# Guidelines for Reproductive Toxicity Risk Assessment

> Briefs, arguments, decisions, and more.

URL: https://www.frixlaw.com/law-library/documents/fr%3A96-27473

## Record

- **Collection:** Federal Register
- **Document type:** Notice
- **Published:** October 31, 1996
- **Citation:** 61 FR 56274

## Text

SUMMARY: The U.S. Environmental Protection Agency (EPA) is today
publishing in final form a document entitled Guidelines for
Reproductive Toxicity Risk Assessment (hereafter ``Guidelines''). These
Guidelines were developed as part of an interoffice guidelines
development program by a Technical Panel of the Risk Assessment Forum.
They were proposed initially in 1988 as separate guidelines for the
female and male reproductive systems. Subsequently, based upon the
public comments and Science Advisory Board (SAB) recommendations,
changes made included combining those two guidelines, integrating the
hazard identification and dose-response sections, assuming as a default
that an agent for which sufficient data were available on only one sex
may also affect reproductive function in the other sex, expansion of
the section on interpretation of female endpoints, and consideration of
the benchmark dose approach for quantitative risk assessment. These
Guidelines were made available again for public comment and SAB review
in 1994. This notice describes the scientific basis for concern about
exposure to agents that cause reproductive toxicity, outlines the
general process for assessing potential risk to humans from exposure to
environmental agents, and addresses Science Advisory Board and public
comments on the 1994 Proposed Guidelines for Reproductive Toxicity Risk
Assessment. Subsequent reviews have included the Agency's Risk
Assessment Forum and interagency comment by members of subcommittees of
the Committee on the Environment and Natural Resources of the Office of
Science and Technology Policy. The EPA appreciates the efforts of all
participants in the process and has tried to address their
recommendations in these Guidelines.

EFFECTIVE DATE: The Guidelines will be effective October 31, 1996.

ADDRESSES: The Guidelines will be made available in the following ways:
(1) The electronic version will be accessible on EPA's Office of
Research and Development home page on the Internet at http://
www.epa.gov/ORD/WebPubs/repro/.
(2) 3\1/2\-inch high-density computer diskettes in WordPerfect 5.1
will be available from ORD Publications, Technology Transfer and
Support Division, National Risk Management Research Laboratory,
Cincinnati, OH; telephone: 513-569-7562; fax: 513-569-7566. Please
provide the EPA No. (EPA/630/R-96/009a) when ordering.
(3) This notice contains the full document. In addition, copies of
the Guidelines will be available for inspection at EPA headquarters in
the Air and Radiation Docket and Information Center and in EPA
headquarters and regional libraries. The Guidelines also will be made
available through the U.S. Government Depository Library program and
for purchase from the National Technical Information Service (NTIS),
Springfield, VA; telephone: 703-487-4650; fax: 703-321-8547. Please
provide the NTIS PB No. (PB97-100093) when ordering.

FOR FURTHER INFORMATION CONTACT: Dr. Eric D. Clegg, National Center for
Environmental Assessment--Washington Office (8623), U.S. Environmental
Protection Agency, 401 M Street, S.W., Washington, DC 20460; telephone:
202-260-8914; e-mail: [email protected].

SUPPLEMENTARY INFORMATION:

A. Application of the Guidelines

The EPA 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. EPA, 1982, 1985b, 1996a) that provide protocols
designed to determine the potential of a test substance to produce
reproductive (including developmental) toxicity in laboratory animals.
Proposed revisions to these testing guidelines are in the final stages
of completion (U.S. EPA, 1996a). The Organization for Economic
Cooperation and Development (OECD) also has issued testing guidelines
(which are under revision) for reproduction studies (OECD, 1993b).
These Guidelines apply within the framework of policies provided by
applicable EPA statutes and do not alter such policies. They do not
imply that one kind of data or another is prerequisite for action
concerning any agent. The Guidelines are not intended, nor can they be
relied upon, to create any rights enforceable by any party in
litigation with the United States. This document is not a regulation
and is not intended to substitute for EPA regulations. These Guidelines
set forth current scientific thinking and approaches for conducting
reproductive toxicity risk assessments. EPA will revisit these
Guidelines as experience and scientific consensus evolve.
The procedures outlined here in the Guidelines provide guidance for
interpreting, analyzing, and using the data from studies that follow
the above testing guidelines (U.S. EPA 1982, 1985b, 1996a). 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 in the future 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. More specific guidance on
developmental effects is provided by the Guidelines for Developmental
Toxicity Risk Assessment (U.S. EPA, 1991). Other health effects
guidance is provided by the Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 1986a, 1996b), the Guidelines for Mutagenicity Risk
Assessment (U.S. EPA, 1986c), and the Proposed Guidelines for
Neurotoxicity Risk Assessment (U.S. EPA, 1995a). These Guidelines and
the four cited above are complementary.
The Agency has sponsored or participated in several conferences
that addressed issues related to evaluations of reproductive toxicity
data which provide some of the scientific bases for these risk
assessment guidelines. Numerous publications from these and other
efforts are available which provide background for these Guidelines
(U.S. EPA, 1982, 1985b, 1995b; Galbraith et al., 1983; OECD, 1983; U.S.
Congress, 1985, 1988; Kimmel, C.A. et al., 1986; Francis and Kimmel,
1988; Burger et al., 1989; Sheehan et al., 1989; Seed et al., 1996).
Also, numerous resources provide background information on the

[[Page 56275]]

physiology, biochemistry, and toxicology of the male and female
reproductive systems (Lamb and Foster, 1988; Working, 1989; Russell et
al., 1990; Atterwill and Flack, 1992; Scialli and Clegg, 1992; Chapin
and Heindel, 1993; Heindel and Chapin, 1993; Paul, 1993; Manson and
Kang, 1994; Zenick et al., 1994; Kimmel, G.L. et al., 1995; Witorsch,
1995). A comprehensive text on reproductive biology also has been
published (Knobil et al., 1994).

