria, combined
with guidance presented in Section III.B. can facilitate such determinations.
The greatest confidence for reproductive hazard identification should be placed on
significant adverse effects on sexual behavior, fertility or pregnancy outcomes, or other
endpoints that are directly related to reproductive function such as menstrual (estrous) cycle
normality, sperm evaluations, reproductive histopathology, reproductive organ weights, and
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possibly reproductive endocrinology (see III.B.3.e. for qualifying statement). Agents
producing effects on these endpoints can be assigned to the "Sufficient Evidence" category
if study quality is adequate.
Less confidence should be placed in results from other measures such as in vitro
tests, data from nonmamnials or structure-activity relationships, but positive results may
trigger followup studies to determine the likelihood and extent to which function might be
affected. Results from these types of studies alone, whether or not they demonstrate an
effect, should be assigned to the "Insufficient Evidence" category.
The absence of effects with test species on the endpoints that are evaluated routinely
(i.e., fertility, histopathology, and organ weights) may constitute sufficient evidence to place
a low priority on the potential reproductive toxicity of a chemical. However, in such cases,
careful consideration should be given to the sensitivity of these endpoints and to the quality
of the data on these endpoints. Consideration should also be given to the possibility of
adverse effects that may not be reflected in these routine measures (e.g., germ-cell
mutation, alterations in estrous cyclicity or sperm measures such as motility or
morphology).
Judging that the health data base indicates a potential reproductive hazard does not
mean that the agent will be a hazard at every exposure level (because of the assumption of
a threshold) or in every situation (e.g., the type and degree of hazard may vary significantly
depending on route and timing of exposure). In the final risk characterization, the
characterization of the health-related data base should always be presented with information
on the dose-response evaluation (e.g., LOAEL, NOAEL, or benchmark dose), exposure
route, timing and duration of exposure, and if available, with the human exposure estimate
(for further discussion, see Section V).
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III.H. DETERMINATION OF THE REFERENCE DOSE OR REFERENCE
CONCENTRATION FOR REPRODUCTIVE TOXICITY
In quantitative risk assessment, the existence of a threshold is usually assumed for
noncarcinogenic health effects. The assumption of a threshold suggests that the application
of adequate uncertainty factors to a NOAEL, LOAEL, or benchmark dose will result in an
exposure level for all humans that is not attended with significant risk. In the absence of a
threshold, it is assumed that some finite risk exists at any level of exposure, with risk
decreasing as exposure decreases. In the absence of data on the responses at low levels of
exposure and associated mechanistic information, a threshold is assumed for reproductive
effects. It is plausible that certain biologic processes (e.g., Sertoli cell barrier selectivity,
metabolic and repair capabilities of the germ cells) may impede the attainment or
maintenance of concentrations of the agent at the target site following exposure to low dose
levels that would be associated with adverse effects.
The RfD or RfC is an estimate of a daily exposure to the human population that is
assumed to be without appreciable risk of deleterious reproductive effects over a lifetime of
exposure. The RfD or RfC is derived by applying uncertainty factors to the NOAEL (or
the LOAEL if a NOAEL is not available) or to the benchmark dose. To date, the Agency
has applied uncertainty factors only to the NOAEL or LOAEL to derive an RfD or RfC.
The Agency is considering the use of the benchmark dose approach as the basis for
derivation of the RfD or RfC and will develop guidance as information is acquired and
analyzed from ongoing Agency studies. Because of the short duration of most studies of
developmental toxicity, a unique value (RfDDT or RfCDT) is determined for adverse
developmental effects. For adverse reproductive effects on endpoints other than those of
developmental toxicity, no special designator is attached.
The effect used for determining the NOAEL, LOAEL, or benchmark dose in
deriving the RfD or RfC is the most sensitive adverse reproductive endpoint (i.e., the
critical effect) from the most appropriate or, in the absence of such information, the most
sensitive mammalian species (see Sections II and III.B.l.). Uncertainty factors for
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reproductive and other forms of systemic toxicity applied to the NOAEL generally include
factors of 3 or 10 each for interspecies variation and for intraspecies variation. Additional
factors may be applied to account for other uncertainties that may exist in the data base.
For example, the standard study design for a reproductive toxicity study calls for a low
dose that demonstrates a NOAEL, but in some cases, the lowest dose administered may
cause significant adverse effects and thus be identified as the LOAEL. In circumstances
where only a LOAEL is available, the use of an additional uncertainty factor of 10 may be
required, depending on the sensitivity of the endpoints evaluated, adequacy of dose levels
tested, or general confidence in the LOAEL. In addition, if a benchmark dose has been
calculated, it may be used to help interpret how close the LOAEL is to a level that would
not be detectable from controls (equivalent to NOAEL), and thus affect the size of the
uncertainty factor to be applied. Additional areas of uncertainty may be identified and
modifying factors used depending on the characterization of the data base (e.g., if the only
data available are from a one-generation reproductive effects study; see Section III.G.), data
on pharmacokinetics, or other considerations that may alter the level of confidence in the
data (U.S. Environmental Protection Agency, 1987). The total size of the uncertainty factor
will vary from agent to agent and requires scientific judgment, taking into account
interspecies differences, variability within species, the slope of the dose-response curve, the
types of reproductive effects observed, the background incidence of the effects, the route of
administration, and pharmacokinetic data.
There is no experience with the application of uncertainty factors to the benchmark
dose approach for calculating the RfD or RfC, and there are several issues that must be
addressed prior to its use for this purpose; for example, which benchmark dose (e.g.,
LEDQ1, LEDQ5, LED10) should be used for calculating the RfD or RfC, and what are the
appropriate uncertainty factors that should be applied to the benchmark dose for deriving
the RfD or RfC? That is, should the uncertainty factor applied to an LED10 be similar to
that applied to a LOAEL, or should the uncertainty factor applied to an LED01 be equal to
or less than that applied to a NOAEL? These questions are being addressed in ongoing
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Agency studies on the calculation of the RfD or RfC using the benchmark dose approach.
As results become available and as further guidance is developed, this information will be
published as a supplement to these Guidelines.
The total uncertainty factor selected is divided into the NOAEL or LOAEL (or the
benchmark dose) for the critical effect in the most appropriate or most sensitive mammalian
species to determine the RfD or RfC. If the NOAEL or LOAEL (or benchmark dose) for
other forms of systemic toxicity is lower than that for reproductive toxicity, this should be
noted in the risk characterization, and this value compared with data from other studies in
which adult animals are exposed. Thus, the reproductive toxicity data should be discussed
in the context of other toxicity data.
The modeling approaches that have been proposed for developmental toxicity are,
for the most part, statistical probability models that do not take into account underlying
biologic processes or mechanisms (Crump, 1984; Rai & Van Ryzin, 1985; Kimmel, C.A.
& Gaylor, 1988; Chen & Kodell, 1989; Faustman et al., 1989; Kodell et al., 1991). These
approaches may also be applicable for modeling reproductive toxicity data and can be
applied to derive dose-response curves for data in the observed dose range, but may or may
not accurately predict risk at low levels of exposure. It has generally been assumed that
there is a biologic threshold for reproductive toxicity, based on known homeo static,
compensatory, or adaptive mechanisms that must be overcome before a toxic endpoint is
manifested and on the rationale that cells and organs of the reproductive system and the
embryo are known to have some capacity for repair of damage. However, a threshold for a
population may not exist because of other endogenous or exogenous factors that may
increase the sensitivity of some individuals in the population. Thus, the addition of a
toxicant may result in an increased risk of adverse effects for some, but not necessarily all
individuals within the population.
Efforts are underway to develop models that are more biologically based. These
models should provide a more accurate estimation of low-dose risk to humans. The
development of biologically based dose-reponse models in reproductive toxicology have
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been impeded by a number of factors, including limited understanding of the biologic
mechanisms underlying reproductive toxicity, intra- and interspecies differences in the types
of reproductive events, lack of appropriate pharmacokinetic data, and inadequate
information on the influence of other types of systemic toxicity on the dose-response curve.
III.I. SUMMARY
The hazard identification/dose-response evaluation of reproductive toxicity data is
incorporated into the final characterization of risk along with information on estimates of
human exposure. The analysis depends on and should describe scientific judgments as to
the accuracy and sufficiency of the health-related data in experimental animals and humans
(if available), the biologic relevance of significant effects, and other considerations
important in the interpretation and application of data to humans. Scientific judgment is
always necessary, and in many cases, interaction with scientists in specific disciplines (e.g.,
reproductive toxicology, developmental toxicology, epidemiology, statistics) is
recommended.
IV. EXPOSURE ASSESSMENT
To obtain a quantitative estimate of risk for the human population, an estimate of
human exposure is required. The Guidelines for Exposure Assessment have been published
separately (U.S. Environmental Protection Agency, 1992) and will not be discussed in detail
here. Rather, issues important to reproductive toxicity risk assessment are addressed. In
general, the exposure assessment describes the magnitude, duration, schedule, and route of
human exposure. This information is usually developed from monitoring data, from
estimates based on modeling of environmental exposures, and from application of
paradigms to exposure data bases. Often quantitative estimates of exposures may not be
available (e.g., workplace or environmental measurements). In such instances, employment
or residential histories also may be used in characterizing exposure in a qualitative sense.
The potential use of biomarkers as indicators of exposure is an area of active interest.
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Studies of occupational populations may provide valuable information on the
potential environmental health risks for certain agents. Exposures among environmentally
exposed human populations tend to be lower (but of longer duration) than those in studies
of occupationally exposed populations and therefore may require more observations to
assure sufficient statistical power. Also, reconstruction of exposures is more difficult in an
environmental study than in those done in workplace settings where industrial hygiene
monitoring may provide more detailed exposure data.
The nature of the exposure may be defined at a particular point or may reflect
cumulative exposure. Each approach makes an assumption about the underlying
relationship between exposure and outcome. For example, a cumulative exposure measure
assumes that total exposure is important, with a greater probability of effect with greater
total exposure or body burden. A dichotomous exposure measure (ever exposed versus
never exposed) assumes an irreversible effect of exposure. Models that define exposure
only at a specific time may assume that only the present exposure is important (Selevan &
Lemasters, 1987). The appropriate exposure model depends on the biologic processes
affected. Thus, a cumulative or dichotomous exposure model may be appropriate if injury
occurs in cells that cannot be replaced or repaired (e.g., Sertoli cells, oocytes); on the other
hand, a concurrent exposure model may be appropriate for cells that are being generated
continually (e.g., spermatids).
There are a number of unique considerations regarding the exposure assessment for
reproductive toxicity. Exposure at different stages of male and female development can
result in different outcomes. Such age-dependent variation has been well documented in
both experimental animal and human studies. Prenatal and neonatal treatment can
irreversibly alter reproductive function in a manner that may not be predicted from adult-
only exposure. Moreover, chemicals that alter sexual differentiation in rodents during these
periods may have similar effects in humans, because the mechanisms underlying these
developmental processes appear to be similar in all mammalian species (Gray, 1991).
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The susceptibility of elderly males and females to chemical insult has not been well
studied. Although procreative competence may not be a major health concern with elderly
individuals, other biologic functions maintained by the gonads (e.g., hormone production)
are of significance (Walker, 1986). An exposure assessment should characterize the
likelihood of exposure of these different subgroups (embryo or fetus, neonate, juvenile,
young adult, older adult) and the risk assessment should factor in the susceptibility of dif-
ferent age groups to the extent possible.
