Guidelines for Reproductive Toxicity Risk Assessment

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

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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

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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

[[Page 56278]]

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

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

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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).

[[Page 56290]]

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

[[Page 56291]]

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 sensitive noninvasive indicators of a compound with

estrogenic action in the female rat is a reduction in food intake and

body weight. Also, growth retardation induced by effects on

extragonadal hormones (e.g., thyroid or growth hormone) can cause a

delay in pubertal development, and induce acyclicity and infertility.

Because of these endocrine-related fluctuations, the weights of the

reproductive organs are poorly correlated with body weight, except in

extreme cases. Thus, actual organ weight data, rather than organ to

body weight ratios, should be reported and evaluated for the female

reproductive system.

Chapin et al. (1993a, b) have studied the influence of food

restriction on female Sprague-Dawley rats and Swiss CD-1 mice when body

weights were 90%, 80%, or 70% of controls. Female rats were resistant

to effects on reproductive function at 80% of control weight whereas

mice showed adverse effects at 80% and a marginal effect at 90%. These

results indicate that differences exist between species (and probably

between strains) in the response of the female rodent reproductive

system to reduced food intake or body weight reduction.

III.B.4.b.2. Ovary. The ovary serves a number of functions that are

critical to reproductive activity, including production and ovulation

of oocytes.

[[Page 56292]]

Estrogen is produced by developing follicles and progesterone is

produced by corpora lutea that are formed after ovulation.

Ovarian Weight

Significant increases or decreases in ovarian weight compared with

controls should be considered an indication of female reproductive

toxicity. Although ovarian function shifts throughout the estrous

cycle, ovarian weight in the normal rat does not show significant

fluctuations. Still, oocyte and follicle depletion, persistent

polycystic ovaries, inhibition of corpus luteum formation, luteal cyst

development, reproductive aging, and altered hypothalamic-pituitary

function may all be associated with changes in ovarian weight.

Therefore, it is important that ovarian gross morphology and histology

also be examined to allow correlation of alterations in those

parameters with changes in ovarian weight. However, not all adverse

histologic alterations in the ovary are concurrent with changes in

ovarian weight. Therefore, a lack of effect on organ weights does not

preclude the need for histologic evaluation.

Histopathology

Histologic evaluation of the three major compartments of the ovary

(i.e., follicular, luteal, and interstitial) plus the epithelial

capsule and ovarian stroma may indicate ovarian toxicity. A number of

pathologic conditions can be detected by ovarian histology (Kurman and

Norris, 1978; Langley and Fox, 1987). Methods are available to quantify

the number of follicles and their stages of maturation (Plowchalk et

al., 1993). These techniques may be useful when a compound depletes the

pool of primordial follicles or alters their subsequent development and

recruitment during the events leading to ovulation.

Adverse Effects

Significant changes in the ovaries in any of the following effects

should be considered adverse:

Increase or decrease in ovarian weight.

Increased incidence of follicular atresia.

Decreased number of primary follicles.

Decreased number or lifespan of corpora lutea.

Evidence of abnormal folliculogenesis or luteinization,

including cystic follicles, luteinized follicles, and failure of

ovulation.

Evidence of altered puberty or premature reproductive

senescence.

III.B.4.b.3. Uterus.

Uterine Weight

An alteration in the weight of the uterus may be considered an

indication of female reproductive organ toxicity. Compounds that

inhibit steroidogenesis and cyclicity can dramatically reduce the

weight of the uterus so that it appears atrophic and small. However,

uterine weight fluctuates three- to four-fold throughout the estrous

cycle, peaking at proestrus when, in response to increased estrogen

secretion, the uterus is fluid filled and distended. This increase in

uterine weight has been used as a basis for comparing relative potency

of estrogenic compounds in bioassays (Kupfer, 1987). As a result of the

wide fluctuations in weight, uterine weights taken from cycling animals

have a high variance, and large compound-related effects are required

to demonstrate a significant effect unless interpreted relative to that

animal's estrous cycle stage. A number of environmental compounds

(e.g., pesticides such as methoxychlor and chlordecone, mycotoxins,

polychlorinated biphenyls, alkylphenols, and phytoestrogens) possess

varying degrees of estrogenic activity and have the potential to

stimulate the female reproductive tract (Barlow and Sullivan, 1982;

Bulger and Kupfer, 1985; Hughes, 1988).