B. Environmental Agents and Reproductive Toxicity

Disorders of reproduction and hazards to reproductive health have
become prominent public health issues. A variety of factors are
associated with reproductive system disorders, including nutrition,
environment, socioeconomic status, lifestyle, and stress. 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.
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
and Pratt, 1990). In their 1988 survey, an estimated 4.9 million women
ages 15-44 (8.4%) had impaired fertility. The proportion of married
couples that was infertile, from all causes, was 7.9%.
Carlsen et al. (1992) have reported from a meta analysis that human
sperm concentration has declined from 113 x 10\6\ per mL of semen prior
to 1960 to 66 x 10\6\ per mL subsequently. When combined with a
reported decline in semen volume from 3.4 mL to 2.75 mL, that suggests
a decline in total number of sperm of approximately 50%. Increased
incidence of human male hypospadias, cryptorchidism, and testicular
cancer have also been reported over the last 50 years (Giwercman et
al., 1993). Several other retrospective studies that examined semen
characteristics from semen donors have obtained conflicting results
(Auger et al., 1995; Bujan et al., 1996; Fisch et al., 1996; Ginsburg
et al., 1994; Irvine et al., 1996; Paulsen et al., 1996; Van Waeleghem
et al., 1996; Vierula et al., 1996). While concerns exist about the
validity of some of those conclusions, the data indicating an increase
in human testicular cancer, as well as possible occurrence of other
plausibly related effects such as reduced sperm production,
hypospadias, and cryptorchidism, suggest that an adverse effect may
have occurred. However, there is no definitive evidence that such
adverse human health effects have been caused by environmental
chemicals.
Endometriosis is a painful reproductive and immunologic disease in
women that is characterized by aberrant location of uterine endometrial
cells, often leading to infertility. It affects approximately five
million women in the United States between 15 and 45 years of age. Very
limited research has suggested a link between dioxin exposure and
development of endometriosis in rhesus monkeys (Rier et al., 1993).
Gerhard and Runnebaum (1992) reported an association in women between
occurrence of endometriosis and elevated blood PCB levels, while a
subsequent small clinical study found no significant correlations
between disease severity in women and serum levels of halogenated
aromatic hydrocarbons (Boyd et al., 1995).
Even though not all infertile couples seek treatment, and
infertility is not the only adverse reproductive effect, it is
estimated that in 1986, Americans spent about $1 billion on medical
care to treat infertility alone (U.S. Congress, 1988). With the
increased use of assisted reproduction techniques in the last 10 years,
that amount has increased substantially.
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; Kline et al., 1989). Methods that detect pregnancy
as early as eight days after conception have shown that 32%-34% of
postimplantation pregnancies end in embryonic or fetal loss (Wilcox et
al., 1988; Zinaman et al., 1996). Approximately 3% of newborn children
have one or more significant congenital malformations at birth, and by
the end of the first post-natal 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).
A variety of developmental alterations may be detected after either
pre- or postnatal exposure. Several of these are discussed in the
Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991),
and developmental neurotoxicity is discussed in the Proposed Guidelines
for Neurotoxicity Risk Assessment (U.S. EPA, 1996a). Relative to
developmental reproductive alterations, 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.
Examples include the actions of estrogens, anti-androgens or dioxin in
interfering with male sexual differentiation (Gill et al., 1979; Gray
et al., 1994, 1995; Giusti et al., 1995; Gray and Ostby, 1995). 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 and Hall, 1984; Schrag and Dixon, 1985b; U.S. Congress, 1985).
For example, developmental exposure to environmental compounds that
possess steroidogenic (Mattison, 1985) or antisteroidogenic (Schardein,
1993) activity affect the onset of puberty and reproductive function in
adulthood.
Numerous agents have been shown to cause reproductive toxicity in
adult male and female laboratory animals and in humans (Mattison, 1985;
Schrag and Dixon, 1985a, b; Waller et al., 1985; Lewis, 1991). In adult
males and females, exposure to agents of abuse, e.g., cocaine, disrupts
normal reproductive function in both test species and humans (Smith,
C.G. and Gilbeau, 1985). Numerous chemicals disrupt the ovarian cycle,
alter ovulation, and impair fertility in experimental animals and
humans. These include agents with steroidogenic activity, certain
pesticides, and some metals (Thomas, 1981; Mattison, 1985). In males,
estrogenic compounds can be testicular toxicants in rodents and humans
(Colborn et al., 1993; Toppari et al., 1995). 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 and
Overstreet, 1981; Working, 1988).

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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 (McNatty, 1979; Scommegna et al.,
1980; Smith, S.K. et al., 1984; Sakai and 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).
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 x 10\6\ sperm per mL 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. However, some men with low
sperm concentrations are able to achieve conception and many subfertile
men have concentrations greater than 20 x 10\6\ illustrating the
importance of sperm quality. Toxic agents may further decrease
production of sperm and increase risk of impaired fertility.