The relationship between time or duration of exposure and observation of male
reproductive effects has particular significance for short-term exposures. Spermatogenesis
is a temporally synchronized process. In humans, germ cells that were spermatozoa,
spermatids, spermatocytes, or spermatogonia at the time of an acute exposure require 1 to 2,
3 to 5, 5 to 8, or 8 to 12 weeks, respectively, to appear in an ejaculate. That timing may
vary somewhat depending on degree of sexual activity. It is possible that an end point may
be examined too early or too late to detect an effect if only a particular cell type was
affected during a relatively brief exposure to an agent. The absence of an effect when
observations were made too late suggests either a reversible effect or no effect. However,
an effect that is reversible at lower exposures might become irreversible with higher or
longer exposures or exposure of a more susceptible individual. Thus, the failure to detect
transient effects because of improper timing of observations may be important. If
information is available on the type of effect expected from a class of agents, it may be
possible to evaluate whether the timing of endpoint measurement relative to the timing of
the short-term exposure is appropriate. Some information on the appropriateness of the
protocol can be obtained if test animal data are available to identify the most sensitive cell
type or the putative mechanism of action for a given agent.
Compared with acute exposures, the link between exposure and outcome may be
more apparent with relatively constant subchronic or longer exposures that are of sufficient
duration to cover all phases of spermatogenesis (Russell et al., 1990). Assessments may be
made at any time after this point as long as exposure remains constant. Time required for
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the agent or metabolite to attain steady-state levels should also be considered. Again,
application of models of exposure (e.g., dichotomous, concurrent, or cumulative) depends
on the suspected target or mechanism of action.
The reversibility of an adverse effect on the male reproductive system can be
affected by the degree and duration of exposure. The degree of stem cell loss is inversely
related to the degree of restoration of sperm production, because repopulation of the
germinal epithelium is dependent on the stem cells (Foote & Berndtson, 1992). For agents
that bioaccumulate, increasing duration of exposure may also increase the extent of damage
to the stem cell population. Damage to other spermatogenic cell types reduces the number
of sperm produced, but recovery should occur when the toxic agent is removed. Less is
known about the effects of toxicity on the Sertoli cells. Temporary impairment of Sertoli
cell function may produce long-lasting effects on spermatogenesis. Destruction of Sertoli
cells or interference with their proliferation before puberty are irreversible effects because
replication ceases after puberty. Sertoli cells are essential for support of the spermatogenic
process and loss of those cells results in a permanent reduction of spermatogenic capability
(Foster, 1992).
When recovery is possible, the duration of the recovery period is determined by the
tune for regeneration (for stem cells) and repopulation of the affected spermatogenic cell
types and appearance of those cells as sperm in the ejaculate. The time required for these
events to occur varies with the species, the pharmacokinetic properties of the agent, the
extent to which the stem cell population has been destroyed, and the degree of sublethal
toxicity inflicted on the stem cells or Sertoli cells. When the stem cell population has been
partially destroyed, humans require longer than mice to reach the same degree of recovery
(Meistrich & Samuels, 1985).
Unique considerations in the assessment of female reproductive toxicity include the
duration and period of exposure as related to the development or stage of reproductive life
(e.g., prenatal, prepubescent, reproductive, or postmenopausal) or considerations of different
physiologic states (e.g., nonpregnant, pregnant, lactating).
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For infertility, a cumulative exposure measure assumes destruction of increasing
numbers of primary oocytes with greater lifetime exposure or increasing body burden.
However, humans may be exposed to varying levels of toxicants within the study period.
Exposures during certain critical points in the reproductive process may affect the outcomes
observed in humans (Lemasters & Selevan, 1984). In test species, perinatal exposure to
androgens or estrogens such as zearalenone, methoxychlor, and DDT (Bulger & Kupfer,
1985; Gray et al., 1985), have been shown to advance puberty and masculinize females.
Similar effects have been reported in humans (both sexes) exposed neonatally to synthetic
estrogens or progestins (Schardein, 1985; Steinberger & Lloyd, 1985). Studies using test
species have also shown that exposure to some environmental agents such as ionizing
radiation (Dobson & Felton, 1983) and glycol ethers (Heindel et al., 1989) can deplete the
pool of primordial follicles and thus significantly shorten the female's reproductive lifespan.
Furthermore, exposure to compounds at different stages of the ovarian cycle can disrupt or
delay follicular recruitment and development (Armstrong, 1986), ovulation (Everett &
Sawyer, 1950; Terranova, 1980), and ovum transport (Cumrnings & Perreault, 1990).
Compounds that delay ovulation can lead to significant alterations in egg viability (Peluso
et al., 1979), fertilizability of the egg (Fugo & Butcher, 1966; Butcher & Fugo, 1967;
Butcher et al., 1975) and a reduction in litter size (Fugo & Butcher, 1966). After ovulation,
single exposures to compounds such as carbendazim also alter the fertilizability of the ova
(Perreault et al., 1992). Thus, knowledge of when acute exposures are administered
relative to the female's lifespan and reproductive cycle can provide insight into how an
agent disrupts reproductive function.
DES is a classic example of an agent causing different effects on the reproductive
system in the developing organism compared with those in adults (McLachlan, 1980).
DES interferes with the development of the Mullerian and Wolffian duct systems and
thereby causes irreversible structural and functional damage to the developing reproductive
system. In adults, the reproductive effects that are caused by the estrogenic activity of DES
do not necessarily result in permanent damage.
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Unique considerations for outcomes of pregnancy are duration and period of
exposure as related to stage of development (i.e., critical periods) and the possibility that
even a single exposure may be sufficient to produce adverse developmental effects.
Repeated exposure is not a necessary prerequisite for developmental toxicity to be
manifested, although it should be considered in cases where there is evidence of cumulative
exposure or where the half-life of the agent is long enough to produce an increasing body
burden over time. For these reasons, it is assumed that, in most cases, a single exposure at
the critical time in development is sufficient to produce an adverse developmental effect.
Therefore, the human exposure estimates used to calculate the MOE for an adverse
developmental effect or to compare to the RfD or RfC are usually based on a single daily
dose that is not adjusted for duration or pattern (e.g., continuous or intermittent) of
exposure. For example, it would be inappropriate to use time-weighted averages or
adjustment of exposure over a different time frame than that actually encountered (such as
the adjustment of a 6-hour inhalation exposure to account for a 24-hour exposure scenario)
unless pharmacokinetic data were available to indicate an accumulation with continuous
exposure. In the case of intermittent exposures, examination of the peak exposures as well
as the average exposure over the time of exposure would be important.
It should be recognized that, based on the definitions used in these Guidelines for
reproductive toxicity, almost any segment of the human population may be at risk for a
reproductive effect. Although the reproductive effects of exposures may be manifested
while the exposure is occurring (e.g., menstrual disorder, decreased sperm count,
spontaneous abortion) some effects may not be detectable until later in life (e.g., premature
reproductive senescence due to oocyte depletion), long after exposure has ceased.
V. RISK CHARACTERIZATION
V.A. OVERVIEW
A risk characterization is a necessary part of any Agency report on risk whether the
report is a preliminary one prepared to support allocation of resources toward further study
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or a comprehensive one prepared to support regulatory decisions. In this final step of a risk
assessment, the risk characterization involves integration of toxicity information from the
hazard identification/dose-response evaluation with the human exposure estimates and
provides an evaluation of the overall quality of the assessment, describes risk in terms of
the nature and extent of harm, and communicates results of the risk assessment to a risk
manager. A risk manager can then use the risk assessment, along with other risk
management elements, to make public health decisions. The information should also assist
others outside the Agency in understanding the scientific basis for regulatory decisions.
Risk characterization is the culmination of the risk assessment process and is
intended to summarize key aspects of the following components of the risk assessment:
1. The nature, reliability and consistency of the data used.
2. The reasons for selection of the key study(ies) and the critical effect(s) and
their relevance to human outcomes.
3. The qualitative and quantitative descriptors of the results of the risk
assessment.
4. The limitations of the available data, the assumptions used to bridge
knowledge gaps in working with those data, and implications of using
alternative assumptions.
5. Discussion of the strengths and weaknesses of the risk assessment and the
level of scientific confidence in the assessment.
6. Identification of the areas of uncertainty, additional data/research needs to
improve confidence in the risk assessment, and the potential impacts of the
new research.
The risk characterization should be limited to the most significant and relevant data,
conclusions and uncertainties. When special circumstances exist that preclude full
assessment, those circumstances should be explained and the related limitations identified.
The following sections describe these aspects of the risk characterization in more
detail, but do not attempt to provide a full discussion of risk characterization. Rather, these
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Guidelines point out issues that are important to risk characterization for reproductive
toxicity. Comprehensive general guidance for risk characterization is provided by Habicht
(1992).
V.B. INTEGRATION OF HAZARD IDENTIFICATION/DOSE-RESPONSE AND
EXPOSURE ASSESSMENTS
In developing the hazard identification/dose-response and exposure assessment
portions of the risk assessment, risk assessors must make judgments concerning human
relevance of the toxicity data, including the appropriateness of the various test animal
models for which data are available, the route, timing and duration of exposure relative to
the expected human exposure. These judgments should be summarized at each stage of the
risk assessment process. When data are not available to make such judgments, as is often
the case, the background information and assumptions discussed in the Overview (Section
I) provide default positions. The rationale behind the use of a default position should be
clearly stated. In integrating the parts of the assessment, risk assessors must determine if
some of these judgments have implications for other portions of the assessment, and
whether the various components of the assessment are compatible.
The description of the relevant data should convey the major strengths and
weaknesses of the assessment that arise from availability and quality of data and the current
limits of understanding of the mechanisms of toxicity. Confidence in the results of a risk
assessment is a function of confidence in the results of these analyses. The hazard
identification/dose response and exposure assessment sections should each have their own
characterization, and these characterizations should be summarized and integrated into the
overall risk characterization. Interpretation of data should be explained, and risk managers
should be given a clear picture of consensus or lack of consensus that exists about
significant aspects of the assessment. When more than one interpretation is supported by
the data, the alternative plausible approaches should be presented along with the strengths,
weaknesses, and impacts of those options. If one interpretation or option has been selected
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over another, the rationale should be given; if not, then both should be presented as
plausible alternatives.
The risk characterization should not only examine the judgements, but also explain
the constraints of available data and the state of knowledge about the phenomena studied in
making them including:
* The qualitative conclusions about the likelihood that the chemical may pose a
specific hazard to human health, the nature of the observed effects, under what conditions
(route, dose levels, time, and duration) of exposure these effects occur, and whether the
health-related data are sufficient and relevant to use in a risk assessment.
* A discussion of the dose-response patterns for the critical effect(s) and their
relationships to the occurrence of other toxicity, data such as the shapes and slopes of the
dose-response curves for the various other endpoints, the rationale behind the determination
of the NOAEL, LOAEL, and benchmark dose, and the assumptions underlying the
estimation of the RfD or RfC.
* Descriptions of the estimates of the range of human exposure (e.g., central
tendency, high end), the route, duration, and pattern of the exposure, relevant
pharmacokinetics, and the size and characteristics of the various populations that might be
exposed.
* The risk characterization of an agent being assessed for reproductive toxicity
should be based on data from the most appropriate species or, if such information is not
available, on the most sensitive species tested. It should also be based on the most
sensitive indicator of an adverse reproductive effect, whether in the male, the female
(nonpregnant or pregnant), or the developing organism, and should be considered in relation
to other forms of toxicity. The relevance of this indicator to human reproductive outcomes
should be described.