When pregnant or postpartum animals are examined, the numbers of

implantation sites or implantation scars should be counted. This

information, along with corpus luteum counts, can be used to calculate

pre- and postimplantation losses.

Histopathology

The histologic appearance of the normal uterus fluctuates with

stage of the estrous cycle and pregnancy. The uterine endometrium is

sensitive to influences of estrogens and progestogens (Warren et al.,

1967), and extended treatment with these compounds leads to hypertrophy

and hyperplasia. Conversely, inhibition of ovarian activity and reduced

steroid secretion results in endometrial hypoplasia and atrophy, as

well as altered vaginal smear cytology. Effects induced during

development may delay or prevent puberty, resulting in persistence of

infantile genitalia.

Adverse Effects

Effects on the uterus that may be considered adverse include

significant dose-related alteration of weight, as well as gross

anatomic or histologic abnormalities. In particular, any of the

following effects should be considered as adverse.

Infantile or malformed uterus or cervix.

Decreased or increased uterine weight.

Endometrial hyperplasia, hypoplasia, or aplasia.

Decreased number of implantation sites.

III.B.4.b.4. Oviducts.

Typically, the oviducts are not weighed or examined histologically

in tests for reproductive toxicity. However, information from visual

and histologic examinations is of value in detecting morphologic

anomalies. Descriptions of pathologic effects within the oviducts of

animals other than humans are not common. Hypoplasia of otherwise well-

formed oviducts and loss of cilia result most commonly from a lack of

estrogen stimulation, and for this reason, this condition may not be

recognized until after puberty. Hyperplasia of the oviductal epithelium

results from prolonged estrogenic stimulation. Anomalies induced during

development have also been described, including agenesis, segmental

aplasia, and hypoplasia.

Anatomic anomalies in the oviduct occurring in excess of control

incidence should be considered as adverse effects. Hypoplasia or

hyperplasia of the oviductal epithelium may be considered as an adverse

effect, particularly if that result is consistent with observations in

the uterine histology.

III.B.4.b.5. Vagina and external genitalia.

Vaginal Weight

Vaginal weight changes should parallel those seen in the uterus

during the estrous cycle, although the magnitude of the changes is

smaller.

Histopathology

In rodents, cytologic changes in the vaginal epithelium (vaginal

smear) may be used to identify the different stages of the estrous

cycle (see Section III.B.4.d.). The vaginal smear pattern may be useful

to identify conditions that would delay or preclude fertility, or

affect sexual behavior. Other histologic alterations that may be

observed include aplasia, hypoplasia, and hyperplasia of the vaginal

epithelial cell lining.

Developmental Effects

Developmental abnormalities, either genetic or related to prenatal

exposure to compounds that disrupt the endocrine balance, include

agenesis, hypoplasia, and dysgenesis. Hypoplasia of the vagina may be

concomitant with hyperplasia of the external genitalia and can be

induced by gonadal or adrenal steroid exposure. In rodents,

[[Page 56293]]

malpositioning of the vaginal and urethral ducts is common in steroid-

treated females. Such developmentally induced lesions are irreversible.

The sex ratio observed at birth may be affected by exposure of

genotypic females in utero to agents that disrupt reproductive tract

development. In cases of incomplete sex reversal because of such

exposures, female rodents may appear more male-like and have an

increased ano-genital distance (Gray and Ostby, 1995).

At puberty, the opening of the vaginal orifice normally provides a

simple and useful developmental marker. However, estrogenic or

antiestrogenic chemicals can act directly on the vaginal epithelium and

alter the age at which vaginal patency occurs without truly affecting

puberty.

Adverse Effects

Significant effects on the vagina that may be considered adverse

include the following:

Increases or decreases in weight

Infantile or malformed vagina or vulva, including

masculinized vulva or increased ano-genital distance

Vaginal hypoplasia or aplasia

Altered timing of vaginal opening

Abnormal vaginal smear cytology pattern

III.B.4.b.6. Pituitary.