C. The Risk Assessment Process and Its Application To Reproductive
Toxicity

Risk assessment is the process by which scientific judgments are
made concerning the potential for toxicity to occur in humans. In 1983,
the National Research Council (NRC) defined risk assessment as
comprising some or all of the following components: hazard
identification, dose-response assessment, exposure assessment, and risk
characterization (NRC, 1983). In its 1994 report, Science and Judgment
in Risk Assessment, the NRC extended its view of the paradigm to
include characterization of each component (NRC, 1994). In addition, it
noted the importance of an interactive approach that deals with
recurring conceptual issues that cut across all stages of risk
assessment. These Guidelines adopt an interactive approach by
organizing the process around the components of hazard
characterization, the quantitative dose-response analysis, the exposure
assessment, and the risk characterization where hazard characterization
combines hazard identification with qualitative consideration of dose-
response relationships, route, timing, and duration of exposure. This
is done because, in practice, hazard identification for reproductive
toxicity and other noncancer health effects include an evaluation of
dose-response relationships, route, timing, and duration of exposure in
the studies used to identify the hazard. Determining a hazard often
depends on whether a dose-response relationship is present (Kimmel,
C.A. et al., 1990). This approach combines the information important in
comparing the toxicity of a chemical to potential human exposure
scenarios identified as part of the exposure assessment. Also, it
minimizes the potential for labeling chemicals inappropriately as
``reproductive toxicants'' on a purely qualitative basis.
In hazard characterization, all available experimental animal and
human data, including observed effects, associated doses, routes,
timing, and duration of exposure, are examined to determine if an agent
causes reproductive toxicity in that species and, if so, under what
conditions. From the hazard characterization and criteria provided in
these Guidelines, the health-related database can be characterized as
sufficient or insufficient for use in risk assessment (Section III.G.).
This approach does not preclude the evaluation and use of the data for
other purposes when adequate quantitative information for setting
reference doses (RfDs) and reference concentrations (RfCs) is not
available.
The next step, the quantitative dose-response analysis (Section
IV), includes determining the no-observed-adverse-effect-level (NOAEL)
and/or the lowest-observed-adverse-effect-level (LOAEL) for each study
and type of effect. Because of the limitations associated with the use
of the NOAEL, the Agency is beginning to use an additional approach,
the benchmark dose approach (Crump, 1984; U.S. EPA. 1995b), for a more
quantitative dose-response evaluation when allowed by the data. The
benchmark dose approach takes into account the variability in the data
and the slope of the dose-response curve, and thus, provides more
complete use of the data for calculation of the RfD or RfC. If the data
are considered sufficient for risk assessment, and if reproductive
toxicity occurs at the lowest toxic dose level (i.e., the critical
effect), an RfD or RfC, based on adverse reproductive effects, could be
derived. This RfD or RfC is derived using the NOAEL or benchmark dose
divided by uncertainty factors to account for interspecies differences
in response, intraspecies variability and deficiencies in the database.
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.
EPA, 1992). However, unique considerations for reproductive toxicity
exposure assessments are detailed in Section V.
A statement of the potential for human risk and the consequences of
exposure can come only from integrating the hazard characterization and
quantitative dose-response analysis with human exposure estimates in
the risk characterization. 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.
In 1992, EPA issued a policy memorandum (Habicht, 1992) and
guidance package on risk characterization to encourage more
comprehensive risk characterizations, to promote greater consistency
and comparability among risk characterizations, and to clarify the role
of professional judgment in characterizing risk. In 1995, the Agency
issued a new risk characterization policy and guidance (Browner, 1995)
that refines and reaffirms the principles found in the 1992 policy and
outlines a process within the Agency for implementation. Although
specific program policies and procedures are still evolving, these
Guidelines discuss attributes of the Agency's risk

[[Page 56277]]

characterization policy as it applies to reproductive toxicity.
Risk assessment is just one component of the regulatory process.
The other component, risk management, uses risk characterization along
with directives of the enabling regulatory legislation and other
factors to decide whether to control exposure to the suspected agent
and the level of control. Risk management decisions also consider
socioeconomic, technical, and political factors. Risk management is not
discussed directly in these guidelines because the basis for
decisionmaking goes beyond scientific considerations alone. However,
the use of scientific information in this process is discussed. For
example, the acceptability of the margin of exposure (MOE) is a risk
management decision, but the scientific bases for generating this value
are discussed here.

Dated: October 15, 1996.
Carol M. Browner,
Administrator.

Contents

List of Tables
Part A. Guidelines for Reproductive Toxicity Risk Assessment
I. Overview
II. Definitions and Terminology
III. Hazard Characterization for Reproductive Toxicants
III.A. Laboratory Testing Protocols
III.A.1. Introduction
III.A.2. Duration of Dosing
III.A.3. Length of Mating Period
III.A.4. Number of Females Mated to Each Male
III.A.5. Single- and Multigeneration Reproduction Tests
III.A.6. Alternative Reproductive Tests
III.A.7. Additional Test Protocols That May Provide Reproductive
Data
III.B. Endpoints for Evaluating Male and Female Reproductive
Toxicity In Test Species
III.B.1. Introduction
III.B.2. Couple-Mediated Endpoints
III.B.2.a. Fertility and Pregnancy Outcomes
III.B.2.b. Sexual Behavior
III.B.3. Male-Specific Endpoints
III.B.3.a. Introduction
III.B.3.b. Body Weight and Organ Weights
III.B.3.c. Histopathologic Evaluations
III.B.3.d. Sperm Evaluations
III.B.3.e. Paternally Mediated Effects on Offspring
III.B.4. Female-Specific Endpoints
III.B.4.a. Introduction
III.B.4.b. Body Weight, Organ Weight, Organ Morphology, and
Histology
III.B.4.b.1. Body weight
III.B.4.b.2. Ovary
III.B.4.b.3. Uterus
III.B.4.b.4. Oviducts
III.B.4.b.5. Vagina and external genitalia
III.B.4.b.6. Pituitary
III.B.4.c. Oocyte Production
III.B.4.c.1. Folliculogenesis
III.B.4.c.2. Ovulation
III.B.4.c.3. Corpus luteum
III.B.4.d. Alterations in the Female Reproductive Cycle
III.B.4.e. Mammary Gland and Lactation
III.B.4.f. Reproductive Senescence
III.B.5. Developmental and Pubertal Alterations
III.B.6. Endocrine Evaluations
III.B.7. In Vitro Tests of Reproductive Function
III.C. Human Studies
III.C.1. Epidemiologic Studies
III.C.1.a. Selection of Outcomes for Study
III.C.1.b. Reproductive History Studies
III.C.1.c. Community Studies and Surveillance Programs
III.C.1.d. Identification of Important Exposures for
Reproductive Effects
III.C.1.e. General Design Considerations
III.C.2. Examination of Clusters, Case Reports, or Series
III.D. Pharmacokinetic Considerations
III.E. Comparisons of Molecular Structure
III.F. Evaluation of Dose-Response Relationships
III.G. Characterization of the Health-Related Database
IV. QUANTITATIVE DOSE-RESPONSE ANALYSIS
V. EXPOSURE ASSESSMENT
VI. RISK CHARACTERIZATION
VI.A. Overview
VI.B. Integration of Hazard Characterization, Quantitative Dose-
Response, and Exposure Assessments
VI.C. Descriptors of Reproductive Risk
VI.C.1. Distribution of Individual Exposures
VI.C.2. Population Exposure
VI.C.3. Margin of Exposure
VI.C.4. Distribution of Exposure and Risk for Different
Subgroups
VI.C.5. Situation-Specific Information
VI.C.6. Evaluation of the Uncertainty in the Risk Descriptors
VI.D. Summary and Research Needs
VII. REFERENCES
PART B. RESPONSE TO SCIENCE ADVISORY BOARD AND PUBLIC COMMENTS
I. INTRODUCTION
II. RESPONSE TO SCIENCE ADVISORY BOARD COMMENTS
III. RESPONSE TO PUBLIC COMMENTS

