EPA/630/R-96/009

October 1996

Guidelines for
Reproductive Toxicity Risk Assessment
Published on October 31, 1996, Federal Register 61(212):56274-56322

These guidelines replace two proposed guidelines: Proposed Guidelines for

Female Reproductive Risk and Proposed Guidelines for Male Reproductive
Risk, both dated June 30, 1988.
 Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC
 DISCLAIMER

This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.

Note: This document represents the final guidelines. A number of editorial corrections have been made
during conversion and subsequent proofreading to ensure the accuracy of this publication.

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 CONTENTS

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Federal Register Preamble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Part A: Guidelines for Reproductive Toxicity Risk Assessment

1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Definitions and Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3. Hazard Characterization for Reproductive Toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1. Laboratory Testing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.2. Duration of Dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.3. Length of Mating Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1.4. Number of Females Mated to Each Male . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1.5. Single- and Multigeneration Reproduction Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1.6. Alternative Reproductive Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.1.7. Additional Test Protocols That May Provide Reproductive Data . . . . . . . . . . . . . 12
3.2. Endpoints for Evaluating Male and Female Reproductive Toxicity in
Test Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2.2. Couple-Mediated Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2.2.1. Fertility and Pregnancy Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2.2.2. Sexual Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2.3. Male-Specific Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.3.2. Body Weight and Organ Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.3.3. Histopathologic Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.3.4. Sperm Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.3.5. Paternally Mediated Effects on Offspring . . . . . . . . . . . . . . . . . . . . . . . 33
3.2.4. Female-Specific Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.4.2. Body Weight, Organ Weight, Organ Morphology, and Histology . . . . . 35
3.2.4.3. Oocyte Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.2.4.4. Alterations in the Female Reproductive Cycle . . . . . . . . . . . . . . . . . . . 42
3.2.4.5. Mammary Gland and Lactation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2.4.6. Reproductive Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.2.5. Developmental and Pubertal Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.2.5.1. Developmental Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

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 CONTENTS (continued)

3.2.5.2. Effects on Puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3.2.5.3. Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.6. Endocrine Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.6.1. Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.2.7. In Vitro Tests of Reproductive Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.3. Human Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.3.1. Epidemiologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.3.1.1. Selection of Outcomes for Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.3.1.2. Reproductive History Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3.1.3. Community Studies and Surveillance Programs . . . . . . . . . . . . . . . . . . 56
3.3.1.4. Identification of Important Exposures for Reproductive
Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.3.1.5. General Design Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.3.2. Examination of Clusters, Case Reports, or Series . . . . . . . . . . . . . . . . . . . . . . . . 61
3.4. Pharmacokinetic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.5. Comparisons of Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.6. Evaluation of Dose-Response Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.7. Characterization of the Health-Related Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

4. Quantitative Dose-Response Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4.1. Utilization of Information in Risk Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

5. Exposure Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

6. Risk Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6.2. Integration of Hazard Characterization, Quantitative Dose-Response,
and Exposure Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.3. Descriptors of Reproductive Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
6.3.1. Distribution of Individual Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
6.3.2. Population Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
6.3.3. Margin of Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.3.4. Distribution of Exposure and Risk for Different Subgroups . . . . . . . . . . . . . . . . . 89
6.3.4.1. Highly Exposed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.3.4.2. Highly Susceptible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
6.3.5. Situation-Specific Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.3.6. Evaluation of the Uncertainty in the Risk Descriptors . . . . . . . . . . . . . . . . . . . . . . 92
6.4. Summary and Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

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 CONTENTS (continued)

Part B: Response to Science Advisory Board and Public Comments

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

2. Response to Science Advisory Board Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

3. Response to Public Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

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 LIST OF TABLES

Table 1. Default assumptions in reproductive toxicity risk assessment . . . . . . . . . . . . . . . . . . . . . . . . 3

Table 2. Couple-mediated endpoints of reproductive toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Table 3. Selected indices that may be calculated from endpoints of reproductive

toxicity in test species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Table 4. Male-specific endpoints of reproductive toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Table 5. Female-specific endpoints of reproductive toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Table 6. Categorization of the health-related database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Table 7. Guide for developing chemical-specific risk characterizations for

reproductive effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

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 GUIDELINES FOR REPRODUCTIVE TOXICITY RISK ASSESSMENT
[FRL-5630-6]

AGENCY: U.S. Environmental Protection Agency

ACTION: Notice of availability of final 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 Agencys 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.

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ADDRESSES: The Guidelines will be made available in the following ways:
(1) The electronic version will be accessible on EPAs Office of Research and Development
home page on the Internet at http://www.epa.gov/ORD/WebPubs/repro/.
(2) 3 -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, Effects Identification and
Characterization Group, National Center for Environmental Assessment-Washington Division (8623D),
U.S. Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460; telephone:
202-564-3297; 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

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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 Agencys 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

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al., 1989; Sheehan et al., 1989; Seed et al., 1996). Also, numerous resources provide background
information on the 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 106 per mL of semen prior to 1960 to 66 x 106 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

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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
postnatal year, about 3% more are recognized to have serious developmental defects (Shepard, 1986).
Of these, it is estimated that 20% are of known genetic transmission, 10% are attributable to known
environmental factors, and the remaining 70% result from unknown causes (Wilson, 1977). Also,
approximately 7.4% of children have low birth weight (i.e., below 2.5 kg) (Selevan, 1981).
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

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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).
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

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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 106 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 20 106, 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.

xiv
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
includes 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 3.7). 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 4), 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

xv
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
5.
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 Agencys risk 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

xvi
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 Signed by EPA Administrator
Carol M. Browner

xvii
PART A: GUIDELINES FOR REPRODUCTIVE TOXICITY RISK ASSESSMENT

1. 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 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

1
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.
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

2
have resulted in developmental toxicity; and (3) Many of the mechanisms controlling important aspects
of reproductive system function are

Table 1. Default assumptions in reproductive toxicity risk assessment

1. An agent that produces an adverse reproductive effect in experimental animals is assumed to

pose a potential threat to humans.

2. Effects of xenobiotics on male and female reproductive processes are assumed generally
to be similar unless demonstrated otherwise. For developmental outcomes, the specific
effects in humans are not necessarily the same as those seen in the experimental species.

3. In the absence of information to determine the most appropriate experimental species, data
from the most sensitive species should be used.

4. In the absence of information to the contrary, an agent that affects reproductive function
in one sex is assumed to adversely affect reproductive function in the other sex.

5. A nonlinear dose-response curve is assumed for reproductive toxicity.

3
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.

4
 2. 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.

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).

5
 3. 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:
C Identify the strengths and limitations of the database, including all available epidemiologic
and experimental animal studies as well as pharmacokinetic and mechanistic information.
C Identify and describe key toxicological studies.
C Describe the type(s) of effects.
C Describe the nature of the effects (irreversible, reversible, transient, progressive, delayed,
residual, or latent effects).
C Describe how much is known about how (through what biological mechanism) the agent
produces adverse effects.
C Discuss the other health endpoints of concern.
C Discuss any nonpositive data in humans or experimental animals.
C Discuss the dose-response data (epidemiologic or experimental animal) available for further
dose-response analysis.
C Discuss the route, level, timing, and duration of exposure in studies as compared to
expected human exposures.
C 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 3.1 and 3.2 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
3.3) and limited discussions of pharmacokinetic and structure-activity factors (Sections 3.4 and 3.5).

6
3.1. LABORATORY TESTING PROTOCOLS
3.1.1. 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.

3.1.2. 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.

7
3.1.3. 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.

3.1.4. 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.

3.1.5. 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

8
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 3.2.2.1) 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.

9
 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 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.

10
3.1.6. 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

11
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.

3.1.7. 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 OECDs 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 NTPs 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).