Pituitary Weight

Alterations in weight of the pituitary gland should be considered

an adverse effect. The discussion on pituitary weight and histology for

males (see Section III.B.3.b.) is pertinent also for females. Pituitary

weight increases normally with age, as well as during pregnancy and

lactation. Changes in pituitary weight can occur also as a consequence

of chemical stimulation. Increased pituitary weight often precedes

tumor formation, particularly in response to treatment with estrogenic

compounds. Increased pituitary size associated with estrogen treatment

may be accompanied by hyperprolactinemia and constant vaginal estrus.

Decreased pituitary weight is less common but may result from decreased

estrogenic stimulation (Cooper et al., 1989).

Histopathology

In histologic evaluations with rats and mice, the relative size of

cell types in the anterior pituitary (acidophils and basophils) has

been reported to vary with the stages of the reproductive cycle and in

pregnancy (Holmes and Ball, 1974). Therefore, the relationship of

morphologic pattern to estrous or menstrual cycle stage or pregnancy

status should be considered in interpreting histologic observations on

the female pituitary.

Adverse Effects

A significant increase or decrease in pituitary weight should be

considered an adverse effect. Significant histopathologic damage in the

pituitary should be considered an adverse effect, but should be shown

to involve cells that control gonadotropin or prolactin production to

be called a reproductive effect.

III.B.4.c. Oocyte Production

III.B.4.c.1. Folliculogenesis. In normal females, all of the

follicles (and the resident oocytes) are present at or soon after

birth. The large majority of these follicles undergo atresia and are

not ovulated. If the population of follicles is depleted, it cannot be

replaced and the female will be rendered infertile. In humans,

depletion of oocytes leads to premature menopause. Ovarian follicle

biology and toxicology have been reviewed by Crisp (1992).

In rodents, lead, mercury, cadmium, and polyaromatic hydrocarbons

have all been implicated in the arrest of follicular growth at various

stages of the life cycle (Mattison and Thomford, 1989). Susceptibility

to oocyte toxicity varies considerably between species (Mattison and

Thorgeirsson, 1978).

Environmental agents that affect gonadotropin-mediated ovarian

steroidogenesis or follicular maturation can prolong the follicular

phase of the estrous or menstrual cycle and cause atresia of follicles

that would otherwise ovulate. Estrogenic as well as antiestrogenic

agents can produce this effect. Also, normal follicular maturation is

essential for normal formation and function of the corpus luteum formed

after ovulation (McNatty, 1979).

III.B.4.c.2. Ovulation. Chemicals can delay or block ovulation by

disrupting the ovulatory surge of luteinizing hormone (LH) or by

interfering with the ability of the maturing follicle to respond to

that gonadotropic signal. Examples for rats include compounds that

interfere with normal central nervous system (CNS) norepinephrine

receptor stimulation such as the pesticides chlordimeform and amitraz

(Goldman et al., 1990, 1991) and compounds that interfere with

norepinephrine synthesis such as the fungicide thiram (Stoker et al.,

1993). Compounds that increase central opioid receptor stimulation also

decrease serum LH and inhibit ovulation in monkeys and rats (Pang et

al., 1977; Smith, C.G., 1983). Delayed ovulation can alter oocyte

viability and cause trisomy and polyploidy in the conceptus (Fugo and

Butcher, 1966; Butcher and Fugo, 1967; Butcher et al., 1969, 1975; Na

et al., 1985). Delayed ovulation induced by exposure to the pesticide

chlordimeform has also been shown to alter fetal development and

pregnancy outcome in rats (Cooper et al., 1994).

III.B.4.c.3. Corpus luteum. The corpus luteum arises from the

ruptured follicle and secretes progesterone, which has an important

role in the estrous or menstrual cycle. Luteal progesterone is also

required for the maintenance of early pregnancy in most mammalian

species, including humans (Csapo and Pulkkinen, 1978). Therefore,

establishment and maintenance of normal corpora lutea are essential to

normal reproductive function. However, with the exception of

histopathologic evaluations that may establish only their presence or

absence, these structures are not evaluated in routine testing.