List of Tables

1. Default Assumptions in Reproductive Toxicity Risk Assessment
2. Couple-Mediated Endpoints of Reproductive Toxicity
3. Selected Indices That May Be Calculated From Endpoints of
Reproductive Toxicity in Test Species
4. Male-Specific Endpoints of Reproductive Toxicity
5. Female-Specific Endpoints of Reproductive Toxicity
6. Categorization of the Health-Related Database
7. Guide for Developing Chemical-Specific Risk Characterizations for
Reproductive Effects

PART A. GUIDELINES FOR REPRODUCTIVE TOXICITY RISK ASSESSMENT

I. Overview

These Guidelines describe the procedures that the EPA follows in
using existing data to evaluate the potential toxicity of environmental
agents to the human male and female reproductive systems and to
developing offspring. These Guidelines focus on reproductive system
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. EPA, 1991) focus specifically on effects of agents on
development 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 mutagenicity or carcinogenicity as well as other
forms of general systemic toxicity. 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. EPA, 1986c) should be
consulted in conjunction with these Guidelines. The Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 1986a, 1996b) 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 having adequate
study

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design and power. 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 default assumptions are made. These default assumptions,
which are summarized in Table 1, should not preclude inquiry into the
relevance of the data to potential human risk and should be invoked
only after examination of the available information indicates that
necessity. 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 judgment as to human risk for each agent, and that information
summarized in the risk characterization.

Table 1.--Default Assumptions in Reproductive Toxicity Risk Assessment
------------------------------------------------------------------------

-------------------------------------------------------------------------
1. An agent that produces an adverse reproductive effect in experimental
animals is assumed to pose a potential threat to humans.
2. Effects of xenobiotics on male and female reproductive processes are
assumed generally to be similar unless demonstrated otherwise. For
developmental outcomes, the specific effects in humans are not
necessarily the same as those seen in the experimental species.
3. In the absence of information to determine the most appropriate
experimental species, data from the most sensitive species should be
used.
4. In the absence of information to the contrary, an agent that affects
reproductive function in one sex is assumed to adversely affect
reproductive function in the other sex.
5. A nonlinear dose-response curve is assumed for reproductive toxicity.
------------------------------------------------------------------------

An agent that produces an adverse reproductive effect in
experimental animal studies is assumed to pose a potential reproductive
threat to humans. This assumption is based on comparisons of data for
agents that are known to cause human reproductive toxicity (Thomas,
1981; Nisbet and Karch, 1983; Kimmel, C.A. et al., 1984, 1990; Hemminki
and 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 developmental
outcomes, it is assumed that the specific outcomes seen in experimental
animal studies are not necessarily the same as those produced in
humans. This latter assumption is made because of the possibility of
species-specific differences in timing of exposure relative to critical
periods of development, pharmacokinetics (including metabolism),
developmental patterns, placentation, or modes of action. However,
adverse developmental outcomes in laboratory mammalian studies are
presumed to predict a hazard for adverse developmental outcome in
humans.
When sufficient data are available (e.g., pharmacokinetic) to allow
a decision, 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
agents known to cause human reproductive toxicity, humans appear to be
as or more sensitive than the most sensitive animal species tested
(Nisbet and Karch, 1983; Kimmel, C.A. et al., 1984, 1990; Hemminki and
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 affects reproductive function 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; and (3) Many of the mechanisms controlling important aspects
of reproductive system function are similar in females and males, and
therefore could be susceptible to the same agents. Information that
would negate this assumption would demonstrate that either a
mechanistic difference existed between the sexes that would preclude
toxic action on the other sex or, on the basis of sufficient testing,
an agent did not produce an adverse reproductive effect when
administered to the other sex. Mechanistic differences could include
functions that do not exist in the other sex (e.g., lactation),
differences in endocrine control of affected organ development or
function, or pharmacokinetic and metabolic differences between sexes.
In a quantitative dose-response analysis, mode of action,
pharmacokinetic, and pharmacodynamic information should be used to
predict the shape of the dose-response curve when sufficient
information of that nature is available. When that information is
insufficient, it has generally been assumed that there is a nonlinear
dose-response for reproductive toxicity. This is based on known
homeostatic, 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 developing organism are
known to have some capacity for repair of damage. However, in a
population, background levels of toxic agents and preexisting
conditions may increase the sensitivity of some individuals in the
population. Thus, exposure to a toxic agent may result in an increased
risk of adverse effects for some, but not necessarily all, individuals
within the population. Although a threshold may exist for endpoints of
reproductive toxicity, it usually is not feasible to distinguish
empirically between a true threshold and a nonlinear low-dose
relationship. The shift to the term nonlinear does not change the RfD/
RfC methodology for reproductive system health effects, including the
use of uncertainty factors.