If data to be used in a risk characterization are from a route of exposure other than
the expected human exposure, then pharmacokinetic data should be used, if available, to
extrapolate across routes of exposure. If such data are not available, the Agency makes
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certain assumptions concerning the amount of absorption likely or the applicability of the
data from one route to another (U.S. Environmental Protection Agency, 1985a, 1986b).
Discussion of some of these issues may be found in the Proceedings of the Workshop on
Acceptability and Interpretation of Dermal Developmental Toxicity Studies (Kimmel, C.A.
& Francis, 1990). and Principles of Route-to-Route Extrapolation for Risk Assessment
(Gerrity et al, 1990).
The level of confidence in the hazard identification/dose-response evaluation should
be stated to the extent possible, including placement of the agent into the appropriate
category regarding the sufficiency of the health-related data (see Section III.G.). A
comprehensive risk assessment ideally includes information on a variety of endpoints that
provide insight into the full spectrum of potential reproductive responses. A profile that
integrates both human and test species data and incorporates both sensitive endpoints (e.g.,
properly performed and fully evaluated histopathology) and functional correlates (e.g.,
fertility) allows more confidence in a risk assessment for a given agent.
Descriptions of the nature of potential human exposures are important for prediction
of specific outcomes and the likelihood of persistence or reversibility of the effect in
different exposure situations with different subpopulations (U.S. Environmental Protection
Agency, 1992). Where possible, several descriptors of exposure such as the nature and
range of populations and their various exposure conditions, central tendencies, and high end
exposure estimates should be presented. Even with similar exposure patterns and levels,
different subpopulations may react differently. For example, the consequences of exposure
to developing individuals versus adults can differ markedly, including whether the effects
are permanent or transient. Other considerations relative to human exposures might include
potential for exposures to other agents, concurrent disease, and nutritional status, and the
possible consequences.
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V.C. DESCRIPTORS OF REPRODUCTIVE RISK
There are a number of ways to describe risk. Some ways that are relevant to
describing reproductive risk are as follows.
V.C.I. Estimation of the Number/Proportion of Individuals Exposed to Levels Above the
RfD or RfC
The RfD or RfC is assumed to be a level below which no significant risk occurs.
Therefore, information from the exposure assessment on the populations below the RfD or
RfC ("not likely to be at risk") and above the RfD or RfC ("may be at risk") may be useful
information for risk managers. Estimating the number of persons potentially removed from
the "at risk" category after a contemplated action is taken may be particularly useful to a
risk manager considering possible actions to ameliorate risk for a population.
V.C.2. Presenting Situation-Specific Exposure Scenarios
Presenting situation-specific scenarios for important exposure situations and
subpopulations in the form of "what if?" questions may be particularly useful to give
perspective to risk managers on possible future events. The question being asked in these
cases is, for any given exposure level, what would be the resulting number or proportion of
individuals that may be exposed to levels above that value?
V.C.3. Margin of Exposure
In the risk characterization, dose-response information and the human exposure
estimates may be combined either by comparing the RfD or RfC and the human exposure
estimate or by calculating the margin of exposure (MOE). The MOE is the ratio of the
NOAEL from the most appropriate or sensitive species to the estimated human exposure
level from all potential sources (U.S. Environmental Protection Agency, 1985a). If a
NOAEL is not available, a LOAEL may be used in the calculation of the MOE, but
consideration for the acceptability would be different than when a NOAEL is used.
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Alternatively, a benchmark dose may be compared with the estimated human exposure level
to obtain an MOE. Considerations for the acceptability of the MOE are similar to those for
the selection of uncertainty factors applied to the NOAEL, LOAEL, or the benchmark dose
for the derivation of an RfD. The MOE is presented along with the characterization of the
data base, including the strengths and weaknesses of the toxicity and exposure data, the
number of species affected, and the dose-response, route, timing and duration information.
The RfD or RfC comparison with the human exposure estimate and the calculation of the
MOE are conceptually similar, but may be used in different regulatory situations.
The choice of approach is dependent on several factors, including the statute
involved, the situation being addressed, the data base used, and the needs of the decision
maker. The RfD, RfC or MOE are considered along with other risk assessment and risk
management issues in making risk management decisions, but the scientific issues that
should be taken into account in establishing them have been addressed here.
V.C.4. Risk Characterization for Highly Exposed Individuals
This measure and the next are examples of specific scenarios. In some situations, it
may be appropriate to combine them.
The purpose of this measure is to describe the upper end of the exposure
distribution, allowing risk managers to evaluate whether certain individuals are at
disproportionately high or unacceptably high risk. The objective is to look at the upper end
of the exposure distribution to derive a realistic estimate of relatively highly exposed
individual(s). The "high end" of the risk distribution has been defined (Habicht, 1992) as
above the 90th percentile of the actual (either measured or estimated) distribution.
Whenever possible, it is important to express the number or proportion of individuals who
comprise the selected highly exposed group and, if data are available, discuss the potential
for exposure at still higher levels.
If population data are absent, it will often be possible to describe a scenario
representing high end exposures using upper percentile or judgment-based values for
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exposure variables. In these instances, caution should be taken not to overestimate the high
end values if a "reasonable" exposure estimate is to be achieved.
V.C.5. Risk Characterization for Highly Sensitive or Susceptible Individuals
The purpose of this measure is to quantify exposure of identified sensitive or
susceptible populations to the agent of concern. Sensitive or susceptible individuals are
those within the exposed population at increased risk of expressing the adverse effect.
Examples might be lactating women, women with reduced oocyte numbers, men with
"borderline" sperm counts, or infants.
In general, not enough is understood about the mechanisms of toxicity to identify
sensitive subgroups for all agents, although factors such as age, nutrition, personal habits
(e.g., smoking, consumption of alcohol, abuse of drugs), or existing disease (e.g., diabetes,
sexually transmitted diseases) may predispose some individuals to be more sensitive to the
reproductive effects of various agents.
V.D. SUMMARY AND RESEARCH NEEDS
These Guidelines summarize the procedures that the U.S. Environmental Protection
Agency will follow in evaluating the potential for agents to cause reproductive toxicity.
They discuss the assumptions that must be made in risk assessment for reproductive toxicity
because of gaps in our knowledge about underlying biologic processes and how these
compare across species. Research to improve the risk assessment process is needed.
Further studies that 1) more completely characterize and define female and male
reproductive endpoints, 2) evaluate the interrelationships among endpoints, 3) examine
quantitative extrapolation between endpoints (e.g., sperm count) and function (e.g., fertility),
4) provide a better understanding of the relationships between reproductive toxicity and
other forms of toxicity, 5) explore pharmacokinetic disposition of the target and, 6) examine
mechanistic phenomena related to pharmacokinetic disposition, will aid in the intepretation
of data and interspecies extrapolation. These types of studies, along with further evaluation
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of a threshold for susceptible populations, should provide methods to more precisely assess
risk.
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VI. REFERENCES
Aafjes, J.H., Vels, J.M., Schenck, E. (1980) Fertility of rats with artificial oligozoospermia.
J. Reprod. Fertil. 58:345-351.
Adler, N.T. & Toner, J.P. (1986) The effect of copulatory behavior on sperm transport and
fertility in rats. In: Komisaruk, B.R., Siegel, H.I., Chang, M.F., Feder, H.H. Reproduction:
Behavioral and Neuroendocrine Perspective. Ann. NY Acad. Sci, . pp. 21-32.
Amann, R.P. (1981) A critical review of methods for evaluation of spermatogenesis from
seminal characteristics. J. Androl. 2:37-58.
Armstrong, D.L. (1986) Environmental stress and ovarian function. Biol. Reprod. 34:29-39.
Atterwill, C.K. & Flack, J.D. (1992) Endocrine Toxicology. Cambridge University Press,
Cambridge.
Axelson, O. (1985) Epidemiologic methods in the study of spontaneous abortions: Sources
of data, methods, and sources of error. In: Hemminki, K., Sorsa, M., Vaino, H.
Occupational Hazards and Reproduction. Hemisphere, Washington, pp. 231-236.
Baird, D.D. & Wilcox, A.J. (1985) Cigarette smoking associated with delayed conception.
J. Am. Med. Assoc. 253:2979-2983.
Baird, D.D., Wilcox, A.J., Weinberg, C.R. (1986) Using time to pregnancy to study
environmental exposures. Am. J. Epidemiol. 124:470-480.
Barlow, S.M. & Sullivan, P.M. (1982) Reproductive Hazards of Industrial Chemicals.
Academic Press, London.
Barsotti, D.A., Abrahamson, L.J., Allen, J.R. (1979) Hormonal alterations in female rhesus
monkeys fed a diet containing 2,3,7,8-TCDD. Bull. Environ. Contam. Toxicol. 21:463-469.
Beach, F.A. (1979) Animal models for human sexuality. In: Ciba Foundation Symposium
No. 62, Sex, Hormones and Behavior. Elsevier-North Holland, London, pp. 113-143.
Berndtson, W.E. (1977) Methods for quantifying mammalian spermatogenesis: A review. J.
Anim. Sci. 44:818-833.
Bernstein, M.E. (1984) Agents affecting the male reproductive system: Effects of structure
on activity. Drug Metab. Rev. 15:941-996.
110
-------
DRAFT-DO NOT QUOTE OR CITE
Biava, C.G., Smuckler, E.A., Whorton, D. (1978) The testicular morphology of individuals
exposed to dibromochloropropane. Exp. Molec. Pathol. 29:448-458.
Blazak, W.F., Ernst, T.L., Stewart, B.E. (1985) Potential indicators of reproductive toxicity,
testicular sperm production and epididymal sperm number, transit time and motility in
Fischer 344 rats. Fund. Appl. Toxicol. 5:1097-1103.
Blazak, W.F., Treinen, K.A., Juniewicz, P.E. (1993) Application of testicular sperm head
counts in the assessment of male reproductive toxicity. In: Chapin, R.E. & Heindel, J.J.
Methods in Toxicology: Male Reproductive Toxicology. Academic Press, San Diego, pp.
86-94.
Bloom, A.D. (1981). Guidelines for reproductive studies in exposed human populations.
Guideline for studies of human populations exposed to mutagenic and reproductive hazards.
Report of Panel II. March of Dimes Birth Defects Foundation, White Plains, NY, pp. 37-
110.
Boyers, S.P., Davis, R.O., Katz, D.F. (1989) Automated semen analysis. Curr. Prob. Obstet.
Gynecol. Fertil. 12:173-200.
Brawer, J.R. & Finch, C.E. (1983) Normal and experimentally altered aging processes in
the rodent hypothalamus and pituitary. In: Walker, R.F. & Cooper, R.L. Experimental and
Clinical Interventions in Aging. Marcel Dekker, New York. pp. 45-65.
Brown, K.G. & Erdreich, L.S. (1989) Statistical uncertainty in the no-observed-effect level.
Fund. Appl. Toxicol. 13:235-244.
Brown-Grant, K., Davidson, J.M., Grieg, F. (1973) Induced ovulation in albino rats exposed
to constant light. J. Endocrinol. 57:7-22.
Bulger, W.H. & Kupfer, D. (1985) Estrogenic activity of pesticides and other xenobiotics
on the uterus and male reproductive tract. In: Thomas, J.A., Korach, K.S., McLachlan, J.A.
Endocrine Toxicology. Raven Press, New York. pp. 1-33.