Additional research is needed to determine the importance of

incorporating endpoints that examine direct effects on luteal function

in routine toxicologic testing.

Adverse Effects

Increased rates of follicular atresia and oocyte toxicity leads to

premature menopause in humans. Altered follicular development,

ovulation failure, or altered corpus luteum formation and function can

result in disruption of cyclicity and reduced fertility, and, in

nonprimates, interference with normal sexual behavior. Therefore,

significant increases in the rate of follicular atresia, evidence of

oocyte toxicity, interference with ovulation, or altered corpus luteum

formation or function should be considered adverse effects.

III.B.4.d. Alterations in the Female Reproductive Cycle. The

pattern of events in the estrous cycle may provide a useful indicator

of the normality of reproductive neuroendocrine and ovarian function in

the nonpregnant female. It also provides a means to interpret hormonal,

histologic, and morphologic measurements relative to stage of the

cycle, and can be useful to monitor the status of mated females.

Estrous cycle normality can be monitored in the rat and mouse by

observing the changes in the vaginal smear cytology (Long and Evans,

1922; Cooper et al., 1993). To be most useful with cycling females,

vaginal smear cytology should be examined daily for at least three

normal estrous cycles prior to treatment, after onset of treatment, and

before necropsy (Kimmel, G.A. et al., 1995). However, practical

[[Page 56294]]

limitations in testing may limit the examination to the period before

mating or necropsy.

Daily vaginal smear data from rodents can provide useful

information on (1) cycle length, (2) occurrence or persistence of

estrus, (3) duration or persistence of diestrus, (4) incidence of

spontaneous pseudopregnancy, (5) distinguishing pregnancy from

pseudopregnancy (based on the number of days the smear remains

leukocytic), and (6) indications of fetal death and resorption by the

presence of blood in the smear after day 12 of gestation. The technique

also can detect onset of reproductive senescence in rodents (LeFevre

and McClintock, 1988). It is useful further to detect the presence of

sperm in the vagina as an indication of mating.

In nonpregnant females, repetitive occurrence of the four stages of

the estrous cycle at regular, normal intervals suggests that

neuroendocrine control of the cycle and ovarian responses to that

control are normal. Even normal, control animals can show irregular

cycles. However, a significant alteration compared with controls in the

interval between occurrence of estrus for a treatment group is cause

for concern. Generally, the cycle will be lengthened or the animals

will become acyclic. Lengthening of the cycle may be a result of

increased duration of either estrus or diestrus. Knowing the affected

phase can provide direction for further investigation.

The persistence of regular vaginal cycles after treatment does not

necessarily indicate that ovulation occurred, because luteal tissue may

form in follicles that have not ruptured. This effect has been observed

after treatment with anti-inflammatory agents (Walker et al., 1988).

However, that effect should be reflected in reduced fertility.

Conversely, subtle alterations of cyclicity can occur at doses below

those that alter fertility (Gray et al., 1989).

Irregular cycles may reflect impaired ovulation. Extended vaginal

estrus usually indicates that the female cannot spontaneously achieve

the ovulatory surge of LH (Huang and Meites, 1975). A number of

compounds have been shown to alter the characteristics of the LH surge

including anesthetics (Nembutal), neurotransmitter receptor binding

agents (Drouva et al., 1982), and the pesticides chlordimeform and

lindane (Cooper et al., 1989; Morris et al., 1990). Persistent or

constant vaginal cornification (or vaginal estrus) may result from one

or several effects. Typically, in the adult, if the vaginal epithelium

becomes cornified and remains so in response to toxicant exposure, it

is the result of the agent's estrogenic properties (i.e., DES or

methoxychlor), or the ability of the agent to block ovulation. In the

latter case, the follicle persists and endogenous estrogen levels bring

about the persistent vaginal cornification. Histologically, the ovaries

in persistent estrus will be atrophied following exposure to estrogenic

substances. In contrast, the ovaries of females in which ovulation has

been blocked because of altered gonadotropin secretion will contain

several large follicles and no corpora lutea. Females in constant

estrus may be sexually receptive regardless of the mechanism

responsible for this altered ovarian condition. However, if ovulation

has been blocked by the treatment, an LH surge may be induced by mating

(Brown-Grant et al., 1973; Smith, E.R. and Davidson, 1974) and a

pregnancy or pseudopregnancy may ensue. The fertility of such matings

is reduced (Cooper et al., 1994). Significant delays in ovulation can

result in increased embryonic abnormalities and pregnancy loss (Fugo

and Butcher, 1966; Cooper et al., 1994).