II. Definitions and Terminology

For the purposes of these Guidelines, the following definitions
will be used: Reproductive toxicity--The occurrence of biologically
adverse effects on the reproductive systems of females or males 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, developmental toxicity, premature reproductive senescence,
or modifications in other functions that are dependent on the integrity
of the reproductive systems.
Fertility--The capacity to conceive or induce conception.

[[Page 56279]]

Fecundity--The ability to produce offspring within a given period
of time. For litter-bearing species, the ability to produce large
litters is also a component of fecundity.
Fertile--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--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 lifespan 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. EPA, 1991).

III. Hazard Characterization for Reproductive Toxicants

Identification and characterization of reproductive hazards can be
based on data from either human or experimental animal studies. Such
data can result from routine or accidental environmental or
occupational exposures or, for experimental animals, controlled
experimental exposures. A hazard characterization should evaluate all
of the information available and should:
Identify the strengths and limitations of the database,
including all available epidemiologic and experimental animal studies
as well as pharmacokinetic and mechanistic information.
Identify and describe key toxicological studies.
Describe the type(s) of effects.
Describe the nature of the effects (irreversible,
reversible, transient, progressive, delayed, residual, or latent
effects).
Describe how much is known about how (through what
biological mechanism) the agent produces adverse effects.
Discuss the other health endpoints of concern.
Discuss any nonpositive data in humans or experimental
animals.
Discuss the dose-response data (epidemiologic or
experimental animal) available for further dose-response analysis.
Discuss the route, level, timing, and duration of exposure
in studies as compared to expected human exposures.
Summarize the hazard characterization, including:

--Major assumptions used,
--Confidence in the conclusions,
--Alternative conclusions also supported by the data,
--Major uncertainties identified, and
--Significant data gaps.

Conduct of a hazard characterization requires knowledge of the
protocols in which data were produced and the endpoints that were
evaluated. Sections III.A. and III.B. present 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. These are followed by presentation of many of the
specific characteristics of human studies (Section III.C.) and limited
discussions of pharmacokinetic and structure-activity factors (Sections
III.D. and III.E.).

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.
Integral parts of the hazard characterization and quantitative
dose-response processes are the 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 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 impact to sperm production is necessary
before an adverse effect on fertility can be detected.

[[Page 56280]]

III.A.4. Number of Females Mated to Each Male
The 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. EPA, 1982, 1985b, 1996a).
However, in some tests that were not designed to conform to EPA test
guidelines (OECD, 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 reproduction protocols have been published by the
Agency pursuant to FIFRA and TSCA and by OECD (U.S. EPA, 1982, 1985b,
1996a; Galbraith et al., 1983; OECD, 1983).
The single-generation reproduction test evaluates effects of
subchronic exposure of peripubertal and adult animals. In the
multigeneration reproduction protocol, F1 and F2 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 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 and increase the likelihood of
detecting histologic lesions. 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., F1, F2). Only the P
generation is mated in a single-generation test, while both the P and
F1 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 can be allowed for up to 3 weeks (U.S. EPA, 1982, 1985b),
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 F1
male and female offspring continue to be exposed after weaning (day 21)
and through the mating period. Treatment of mated F1 females is
continued throughout gestation and lactation. More than one litter may
be produced from either P or F1 animals. Depending on the route of
exposure of lactating females, it is important to consider that
offspring may be exposed 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, as well as via the milk.
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
from males, and histopathology of the vagina, uterus, cervix, ovaries,
and mammary glands from females. Uterine and ovarian weights also are
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 and 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. For
example, adverse transgenerational effects on reproductive system
development by agents that disrupt endocrine control of sexual
differentiation would be missed. 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. These limitations are shared
with the shorter-term ``screening'' protocols described below. Because
of these limitations, a comprehensive reproductive risk assessment
should include results from a two-generation test or its equivalent. A
further recommendation from the workshop was to include sperm analyses
and estrous cycle normality as endpoints in reproductive effects
studies. These endpoints have been included in the proposed revisions
to the EPA test guideline (U.S. EPA, 1996a).
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 always, observed.
Qualitative predictions of increased risk of the filial generations
could be strengthened by

[[Page 56281]]

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 decreased
with succeeding generations. When a decrease occurs, one explanation
may be that the animals in the F1 and F2 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 and 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 and Kimmel, 1988).
A continuous breeding protocol, Fertility (or Reproductive)
Assessment by Continuous Breeding (FACB or RACB), has been developed by
the National Toxicology Program (NTP) (Lamb and Chapin, 1985; Morrissey
et al., 1989; Gulati et al., 1991). As originally described, this
protocol (FACB) was a one-generation test. However, in the current
design (RACB), dosing is extended into the F1 generation to make
it compatible with the EPA workshop recommendations for a two-
generation design (Francis and 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 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 and then discarded. 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 improves the 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 were allowed to grow and reproduce (as they are routinely 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 and their
utility has been examined using model compounds in the mouse (Morrissey
et al., 1989).
Another test method 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 F1 generation is mated in a
continuous breeding 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 F1 generations at all doses, and
terminal studies on both generations include comprehensive assessment
of sperm measures (number, 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 or antiandrogens.
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 protocol (Scala et al., 1992; Tanaka et al., 1992; OECD, 1993a), a
tripartite protocol developed by the International Conference on
Harmonization (International Conference on Harmonization of Technical
Requirements of Pharmaceuticals for Human Use, 1994; Manson, 1994), and
the NTP's Short-Term Reproductive and Developmental Toxicity Screen
(Harris, M.W. 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
multigeneration reproduction protocols.
The male dominant lethal test was designed to detect mutagenic
effects in the male spermatogenic process that are lethal to the
offspring. A female dominant lethal protocol has also been used to
detect equivalent effects on oogenesis (Generoso and Piegorsch, 1993).
A review of the male dominant lethal 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 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. Dose levels used with the acute protocol are usually higher
than those used with the subchronic protocol. 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).