Burch, T.K., Macisco, J.J., Parker, M.P. (1967) Some methodologic problems in the
analysis of menstrual data. Int. J. Fertil. 12:67-76.
Burger, E.J., Tardiff, R.G., Scialli, A.R., Zenick, H. (1989) Sperm Measures and
Reproductive Success. Alan R. Liss, New York.
Butcher, R.L. & Fugo, N.W. (1967) Overripeness and the mammalian ova. II. Delayed
ovulation and chromosome anomalies. Fertil. Steril. 18:297-302.
Ill
-------
DRAFT-DO NOT QUOTE OR CITE
Butcher, R.L., Blue, J.D., Fugo, N.W. (1969) Overripeness and the mammalian ova. III.
Fetal development at midgestation and at term. Fertil. Steril. 20:223-231.
Butcher, R.L., Collins, W.E., Fugo, N.W. (1975) Altered secretion of gonadotropins and
steroids resulting from delayed ovulation in the rat. Endocrinol. 96:576-586.
Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E. (1992) Evidence for decreasing
quality of semen during past 50 years. Br. Med. J. 305:609-613.
Cassidy, S.L., Dix, K.M., Jenkins, T. (1983) Evaluation of a testicular sperm head counting
technique using rats exposed to dimethoxyethyl phthalate (DMEP), glycerol alpha-
monochlorohydrin (GMCH), epichlorohydrin (ECH), formaldehyde (FA), or methyl
methanesulphonate (MMS). Arch. Toxicol. 53:71-78.
Chapin, R.E. (1988) Morphologic evaluation of seminiferous epithelium of the testis. In:
Lamb, J.C. & Foster, P.M.D. Physiology and Toxicology of Male Reproduction. Academic
Press, New York. pp. 155-177.
Chapin, R.E. & Heindel, J.J. (1993) Methods in Toxicology: Male Reproductive
Toxicology. Academic Press, San Diego.
Chapin, R.E., Gulati, O.K., Barnes, L.H., Teague, J.L. (1993a) The effects of feed
restriction on reproductive function in Sprague-Dawley rats. Fund. Appl. Toxicol. 20:23-29.
Chapin, R.E., Gulati, D.K., Fail, P.A., Hope, E., Russell, S.R., Heindel, J.J., George, J.D.,
Grizzle, T.B., Teague, J.L. (1993b) The effects of feed restriction on reproductive function
in Swiss CD-I mice. Fund. Appl. Toxicol. 20:15-22.
Chapman, R.M. (1983) Gonadal injury resulting from chemotherapy. In: Mattison, D.R.
Reproductive Toxicology. Alan R. Liss, New York. pp. 149-161.
Chen, J.J. & Kodell, R.L. (1989) Quantitative risk assessment for teratological effects. J.
Am. Stat. Assoc. 84:966-971.
Colborn, T., vom Saal, F.S., Soto, A.M. (1993) Developmental effects of endocrine-
disrupting chemicals in wildlife and humans. J. Nat. Inst. Environ. Health Sci. 101:378-384.
Collins, T.F.X. (1978) Multigeneration reproduction studies. In: Wilson, J.G. & Fraser, F.C.
Handbook of Teratology. Plenum Press, New York. pp. 191-214.
Cooper, R.L. & Walker, R.F. (1979) Potential therapeutic consequences of age-dependent
changes in brain physiology. Interdis. Topics Gerontol. 15:54-76.
112
-------
DRAFT-DO NOT QUOTE OR CITE
Cooper, R.L., Conn, P.M., Walker, R.F. (1980) Characterization of the LH surge in middle-
aged female rats. Biol. Reprod. 23:611-615.
Cooper, R.L., Chadwick, R.W., Rehnberg, G.L., Goldman, J.M., Booth, K.C., Hein, J.F.,
McElroy, W.K. (1989) Effect of lindane on hormonal control of reproductive function in
the female rat. Toxicol. Appl. Pharmacol. 99:384-394.
Cooper, R.L., Goldman, J.M., Vandenbergh, J.G. (1993) Monitoring of the estrous cycle in
the laboratory rodent by vaginal lavage. In: Heindel, J.J. & Chapin, R.E. Methods in
Toxicology: Female Reproductive Toxicology. Academic Press, San Diego, pp. 45-56.
Crump, K.S. (1984) A new method for determining allowable daily intakes. Fund. Appl.
Toxicol. 4:854-871.
Csapo, A.I. & Pulkkinen, M. (1978) Indispensability of the human corpus luteum in the
maintenance of early pregnancy: lutectomy evidence. Obstet. Gynecol. Surv. 33:69.
Cummings, A.M. & Gray, L.E. (1987) Methoxychlor affects the decidual cell response of
the uterus but not other progestational parameters in female rats. Toxicol. Appl. Pharmacol.
90:330-336.
Cummings, A.M. & Perreault, S.D. (1990) Methoxychlor accelerates embryo transport
through the rat reproductive tract. Toxicol. Appl. Pharmacol. 102:110-116.
Davis, D.L., Friedler, G., Mattison, D., Morris, R. (1992) Male-mediated teratogenesis and
other reproductive effects: Biologic and epidemiologic findings and a plea for clinical
research. Reprod. Toxicol. 6:289-292.
Deane, M., Swan, S.H., Harris, J.A., Epstein, D.M., Neutra, R.R. (1989) Adverse pregnancy
outcomes in relation to water contamination, Santa Clara County, California, 1981-1983.
Am. J. Epidemiol. 129:894-904.
de Boer, P., van der Hoeven, F.A., Chardon, J.A.P. (1976) The production, morphology,
karyotypes and transport of spermatozoa from tertiary trisomic mice and the consequences
for egg fertilization. J. Reprod. Fertil. 48:249-256.
Dixon, R.L. & Hall, J.L. (1984) Reproductive toxicology. In: Hayes, A.W. Principles and
Methods of Toxicology. Raven Press, New York. pp. 107-140.
Dobbins, J.G., Eifler, C.W., Buffler, P.A. (1978). The use of parity survivorship analysis in
the study of reproductive outcomes. Presented at the Society for Epidemiologic Research
Conference, Seattle, WA: June, 1978.
113
-------
DRAFT-DO NOT QUOTE OR CITE
Dobson, R.L. & Felton, J.S. (1983) Female germ cell loss from radiation and chemical
exposure. Am. J. Ind. Med. 4:175-190.
Drouva, S.V., Laplante, E., Kordon, C. (1982) Alpha 1-adrenergic receptor involvement in
the LH surge in ovariectomized estrogen-primed rats. Eur. J. Pharmacol. 81:341-344.
Egnatz, D.G., Ott, M.G., Townsend, J.C., Olson, R.D., Johns, D.B. (1980) DBCP and
testicular effects in chemical workers; an epidemiological survey in Midland. J. Occup.
Med. 22:727-732.
Epidemiology Workgroup for the Interagency Regulatory Liaison Group (1981). Guidelines
for documentation of epidemiologic studies. Amer. J. Epidemiol., 114:609-613.
Everett, J.W. & Sawyer, C.H. (1950) A 24-hour periodicity in the "LH-release apparatus"
of female rats disclosed by barbiturate sedation. Endocrinol. 47:198-218.
Everson, R.B., Sandier, D.P., Wilcox, A.J., Schreinemachers, D., Shore, D.L., Weinberg, C.
(1986) Effect of passive exposure to smoking on age at natural menopause. Br. Med. J.
293:792.
Fabia, J. & Thuy, T.D. (1974) Occupation of father at time of children dying of malignant
disease. Br. J. Prev. Soc. Med. 28:98-100.
Faustman, E.M., Wellington, D.G., Smith, W.P., Kimmel, C.A. (1989) Characterization of a
developmental toxicity dose-response model. Environ. Health 79:229-241.
Fawcett, D.W. (1986) Bloom and Fawcett: A Textbook of Histology. W. B. Saunders,
Philadelphia, PA.
Filler, R. (1993) Methods for evaluation of rat epididymal sperm morphology. In: Chapin,
R.E. & Heindel, J.J. Methods in Toxicology: Male Reproductive Toxicology. Academic
Press, San Diego, pp. 334-343.
Finch, C.E., Felicio, L.S., Mobbs, C.V. (1984) Ovarian and steroidal influences on
neuroendocrine aging processes in female rodents. Endocrinol. Rev. 5:467-497.
Fink, G. (1988) Gonadotropin secretion and its control. In: Knobil, E. & Neill, J.D. The
Physiology of Reproduction. Raven Press, New York. pp. 1349-1377.
Foote, R.H. & Berndtson, W.E. (1992) The Germinal Cells. In: Scialli, A.R. & Clegg, E.D.
Reversibility in Testicular Toxicity Assessment. CRC Press, Boca Raton, pp. 1-55.
114
-------
DRAFT-DO NOT QUOTE OR CITE
Foote, R.H., Schermerhorn, E.G., Simkin, M.E. (1986) Measurement of semen quality,
fertility, and reproductive hormones to assess dibromochloropropane (DBCP) effects in live
rabbits. Fund. Appl. Toxicol. 6:628-637.
Forsberg, J.G. (1981) Permanent changes induced by DBS at critical stages in human and
model systems. Biol. Res. Pregnancy 2:168-175.
Foster, P.M.D. (1992) The Sertoli cell. In: Scialli, A.R. & Clegg, E.D. Reversibility in
Testicular Toxicity Assessment. CRC Press, Boca Raton, pp. 57-86.
Francis, E.Z. & Kimmel, G.L. (1988) Proceedings of the workshop on one- vs two-
generation reproductive effects studies. J. Amer. Coll. Toxicol. 7:911-925.
Franken, D.R., Burkman, L.J., Coddington, C.C., Oehninger, S., Hodgen, G.D. (1990)
Human hemizona attachment assay. In: Acosta, A.A., Swanson, R.J., Ackerman, S.B.,
Kruger, T.F., VanZyl, J.A., Menkveld, R. Human Spermatozoa in Assisted Reproduction.
Williams & Wilkins, Baltimore, pp. 355-371.
Fugo, N.W. & Butcher, R.L. (1966) Overripeness and the mammalian ova. I. Overripeness
and early embryonic development. Fertil. Steril. 17:804-814.
Gaffey, W.R. (1976) A critique of the standard mortality ratio. J. Occup. Med. 18:157-160.
Galbraith, W.M., Voytek, P., Ryon, M.S. (1983) Assessment of risks to human reproduction
and development of the human conceptus from exposure to environmental substances. In:
Christian, M.S., Galbraith, W.M., Voytek, P., Mehlman, M.A. Advances in Modern
Environmental Toxicology. Princeton Scientific Publ., Princeton, pp. 41-153.
Gaylor, D.W. (1983) The use of safety factors for controlling risk. J. Toxicol. Environ.
Health 11:329-336.
Gaylor, D.W. (1989) Quantitative risk analysis for quantal reproductive and developmental
effects. Environ. Health 79:243-246.
Gelletti, F. & Klopper, A. (1964) The effect of progesterone on the quantity and
distribution of body fat in the female rat. Acta Endocrinol. 46:379-386.
Generoso, W.M., Rutledge, J.C., Cain, K.T., Hughes, L.A., Braden, P.W. (1987) Exposure
of female mice to ethylene oxide within hours after mating leads to fetal malformation and
death. Mutat. Res. 176:269-274.
115
-------
DRAFT-DO NOT QUOTE OR CITE
Gerrity, T.R., Henry, C.J., Bronaugh, R., et al. (1990) Summary report of the workshops on
principles of route-to-route extrapolation for risk assessment. In: Gerrity, T.R. & Henry,
C.J. Principles of Route-To-Route Extrapolation for Risk Assessment. Elsevier Science
Publ. Co., New York. pp. 1-12.