12
 A review of the male dominant lethal test has been published as part of the EPAs 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
3.2.2 for discussions of these endpoints).
Pre- or postimplantation loss in the dominant lethal test is often considered evidence that the
agent has induced mutagenic damage to the male germ cell (U.S. 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 agents 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

13
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.

3.2. ENDPOINTS FOR EVALUATING MALE AND FEMALE REPRODUCTIVE

TOXICITY IN TEST SPECIES
3.2.1. 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.

14
 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 3.3.
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.

3.2.2. 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.

3.2.2.1. 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 Agencys Guidelines for Developmental
Toxicity Risk Assessment (U.S. EPA, 1991). Also included in Table 2 are measures that may be
obtained from other types of studies (e.g., single-generation reproduction studies, developmental
toxicity studies, dominant lethal studies) in which offspring are not retained to evaluate subsequent
reproductive performance.

15
 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.

16
Table 2. Couple-mediated endpoints of reproductive toxicity

Multigeneration studies Other reproductive endpoints

Mating rate, time to mating (time to pregnancy*) Ovulation rate
Pregnancy rate* Fertilization rate
Delivery rate* Preimplantation loss
Gestation length* Implantation number
Litter size (total and live) Postimplantation loss*
Number of live and dead offspring (fetal death rate*) Internal malformations and
Offspring gender* (sex ratio) variations*
Birth weight* Postnatal structural and
Postnatal weights* functional development*
Offspring survival*
External malformations and variations*
Offspring reproduction*

*Endpoints that can be obtained with humans.

17
Table 3. Selected indices that may be calculated from endpoints of reproductive toxicity in
test species

MATING INDEX

Number of males or females mating 100

Number of males or females cohabited

Note: Mating is used to indicate that evidence of copulation (observation or other evidence of
ejaculation such as vaginal plug or sperm in vaginal smear) was obtained.

FERTILITY INDEX

Number of cohabited females becoming pregnant 100

Number of nonpregnant couples cohabited

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

Number of females delivering live young 100

Number of females with evidence of pregnancy

LIVE BIRTH INDEX

Number of live offspring 100

Number of offspring delivered

SEX RATIO

Number of male offspring

Number of female offspring

4-DAY SURVIVAL INDEX (VIABILITY INDEX)

Number of live offspring at lactation day 4 100

Number of live offspring delivered

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

18
Table 3. Selected indices that may be calculated from endpoints of reproductive toxicity in
test species (continued)

LACTATION INDEX (WEANING INDEX)

Number of live offspring at day 21 100

Number of live offspring born

Note: If litters were standardized to equalize numbers of offspring per litter, number of
offspring after standardization should be used instead of number born alive. When no standardization is
done, measure is called weaning index. When standardization is done, measure is called lactation
index.

PREWEANING INDEX

Number of live offspring born -

Number of offspring weaned 100
Number of live offspring born

Note: If litters were standardized to equalize numbers of offspring per litter, then number of
offspring remaining after standardization should be used instead of number born.

19
 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 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 3.1.3 and 3.1.4).
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.

20
 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 4) 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 4).
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.

21
 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 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

22
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.

3.2.2.1.1. 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.

3.2.2.2. 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

23
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.

24
3.2.2.2.1. 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.

3.2.3. Male-Specific Endpoints

3.2.3.1. Introduction
The following sections (3.2.3 and 3.2.4) 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 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.

3.2.3.2. 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 3.2.2). 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

25
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

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.

26
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
animals 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.

27
3.2.3.2.1. 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.

3.2.3.3. Histopathologic Evaluations

Histopathologic evaluations of test animal tissues have a prominent role in male reproductive
risk assessment. Organs that are often evaluated include the testes, epididymides, prostate, seminal
vesicles (often including coagulating glands), and pituitary. Tissues from lower dose exposures are
often not examined histologically if the high dose produced no difference from controls. Histologic
evaluations can be especially useful by (1) providing a relatively sensitive indicator of damage; (2)
providing information on toxicity 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,

28
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).

3.2.3.3.1. 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

29
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.

3.2.3.4. 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.

3.2.3.4.1. 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 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

30
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.

3.2.3.4.2. 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 agents 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

31
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.

3.2.3.4.3. 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

32
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).
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 reanalyzed 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

33
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.

3.2.3.4.4. 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.

3.2.3.5. 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

34
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.

3.2.4. Female-Specific Endpoints

3.2.4.1. 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 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.

35
 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 agents effect(s). Areas that may be examined in evaluations of the female reproductive
system are listed in Table 5.
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

36
cyclicity and their interpretation. Then, considerations relevant to prepubertal, pregnant, lactating, and
aging females are presented.

3.2.4.2. Body Weight, Organ Weight, Organ Morphology, and Histology

3.2.4.2.1. 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

37
Table 5. Female-specific endpoints of reproductive toxicity

Organ weights Ovary, uterus, vagina, pituitary

Visual examination Ovary, uterus, vagina, pituitary, oviduct, mammary

and histopathology gland

Estrous (menstrual*) Vaginal smear cytology

cycle normality

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.

38
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.

3.2.4.2.2. Ovary. The ovary serves a number of functions that are critical to reproductive activity,
including production and ovulation of oocytes. Estrogen is produced by developing follicles and
progesterone is produced by corpora lutea that are formed after ovulation.

3.2.4.2.2.1. 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.

3.2.4.2.2.2. 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

39
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.

3.2.4.2.2.3. Adverse effects. Significant changes in the ovaries in any of the following effects should
be considered adverse:
C Increase or decrease in ovarian weight
C Increased incidence of follicular atresia
C Decreased number of primary follicles
C Decreased number or lifespan of corpora lutea
C Evidence of abnormal folliculogenesis or luteinization, including cystic follicles,
luteinized follicles, and failure of ovulation
C Evidence of altered puberty or premature reproductive senescence

3.2.4.2.3. Uterus
3.2.4.2.3.1. 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 fourfold 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 animals 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.

3.2.4.2.3.2. 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

40
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.

3.2.4.2.3.3. 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.
C Infantile or malformed uterus or cervix
C Decreased or increased uterine weight
C Endometrial hyperplasia, hypoplasia, or aplasia
C Decreased number of implantation sites

3.2.4.2.4. 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.

3.2.4.2.5. Vagina and external genitalia

3.2.4.2.5.1. Vaginal weight. Vaginal weight changes should parallel those seen in the uterus during
the estrous cycle, although the magnitude of the changes is smaller.

3.2.4.2.5.2. 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 3.2.4.4). The vaginal
smear pattern may be useful to identify conditions that would delay or preclude fertility, or affect sexual

41
behavior. Other histologic alterations that may be observed include aplasia, hypoplasia, and
hyperplasia of the vaginal epithelial cell lining.

3.2.4.2.5.3. Developmental effects. Developmental abnormalities, either genetic or related to

prenatal exposure to compounds that disrupt the endocrine balance, include agenesis, hypoplasia, and
dysgenesis. Hypoplasia of the vagina may be concomitant with hyperplasia of the external genitalia and
can be induced by gonadal or adrenal steroid exposure. In rodents, malpositioning of the vaginal and
urethral ducts is common in steroid-treated females. Such developmentally induced lesions are
irreversible.
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.

3.2.4.2.5.4. Adverse effects. Significant effects on the vagina that may be considered adverse include
the following:
C Increases or decreases in weight
C Infantile or malformed vagina or vulva, including masculinized vulva or increased ano-
genital distance
C Vaginal hypoplasia or aplasia
C Altered timing of vaginal opening
C Abnormal vaginal smear cytology pattern

3.2.4.2.6. Pituitary
3.2.4.2.6.1. 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 3.2.3.2) 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

42
accompanied by hyperprolactinemia and constant vaginal estrus. Decreased pituitary weight is less
common but may result from decreased estrogenic stimulation (Cooper et al., 1989).

3.2.4.2.6.2. 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.