[[Page 56282]]

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. EPA, 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 toxicity tests may have been conducted before a detailed
reproduction study is initiated. In the subchronic toxicity test with
rats, exposure usually begins at 6-8 weeks of age and is continued for
90 days (U.S. EPA, 1982, 1985b). Initiation of exposure at 8 weeks of
age (compared with 6) 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 females, 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, data from 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 pathologic conditions is often
increased in the reproductive tracts of aged control animals.
Therefore, findings should be interpreted carefully.

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,
secondary 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
can have a contributing role if both partners are exposed. Thus,
exposure of 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, 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 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 only 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 2. Many endpoints that are
pertinent for developmental toxicity are also listed and discussed in
the Agency's Guidelines for Developmental Toxicity Risk Assessment
(U.S. EPA, 1991). Also included in Table 2 are measures that

[[Page 56283]]

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.

Table 2.--Couple-Mediated End-points of Reproductive Toxicity
------------------------------------------------------------------------

-------------------------------------------------------------------------
Multigeneration studies:
Mating rate, time to mating (time to pregnancy*)
Pregnancy rate*
Delivery rate*
Gestation length*
Litter size (total and live)
Number of live and dead offspring (Fetal death rate*)
Offspring gender* (sex ratio)
Birth weight*
Postnatal weights*
Offspring survival*
External malformations and variations*
Offspring reproduction*
Other reproductive endpoints:
Ovulation rate
Fertilization rate
Preimplantation loss
Implantation number
Postimplantation loss*
Internal malformations and variations*
Postnatal structural and functional development*
------------------------------------------------------------------------
*Endpoints that can be obtained with humans.

Some of the endpoints identified above are used to calculate ratios
or indices (NRCl, 1977; Collins, 1978; Schwetz et al., 1980; U.S. EPA,
1982, 1985b; Dixon and 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 3.

[[Page 56284]]

Table 3.--Selected Indices That May Be Calculated From Endpoints of
Reproductive Toxicity in Test Species

Mating Index
[GRAPHIC] [TIFF OMITTED] TN31OC96.000

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
[GRAPHIC] [TIFF OMITTED] TN31OC96.001

Note: Because both sexes are often exposed to an agent,
distinction between sexes often is 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 (Pregnancy) Index
[GRAPHIC] [TIFF OMITTED] TN31OC96.002

Live Birth Index
[GRAPHIC] [TIFF OMITTED] TN31OC96.003

Sex Ratio
[GRAPHIC] [TIFF OMITTED] TN31OC96.004

4-Day Survival Index (Viability Index)
[GRAPHIC] [TIFF OMITTED] TN31OC96.005

Note: This definition assumes that no standardization of litter
size is done until after the day 4 determination is completed.

Lactation Index (Weaning Index)
[GRAPHIC] [TIFF OMITTED] TN31OC96.006

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
[GRAPHIC] [TIFF OMITTED] TN31OC96.007

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.

------------------------------------------------------------------------

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. Therefore, mating rate and fertility data from the first estrous
cycle after initiation of cohabitation should be more discriminating
than measurements involving multiple cycles. 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 after the start of
cohabitation (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 the start of
cohabitation. If the

[[Page 56285]]

stage of the estrous cycle at the time of cohabitation is known, the
component of the variance due to variation in stage at cohabitation can
be removed in the data 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 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 increased 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 and Perreault, 1990). Preimplantation
loss (described below) occurs in untreated as well as treated rodents
and contributes to the normal 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
can 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 oolemma, 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 oocyte
maturation and normal zygote formation after sperm-egg fusion by
affecting meiosis (Perreault et al., 1992; Zuelke and Perreault, 1995).
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 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
Supplementary 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 IV) 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 IV).
Both the blastocyst and the uterus must be ready for implantation,
and their synchronous development is critical (Cummings and 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 or inhibit
protein synthesis, mitosis, or cell differentiation can block
implantation and cause embryo death.
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 constitutes a maternal health threat for humans as
well as test species. 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. Postimplantation loss can be determined also by
examining uteri from postparturient females. Preimplantation loss is
the (number of corpora lutea minus number of implantation sites)/number
of corpora lutea. Postimplantation loss, determined following delivery
of a litter, is the (total number of implantation sites minus number of
full-term pups)/number of implantation sites.
Offspring gender in mammals 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. Further, alteration of the external sexual
characteristics of offspring by agents that disrupt sexual development
may produce apparent

[[Page 56286]]

effects on sex ratios. Although not examined routinely, these factors
provide the most likely explanations for alterations in the sex ratio.
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 (Tyl, 1987).
Individual pups in large litters tend to be smaller than 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. Multivariate
analyses may be used to adjust pup weights for litter size (e.g.,
analysis of covariance, multiple regression). 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 and the Proposed Guidelines
for Neurotoxicity Risk Assessment (U.S. EPA, 1991, 1995a). These
documents should be consulted for additional information on those
parameters.

Adverse Effects

Table 2 lists couple-mediated endpoints that may be measured in
reproduction studies. Table 3 presents examples of indices that may be
calculated from couple-mediated reproductive toxicity data. Significant
detrimental effects on any of those endpoints or on indices derived
from those data 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.
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 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 studies on
effects of drugs on sexual behavior or 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 suggests 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 and 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 and 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 that can be
reflected in reduced numbers of females with vaginal plugs or vaginal
sperm, alterations in lordosis behavior, and increased time to mating
after start of cohabitation. In the male, measures include latency
periods to first mount, mount with intromission, and first ejaculation,
number of mounts with intromission to ejaculation, and the
postejaculatory 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 central or peripheral neurotoxicity.
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.