Giwercman, A., Carlsen, E., Keiding, N., Skakkebaek, N.E. (1993) Evidence for increasing
incidence of abnormalities of the human testis: A review. Envir. Health Perspect. 101:65-
71.
Goldman, J.M., Cooper, R.L., Laws, S.C., Rehnberg, G.L., Edwards, T.L., McElroy, W.K.,
Hein, J.F. (1990) Chlordimeform-induced alterations in endocrine regulation within the
male rat reproductive system. Toxicol. Appl. Pharmacol. 104:25-35.
Goldman, J.M., Cooper, R.L., Edwards, T.L., Rehnberg, G.L., McElroy, W.K., Hein, J.F.
(1991) Suppression of the luteinizing hormone surge by chlordimeform in ovariectomized,
steroid-primed female rats. Pharmacol. Toxicol. 68:131-136.
Gorski, R.A. (1979) The neuroendocrinology of reproduction: An overview. Biol. Reprod.
20:111-127.
Gray, L.E. (1991) Delayed effects on reproduction following exposure to toxic chemicals
during critical periods of development. In: Cooper, R.L., Goldman, J.M., Harbin, T.J. Aging
and Environmental Toxicology: Biological and Behavioral Perspectives. Johns Hopkins
University Press, Baltimore, pp. 183-210.
Gray, L.E., Ferrell, J.M., Ostby, J.S. (1985) Alteration of behavioral sex differentiation by
exposure to estrogenic compounds during a critical neonatal period: Effects of zearalenone,
methoxychlor, and estradiol in hamster. Toxicol. Appl. Pharmacol. 80:127-136.
Gray, L.E., Ostby, J., Sigmon, R., Ferrell, J., Linder, R., Cooper, R., Goldman, J., Laskey,
J. (1988) The development of a protocol to assess reproductive effects of toxicants in the
rat. Reprod. Toxicol. 2:281-287.
Gray, L.E., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., Slott,
V., Laskey, J. (1989) A dose-response analysis of methoxychlor-induced alterations of
reproductive development and function in the rat. Fund. Appl. Toxicol. 12:92-108.
Gray, L.E., Ostby, J., Linder, R., Goldman, J., Rehnberg, G., Cooper, R. (1990)
Carbendazim-induced alterations of reproductive development and function in the rat and
hamster. Fund. Appl. Toxicol. 15:281-297.
116
-------
DRAFT-DO NOT QUOTE OR CITE
Green, S., Auletta, A., Fabricant, R., Kapp, M., Sheu, C., Springer, J., Whitfield, B. (1985)
Current status of bioassays in genetic toxicology: The dominant lethal test. Mutat. Res.
154:49-67.
Greenland, S. (1987) Quantitative methods in the review of epidemiologic literature.
Epidem. Rev. 9:1-30.
Gulati, D.K., Hope, E., Teague, J., Chapin, R.E. (1991) Reproductive toxicity assessment by
continuous breeding in Sprague-Dawley rats: A comparison of two study designs. Fund.
Appl. Toxicol. 17:270-279.
Gustafsson, J.-A., Mode, A., Norstedt, G., Hokfelt, T., Sonnenschein, C., Eneroth, P., Skett,
P. (1980) The hypothalamo-pituitary-liver axis: A new hormonal system in control of
hepatic steroid and drug metabolism. Biochem. Act. Hormones 14:47-89.
Habicht, F.H. (1992). Guidance on Risk Characterization for Risk Managers and Risk
Assessors. U.S. EPA, Memorandum to Assistant Administrators and Regional
Administrators, February 26, 1992.
Hakulinen, T., Salonen, T., Teppo, L. (1976) Cancer in the offspring of fathers in
hydrocarbon-related occupations. Br. J. Prev. Soc. Med. 30:130-140.
Harris, M.W., Chapin, R.E., Lockhart, A.C., Jokinen, M.P., Allen, J.D., Haskins, E.A.
(1992) Assessment of a short-term reproductive and developmental toxicity screen. Fund.
Appl. Toxicol. 19:186-196.
Haschek, W.M. & Rousseaux, C.G. (1991) Handbook of Toxicologic Pathology. Academic
Press, New York.
Hatch, M. & Kline, J. (1981). Spontaneous abortion and exposure to the herbicide 2,4,5-T:
A pilot study. U.S. Environmental Protection Agency, Washington, D.C. EPA-560/6-81-
006.
Heindel, J.J. & Chapin, R.E. (1993) Methods in Toxicology: Female Reproductive
Toxicology. Academic Press, San Diego.
Heindel, J.J., Thomford, P.J., Mattison, D.R. (1989) Histological assessment of ovarian
follicle number in mice as a screen of ovarian toxicity. In: Hirshfield, A.N. Growth Factors
and the Ovary. Plenum Press, New York. pp. 421-426.
117
-------
DRAFT-DO NOT QUOTE OR CITE
Hemminki, K. & Vineis, P. (1985) Extrapolation of the evidence on teratogenicity of
chemicals between humans and experimental animals: Chemicals other than drugs. Terat.
Carcin. Mutagen. 5:251-318.
Hemminki, K., Mutanen, P., Luoma, K., Saloniemi, I. (1980) Congenital malformations by
the parental occupation in Finland. Int. Arch. Occup. Environ. Health 46:93-98.
Hemminki, K., Saloniemi, I., Salonen, T. (1981) Childhood cancer and paternal occupation
in Finland. J. Epidemiol. Community Health 35:11-15.
Hertig, A.T. (1967) The overall problem in man. In: Benirschke, K. Comparative Aspects
of Reproductive Failure. Springer-Verlag, New York. pp. 11-41.
Hervey, E. & Hervey, G.R. (1967) The effects of progesterone on body weight and
composition in the rat. J. Endocrinol. 37:361-384.
Hess, R.A. (1990) Quantitative and qualitative characteristics of the stages and transitions in
the cycle of the rat seminiferous epithelium: Light microscopic observations of perfusion-
fixed and plastic-embedded testes. Biol. Reprod. 43:525-542.
Hess, R.A. & Moore, B.J. (1993) Histological methods for evaluation of the testis. In:
Chapin, R.E. & Heindel, J.J. Methods in Toxicology: Male Reproductive Toxicology.
Academic Press, San Diego, pp. 52-85.
Hess, R.A., Moore, B.J., Forrer, J., Linder, R.E., Abuel-Atta, A.A. (1991) The fungicide
Benomyl (methyl l-(butylcarbamoyl)-2-benzimidazolecarbamate) causes testicular
dysfunction by inducing the sloughing of germ cells and occlusion of efferent ductules.
Fund. Appl. Toxicol. 17:733-745.
Heywood, R. & James, R.W. (1985) Current laboratory approaches for assessing male
reproductive toxicity. In: Dixon, R.L. Reproductive Toxicology. Raven Press, New York.
pp. 147-160.
Holloway, A.J., Moore, H.D.M., Foster, P.M.D. (1990a) The use of in vitro fertilization to
detect reductions in the fertility of male rats exposed to 1,3-dinitrobenzene. Fund. Appl.
Toxicol. 14:113-122.
Holloway, A.J., Moore, H.D.M., Foster, P.M.D. (1990b) The use of rat in vitro fertilization
to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol
monomethyl ether. Reprod. Toxicol. 4:21-27.
118
-------
DRAFT-DO NOT QUOTE OR CITE
Holmes, R.L. & Ball, J.N. (1974) The Pituitary Gland: A Comparative Account. Cambridge
University Press, Cambridge.
Hood, R.D. (1989) Paternally mediated effects. In: Hood, R.D. Developmental Toxicity:
Risk Assessment and the Future. Van Nostrand Reinhold, New York. pp. 77-79.
Huang, H.H. & Meites, J. (1975) Reproductive capacity of aging female rats.
Neuroendocrinol. 17:289-295.
Hugenholtz, A.P. & Bruce, W.R. (1983) Radiation induction of mutations affecting sperm
morphology in mice. Mutat. Res. 107:177-185.
Hughes, C.L. (1988) Phytochemical mimicry of reproductive hormones and modulation of
herbivore fertility by phytoestrogens. Environ. Health 78:171-175.
Hurtt, M.E. & Zenick, H. (1986) Decreasing epididymal sperm reserves enhances the
detection of ethoxyethanol-induced spermatotoxicity. Fund. Appl. Toxicol. 7:348-353.
Joffe, M. (1985) Biases in research on reproduction and women's work. Int. J. Epidemiol.
14:118-123.
Jones, T.C., Mohr, U., Hunt, R.D. (1987) Genital System. Springer-Verlag, New York.
Katz, D.F. & Overstreet, J.W. (1981) Sperm motility assessment by videomicrography.
Fertil. Steril. 35:188-193.
Katz, D.F., Diel, L., Overstreet, J.W. (1982) Differences in the movement of
morphologically normal and abnormal human seminal spermatozoa. Biol. Reprod. 26:566-
570.
Kimmel, C.A. (1990) Quantitative approaches to human risk assessment for noncancer
health effects. Neurotoxicol. 11:189-198.
Kimmel, C.A. & Francis, E.Z. (1990) Proceedings of the Workshop on the Acceptability
and Interpretation of Dermal Developmental Toxicity Studies. Fund. Appl. Toxicol. 14:386-
398.
Kimmel, C.A. & Gaylor, D.W. (1988) Issues in qualitative and quantitative risk analysis for
developmental toxicology. Risk Anal. 8:15-20.
119
-------
DRAFT-DO NOT QUOTE OR CITE
Kimmel, C.A., Holson, J.F., Hogue, C.J., Carlo, G.L. (1984). Reliability of experimental
studies for predicting hazards to human development. National Center for Toxicological
Research, Jefferson, AR. NCTR Technical Report for Experiment No. 6015.
Kimmel, C.A., Kimmel, G.L., Frankos, V. (1986) Interagency Regulatory Liason Group
workshop on reproductive toxicity risk assessment. Environ. Health 66:193-221.
Kimmel, C.A., Rees, D.C., Francis, E.Z. (1990) Proceedings of the Workshop on the
Qualitative and Quantitative Comparability of Human and Animal Developmental
Neurotoxicity. Neurotoxicol. Teratol. 12:173-292.
Kimmel, G.L., Clegg, E.D., Crisp, T.M. (In press) Reproductive toxicity testing: a risk
assessment perspective. In: Witorsch, R.J. Reproductive Toxicology. Raven Press, New
York.
Kissling, G. (1981) A generalized model for analysis of non-independent observations.
Thesis. University of North Carolina.
Kleinbaum, D.G., Kupper, L.L., Morgenstern, H. (1982) Epidemiologic Research: Principle
and Quantitative Methods. Lifetime Learning Publications, London.
Knobil, E. & Neill, J.D. (1988) The Physiology of Reproduction. Raven Press, New York.
Kodell, R.L., Howe, R.B., Chen, J.J., Gaylor, D.W. (1991) Mathematical modeling of
reproductive and developmental toxic effects for quantitative risk assessment. Risk Anal.
11:583-590.
Kupfer, D. (1987) Critical evaluation of methods for detection and assessment of estrogenic
compounds in mammals: Strengths and limitations for application to risk assessment.
Reprod. Toxicol. 2:147-153.
Kurman, R. & Norris, H.J. (1978) Germ cell tumors of the ovary. Pathol. Annu. 13:291.