Persistent diestrus indicates temporary or permanent cessation of

follicular development and ovulation, and thus at least temporary

infertility. Prolonged vaginal diestrus, or anestrus, may be indicative

of agents (e.g., polyaromatic hydrocarbons) that interfere with

follicular development or deplete the pool of primordial follicles

(Mattison and Nightingale, 1980) or agents such as atrazine that

interrupt gonadotropin support of the ovary (Cooper et al., 1996).

Pseudopregnancy is another altered endocrine state reflected by

persistent diestrus. A pseudopregnant condition also has been shown to

result in rats following single or multiple doses of atrazine (Cooper

et al., 1996). The ovaries of anestrous females are atrophic, with few

primary follicles and an unstimulated uterus (Huang and Meites, 1975).

Serum estradiol and progesterone are abnormally low.

Adverse Effects

Significant evidence that the estrous cycle (or menstrual cycle in

primates) has been disrupted should be considered an adverse effect.

Included should be evidence of abnormal cycle length or pattern,

ovulation failure, or abnormal menstruation.

III.B.4.e. Mammary Gland and Lactation. The mammary glands of

normal adults change dramatically during the period around parturition

because of the sequential effects of a number of gonadal and

extragonadal hormones. Milk letdown is dependent on the suckling

stimulus and the release of oxytocin from the posterior pituitary.

Thus, mammary tissue is highly endocrine dependent for development and

function (Wolff, 1993; Imagawa et al., 1994; Tucker, 1994).

Mammary gland size, milk production and release, and histology can

be affected adversely by toxic agents, and many exogenous chemicals and

drugs are transferred into milk (American Academy of Pediatrics

Committee on Drugs, 1994; Oskarsson et al., 1995; Sonawane, 1995).

Reduced growth of young could be caused by reduced milk availability,

palatability or quality, by ingestion of a toxic agent secreted into

the milk, or by other factors unrelated to lactational ability (e.g.,

deficient suckling ability or deficient maternal behavior). Perinatal

exposure to steroid hormones and other chemicals can alter mammary

gland morphology and tumor potential in adulthood. Because of the

tendency for mobilization of lipids from adipose tissue and secretion

of those lipids into milk by lactating females, milk may contain

lipophilic agents at concentrations equal to or higher than those

present in the blood or organs of the dam. Thus, suckling offspring may

be exposed to elevated levels of such agents.

Techniques for measuring mammary tissue development, nucleic acid

content, milk production and milk composition in rodents are discussed

by Tucker (1994). During lactation, the mammary glands can be dissected

and weighed only with difficulty. RNA content of the mammary glands may

be measured as an index of lactational potential. More direct estimates

of milk production may be obtained by measuring litter weights of milk-

deprived pups taken before and after nursing. Milk from the stomachs of

pups treated similarly can also be weighed at necropsy. Cleared and

stained whole mounts of the mammary gland can be prepared at necropsy

for histologic examination. The DNA, RNA, and lipid content of the

mammary gland and the composition of the milk have been measured

following toxicant administration as indicators of toxicity to this

target organ.

Significant reductions in milk production or negative effects on

milk quality, whether measured directly or reflected in impaired

development of young, should be considered adverse reproductive

effects.

III.B.4.f. Reproductive Senescence. With advancing age, there is a

loss of the regular ovarian cycles and associated normal cyclical

changes in the uterine and vaginal epithelium that

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are typical of the young-adult female rat (Cooper and Walker, 1979).

Although the mechanisms responsible for this loss of cycling are not

thoroughly understood, age-dependent changes occur within the

hypothalamic-pituitary control of ovulation (Cooper et al., 1980; Finch

et al., 1984). Cumulative exposure to estrogen secreted by the ovary

may play a role, as treatment with estrogens during adulthood can

accelerate the age-related loss of ovarian function (Brawer and Finch,

1983). In contrast, the principal cause of the loss of ovarian cycling

in humans appears to be the depletion of oocytes (Mattison, 1985).