3.2.4.2.6.3. 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.

3.2.4.3. Oocyte Production

3.2.4.3.1. 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).

3.2.4.3.2. 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

43
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).

3.2.4.3.3. 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.

3.2.4.3.3.1. 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.

3.2.4.4. 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

44
(Kimmel, G.A. et al., 1995). However, practical 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 agents 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

45
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.

3.2.4.4.1. 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.

3.2.4.5. 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

46
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.

3.2.4.6. Reproductive Senescence

With advancing age, there is a loss of the regular ovarian cycles and associated normal cyclical
changes in the uterine and vaginal epithelium that are typical of the young-adult female rat (Cooper 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.

3.2.4.6.1. 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

47
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.

3.2.5. Developmental and Pubertal Alterations

3.2.5.1. 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.

3.2.5.2. Effects on Puberty

48
 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).

3.2.5.3. 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.

3.2.6. 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

49
and toxicology has been presented to a substantial degree as a necessary component in Section 3.2.4
(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).
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

50
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 3.2.5. 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;

51
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.

3.2.6.1. Adverse Effects

In the absence of endocrine data, significant effects on reproductive system anatomy, sexual
behavior, pituitary, uterine or accessory sex gland 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 gametogenesis, gamete maturation, mating ability, or fertility. Such
effects, if compatible with other available information, may be considered adverse and may be used to
establish a NOAEL, LOAEL, or benchmark dose. Furthermore, endocrine data may facilitate

52
identification of sites or mechanisms of toxicant action, especially when obtained after short-term
exposures.

3.2.7. In Vitro Tests of Reproductive Function

Numerous in vitro tests are available and under development to measure or detect chemically
induced changes in various aspects of both male and female reproductive systems (Kimmel, G.L. et al.,
1995). These include in vitro fertilization using isolated gametes, whole organ (e.g., testis, ovary)
perfusion, culture of isolated cells from the reproductive organs (e.g., Leydig cells, Sertoli cells,
granulosa cells, oviductal or epididymal epithelium), co-culture of several populations of isolated cells,
ovaries, quarter testes, seminiferous tubule segments, various receptor binding assays on reproductive
cells and transfected cell lines, and others.
Tests of sperm properties and function that have been applied to reproductive toxicology
include penetration of sperm through viscous medium (Yeung et al., 1992), in vitro capacitation and
fertilization assays (Holloway et al., 1990a,b; Perreault and Jeffay, 1993; Slott et al., 1995), and
evaluation of sperm nuclear integrity (Darney, 1991). In addition, evaluation of human sperm function
may include sperm penetration of cervical mucus, ability of sperm to undergo an acrosome reaction,
and ability to penetrate zona pellucida-free hamster oocytes or bind to human hemi-zona pellucidae
(Franken et al., 1990; Liu and Baker, 1992).
The diagnostic information obtained from such tests may help to identify potential effects on the
reproductive systems. However, each test bypasses essential components of the intact animal system
and therefore, by itself, is not capable of predicting exposure levels that would result in toxicity in intact
animals. While it is desirable to replace whole animal testing to the extent possible with in vitro tests,
the use of such tests currently is to screen for toxicity potential and to study mechanisms of action and
metabolism (Perreault, 1989; Holloway et al., 1990a,b).

3.3. HUMAN STUDIES

In principle, human data are scientifically preferable for risk assessment since test animal to
human extrapolation is not required. At this time, reproductive data for humans are available for only a
limited number of toxicants. Many of these are from occupational settings in which exposures tend to
be higher than in environmental settings. As more data become available, expanding the number of
agents and endpoints studied and improving exposure assessment, more risk assessments will include
these data. The following describes the methods of generation and evaluation of human data and the
relative weight the various types of human data should be given in risk assessments.

53
 Human studies include both epidemiologic studies and other reports of individual cases or
clusters of events. Typical epidemiologic studies include (1) cohort studies in which groups are defined
by exposure and health outcomes are examined; (2) case-referent studies in which groups are defined
by health status and prior exposures are examined; (3) cross-sectional studies in which exposure and
outcome are determined at the same time; and 4) ecologic studies in which exposure is presumed based
typically on residence. Greatest weight should be given to carefully designed epidemiologic studies with
more precise measures of exposure, because they can best evaluate exposure-response relationships.
This assumes that human exposures occur in broad enough ranges for observable differences in
response to occur. Epidemiologic studies in which exposure is presumed, based on occupational title
or residence (e.g., some case-referent and all ecologic studies), may contribute data for hazard
characterization, but are of limited use for quantitative risk determination because of the generally broad
categorical groupings of exposure. Reports of individual cases or clusters of events may generate
hypotheses of exposure-outcome associations, but require further confirmation with well-designed
epidemiologic or laboratory studies. These reports of cases or clusters may support associations
suggested by other human or test animal data, but cannot stand by themselves in risk assessments.

3.3.1. Epidemiologic Studies

Good epidemiologic studies provide valuable data for assessment of human risk. As there are
many different designs for epidemiologic studies, simple rules for their evaluation do not exist. Risk
assessors should seek the assistance of professionals trained in epidemiology when conducting a
detailed analysis. The following is an overview of key issues to consider in evaluation for risk
assessment of reproductive effects.

3.3.1.1. Selection of Outcomes for Study

As already discussed, a number of endpoints can be considered in the evaluation of adverse
reproductive effects. However, some of the outcomes are not easily observed in humans, such as early
embryonic loss, reproductive capacity of the offspring, and invasive evaluations of reproductive function
(e.g., testicular biopsies). Currently, the most feasible endpoints for epidemiologic studies are (1)
indirect measures of fertility/infertility; (2) reproductive history studies of some pregnancy outcomes
(e.g., embryonic/fetal loss, birth weight, sex ratio, congenital malformations, postnatal function, and
neonatal growth and survival); (3) semen evaluations; (4) menstrual history; and (5) blood or urinary
hormone measures. Factors requiring control in the design or analysis (such as effect modifiers and
confounders, described below) may vary depending on the specific outcomes selected for study.

54
 The reproductive outcomes available for epidemiologic examination are limited by a number of
factors, including the relative magnitude of the exposure, the size and demographic characteristics of the
population, and the ability to observe the outcome in humans. Use of improved methods for identifying
some outcomes, such as embryonic loss detected by more sensitive urinary hCG (human chorionic
gonadotropin) assays, change the spectrum of outcomes available for study (Wilcox et al., 1985;
Sweeney et al., 1988; Zinaman et al., 1996). Other, less accessible, endpoints may require invasive
techniques to obtain samples (e.g., histopathology) or may have high intra- or interindividual variability
(e.g., serum hormone levels, sperm count).
Demographic characteristics of the population, such as marital status, age, education,
socioeconomic status (SES), and prior reproductive history are associated with the probability of
whether couples will attempt to have children. Differences in birth control practices would also affect
the number of outcomes available for study.
In addition to the above-mentioned factors, reproductive endpoints may be envisioned as
effects recognized at various points in a continuum starting before conception and continuing through
death of the progeny. Many studies, however, are limited to evaluating endpoints at a particular time in
this continuum. For example, in a study of defects observed at live birth, a malformed stillbirth would
not be included, even though the etiology could be identical (Bloom, 1981). Also, a different spectrum
of outcomes could result from differences in timing or in level of exposure (Selevan and Lemasters,
1987).

3.3.1.1.1. Human reproductive endpoints. The following section discusses various human male and
female reproductive endpoints. These outcomes may be an indicator of sub- or infertility. These are
followed by a discussion of reproductive history studies.