Kwa, S.-L. & Fine, L.J. (1980) The association between parental occupation and childhood
malignancy. J. Occup. Med. 22:792-794.
La Bella, F.S., Dular, R., Lemons, P., Vivian, S., Queen, M. (1973a) Prolactin secretion is
specifically inhibited by nickel. Nature 245:330-332.
La Bella, F.S., Dular, R., Vivian, S., Queen, G. (1973b) Pituitary hormone releasing activity
of metal ions present in hypothalamic extracts. Biochem. Biophys. Res. Commun. 52:786-
791.
120
-------
DRAFT-DO NOT QUOTE OR CITE
Lamb, J.C. (1985) Reproductive toxicity testing: Evaluating and developing new testing
systems. J. Amer. Coll. Toxicol. 4:163-171.
Lamb, J.C. & Chapin, R.E. (1985) Experimental models of male reproductive toxicology.
In: Thomas, J.A., Korach, K.S., McLachlan, J.A. Endocrine Toxicology. Raven Press, New
York. pp. 85-115.
Lamb, J.C. & Foster, P.M.D. (1988) Physiology and Toxicology of Male Reproduction.
Academic Press, New York.
Lamb, J.C., Jameson, C.W., Choudhury, H., Gulati, D.K. (1985) Fertility assessment by
continuous breeding: Evaluation of diethylstilbestrol and a comparision of results from two
laboratories. J. Amer. Coll. Toxicol. 4:173-183.
Langley, F.A. & Fox, H. (1987) Ovarian tumors. Classification, histogenesis, etiology. In:
Fox, H. Haines and Taylor's Obstetrical and Gynaecologic Pathology. Churchill
Livingstone, Edinburgh, pp. 542-555.
Lantz, G.D., Cunningham, G.R., Huckins, C., Lipshultz, L.I. (1981) Recovery from severe
oligospermia after exposure to dibromochloropropane. Fertil. Steril. 35:46-53.
LeFevre, J. & McClintock, M.K. (1988) Reproductive senescence in female rats: A
longitudinal study of individual differences in estrous cycles and behavior. Biol. Reprod.
38:780-789.
Lemasters, G.K. & Pinney, S.M. (1989) Employment status as a confounder when assessing
occupational exposures and spontaneous abortion. J. Clin. Epidemiol. 42:975-981.
Lemasters, G.K. & Selevan, S.G. (1984) Use of exposure data in occupational reproductive
studies. Scan. J. Work. Environ. Health 10:1-6.
Lemasters, G.K., Hagen, A., Samuels, S. (1985) Reproductive outcomes in women exposed
to solvents in 36 reinforced plastic companies. 1. Menstrual dysfunction. J. Occup. Med.
27:490-494.
Leridon, H. (1977) Human Fertility: The Basic Components. The University of Chicago
Press, Chicago.
Le Vier, R.R. & Jankowiak, M.E. (1972) The hormonal and antifertility activity of 2,6-cis-
diphenylhexamethylcyclotetra-siloxane in the female rat. Biol. Reprod. 7:260-266.
121
-------
DRAFT-DO NOT QUOTE OR CITE
Levine, R.J. (1983) Methods for detecting occupational causes of male infertility:
Reproductive history versus semen analysis. Scand. J. Work Environ. Health 9:371-376.
Levine, R.J., Symons, M.J., Balogh, S.A., Arndt, D.M., Kaswandik, N.R., Gentile, J.W.
(1980) A method for monitoring the fertility of workers: I. Method and pilot studies. J.
Occup. Med. 22:781-791.
Levine, R.J., Symons, M.J., Balogh, S.A., Milby, T.H., Whorton, M.D. (1981) A method
for monitoring the fertility of workers: II. Validation of the method among workers exposed
to dibromochloropropane. J. Occup. Med. 23:183-188.
Levine, R.J., Blunden, P.B., DalCorso, R.D., Starr, T.B., Ross, C.E. (1983) Superiority of
reproductive histories to sperm counts in detecting infertility at a dibromochloropropane
manufacturing plant. J. Occup. Med. 25:591-597.
Lewis, R.J. (1991) Reproductively Active Chemicals: A Reference Guide. Van Nostrand
Reinhold, New York.
Linder, R.E., Hess, R.A., Strader, L.F. (1986) Testicular toxicity and infertility in male rats
treated with 1,3-dinitrobenzene. J. Toxicol. Environ. Health 19:477-489.
Linder, R.E., Strader, L.F., Barbee, R.R., Rehnberg, G.L., Perreault, S.D. (1990)
Reproductive Toxicity of a Single Dose of 1,3-Dinitrobenzene in 2 Ages of Young Adult
Male Rats. Fund. Appl. Toxicol. 14:284-298.
Lipshultz, L.I., Ross, C.E., Whorton, D., Thomas, M., Smith, R., Joyner, R.E. (1980)
Dibromochloropropane and its effect on testicular function in man. J. Urol. 124:464-468.
Long, J.A. & Evans, H.M. (1922) The oestrous cycle in the rat and its associated
phenomena. Mem. Univ. Calif. 6:1-111.
Mackeprang, M., Hay, S., Lunde, A.S. (1972) Completeness and accuracy of reporting of
malformations on birth certificates. HSMHA Health Reports 84:43-49.
Manson, J.M. (1994) Testing of pharmaceutical agents for reproductive toxicity. In:
Kimmel, C.A. & Buelke-Sam, J. Developmental Toxicology. Raven Press, New York. p.
379.
Manson, J.M. & Kang, Y.J. (In press) Test methods for assessing female reproductive and
developmental toxicology. In: Hayes, A.W. Principles and Methods of Toxicology. Raven
Press, New York.
122
-------
DRAFT-DO NOT QUOTE OR CITE
Mattison, D.R. (1985) Clinical manifestations of ovarian toxicity. In: Dixon, R.L.
Reproductive Toxicology. Raven Press, New York. pp. 109-130.
Mattison, D.R. & Nightingale, M.R. (1980) The biochemical and genetic characteristics of
murine ovarian aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity and its relationship
to primary oocyte destruction by polycyclic aromatic hydrocarbons. Toxicol. Appl.
Pharmacol. 56:399-408.
Mattison, D.R. & Thomford, P.J. (1989) The mechanisms of action of reproductive
toxicants. Toxicol. Pathol. 17:364-376.
Mattison, D.R. & Thorgeirsson, S.S. (1978) Gonadal aryl hydrocarbon hydroxylase in rats
and mice. Cancer Res. 38:1368-1373.
McLachlan, J.A. (1980) Estrogens in the Environment. Elsevier North Holland, New York.
McMichael, A.J. (1976) Standardized mortality ratios and the healthy worker effect:
Scratching beneath the surface. J. Occup. Med. 18:165-168.
McNatty, K.P. (1979) Follicular determinants of corpus luteum function in the human
ovary. Adv. Exp. Med. Biol. 112:465-481.
Meistrich, M.L. (1982) Quantitative correlation between testicular stem cell survival, sperm
production, and fertility in the mouse after treatment with different cytotoxic agents. J.
Androl. 3:58-68.
Meistrich, M.L. (1986) Critical components of testicular function and sensitivity to
disruption. Biol. Reprod. 34:17-28.
Meistrich, M.L. & Brown, C.C. (1983) Estimation of the increased risk of human infertility
from alterations in semen characteristics. Fertil. Steril. 40:220-230.
Meistrich, M.L. & Samuels, R.C. (1985) Reduction in sperm levels after testicular
irradiation of the mouse: A comparison with man. Rad. Res. 102:138-147.
Meistrich, M.L. & van Beek, M.E.A.B. (1993) Spermatogonial stern cells: assessing their
survival and ability to produce differentiated cells. In: Chapin, R.E. & Heindel, J.J. Methods
in Toxicology: Male Reproductive Toxicology. Academic Press, San Diego, pp. 106-123.
Meyer, C.R. (1981) Semen quality in workers exposed to carbon disulfide compared to a
control group from the same plant. J. Occup. Med. 23:435-439.
123
-------
DRAFT-DO NOT QUOTE OR CITE
Milby, T.H. & Whorton, D. (1980) Epidemiological assessment of occupationally related
chemically induced sperm count suppression. J. Occup. Med. 22:77-82.
Milby, T.H., Whorton, M.D., Stubbs, H.A., Ross, C.E., Joyner, R.E., Lipshultz, L.I. (1981)
Testicular function among epichlorohydrin workers. Br. J. Ind. Med. 38:372-377.
Morris, I.D., Bardin, C.W., Gunsalus, G., Ward, J.A. (1990) Prolonged suppression of
spermatogenesis by oestrogen does not preserve the seminiferous epithelium in
Procarbazine-treated rats. Int. J. Androl. 13:180-189.
Morrissey, R.E., Lamb, J.C., Morris, R.W., Chapin, R.E., Gulati, D.K., Heindel, J.J. (1989)
Results and evaluations of 48 continuous breeding reproduction studies conducted in mice.
Fund. Appl. Toxicol. 13:747-777.
Mosher, W.D. & Pratt, W.F. (1990). Fecundity and infertility in the United States, 1965-88.
Report 192, National Center for Health Statistics, Hyattsville, MD.
Mukhtar, H., Philpot, R.M., Lee, I.P., Bend, J.R. (1978) Developmental aspects of epoxide-
metabolizing enzyme activities in adrenals, ovaries, and testes of the rat. In: Mahlum, D.D.,
Sikov, M.R., Hackett, P.L., Andrew, F.D. Developmental Toxicology of Energy Related
Pollutants. Technical Information Center, U.S. Department of Energy, Springfield, VA. pp.
89-104.
Na, J.Y., Garza, F., Terranova, P.P. (1985) Alterations in follicular fluid steroids and
follicular hCG and FSH binding during atresia in hamsters. Proc. Soc. Exp. Biol. Med.
179:123-127.
Nagao, T. & Fujikawa, K. (1990) Genotoxic potency in mouse spermatogonial stem cells of
triethylenemelamine, mitomycin-C, ethylnitrosourea, procarbazine, and propyl
methanesulfonate as measured by Fl congenital defects. Mutat. Res. 229:123-128.
Nakai, M., Moore, B.J., Hess, R.A. (1993) Epithelial reorganization and irregular growth
following carbendazim-induced injury of the efferent ductules of the rat testis. Anat. Rec.
235:51-60.
National Research Council. (1977) Reproduction and teratogenicity tests. In: Principles and
Procedures for Evaluating the Toxicity of Household Substances. National Academy Press,
Washington, DC.
National Research Council. (1983) Risk Assessment in the Federal Government: Managing
the Process. National Academy Press, Washington.
124
-------
DRAFT-DO NOT QUOTE OR CITE
National Research Council. (1989) Biologic Markers in Reproductive Toxicity. National
Academy Press, Washington, DC.
Nestor, A. & Handel, M.A. (1984) The transport of morphologically abnormal sperm in the
female reproductive tract of mice. Gamete Res. 10:119-125.
Nisbet, I.C.T. & Karch, N.J. (1983). Chemical hazards to human reproduction, Park Ridge,
N.J.: Noyes Data Corp.
Organization for Economic Cooperation and Development (1983). First addendum to OECD
guideline 415 for testing of chemicals, "One-Generation Reproduction Toxicity".
Organization for Economic Cooperation and Development (1993a). Draft guidelines for
testing chemicals: Combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test. #422.