Prenatal or postnatal treatment of females with estrogens or

estrogenic pesticides can also cause impaired ovulation and sterility

(Gorski, 1979). These observations imply that alterations in ovarian

function may not be noticeable immediately after treatment but may

become evident at puberty or influence the age at which reproductive

senescence occurs.

Adverse Effects

Significant effects on measures showing a decrease in the age of

onset of reproductive senescence in females should be considered

adverse. Cessation of normal cycling, which is measured by vaginal

smear cytology, ovarian histopathology, or an endocrine profile that is

consistent with this interpretation, should be included as an adverse

effect.

III.B.5. Developmental and Pubertal Alterations

Developmental Effects

Alterations of reproductive differentiation and development,

including those produced by endocrine system disruption, can result in

infertility, functional and morphologic alterations of the reproductive

system, and cancer (Steinberger and Lloyd, 1985; Gray, 1991). Prenatal

and postnatal exposure to toxicants can produce changes that may not be

predicted from effects seen in adults, and those effects are often

irreversible. Adverse developmental outcomes in either sex can result

from exposure to toxic agents in utero, through contact with exposed

dams, or in milk. Dosing of dams during lactation also can result in

developmental effects through impaired nursing capability of the dams.

Effects observed in rodents following developmental exposure to

agents can include alterations in the genitalia (including ano-genital

distance), inhibited (female) or retained (male) nipple development,

impaired sexual behavior, delay or acceleration of the onset of

puberty, and reduced fertility (Gray et al., 1985, 1994, 1995; Gray and

Ostby, 1995; Kelce et al., 1995). Effects may include altered sexual

behavior or ability to produce gametes normally that are not observed

until after puberty. Hepatic enzyme systems for steroid metabolism that

are imprinted during development may be altered in males. Testis

descent from the abdominal cavity into the scrotum may be delayed or

may not occur. Generally, the type of effect seen may differ depending

on the stage of development at which the exposure occurred.

Many of these effects have been detected in human females and males

exposed prenatally to diethylstilbestrol (DES), other estrogens,

progestins, androgens, and anti-androgens (Giusti et al., 1995;

Harrison et al., 1995). Accelerated reproductive aging and tumors of

the reproductive tract have been observed in laboratory animal and

human females after pre- or perinatal exposure to hormonally active

agents. However, capability to alter sexual differentiation is not

limited to agents with known direct hormonal activity. Other agents,

for which the mode of action is not known (e.g., busulfan, nitrofen),

or which affect the endocrine system indirectly (e.g., PCBs, dioxin),

may act via different mechanisms during critical periods of development

to alter sexual differentiation and reproductive system development.

Effects on Puberty

In female rats and mice, the age at vaginal opening is the most

commonly measured marker of puberty. This event results from an

increase in the blood level of estradiol. The ages and weights of

females at the first cornified (estrous) vaginal smear, the first

diestrous smear, and the onset of vaginal cycles have also been used as

endpoints for onset of puberty. In males, preputial separation or

appearance of sperm in expressed urine or ejaculates can serve as

markers of puberty. Body weight at puberty may provide a means to

separate specific delays in puberty from those that are related to

general delays in development. Agents may differentially affect the

endpoints related to puberty onset, so it is useful to have information

on more than one marker.

Puberty can be accelerated or delayed by exogenous agents, and both

types of effects may be adverse (Gray et al., 1989, 1995; Gray and

Ostby, 1995; Kelce et al., 1995). For example, an acceleration of

vaginal opening may be associated with a delay in the onset of

cyclicity, infertility, and with accelerated reproductive aging

(Gorski, 1979). Delays in pubertal development in rodents are usually

related to delayed maturation or inhibition of function of the

hypothalamic-pituitary axis. Adverse reproductive outcomes have been

reported in rodents when puberty is altered by a week or more, but the

biologic relevance of a change in these measures of a day or two is

unknown (Gray, 1991).