3.3.1.1.1.1. Male endpoints - semen evaluations. The use of semen analysis was discussed in
Section 3.2.3.4. Most epidemiologic studies of potential effects of agents on semen characteristics
have been conducted in occupational groups and patients receiving drug therapy. Obtaining a high level
of participation in the workforce has been difficult, because social and cultural attitudes concerning sex
and reproduction may affect cooperation of the study groups. Increased participation may occur in
men who are planning to have children or who are concerned about existing reproductive problems or
possible ill effects of their exposures. Unless controlled, such biased participation may yield
unrepresentative estimates of risk associated with exposure, resulting in data that are less useful for risk
assessment. While some studies have response rates greater than 70% (Ratcliffe et al., 1987; Welch et

55
al., 1988), response rates are often less than 70% in such studies and may be even lower in the
comparison group (Egnatz et al., 1980; Lipshultz et al., 1980; Milby and Whorton, 1980; Lantz et al.,
1981; Meyer, 1981; Milby et al., 1981; Rosenberg et al., 1985; Ratcliffe et al., 1989). Some of the
low response rates may be caused by inclusion of vasectomized men in the total population, although
this could vary widely by population (Milby and Whorton, 1980). Participation in the comparison
group may be biased toward those with preexisting reproductive problems. The response rate may be
improved substantially with proper education and payment of subjects (Ratcliffe et al., 1986, 1987).
Several factors may influence the semen evaluation, including the period of abstinence
preceding collection of the sample, health status, and social habits (e.g., alcohol, recreational drugs,
smoking). Data on these factors may be collected by interview, subject to the limitations described for
pregnancy outcome studies.
Reports of studies with semen analyses have rarely included an evaluation of endocrine status
(hormone levels in blood or urine) of exposed males (Lantz et al., 1981; Ratcliffe et al., 1989).
Conversely, studies that have examined endocrine status typically do not have data on semen quality
(Mason, 1990; McGregor and Mason, 1991; Egeland et al., 1994).

3.3.1.1.1.2. Female endpoints. Reproductive effects may result from a variety of exposures. For
example, environmental exposures may be toxic to the oocyte, producing a loss of primary oocytes that
irreversibly affects the womans fecundity. The exposures of importance may occur during the prenatal
period, and beyond. Oocyte depletion is difficult to examine directly in women because of the
invasiveness of the tests required; however, it can be studied indirectly through evaluation of the age at
reproductive senescence (menopause) (Everson et al., 1986).
Numerous diagnostic methods have been developed to evaluate female reproductive
dysfunction. Although these methods have been used rarely for occupational or environmental
toxicologic evaluations, they may be helpful in defining biologic parameters and the mechanisms related
to female reproductive toxicity. If clinical observations are able to link exposures to the reproductive
effect of concern, these data will aid the assessment of adverse female reproductive toxicity. The
following clinical observations include endpoints that may be reported in case reports or epidemiologic
research studies.
Reproductive dysfunction also can be studied by the evaluation of irregularities of menstrual
cycles. However, menstrual cyclicity is affected by many parameters such as age, nutritional status,
stress, exercise level, certain drugs, and the use of contraceptive measures that alter endocrine
feedback. Vaginal bleeding at menstruation is a reflection of withdrawal of steroidogenic support,

56
particularly progesterone. Vaginal bleeding can occur at midcycle, in early miscarriage, after
withdrawal of contraceptive steroids, or after an inadequate luteal phase. The length of the menstrual
cycle, particularly the follicular phase (before ovulation), can vary between individuals and may make it
difficult to determine significant effects on length in populations of women (Burch et al., 1967; Treloar et
al., 1967). Human vaginal cytology may provide information on the functional state of reproductive
cycles. Cytologic evaluations, along with the evaluation of changes in cervical mucus viscosity, can be
used to estimate the occurrence of ovulation and determine different stages of the reproductive cycle
(Kesner et al., 1992). Menstrual dysfunction data have been used to examine adverse reproductive
effects in women exposed to potentially toxic agents occupationally (Lemasters, 1992),
Reports of prospective clinical evaluations of menstrual function (Kesner et al., 1992; Wright et
al., 1992), have shown urinary endocrine measures to be practical and useful. The endocrine status of
a woman can be evaluated by the measurement of hormones in blood and urine. Progesterone can also
be measured in saliva. Because the female reproductive endocrine milieu changes in a cyclic pattern,
single sample analysis does not provide adequate information for evaluating alterations in reproductive
function. Still, a single sample for progesterone determination some 7 to 9 days after the estimated
midcycle surge of gonadotropins in a regularly cycling woman may provide suggestive evidence for the
presence of a functioning corpus luteum and prior follicular maturation and ovulation. Clinically
abnormal levels of gonadotropins, steroids, or other biochemical parameters may be detected from a
single sample. However, a much stronger design involves collection of multiple samples and their
observation in conjunction with events in the menstrual cycle.
The day of ovulation can be estimated by the biphasic shift in basal body temperature.
Ovulation can also be detected by serial measurement of hormones in the blood or urine and analyses
of estradiol and gonadotropin status at midcycle. After ovulation, luteal phase function can be assessed
by analysis of progesterone secretion and by evaluation of endometrial histology. Tubal patency, which
could be affected by abnormal development, endometriosis or infection, is an endpoint that can be
observed in clinical evaluations of reproductive function (Forsberg, 1981). These latter evaluations of
endometrial histology and tubal patency are less likely to be present in epidemiologic studies or
surveillance programs because of the invasiveness of the procedures.

3.3.1.2. Reproductive History Studies

3.3.1.2.1. Measures of fertility. Subfertility may be thought of as nonevents: a couple is unable to
have children within a specific time frame. Therefore, the epidemiologic measurement of reduced
fertility or fecundity is typically indirect and is accomplished by comparing birth rates or time intervals

57
between births or pregnancies. These outcomes have been examined using several methods: the
Standardized Birth Ratio (SBR; also referred to as the Standardized Fertility Ratio) and the length of
time to pregnancy or birth. In these evaluations, the couples joint ability to procreate is estimated. The
SBR compares the number of births observed to those expected based on the person-years of
observation preferably stratified by factors such as time period, age, race, marital status, parity, and (if
possible) contraceptive use (Wong et al., 1979; Levine et al., 1980, 1981, 1983; Levine, 1983; Starr
et al., 1986). The SBR is analogous to the Standardized Mortality Ratio (SMR), a measure frequently
used in studies of occupational cohorts and has similar limitations in interpretation (Gaffey, 1976;
McMichael, 1976; Tsai and Wen, 1986). The SBR was found to be less sensitive in identifying an
effect when compared to semen analyses (Welch et al., 1991). These data can also be analyzed using
Poisson regression.
Analysis of the time between recognized pregnancies or live births is a more recent approach to
indirect measurement of fertility (Dobbins et al., 1978; Baird and Wilcox, 1985; Baird et al., 1986;
Weinberg and Gladen, 1986; Rowland et al., 1992). Because the time between births increases with
increasing parity (Leridon, 1977), comparisons within birth order (parity) are more appropriate. A
statistical method (Cox regression) can stratify by birth or pregnancy order to help control for
nonindependence of these events in the same woman or couple.
Fertility may also be affected by alterations in sexual behavior. However, data linking toxic
exposures to these alterations in humans are limited and are not obtained easily in epidemiology studies
(see Section 3.3.1.4).