Organization for Economic Cooperation and Development (1993b). First amendment to
OECD guidelines 416 "Two Generation Reproduction Toxicity".
Overstreet, J.W. (1984) Laboratory tests for human male reproductive risk assessment. In:
Legator, M.S., Rosenberg, M., Zenick, H. Environmental Influences on Fertility, Pregnancy
and Development. Strategies for Measurement and Evaluation. Alan R. Liss, New York. pp.
67-82.
Pang, C.N., Zimmerman, E., Sawyer, C.H. (1977) Morphine inhibition of preovulatory
surges of plasma luteinizing hormone and follicle stimulating hormone in the rat.
Endocrinol. 101:1726-1732.
Papier, C.M. (1985) Parental occupation and congenital malformations in a series of 35,000
births in Israel. Prog. Clin. Biol. Res. 163:291-294.
Pease, W., Vandenberg, J., Hooper, K. (1991) Comparing alternative approaches to
establishing regulatory levels for reproductive toxicants: DBCP as a case study. Environ.
Health 91:141-155.
Peluso, J.J., Bolender, D.L., Perri, A. (1979) Temporal changes associated with the
degeneration of the rat oocyte. Biol. Reprod. 20:423-430.
Perreault, S.D. (1989) Impaired gamete function: Implications for reproductive toxicology.
In: Working, P.K. Toxicology of the Male and Female Reproductive Systems. Hemisphere,
New York. pp. 217-229.
125
-------
DRAFT-DO NOT QUOTE OR CITE
Perreault, S.D., Jeffay, S., Poss, P., Laskey, J.W. (1992) Use of the fungicide carbendazim
as a model compound to determine the impact of acute chemical exposure during oocyte
maturation and fertilization on pregnancy outcome in the hamster. Toxicol. Appl.
Pharmacol. 114:225-231.
Peters, J.M., Preston-Martin, S., Yu, M.C. (1981) Brain tumors in children and occupational
exposure of the parents. Science 213:235-237.
Plowchalk, D.R., Smith, B.J., Mattison, D.R. (1993) Assessment of toxicity to the ovary
using follicle quantitation and morphometrics. In: Heindel, J.J. & Chapin, R.E. Methods in
Toxicology: Female Reproductive Toxicology. Academic Press, San Diego, pp. 57-68.
Rai, K. & Van Ryzin, J. (1985) A dose response model for teratological experiments
involving quantal responses. Biometrics 41:1-10.
Ratcliffe, J.M., Clapp, D.E., Schrader, S.M., Turner, T.W., Oser, J., Tanaka, S., Hornung,
R.W., Halperin, W.E. (1986). Semen quality in 2-ethoxyethanol-exposed workers. Health
Hazard evaluation report, HETA 84-415-1688. Department of Health and Human Services,
National Institute for Occupational Safety and Health, Cincinnati, Ohio.
Ratcliffe, J.M., Schrader, S.M., Steenland, K., Clapp, D.E., Turner, T., Hornung, R.W.
(1987) Semen quality in papaya workers with long term exposure to ethylene dibromide.
Br. J. Ind. Med. 44:317-326.
Redi, C.A., Garagna, S., Pellicciari, C, Manfredi-Romanini, M.G., Capanna, E., Winking,
H., Gropp, A. (1984) Spermatozoa of chromosomally heterozygous mice and their fate in
male and female genital tracts. Gamete Res. 9:273-286.
Robaire, B., Smith, S., Hales, B.F. (1984) Suppression of spermatogenesis by testosterone
in adult male rats: Effect on fertility, pregnancy outcome and progeny. Biol. Reprod.
31:221-230.
Rosenberg, M.J., Wyrobeck, A.J., Ratcliffe, J., Gordon, L.A., Watchmaker, G., Fox, S.H.,
Moore, D.H. (1985) Sperm as an indicator of reproductive risk among petroleum refinery
workers. Br. J. Ind. Med. 42:123-127.
Rothman, K.J. (1986) Modern epidemiology. Little, Brown, Boston.
Rubin, H.B. & Henson, D.E. (1979) Effects of drugs on male sexual function. In: Advances
in Behavioral Pharmacology. Academic Press, New York. pp. 65-86.
126
-------
DRAFT-DO NOT QUOTE OR CITE
Russell, L.D. (1983) Normal testicular structure and methods of evaluation under
experimental and disruptive conditions. In: Clarkson, T.W., Nordberg, G.F., Sager, P.R.
Reproductive and Developmental Toxicity of Metals. Plenum Publishing Co., New York.
pp. 227-252.
Russell, L.D., Malone, J.P., McCurdy, D.S. (1981) Effect of microtubule disrupting agents,
colchicine and vinblastine, on seminiferous tubule structure in the rat. Tiss. Cell 13:349-
367.
Russell, L.D., Ettlin, R., Sinha Hikim, A.P., Clegg, E.D. (1990) Histological and
Histopathological Evaluation of the Testis. Cache River Press, Clearwater, FL.
Sakai, C.N. & Hodgen, G.D. (1987) Use of primate folliculogenesis models in
understanding human reproductive biology and applicablity to toxicology. Reprod. Toxicol.
1:207-222.
Scala, R.A., Bevan, C., Beyer, B.K. (1992) An Abbreviated Repeat Dose and
Reproductive/Developmental Toxicity Test for High Production Volume Chemicals. Regul.
Toxicol. Pharmacol. 16:73-80.
Schardein, J.L., Schwetz,B.B., Kenel, M.F. (1985) Species sensitivities and prediction of
teratogenic potential. Environ. Health Perspect. 61:55-62.
Schrag, S.D. & Dixon, R.L. (1985a) Occupational exposures associated with male
reproductive dysfunction. Ann. Rev. Pharmacol. Toxicol. 25:567-592.
Schrag, S.D. & Dixon, R.L. (1985b) Reproductive effects of chemical agents. In: Dixon,
R.L. Reproductive Toxicology. Raven Press, New York. pp. 301-319.
Schwetz, B.A., Rao, K.S., Park, C.N. (1980) Insensitivity of tests for reproductive
problems. J. Environ. Pathol. Toxicol. 3:81-98.
Scialli, A.R. & Clegg, E.D. (1992) Reversibility in Testicular Toxicity Assessment. CRC
Press, Boca Raton.
Scommegna, A., Vorys, N., Givens, J.R. (1980) Chapter 15: Menstrual dysfunction. In:
Gold, J.J. & Josimovich, J.B. Gynecologic Endocrinology. Harper and Row, Hagerstown,
Maryland.
Selevan, S.G. (1980) Evaluation of data sources for occupational pregnancy outcome
studies. University of Cincinnati from University Microfilms, Ann Arbor.
127
-------
DRAFT-DO NOT QUOTE OR CITE
Selevan, S.G. (1981) Design considerations in pregnancy outcome studies of occupational
populations. Scand. J. Work Environ. Health 7:76-82.
Selevan, S.G. (1985) Design of pregnancy outcome studies of industrial exposure. In:
Hemminki, K., Sorsa, M., Vainio, H. Occupational Hazards and Reproduction. Hemisphere,
Washington, DC. pp. 219-229.
Selevan, S.G. & Lemasters, G.K. (1987) The dose response fallacy in human reproductive
studies of toxic exposure. J. Occup. Med. 29:451-454.
Selevan, S.G., Edwards, B., Samuels, S. (1982) Interview data from both parents on
pregnancies and occupational exposures. How do they compare? Am. J. Epidemiol.
116:583.
Sever, L.E. & Hessol, N.A. (1984) Overall design considerations in male and female
occupational reproductive studies. In: Lockey, J.E., Lemasters, G.K., Keye, W.R.
Reproduction: The New Frontier in Occupational and Environmental Research. Alan R.
Liss, Inc., New York. pp. 15-48.
Sheehan, D.M., Young, J.F., Slikker, W., Gaylor, D.W., Mattison, D.R. (1989) Workshop
on risk assessment in reproductive and developmental toxicology: Addressing the
assumptions and identifying the research needs. Regul. Toxicol. Pharmacol. 10:110-122.
Shepard, T.H. (1986) Human teratogenicity. Adv. Pediatrics 33:225-268.
Silverman, J., Kline, J., Hutzler, M. (1985) Maternal employment and the chromosomal
characteristics of spontaneously aborted conceptions. J. Occup. Med. 27:427-438.
Skett, P. (1988) Biochemical basis of sex differences in drug metabolism. Pharm. Thera.
38:269-304.
Slott, V.L., Suarez, J.D., Simmons, I.E., Perreault, S.D. (1990) Acute inhalation exposure to
epichlorohydrin transiently decreases rat sperm velocity. Fund. Appl. Toxicol. 15:597-606.
Slott, V.L., Suarez, J.D., Perreault, S.D. (1991) Rat sperm motility analysis: Methodologic
considerations. Reprod. Toxicol. 5:449-458.
Smith, B.J., Plowchalk, D.R., Sipes, I.G., Mattison, D.R. (1991) Comparison of random and
serial sections in assessment of ovarian toxicity. Reprod. Toxicol. 5:379-383.
128
-------
DRAFT-DO NOT QUOTE OR CITE
Smith, C.G. (1983) Reproductive toxicity: hypothalamic-pituitary mechanisms. Am. J. Ind.
Med. 4:107-112.
Smith, C.G. & Gilbeau, P.M. (1985) Drug abuse effects on reproductive hormones. In:
Thomas, J.A., Korach, K.S., McLachlan, J.A. Endocrine Toxicology. Raven Press, New
York. pp. 249-267.
Smith, E.R. & Davidson, J.M. (1974) Luteinizing hormone releasing factor in rats exposed
to constant light: Effects of mating. Neuroendocrinol. 14:129-138.
Smith, P.E. (1939) The effect on the gonads of the ablation and implantation of the
hypophysis and the potency of the hypophsis under various conditions. In: Allen, E. Sex
and Internal Secretions. Williams and Wilkins., Baltimore, Maryland.
Smith, S.K., Lenton, E.A., Landgren, B.M., Cooke, I.D. (1984) The short luteal phase and
infertility. Br. J. Obstet. Gynaecol. 91:1120-1122.
Sonawane, B.R. & Yaffe, S.J. (1983) Delayed effects of drug exposure during pregnancy:
Reproductive function. Biol. Res. Pregnancy 4:48-55.
Starr, T.B., Dalcorso, R.D., Levine, R.J. (1986) Fertility of workers: A comparision of
logistic regression and indirect standardization. Am. J. Epidemiol. 123:490-498.
Stein, A. & Hatch, M. (1987) Biological markers in reproductive epidemiology: Prospects
and precautions. Environ. Health 74:67-75.
Stein, Z., Kline, J., Shrout, P. (1985) Power in surveillance. In: Hemminki, K., Sorsa, M.,
Vainio, H. Occupational Hazards and Reproduction. Hemisphere, Washington, DC. pp. 203-
208.
Steinberger, E. & Lloyd, J.A. (1985) Chemicals affecting the development of reproductive
capacity. In: Dixon, R.L. Reproductive Toxicology. Raven Press, New York, New York. .
Stevens, K.R. & Gallo, M.A. (1989) Practical considerations in the conduct of chronic
toxicity studies. In: Hayes, A.W. Principles and Methods of Toxicology. Raven Press, New
York. pp. 237-250.
Stiratelli, R., Laird, N., Ware, J.H. (1984) Random-effects models for serial observations
with binary responses. Biometrics 40:961-971.