Adverse Effects

Effects induced or observed during the pre- or perinatal period

should be judged using guidance from the Guidelines for Developmental

Toxicity Risk Assessment (U.S. EPA, 1991) as well as from these

Guidelines. Significant effects on ano-genital distance or age at

puberty, either early or delayed, should be considered adverse as

should malformations of the internal or external genitalia. Included as

adverse effects for females should be effects on nipple development,

age at vaginal opening, onset of cyclic vaginal smears, onset of estrus

or menstruation, or onset of an endocrine or behavioral pattern

consistent with estrous or menstrual cyclicity. Included as adverse

effects for males should be delay or failure of testis descent, as well

as delays in age at preputial separation or appearance of sperm in

expressed urine or ejaculates.

III.B.6. Endocrine Evaluations

Toxic agents can alter endocrine system function by affecting any

part of the hypothalamic-pituitary-gonadal-reproductive tract axis.

Effects may be induced in either sex by altering hormone synthesis,

storage, release, transport, or clearance, as well as by altering

hormone receptor recognition or postreceptor responses. The involvement

of the endocrine system in female reproductive physiology and

toxicology has been presented to a substantial degree as a necessary

component in Section III.B.4. (Female-specific Endpoints). The

information in that section should be considered together with the

following material.

The male reproductive system can be affected adversely by

disruption of the normal endocrine balance. In adults, effects that

result in interference with normal concentrations or action of LH and/

or follicle stimulating hormone (FSH) can decrease or abolish

spermatogenesis, affect secondary sex organ (e.g., epididymis) and

accessory sex gland (e.g., prostate, seminal vesicle) function, and

impair sexual behavior (Sharpe, 1994). In mammals, a female

reproductive tract develops unless androgen is produced and utilized

normally by the fetus (Byskov and Hoyer, 1994; George and Wilson,

1994).

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Therefore, the consequences of disruption of the normal endocrine

pattern during development of the male reproductive system pre- and

postnatally are of particular concern. Differentiation and development

of the male reproductive system are especially sensitive to substances

that interfere with the production or action of androgens (testosterone

and dihydrotestosterone). Sexual differentiation of the CNS can be

affected also. Therefore, interference with normal production or

response to androgens can result in a range of abnormal effects in

genotypic males ranging from a pseudohermaphrodite condition to

reduction in sperm production or altered sexual behavior. Chemicals

with estrogenic or anti-androgenic activity have been identified that

are capable, with sufficient exposure levels, of causing effects of

these types in males (Gray et al., 1994; Harrison et al., 1995; Kelce

et al., 1995). While sensitivity may differ, it is likely that

mechanisms of action for these endocrine disrupting agents will be

consistent across mammalian species. Chemicals with the ability to

interact with the Ah receptor (e.g., dioxin or PCBs) may also disrupt

reproductive system development or function (Brouwer et al., 1995;

Safe, 1995). Several of the effects seen with exposure of male and

female rats and hamsters differ from those caused by estrogens,

indicating a different mechanism of action.

The developing nervous system can be a target of chemicals. In

rats, sexual differentiation of the CNS can be modified by hormonal

treatments or exposure to environmental agents that mimic or interfere

with the action of certain hormones. Prior to gender differentiation,

the brain is inherently female or at least bipotential (Gorski, 1986).

Thus, the functional and structural sex differences in the CNS are not

due directly to sex differences in neuronal genomic expression, but

rather are imprinted by the gonadal steroid environment during

development.

Chemicals with endocrine activity have been shown to masculinize

the CNS of female rats. Examples include chlordecone (Gellert, 1978),

DDT (Bulger and Kupfer, 1985), and methoxychlor (Gray et al., 1989).

Exposure of newborn female rats to these agents during the critical

period of sexual differentiation can alter the timing of puberty and

perturb subsequent reproductive function, presumably by altering the

development of the neural mechanisms that regulate gonadotropin

secretion.

In females, the situation is more complex than in males due to the

female cycle, the fertilization process, gestation and lactation. All

of the functions of the female reproductive system are under endocrine

control, and therefore can be susceptible to disruption by effects on

the reproductive endocrine system.

As with males, disturbance of the normal endocrine patterns during

development can result in abnormal development of the female

reproductive tract at exposure levels that tend to be lower than those

affecting adult females (Gellert, 1978; Brouwer et al., 1995).