3.3.1.2.2. Developmental outcomes. Developmental outcomes examined in human studies of

parental exposures may include embryo or fetal loss, congenital malformations, birth weight effects, sex
ratio at birth, and possibly postnatal effects (e.g., physical growth and development, organ or system
function, and behavioral effects of exposure). Developmental effects are discussed in more detail in the
Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991). As mentioned above,
epidemiologic studies that focus on only one type of developmental outcome or exposures to only one
parent may miss a true effect of exposure.
Evidence of a dose-response relationship is usually an important criterion in the assessment of
exposure to a potentially toxic agent. However, traditional dose-response relationships may not always
be observed for some endpoints (Wilson, 1973; Selevan and Lemasters, 1987). For example, with
increasing dose, a pregnancy might end in embryo or fetal loss, rather than a live birth with
malformations. A shift in the patterns of outcomes could result from differences either in level of

58
exposure or in timing (Wilson, 1973; Selevan and Lemasters, 1987) (for a more detailed description,
see Section 3.3.1.4). Therefore, a risk assessment should, when possible, attempt to look at the
relationship of different reproductive endpoints and patterns of exposure.
In addition to the above effects, exposure may produce genetic damage to germ cells.
Outcomes resulting from germ-cell mutations could include reduced probability of fertilization and
increased probability of embryo or fetal loss and postnatal developmental effects. Based on studies
with test species, germ cells or early zygotes are critical targets of potentially toxic agents. Germ-cell
mutagenicity could be expressed also as genetic diseases in future generations. Unfortunately, these
studies are difficult to conduct in human populations because of the long time between exposure and
outcome and the large study groups needed. For more information and guidance on the evaluation of
these data, refer to the Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986c).

3.3.1.3. Community Studies and Surveillance Programs

Epidemiologic studies may be based on broad populations such as a community, a nationwide
probability sample, or surveillance programs (such as birth defects registries). Some studies have
examined the effects of environmental exposures such as potential toxic agents in outdoor air, food,
water, and soil. These studies may assume certain exposures through these routes due to residence
(ecologic studies). The link between environmental measurements and critical periods of exposure for a
given reproductive effect may be difficult to make. Other studies may go into more detail, evaluating
the above routes and also indoor air, house dust, and occupational exposures on an individual basis
(Selevan, 1991). Such environmental studies, relating individual exposures to health outcomes should
have less misclassification of exposure.
Exposure definition in community studies has some limitations in the assessment of exposure-
effect relationships. For example, in many community-based studies, it may not be possible to
distinguish maternally mediated effects from paternally mediated effects since both parents spend time in
the same home environment. In addition, the presumably lower exposure levels (compared with
industrial settings) may require very large groups for the study. A number of case-referent studies have
examined the relationship between broad classes of parental occupation in certain communities or
countries and embryo/fetal loss (Silverman et al., 1985; McDonald et al., 1989; Lindbohm et al.,
1991), birth defects (Hemminki et al., 1980; Kwa and Fine, 1980; Papier, 1985), and childhood
cancer (Fabia and Thuy, 1974; Hemminki et al., 1981; Peters et al., 1981; Gardner et al., 1990a,b).
In these reports, jobs are classified typically into broad categories based on the probability of exposure
to certain classes or levels of exposure. Such studies are most helpful in the identification of topics for

59
additional study. However, because of the broad groupings of types or levels of exposure, these
studies are not typically useful for risk assessment of any one particular agent.
Surveillance programs may also exist in occupational settings. In this case, reproductive
histories (including menstrual cycles) or semen evaluations could be followed to monitor reproductive
effects of exposures. With adequate exposure information, these could yield very useful data for risk
assessment. Reproductive histories tend to be easier and less costly to collect, whereas, a semen
evaluation program would be rather costly. Success with such programs in the workplace will be
determined by the confidence the worker has that reproductive data are kept confidential and will not
affect employment status (Samuels, 1988; Lemasters and Selevan, 1993).

3.3.1.4. Identification of Important Exposures for Reproductive Effects

For all examinations of the relationship between reproductive effects and potentially toxic
exposures, defining the exposure that produces the effect is crucial. Preconceptional exposures of
either parent and in utero exposures have been associated with the more commonly examined
outcomes (e.g., fetal loss, malformations, low birth weight, and measures of in- or subfertility). These
exposures, plus postnatal exposure via breast milk, food, and the environment, may also be associated
with postnatal developmental effects (e.g., changes in growth or in behavioral and cognitive function).
A number of factors affect the intensity and duration of exposure. General environmental
exposures are typically lower than those found in industrial or agricultural settings. However, this
relationship may change as exposures are reduced in workplaces and as more is learned about
environmental exposures (e.g., indoor air exposures, home pesticide usage). Larger populations are
necessary to achieve sufficient power in settings with lower exposures which are likely to have lower
measures of risk (Lemasters and Selevan, 1984). In addition, exposure to individuals may change as
they move in and out of areas with differing levels and types of exposures, thus affecting the number of
exposed and comparison events for study.
Data on exposure from human studies are frequently qualitative, such as employment or
residence histories. More quantitative data may be difficult to obtain because of the nature of certain
study designs (e.g., retrospective studies) and limitations in estimates of historic exposures. Many
reproductive effects result from exposures during certain critical times. The appropriate exposure
classification depends on the outcomes studied, the biologic mechanism affected by exposure, and the
biologic half-life of the agent. The half-life, in combination with the patterns of exposure (e.g.,
continuous or intermittent) affects the individuals body burden and consequently the actual dose during
the critical period. The probability of misclassification of exposure status may affect the ability to

60
recognize a true effect in a study (Selevan, 1981; Hogue, 1984; Lemasters and Selevan, 1984; Sever
and Hessol, 1984; Kimmel, C.A. et al., 1986). As more prospective studies are done, better estimates
of exposure should be developed.

3.3.1.5. General Design Considerations

The factors that enhance a study and thus increase its usefulness for risk assessment have been
noted in a number of publications (Selevan, 1980; Bloom, 1981; Hatch and Kline, 1981; Wilcox,
1983; Sever and Hessol, 1984; Axelson, 1985; Tilley et al., 1985; Kimmel, C.A. et al., 1986; Savitz
and Harlow, 1991). Some of the more prominent factors are discussed below.

3.3.1.5.1. The power of the study. The power, or ability of a study to detect a true effect, is
dependent on the size of the study group, the frequency of the outcome in the general population, and
the level of excess risk to be identified. In a cohort study, common outcomes, such as recognized fetal
loss, require hundreds of pregnancies to have a high probability of detecting a modest increase in risk
(e.g., 133 pregnancies in both exposed and unexposed groups to detect a twofold increase; alpha = 0.05,
power = 80%), while less common outcomes, such as the total of all malformations recognized at birth,
require thousands of pregnancies to have the same probability (e.g., more than 1,200 pregnancies in
both exposed and unexposed groups) (Bloom, 1981; Selevan, 1981, 1985; Sever and Hessol, 1984;
Stein, Z. et al., 1985; Kimmel, C.A. et al., 1986). Semen evaluation may require fewer subjects
depending on the sperm parameters evaluated, especially when each man is used as his own control
(Wyrobek, 1982, 1984). In case-referent studies, study sizes are dependent upon the frequency of
exposure within the source population. The confidence one has in the results of a study showing no
effect is related directly to the power of the study to detect meaningful differences in the endpoints.
Power may be enhanced by combining populations from several studies using a meta-analysis
(Greenland, 1987). The combined analysis could increase confidence in the absence of risk for agents
showing no effect. However, caution must be exercised in the combination of potentially dissimilar
study groups.
Results of a negative study should be carefully evaluated, examining the power of the study and
the degree of concordance or discordance between that study and other studies (including careful
examination of comparability in the details such as similarity of adverse endpoints and study design).
The consistency among results of different studies could be evaluated by comparing statistical
confidence intervals for the effects found in different studies. Studies with lower power will tend to yield

61
wider confidence intervals. If the confidence intervals from a negative study and a positive study
overlap, then there may be no conflict between the results of the two studies.