Adverse Effects

Effects on sexual behavior (within the limited definition of these
Guidelines) should be considered as adverse reproductive effects.
Included is evidence of impaired sexual receptivity and copulatory
behavior. Impairment that is secondary to more generalized physical
debilitation (e.g., impaired rear leg motor activity or general
lethargy) should not be considered an adverse reproductive effect,
although such conditions represent adverse systemic 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.
The Agency may obtain data on the potential male reproductive
toxicity of

[[Page 56287]]

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 4.

Table 4.--Male-Specific Endpoints of Reproductive Toxicity
------------------------------------------------------------------------

------------------------------------------------------------------------
Organ weights................ Testes, epididymides, seminal vesicles,
prostate, pituitary.
Visual examination and Testes, epididymides, seminal vesicles,
histopathology. prostate, pituitary.
Sperm evaluation *........... Sperm number (count) and quality
(morphology, motility)
Sexual behavior *............ Mounts, intromissions, ejaculations.
Hormone levels *............. Luteinizing hormone, follicle stimulating
hormone, testosterone, estrogen,
prolactin.
Developmental effects........ Testis descent*, preputial separation,
sperm production*, ano-genital distance,
structure of external genitalia*.
------------------------------------------------------------------------
* Reproductive endpoints that can be obtained or estimated relatively
noninvasively with humans.

III.B.3.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-containing food or water because of
reduced palatability, treatment-induced anorexia, or systemic toxicity.
Less than severe reductions in adult body weight induced by restricted
nutrition have shown 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 nonreproductive
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 or other severe systemic
debilitation, it should be noted that an adverse effect on a
reproductive endpoint occurred, but the effect may have resulted from a
more generalized toxic effect. Regardless, adverse effects would have
been 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 usually remains
quite stable (Stevens and 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 absolute 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; Ku et al., 1993). This
contradiction may arise from several factors, including a delay before
cell deaths are reflected in a weight decrease (due to preceding 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, while testis weight
measurements may not reflect certain adverse testicular effects and do
not indicate the nature of an effect, 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. 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 difficult in rodents. However, the seminal vesicle
and prostate can be separated and results may be reported for these
glands separately or together, with or without their secretory fluids.
Differential loss of secretory fluids prior to weighing could produce
artifactual weights. 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.

Adverse Effects

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 animal tissues have a prominent role in male reproductive risk
assessment. Organs that are often evaluated include

[[Page 56288]]

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 from a variety of protocols; and (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 and 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 or
degenerating, if multinucleated giant cells are obvious, or if sloughed
cells are present in the tubule lumen. More subtle lesions, such as
retained spermatids or missing germ cell types, 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
book is available (Russell et al., 1990) which 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 alterations that may accompany certain
disease states (Fawcett, 1986; Jones et al., 1987; Haschek and
Rousseaux, 1991). Compared with the testes, less is known about
structural 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. Evaluation should include
information on the caput, corpus, and cauda segments of the epididymis.
Presence of debris and sloughed cells in the epididymal lumen are
valuable indicators of damage to the germinal epithelium or the
excurrent ducts. The presence of lesions such as sperm granulomas,
leucocyte infiltration (inflammation) or absence of clear cells in the
cauda epididymal epithelium should be noted. 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 standardized tissue preparation
techniques and a standardized quantification system, the evaluation of
histopathologic data would be facilitated by the presentation of the
evaluation criteria and procedure by 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).

Adverse Effects

Significant and biologically meaningful 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 evaluations 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 testicular
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 in
humans. Brief descriptions of these measures are provided below,
followed by a discussion of the use of 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 (Seed
et al., 1996). 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

[[Page 56289]]

ejaculates, both sperm concentration (number of sperm/mL of ejaculate)
and total sperm per ejaculate (sperm concentration x volume) should be
evaluated.
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 variation 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 when allowed by protocol), 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, but the samples for
sperm motility and morphology may be derived also from the vas
deferens. 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 and 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 and van Beek, 1993). If evaluation was conducted when
the effect of a lesion would be reflected adequately in the spermatid
count, then 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
the spermatid count. Also, spermatid counts reported from some
laboratories have large coefficients of variation that may reduce the
statistical power and thus the usefulness of that measure.
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, vas
deferens, 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. Thus, the link between
sperm morphology and mutagenicity is not necessarily sensitive or
specific.
An increase in abnormal sperm morphology has been considered
evidence that the agent has gained access to the germ cells (U.S. EPA,
1986c). Exposure of males to toxic agents may lead to sperm
abnormalities in their progeny (Wyrobek and Bruce, 1978; Hugenholtz and
Bruce, 1983; Morrissey et al., 1988a, b). 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, midpiece, and tail defects in either stained preparations by
bright field microscopy (Filler, 1993) or fixed, unstained preparations
by phase contrast microscopy (Linder et al., 1992; Seed et al., 1996).
Such an approach may be adequate for mice 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 categorize the types of abnormalities
observed and quantify 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
in 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 et al., 1994). 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 and 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 (Morrissey et al., 1988a, b; Toth
et al., 1989b, 1991b), and sperm motility assessments are an integral
part of some reproductive toxicity tests (Gray et al., 1988; Morrissey
et al., 1989; U.S. EPA, 1996a).