129
-------
DRAFT-DO NOT QUOTE OR CITE
Swan, S.H., Shaw, G., Harris, J.A., Neutra, R.R. (1989) Congenital cardiac anomalies in
relation to water contamination, Santa Clara County, California, 1981-1983. Am J
Epidemiol. 129:885-893.
Sweeney, A.M., Meyer, M.R., Aarons, J.H., Mills, J.L., LaPorte, R.E. (1988) Evaluation of
methods for the prospective identification of early fetal losses in environmental
epidemiology studies. Am. J. Epidemiol. 127:843-850.
Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., Imaida, K., Takahashi,
M., Hayashi, Y., Kurokawa, Y., Tobe, M. (1992) Combined Repeat Dose and
Reproductive/Developmental Toxicity Screening Test (OECD): Familiarization using
cyclophosphamide. Fund. Appl. Toxicol. 18:89-95.
Terranova, P.F. (1980) Effects of phenobarbital-induced ovulatory delay on the follicular
population and serum levels of steroids and gonadotropins in the hamster: A model for
atresia. Biol. Reprod. 23:92-99.
Thomas, J.A. (1981) Reproductive hazards and environmental chemicals: A review. Toxic
Subst. J. 2:318-348.
Thomas, J.A. (1991) Toxic responses of the reproductive system. In: Amdur, M.O., Doull,
J., Klaassen, C.D. Casarett and Doull's Toxicology. Pergamon Press, New York. pp. 484-
520.
Tilley, B.C., Barnes, A.B., Bergstrahl, E., Labarthe, D., Noller, K.L., Colton, T., Adam, E.
(1985) A comparision of pregnancy history recall and medical records: Implications for
retrospective studies. Am. J. Epidemiol. 121:269-281.
Toth, G.P., Stober, J.A., Read, E.J., Zenick, H., Smith, M.K. (1989a) The automated
analysis of rat sperm motility following subchronic epichlorohydrin administration:
Methodologic and statistical considerations. J. Androl. 10:401-415.
Toth, G.P., Zenick, H., Smith, M.K. (1989b) Effects of epichlorohydrin on male and female
reproduction in Long-Evans rats. Fund. Appl. Toxicol. 13:16-25.
Toth, G.P., Stober, J.A., Zenick, H., Read, E.J., Christ, S.A., Smith, M.K. (1991)
Correlation of sperm motion parameters with fertility in rats treated subchronically with
epichlorohydrin. J. Androl. 12:54-61.
Treloar, A.E., Boynton, R.E., Borghild, G.B., Brown, B.W. (1967) Variation in the human
menstrual cycle through reproductive life. Int. J. Fertil. 12:77-126.
130
-------
DRAFT-DO NOT QUOTE OR CITE
Tsai, S.P. & Wen, C.P. (1986) A review of methodological issues of the standardized
mortality ratio (SMR) in occupational cohort studies. Int. J. Epidemiol. 15:8-21.
U.S. Congress (1985). Reproductive Health Hazards in the Workplace. Office of
Technology Assessment, OTA-BA-266, U.S. Government Printing Office, Washington, DC.
U. S. Congress (1988). Infertility: Medical and Social Choices. Office of Technology
Assessment, OTA-BA-358, U.S. Government Printing Office, Washington, DC.
U.S. Environmental Protection Agency (1982). Reproductive and Fertility Effects. Pesticide
Assessment Guidelines, Subdivision F. Hazard Evaluation: Human and Domestic Animals.
Office of Pesticides and Toxic substances, Washington, D.C. EPA-540/9-82-025.
U.S. Environmental Protection Agency (1985a). Hazard Evaluation Division Standard
Evaluation Procedure. Teratology Studies. Office of Pesticide Programs, Washington, DC.
pp. 22-23.
U.S. Environmental Protection Agency (1985b). Toxic Substances Control Act Test
Guidelines: Final Rules. Federal Register 50 (188):39426-39436.
U.S. Environmental Protection Agency (1986a). Guidelines for Carcinogen Risk
Assessment. Federal Register. 51(185):33992-34003.
U.S. Environmental Protection Agency (1986b). Guidelines for Estimating Exposures.
Federal Register 51(185):34042-34054.
U.S. Environmental Protection Agency (1986c). Guidelines for Mutagenicity Risk
Assessment. Federal Register 51(185):34006-34012.
U.S. Environmental Protection Agency (1987). Reference Dose (RfD): Description and Use
in Health Risk Assessments. Intergrated Risk Information System (IRIS): Appendix A.
Integrated Risk Information System Documentation, Vol. 1. EPA/600/8-66/032a.
U.S. Environmental Protection Agency. (1991) Guidelines for Developmental Toxicity Risk
Assessment. Fed. Reg. 56(234):63798-63826.
U.S. Environmental Protection Agency (1992). Guidelines for Exposure Assessment.
Federal Register 57(104):22888-22938.
Wade, G.N. (1972) Gonadal hormones and behavioral regulation of body weight. Physiol.
Behav. 8:523-534.
131
-------
DRAFT-DO NOT QUOTE OR CITE
Walker, R.F. (1986) Age factors potentiating drug toxicity in the reproductive axis.
Environ. Health 70:185-191.
Walker, R.F., Schwartz, L.W., Manson, J.M. (1988) Ovarian effects of an anti-
inflammatory-irnmunomodulatory drug in the rat. Toxicol. Appl. Pharmacol. 94:266-275.
Waller, D.P., Killinger, J.M., Zaneveld, L.J.D. (1985) Physiology and toxicology of the
male reproductive tract. In: Thomas, J.A., Korach, K.S., McLachlan, J.A. Endocrine
Toxicology. Raven Press, New York. pp. 269-333.
Wang, G.H. (1923) The relation between the "spontaneous" activity and the oestrous cycle
in the white rat. Comp. Psychol. Monographs 2:1-27.
Warren, J.C., Cheatum, S.G., Greenwald, G.S., Barker, K.L. (1967) Cyclic variation of
uterine metabolic activity in the golden hamster. Endocrinol. 80:714-718.
Weinberg, C.R. & Gladen, B.C. (1986) The beta-geometric distribution applied to
comparative fecundability studies. Biometrics 42:547-560.
Whorton, D. & Milby, T.H. (1980) Recovery of testicular function among DBCP workers.
J. Occup. Med. 22:177-179.
Whorton, D., Krauss, R.M., Marshall, S., Milby, T.H. (1977) Infertility in male pesticide
workers. Preliminary communication. Lancet 2(8051): 1259-1261.
Whorton, D., Milby, T.H., Krauss, R.M., Stubbs, H.A. (1979) Testicular function in DBCP
exposed pesticide workers. J. Occup. Med. 21:161-166.
Wilcox, A.J. (1983) Surveillance of pregnancy loss in human populations. Am. J. Ind. Med.
4:285-291.
Wilcox, A.J., Weinburg, C.R., Wehmann, R.E., Armstrong, E.G., Canfield, R.E., Nisula,
B.C. (1985) Measuring early pregnancy loss: laboratory and field methods. Fertil. Steril.
44:366-374.
Williams, J., Gladen, B.C., Schrader, S.M., Turner, T.W., Phelps, J.L., Chapin, R.E. (1990)
Semen analysis and fertility assessment in rabbits: Statistical power and design
considerations for toxicology studies. Fund. Appl. Toxicol. 15:651-665.
Wilson, J.G. (1973) Environment and birth defects. Academic Press, New York.
132
-------
DRAFT-DO NOT QUOTE OR CITE
Wilson, J.G. (1977) Embryotoxicity of drugs in man. In: Wilson, J.G. & Fraser, F.C.
Handbook of Teratology. Plenum Press, New York. pp. 309-355.
Wilson, J.G., Scott, W.J., Ritter, E.J., Fradkin, R. (1975) Comparative distribution and
embryotoxicity of hydroxyurea in pregnant rats and rhesus monkeys. Teratol. 11:169-178.
Wilson, J.G., Ritter, E.J., Scott, W.J., Fradkin, R. (1977) Comparative distribution and
embryotoxicity of acetylsalicylic acid in pregnant rats and rhesus monkeys. Toxicol. Appl.
Pharmacol. 41:67-78.
Wong, O., Utidjian, H.M.D., Karten, V.S. (1979) Retrospective evaluation of reproductive
performance of workers exposed to ethylene dibromide. J. Occup. Med. 21:98-102.
Working, P.K. (1988) Male reproductive toxicity: Comparison of the human to animal
models. Environ. Health 77:37-44.
Working, P.K. (1989) Toxicology of the Male and Female Reproductive Systems.
Hemisphere, New York.
Working, P.K. & Hurtt, M. (1987) Computerized videomicrographic analysis of rat sperm
motility. J. Androl. 8:330-337.
Wyrobek, A.J. (1982) Sperm assays as indicators of chemically-induced germ cell damage
in man. In: . Mutagenicity: New Horizons in Genetic Toxicology. Academic Press, New
York. pp. 337-349.
Wyrobek, A.J. (1984). Identifying agents that damage human spermatogenesis:
Abnormalities in sperm concentration and morphology. In: Monitoring Human Exposure to
Carcinogenic and Mutagenic agents. Proceedings of a joint symposium held in Espoo,
Finland. Dec. 12-15, 1983. International Agency for Research on Cancer, Lyon, France.
Wyrobek, A.J. & Bruce, W.R. (1978) The induction of sperm-shape abnormalities in mice
and humans. In: Hollander, A. & de Serres, F.J. Chemical Mutagens: Principles and
Methods for Their Detection. Plenum Press, New York.
Wyrobek, A.J., Gordon, L.A., Burkhart, J.G., Francis, M.W., Kapp, R.W., Letz, G.,
Mailing, H.V., Topham, J.C., Whorton, D.M. (1983a) An evaluation of the mouse sperm
morphology test and other sperm tests in nonhuman mammals. Mutat. Res. 115:1-72.
Wyrobek, A.J., Gordon, L.A., Burkhart, J.G., Francis, M.W., Kapp, R.W., Jr., Letz, G.,
Mailing, H., V, Topham, J.C., Whorton, D.M. (1983b) An evaluation of human sperm as
133
-------
DRAFT-DO NOT QUOTE OR CITE
indicators of chemically induced alterations of spermatogenic function. Mutat. Res. 115:73-
148.
Wyrobek, A.J., Watchmaker, G., Gordon, L. (1984) An evaluation of sperm tests as
indicators of germ-cell damage in men exposed to chemical or physical agents. In: Lockey,
I.E., Lemasters, G.K., Keye, W.R. Reproduction: The New Frontier in Occupational and
Environmental Health Research. Alan R. Liss, New York. pp. 385-407.
Zack, M., Cannon, S., Lloyd, D., Heath, C.W., Falleta, J.M., Jones, B., Housworth, J.,
Crowley, S. (1980) Cancer in children of parents exposed to hydrocarbon-related industries
and occupations. Am. J. Epidemiol. 3:329-336.
Zenick, H. & Clegg, E.D. (1986) Issues in risk assessment in male reproductive toxicology.
J. Amer. Coll. Toxicol. 5:249-259.
Zenick, H., Blackburn, K., Hope, E., Baldwin, D.J. (1984) Evaluating male reproductive
toxicity in rodents: A new animal model. Terat. Carcin. Mutagen. 4:109-128.
Zenick, H., Clegg, E.D., Perreault, S.D., Klinefelter, G.R., Gray, L.E. (In press) Assessment
of male reproductive toxicity: A risk assessment approach. In: Hayes, A.W. Principles and
Methods of Toxicology. Raven Press, New York.
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