3.3.1.5.2. Potential bias in data collection. Bias may result from the way the study group is
selected or information is collected (Rothman, 1986). Selection bias may occur when an individuals
willingness to participate varies with certain characteristics relating to exposure or health status. In
addition, selection bias may operate in the identification of subjects for study. For example, in studies
of very early pregnancy loss, use of hospital records to identify the study group will under-ascertain
events, because women are not always hospitalized for these outcomes. More weight would be given
in a risk assessment to a study in which a more complete list of pregnancies is obtained by, for example,
collecting biologic data (e.g., human chorionic gonadotropin [hCG] measurements) of pregnancy status
from study members. The representativeness of these data may be affected by selection factors related
to the willingness of different groups of women to continue participation over the total length of the
study. Interview data result in more complete ascertainment than hospital records; however this
strategy carries with it the potential for recall bias, discussed in further detail below. Other examples of
different levels of ascertainment of events include: (1) use of hospital records to study congenital
malformations since hospital records contain more complete data on malformations than do birth
certificates (Mackeprang et al., 1972; Snell et al., 1992) and (2) use of sperm bank or fertility clinic
data for semen studies. Semen data from either source are selected data because semen donors are
typically of proven fertility, and men in fertility clinics are part of a subfertile couple who are actively
trying to conceive. Thus, studies using the different record sources to identify reproductive outcomes
need to be evaluated for ascertainment patterns prior to use in risk assessment.
Studies of women who work outside the home present the potential for additional bias because
some factors that influence employment status may also affect reproductive endpoints. For example,
because of child-care responsibilities, women may terminate employment, as might women with a
history of reproductive problems who wish to have children and are concerned about workplace
exposures (Joffe, 1985; Lemasters and Pinney, 1989). Thus, retrospective studies of female exposure
that do not include terminated women workers may be of limited use in risk assessment because the
level of risk for these outcomes is likely to be overestimated (Lemasters and Pinney, 1989).
Information bias may result from misclassification of characteristics of individuals or events
identified for study. Recall bias, one type of information bias, may occur when respondents with
specific exposures or outcomes recall information differently than those without the exposures or
outcomes. Interview bias may result when the interviewer knows a priori the category of exposure

62
(for cohort studies) or outcome (for case-referent studies) in which the respondent belongs. Use of
highly structured questionnaires and/or blinding of the interviewer reduces the likelihood of such bias.
Studies with lower likelihood of such bias should carry more weight in a risk assessment.
When data are collected by interview or questionnaire, the appropriate respondent depends on
the type of data or study. For example, a comparison of husband-wife interviews on reproduction
found the wives responses to questions on pregnancy-related events to be more complete and valid
than those of the husbands, and the individuals self-report of his/her occupational exposures and health
characteristics more reliable than his/her mates report (Selevan, 1980; Selevan et al., 1982). Studies
based on interview data from the appropriate respondents would carry more weight than those from
proxy respondents.
Data from any source may be prone to errors or bias. All types of bias are difficult to assess;
however, validation with an independent data source (e.g., vital or hospital records), or use of
biomarkers of exposure or outcome, where possible, may suggest the degree of bias present and
increase confidence in the results of the study. Those studies with a low probability of biased data
should carry more weight (Axelson, 1985; Stein, A. and Hatch, 1987; Weinberg et al., 1994).
Differential misclassification (i.e., when certain subgroups are more likely to have misclassified
data than others) may either raise or lower the risk estimate. Nondifferential misclassification will bias
the results toward a finding of no effect (Rothman, 1986).

3.3.1.5.3. Collection of data on other risk factors, effect modifiers, and confounders. Risk
factors for reproductive toxicity include such characteristics as age, smoking, alcohol or caffeine
consumption, drug use, and past reproductive history. Groups of individuals may represent susceptible
subpopulations based on genetic, acquired (e.g., behavioral), or developmental characteristics (e.g.,
greater effect of childhood exposures). Known and potential risk factors should be examined to
identify those that may be confounders or effect modifiers. An effect modifier is a factor that produces
different exposure-response relationships at different levels of that factor. For example, age would be
an effect modifier if the risk associated with a given exposure changed with age (e.g., if older men had
semen changes with exposure while younger ones did not). A confounder is a variable that is a risk
factor for the outcome under study and is associated with the exposure under study, but is not a
consequence of the exposure. A confounder may distort both the magnitude and direction of the
measure of association between the exposure of interest and the outcome. For example, smoking might
be a confounder in a study of the association of socioeconomic status and fertility because smoking may
be associated with both.

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 Both effect modifiers and confounders need to be controlled in the study design and/or analysis
to improve the estimate of the effects of exposure (Kleinbaum et al., 1982). A more in-depth
discussion may be found elsewhere (Epidemiology Workgroup for the Interagency Regulatory Liaison
Group, 1981; Kleinbaum et al., 1982; Rothman, 1986). The statistical techniques used to control for
these factors require careful consideration in their application and interpretation (Kleinbaum et al.,
1982; Rothman, 1986). Studies that fail to account for these important factors should be given less
weight in a risk assessment.

3.3.1.5.4. Statistical factors. As in studies of test animals, pregnancies experienced by the same
woman are not fully independent events. For example, women who have had fetal loss are reported to
be more likely to have subsequent losses (Leridon, 1977). In test animal studies, the litter can be used
as the unit of measure to deal with nonindependence of response within the litter. In studies of humans,
pregnancies are sequential, requiring analyses which consider nonindependence of events
(Epidemiology Workgroup for the Interagency Regulatory Liaison Group, 1981; Kissling, 1981;
Selevan, 1981; Zeger and Liang, 1986). If more than one pregnancy per woman is included, as is
often necessary with small study groups, the use of nonindependent observations overestimates the true
size of the groups being compared, thus artificially increasing the probability of reaching statistical
significance (Stiratelli et al., 1984). Analysis problems may occur when (1) prior adverse outcomes are
due to the same exposures or (2) when prior adverse outcomes could result in changes in behaviors
that could reduce exposures. Some approaches to deal with these issues have been suggested
(Kissling, 1981; Stiratelli et al., 1984; Selevan, 1985; Zeger and Liang, 1986). These approaches
include selecting one pregnancy per family (Selevan, 1985) or using generalized estimating equations
(Zeger and Liang, 1986).

3.3.2. Examination of Clusters, Case Reports, or Series

The identification of cases or clusters of adverse reproductive effects is generally limited to
those identified by the individuals involved or clinically by their physicians. The likelihood of
identification varies with the gender of the exposed person. Identification of subfecundity in either
gender is difficult. This might be thought of as identification of a nonevent (e.g., lack of pregnancies or
children), and thus is much harder to recognize than are some developmental effects, including
malformations, resulting from in utero exposure.
The identification of cases or clusters of adverse male reproductive outcomes may be limited
because of cultural norms that may inhibit the reporting of impaired fecundity in men. Identification is

64
also limited by the decreased likelihood of recognizing adverse developmental effects in their offspring
as resulting from paternal exposure rather than maternal exposure. Thus far, only one agent causing
human male reproductive toxicity, dibromochloropropane (DBCP), has been identified after
observation of a cluster of infertility that resulted from male subfecundity. This cluster was identified
because of an atypically high level of communication among the workers wives (Whorton et al., 1977,
1979; Biava et al., 1978; Whorton and Milby, 1980).
Adverse effects identified in females through clusters and case reports have, thus far, been
limited to adverse pregnancy outcomes such as fetal loss and congenital malformations. Identification of
other effects, such as subfertility/subfecundity or menstrual cycle disorders, may be more difficult, as
noted above.
Case reports may have importance in the recognition of agents that cause reproductive toxicity.
However, they are probably of greatest use in suggesting topics for further investigation. Reports of
clusters and case reports/series are best used in risk assessment in conjunction with strong laboratory
data to suggest that effects observed in test animals also occur in humans.