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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 experimental variables, including
abstinence interval, method of sample collection and handling, elapsed
time between sampling and observation, the temperature at which the
sample is stored and analyzed, the extent of sperm dilution, the nature
of the dilution medium, and the microscopic chamber employed for the
observations (Slott et al., 1991; Toth et al., 1991a; Chapin et al.,
1992; Schrader et al., 1992; Weir and Rumberger, 1995; Seed et al.,
1996).
Sperm motility can be evaluated in fresh samples under phase
contrast microscopy, or sperm images can be recorded and stored in
video or digital format and analyzed later, either manually or by
computer-aided semen analysis (Linder et al., 1986; Boyers et al.,
1989; Toth et al., 1989a; Yeung et al., 1992; Slott and Perreault,
1993). For manual assessments, the percentage of motile and
progressively motile sperm can be estimated and a simple scale used to
describe the vigor of the sperm motion.
The recent application of video and/or digital technology to sperm
analysis allows a more detailed evaluation of sperm motion including
information about the individual sperm tracks. It also provides
permanent storage of the sperm tracks which can be re-analyzed as
necessary (manually or computer-assisted). With computer-assisted
technology, information about sperm velocity (straight-line and
curvilinear) as well as the amplitude and frequency of the track are
obtained rapidly and efficiently on large numbers of sperm. Using this
technology, chemically induced alterations in sperm motion have been
detected (Toth et al., 1989a, 1992; Slott et al., 1990; Klinefelter et
al., 1994a), and such changes have been related to the fertility of the
exposed animals (Toth et al., 1991a; Oberlander et al., 1994; Slott et
al., 1995). These preliminary studies indicate that significant
reductions in sperm velocity are associated with infertility, even when
the percentage of motile sperm is not affected. The ability to
distinguish between the proportion of sperm showing any type of motion
and those with progressive motility is important (Seed et al., 1996).
Changes in endpoints that measure effects on spermatogenesis and
sperm maturation have been related to fertility in several test
species, but the ability to predict infertility from these data (in the
absence of fertility data) is not reliable. This is in part due to the
observation, in both test species and humans, that fertility is
dependent not only on having adequate numbers of sperm, but also on the
degree to which those sperm are normal. If sperm quality is high, then
sperm number must be substantially reduced before fertility is
affected. For example, in a rat model that employs artificial
insemination of differing numbers of good quality sperm, sperm numbers
can be reduced substantially before fertility is affected (Klinefelter
et al., 1994b). In humans, the distribution of sperm counts for fertile
and infertile men overlap, with the mean for fertile men being higher
(Meistrich and Brown, 1983), but fertility is likely to be impaired
when counts drop below 20 million/mL (WHO, 1992). Similarly, if sperm
numbers are normal in rodents, a relatively large effect on sperm
motility is required before fertility is affected. For example, rodent
sperm velocity must be substantially reduced, in the presence of
adequate numbers of sperm, before fertility is affected (Toth et al.,
1991a; Slott et al., 1995). These models also show that relatively
modest changes in sperm numbers or quality may not cause infertility,
but can nevertheless be predictive of infertility. On the other hand,
fertility may be impaired by smaller decrements in both number and
motility (or other qualitative characteristics).
Thus, the process of reproductive risk assessment is facilitated by
having information on a variety of sperm measures and reproductive
organ histopathology in addition to fertility. Specific information
about reproductive organ and gamete function can then be used to
evaluate the occurrence and extent of injury, and the probable site of
toxicity in the reproductive system. The more information that is
available from supplementary endpoints, the more the risk assessment
can be based on science rather than uncertainty.

Adverse Effects

Human male fertility is generally lower than that of test species
and may be more susceptible to damage from toxic agents (see
Supplementary Information). Therefore, the conservative approach should
be taken that, within the limits indicated in the sections on those
parameters, statistically significant changes in measures of sperm
count, morphology, or motility as well as number of normal sperm should
be considered adverse effects.
III.B.3.e. 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
(Davis et al., 1992; Colie, 1993; Savitz et al., 1994; Qiu et al.,
1995). Paternally mediated effects include pre- and postimplantation
loss, growth and behavioral deficits, and malformations. A large
proportion of the chemicals reported to cause paternally mediated
effects have genotoxic activity, and are considered to exert this
effect via transmissible genetic alterations. Low doses of
cyclophosphamide have resulted in induction of single strand DNA breaks
during rat spermatogenesis which, due in part to absence of subsequent
DNA repair capability, remain at fertilization (Qiu et al., 1995). The
results of such damage have been observed in the F2 generation
offspring (Hales et al., 1992). Other mechanisms of induction of
paternally mediated effects are also possible. 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 fertilization or early development. With humans,
the possibility 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 or in
humans 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.B.4.a. 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 et al., 1994). 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

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puberty have not been examined, nor have the long-term consequences of
exposure on reproductive senescence. Thus, the amount of information
obtained routinely to detect toxic effects on the female reproductive
system has been 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.
Adverse 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 and Jankowiak, 1972; Barsotti et al., 1979; Sonawane
and Yaffe, 1983; Cummings and 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 agent's effect(s). Areas
that may be examined in evaluations of the female reproductive system
are listed in Table 5.

Table 5.--Female-Specific Endpoints of Reproductive Toxicity
----------------------------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------------------------
Organ weights............................................................... Ovary, uterus, vagina, pituitary.
Visual examination and histopathology....................................... Ovary, uterus, vagina, pituitary,
oviduct, mammary gland.
Estrous (menstrual *) cycle normality....................................... Vaginal smear cytology.
Sexual behavior............................................................. Lordosis, time to mating, vaginal
plugs, or sperm.
Hormone levels *............................................................ LH, FSH, estrogen, progesterone,
prolactin.
Lactation *................................................................. Offspring growth, milk quantity
and quality.
Development................................................................. Normality of external genitalia *,
vaginal opening, vaginal smear
cytology, onset of estrous
behavior (menstruation *).
Senescence.................................................................. Vaginal smear cytology, ovarian
histology (menopause *).
----------------------------------------------------------------------------------------------------------------
* Endpoints that can be obtained relatively noninvasively with humans.

Reproductive function in the female is controlled through complex
interactions involving the central nervous system (particularly the
hypothalamus), pituitary, ovaries, the reproductive tract, and the
secondary sexual organs. Other nongonadotrophic 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 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 used below 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.B.4.b. Body Weight, Organ Weight, Organ Morphology, and Histology
III.B.4.b.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 body weight fluctuates
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 (Galletti and Klopper, 1964;
Hervey and Hervey, 1967). Food consumption is elevated during
pregnancy, in part because of the elevated serum progesterone level.
One of the most sensitiv

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Source: Frix Law Library, https://www.frixlaw.com/law-library/documents/fr%3A96-27473. Public record. Not legal advice.