Consistent with the differentiation mechanism described above, exposure

of genotypic females to androgens causes formation of

pseudohermaphrodite reproductive tracts with varying degrees of

severity as well as alteration of brain imprinting. However, exposure

to estrogenic substances during development also results in adverse

effects on anatomy and function including, in rats, malformations of

the genitalia. Exposure of human females to diethylstilbestrol in utero

has been shown to cause an increased incidence of vaginal clear cell

adenoma (Giusti et al., 1995). Dioxin, presumably acting through the Ah

receptor, also disrupts development of the female reproductive system

(Gray and Ostby, 1995).

Endpoints can be included in standardized toxicity testing that are

capable of detecting, but are not specific for, effects of reproductive

endocrine system disruption. For effects of exposure on adults,

endpoints can be incorporated into the subchronic toxicity protocol or

into reproductive toxicity protocols. For effects that are induced

during development, protocols that include exposure throughout the

development process and allow evaluation of the offspring

postpubertally are needed. Data from specialized testing, including in

vitro screening tests, may be useful to evaluate further the site,

timing, and mechanism of action.

Endpoints that can detect endocrine-related effects with adult-only

exposure in standardized testing include evaluation of fertility,

reproductive organ appearance, weights, and histopathology, oocyte

number, cycle normality and mating behavior. Endpoints that can detect

effects induced by endocrine system disruption during development

include, in addition to those identified for adult-exposed animals, the

reproductive developmental endpoints identified in Section III.B.5.

Significant effects on any of these measures may be considered to be

adverse if the results are consistent and biologically plausible.

Levels of the reproductive hormones are not available routinely

from toxicity testing. However, measurements of the reproductive

hormones in males offer useful supplemental information in assessing

potential reproductive toxicity for test species (Sever and Hessol,

1984; Heywood and James, 1985; NRC, 1989). Such measurements have

increased importance with humans where invasiveness of approaches must

be limited. The reproductive hormones measured often are circulating

levels of LH, FSH, and testosterone. Other useful measures that may be

available include prolactin, inhibin, and androgen binding protein

levels. In addition, challenge tests with exogenous agents (e.g.,

gonadotropin releasing hormone, LH, or human chorionic gonadotropin)

may provide insight into the functional responsiveness of the pituitary

or Leydig cells.

Interpretation of endocrine effects is facilitated if information

is available on a battery of hormones. However, in evaluating such

data, it is important to consider that serum hormones such as FSH, LH,

prolactin, and androgens exhibit cyclic variations within a 24-hour

period (Fink, 1988). Thus, the time of sampling should be controlled

rigorously to avoid excessive variability (Nett, 1989). Sequential

sampling can allow detection of treatment-related changes in circadian

and pulsatile rhythms.

The pattern seen in levels of reproductive system hormones can

provide useful information about the possible site and type of effect

on reproductive system function. For example, if a compound acts at the

level of the hypothalamus or pituitary, then serum LH and FSH may be

decreased, leading to decreased testosterone levels. On the other hand,

severe interference with Sertoli cell function or spermatogenesis would

be expected to elevate serum FSH levels. An agent having antiandrogenic

activity in adults might elevate serum LH and testosterone. Testis

weight might be unaffected, while the weight and size of the accessory

sex glands may be reduced. The endocrine profile presented by exposure

to specific antiandrogens can differ markedly because of differences in

tissue specificity and receptor kinetics, as well as age at which

exposure occurred.

Adverse Effects

In the absence of endocrine data, significant effects on

reproductive system anatomy, sexual behavior, pituitary, uterine or

accessory sex gland

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weights or histopathology, female cycle normality, or Leydig cell

histopathology may suggest disruption of the endocrine system. In those

instances, additional testing for endocrine effects may be indicated.

Significant alterations in circulating levels of estrogen,

progesterone, testosterone, prolactin, LH, or FSH may be indicative of

existing pituitary or gonadal injury. When significant alterations from

control levels are observed in those hormones, the changes should be

considered cause for concern because they are likely to affect, occur

in concert with, or result from alterations in game

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