3.4. PHARMACOKINETIC CONSIDERATIONS

Extrapolation of toxicity data between species can be aided considerably by the availability of
data on the pharmacokinetics of a particular agent in the species tested and, when available, in humans.
Information on absorption, half-life, steady-state or peak plasma concentrations, placental metabolism
and transfer, comparative metabolism, and concentrations of the parent compound and metabolites in
target organs may be useful in predicting risk for reproductive toxicity. Information on the variability
between humans and test species also may be useful in evaluating factors such as age-related
differences in the balance between activation and deactivation of a toxic agent. These types of data
may be helpful in defining the sequence of events leading to an adverse effect and the dose-response
curve, developing a more accurate comparison of species sensitivity, including that of humans (Wilson
et al., 1975, 1977), determining dosimetry at target sites, and comparing pharmacokinetic profiles for
various dosing regimens or routes of exposure. EPAs Office of Prevention, Pesticides, and Toxic
Substances has published protocols for metabolism studies that may be adapted to provide information
useful in reproductive toxicity risk assessment for a suspect agent. Pharmacokinetic studies in
reproductive toxicology are most useful if the data are obtained with animals that are at the same
reproductive status and stage of life (e.g., pregnant, nonpregnant, embryo or fetus, neonate,
prepubertal, adult) at which reproductive insults are expected to occur in humans.

65
 Specific guidance regarding both the development and application of pharmacokinetic data was
agreed on by the participants of the Workshop on Dermal Developmental Toxicity Studies (Kimmel,
C.A. and Francis, 1990). This guidance is also applicable to nondermal reproductive toxicity studies.
Participants of the Workshop concluded that absorption data are needed both when a dermal study
does or does not show effects. The results of a dermal study showing no effects and without blood
level data are potentially misleading and are inadequate for risk assessment, especially if interpreted as a
negative study. In studies where adverse effects are detected, regardless of the route of exposure,
pharmacokinetic data can be used to establish the internal dose in maternal and paternal animals for risk
extrapolation purposes.
The existence of a Sertoli cell barrier (formerly called the blood-testis barrier) in the
seminiferous tubules may influence the pharmacokinetics of an agent with potential to cause testicular
toxicity by restricting access of compounds to the adluminal compartment of seminiferous tubules. The
Sertoli cell barrier is formed by tight junctions between Sertoli cells and divides the seminiferous
epithelium into basal and adluminal compartments (Russell et al., 1990). The basal compartment
contains the spermatogonia and primary spermatocytes to the preleptotene stage, whereas more
advanced germ cells are located on the adluminal side. This selectively permeable barrier is most
effective in limiting the access of large, hydrophilic molecules in the intertubular lymph to cells on the
adluminal side. An analogous barrier in the ovary has not been found, although the zona pellucida and
granulosa cells may modulate access of chemicals to oocytes (Crisp, 1992).
The reproductive organs appear to have a wide range of metabolic capabilities directed at both
steroid and xenobiotic metabolism. However, there are substantial differences between compartments
within the organs in types and levels of enzyme activities (Mukhtar et al., 1978). Recognition of these
differences can be important in understanding the potential of agents to have specific toxic effects.
Most pharmacokinetic studies have incompletely characterized the distribution of toxic agents
and their subsequent metabolic fate within the reproductive organs. Generalizations based on hepatic
metabolism are not necessarily adequate to predict the fate of the agent in the testis, ovary, placenta, or
conceptus. For example, the metabolic profile for a given agent may differ in the male between the liver
and the testis and in the female between the maternal liver, ovary, and placenta. Detailed interspecies
comparisons of the metabolic capabilities of the testis, ovary, placenta, and conceptus also have not
been conducted. For some xenobiotics, significant differences in metabolism have been identified
between males and females (Harris, R.Z. et al., 1995). This is, in part, attributable to organizational
effects of the gonadal steroids in the developing liver (Gustafsson et al., 1980; Skett, 1988). Also, in
adults, the sex steroids have been shown to affect the activity of a number of enzymes involved in the

66
metabolism of administered compounds. Thus, the blood levels of a toxic agent, as well as the final
concentration in the target tissue, may differ significantly between sexes. If data are to be used
effectively in interspecies comparisons and extrapolations for these target systems, more attention
should be directed to the pharmacokinetic properties of chemicals in the reproductive organs and in
other organs that are affected by reproductive hormones.

3.5. COMPARISONS OF MOLECULAR STRUCTURE

Comparisons of the chemical or physical properties of an agent with those of agents known to
cause reproductive toxicity may provide some indication of a potential for reproductive toxicity. Such
information may be helpful in setting priorities for testing of agents or for evaluation of potential toxicity
when only minimal data are available. Structure-activity relationships (SAR) have not been well studied
in reproductive toxicology, and have had limited success in predicting reproductive toxicity. The early
literature has been reviewed and a set of classifications offered relating structure to reported male
reproductive system activity (Bernstein, 1984). Data are available that suggest structure-activity
relationships with limited utility in risk assessment for certain classes of chemicals (e.g., glycol ethers,
some estrogens, androgens, other steroids, substituted phenols, retinoids, phthalate esters, short-chain
halogenated hydrocarbon pesticides, alkyl-substituted polychlorinated dibenzofurans, PCBs,
vinylcyclohexene and related olefins, halogenated propanes, metals, and azo dyes). McKinney and
Waller (1994) have studied the qualitative SAR properties of PCBs with respect to their recognition by
thyroxine, Ah and estrogen receptors. Although generally limited in scope and in need of validation,
such relationships provide hypotheses that can be tested.
In spite of the limited information available on SAR in reproductive toxicology, under certain
circumstances (e.g., in the case of new chemicals), this procedure can be used to evaluate the potential
for toxicity when little or no other data are available.

3.6. EVALUATION OF DOSE-RESPONSE RELATIONSHIPS

The description and evaluation of dose-response relationships is a critical component of the
hazard characterization. Evidence for a dose-response relationship is an important criterion in
establishing a toxic reproductive effect. It includes the evaluation of data from both human and
laboratory animal studies. When possible, pharmacokinetic data should be used to determine the
effective dose at the target organ(s). When adequate dose-response data are available in humans and
with a sufficient range of exposure, dose-response relationships in humans may be examined. Because

67
quantitative data on human dose-response relationships are available infrequently, the dose-response
evaluation is usually based on the assessment of data from tests performed in laboratory animals.
The dose-response relationships for individual endpoints, as well as the combination of
endpoints, must be examined in data interpretation. Dose-response evaluations should consider the
effects that competing risks between different endpoints may have on outcomes observed at different
exposure levels. For example, an agent may interfere with cell function in such a manner that, at a low
dose level, an increase in abnormal sperm morphology is observed. At higher doses, cell death may
occur, leading to a decrease in sperm counts and a possible decrease in proportion of abnormal sperm.
When data on several species are available, the selection of the data for the dose-response
evaluation is based ideally on the response of the species most relevant to humans (e.g., comparable
physiologic, pharmacologic, pharmacokinetic, and pharmacodynamic processes), the adequacy of
dosing, the appropriateness of the route of administration, and the endpoints selected. However,
availability of information on many of those components is usually very limited. For dose-response
assessment, no single laboratory animal species can be considered the best in all situations for
predicting risk of reproductive toxicity to humans. However, in some cases, such as in the assessment
of physiologic parameters related to menstrual disorders, higher nonhuman primates are considered
generally similar to the human. In the absence of a clearly most relevant species, data from the most
sensitive species (i.e., the species showing a toxic effect at the lowest administered dose) are used,
because humans are assumed to be at least as sensitive generally as 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).
The evaluation of dose-response relationships includes the identification of effective dose levels
as well as doses that are associated with low or no increased incidence of adverse effects compared
with controls. Much of the focus is on the identification of the critical effect(s) (i.e., the adverse effect
occurring at the lowest dose level) and the LOAEL and NOAEL or benchmark dose associated with
the effect(s) (see Section 4).
Generally, in studies that do not evaluate reproductive toxicity, only adult male and nonpregnant
females are examined. Therefore, the possibility that pregnant females may be more sensitive to the
agent is not tested. In studies in which reproductive toxicity has been evaluated, the effective dose
range should be identified for both reproductive and other forms of systemic toxicity, and should be
compared with the corresponding values from other adult toxicity data to determine if the pregnant or
lactating female may be more sensitive to an agent.

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 In addition to identification of the range of doses that is effective in producing reproductive and
other forms of systemic toxicity for a given agent, the route of exposure, timing and duration of
exposure, species specificity of effects, and any pharmacokinetic or other considerations that might
influence the comparison with human exposure scenarios should be identified and evaluated. This
information should always accompany the characterization of the health-related database (discussed in
the next section).
Because the developing organism is changing rapidly and is vulnerable at a number of stages, an
assumption is made with developmental effects that a single exposure at a critical time in development
may produce an adverse effect (U.S. EPA, 1991). Therefore, with inhalation exposures, the daily dose
is usually not adjusted to a 24-hour equivalent duration with developmental toxicity unless appropriate
pharmacokinetic data are available. However, for other reproductive effects, daily doses by the
inhalation route may be adjusted for duration of exposure. The Agency is planning to review these
stances to determine the most appropriate approach for the future.

3.7. CHARACTERIZATION OF THE HEALTH-RELATED DATABASE

This section describes evaluation of the health-related database on a particular chemical and
provides criteria for judging the potential for that chemical to produce reproductive toxicity under the
exposure conditions inherent in the database. This determination provides the basis for judging whether
the available data are sufficient to characterize a hazard and to conduct quantitative dose-response
analyses. It also should provide a summary and evaluation of the existing data and identify data gaps
for an agent that is judged to have insufficient information to proceed with a quantitative dose-response
analysis. Characterizing the available evidence in this way clarifies the strengths and uncertainties in a
particular database. It does not address the level of concern, nor does it completely address
determining relevance of available data for estimating human risk. Issues concerning relevance of
mechanisms of action and types of effects observed should be included in the hazard characterization.
Both level of concern and relevance are discussed further as part of the final characterization of risk,
taking into account the information concerning potential human exposure. Data from all potentially
relevant studies, whether indicative of potential hazard or not, should be included in the hazard
characterization.
A complex interrelationship exists among study design, statistical analysis, and biologic
significance of the data. Thus, substantial scientific judgment, based on experience with reproductive
toxicity data and with the principles of study design and statistical analysis, may be required to evaluate
the database adequately. In some cases, a database may contain conflicting data. In these instances,

69
the risk assessor must consider each studys strengths and weaknesses within the context of the overall
database to characterize the evidence for assessing the potential hazard for reproductive toxicity.
Scientific judgment is always necessary and, in many cases, interaction with scientists in specific
disciplines (e.g., reproductive toxicology, epidemiology, genetic toxicology, statistics) is recommended.
A scheme for judging the available evidence on the reproductive toxicity of a particular agent is
presented below (Table 6). The scheme contains two broad categories, Sufficient and Insufficient,
which are defined in Table 6. Data from all available studies, whether or not indicative of potential
concern, are evaluated and used in the hazard characterization for reproductive toxicity. The primary
considerations are the human data, if available, and the experimental animal data. The judgment of
whether data are sufficient or insufficient should consider a variety of parameters that contribute to the
overall quality of the data, such as the power of the studies (e.g., sample size and variation in the data),
the number and types of endpoints examined, replication of effects, relevance of route and timing of
exposure for both human and experimental animal studies, and the appropriateness of the test species
and dose selection in experimental animal studies. In addition, pharmacokinetic data and structure-
activity considerations, data from other toxicity studies, as well as other factors that may affect the
overall decision about the evidence, should be taken into account.
In general, the characterization is based on criteria defined by these Guidelines as the minimum
evidence necessary to characterize a hazard and conduct dose-response analyses. Establishing the
minimum human evidence to proceed with quantitative analyses based on the human data is often
difficult because there may be considerable variations in study designs and study group selection. The
body of human data should contain convincing evidence as described in the Sufficient Human
Evidence category. Because the human data necessary to judge whether or not a causal relationship
exists are generally limited, few agents can be classified in this category. Agents that have been tested
in laboratory animals according to EPAs two-generation reproductive effects test guidelines (U.S.
EPA, 1982, 1985b, 1996a), but not limited to such designs (e.g., a continuous breeding study with two
generations), generally would be included in the Sufficient Experimental Animal Evidence/Limited
Human Data category. There are occasions in which more limited data regarding the potential
reproductive toxicity of an agent (e.g., a one-generation reproductive effects study, a standard
subchronic or chronic toxicity study in which the reproductive organs were well examined) are
available. If reproductive toxicity is observed in these limited studies, the data may be used to the
extent possible to reach a decision regarding hazard to the reproductive system, including determination
of an RfD or RfC. In cases in which such limited data are available, it would be appropriate to adjust
the uncertainty factor to reflect the attendant increased uncertainty regarding the use of these data until

70
more definitive data are developed. Identification of the increased uncertainty and justification for the
adjustment of the uncertainty factor should be stated clearly.

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Table 6. Categorization of the health-related database

SUFFICIENT EVIDENCE
The Sufficient Evidence category includes data that collectively provide enough information to
judge whether or not a reproductive hazard exists within the context of effect as well as dose, duration,
timing, and route of exposure. This category may include both human and experimental animal
evidence.

Sufficient Human Evidence

This category includes agents for which there is convincing evidence from epidemiologic studies
(e.g., case control and cohort) to judge whether exposure is causally related to reproductive toxicity. A
case series in conjunction with other supporting evidence also may be judged as Sufficient Evidence.
An evaluation of epidemiologic and clinical case studies should discuss whether the observed effects
can be considered biologically plausible in relation to chemical exposure.

Sufficient Experimental Animal Evidence/Limited Human Data

This category includes agents for which there is sufficient evidence from experimental animal
studies and/or limited human data to judge if a potential reproductive hazard exists. Generally, agents
that have been tested according to EPAs two-generation reproductive effects test guidelines (but not
limited to such designs) would be included in this category. The minimum evidence necessary to
determine if a potential hazard exists would be data demonstrating an adverse reproductive effect in a
single appropriate, well-executed study in a single test species. The minimum evidence needed to
determine that a potential hazard does not exist would include data on an adequate array of endpoints
from more than one study with two species that showed no adverse reproductive effects at doses that
were minimally toxic in terms of inducing an adverse effect. Information on pharmacokinetics,
mechanisms, or known properties of the chemical class may also strengthen the evidence.

INSUFFICIENT EVIDENCE
This category includes agents for which there is less than the minimum sufficient evidence
necessary for assessing the potential for reproductive toxicity. Included are situations such as when no
data are available on reproductive toxicity; as well as for data bases from studies on test animals or
humans that have a limited study design or conduct (e.g., small numbers of test animals or human
subjects, inappropriate dose selection or exposure information, other uncontrolled factors); data from
studies that examined only a limited number of endpoints and reported no adverse reproductive effects;
or data bases that were limited to information on structure-activity relationships, short-term or in vitro
tests, pharmacokinetic data, or metabolic precursors.

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 Because it is more difficult, both biologically and statistically, to support a finding of no apparent
hazard, more data are generally required to support this conclusion than a finding for a potential hazard.
For example, to judge whether a hazard for reproductive toxicity could exist for a given agent, the
minimum evidence could be data from a single appropriate, well-executed study in a single test species
that demonstrates an adverse reproductive effect, or suggestive evidence from adequately conducted
clinical or epidemiologic studies. As in all situations, it is important that the results be biologically
plausible and consistent. On the other hand, to judge whether an agent is unlikely to pose a hazard for
reproductive toxicity, the minimum evidence would include data on an array of endpoints and from
studies with more than one species that showed no reproductive effects at doses that were otherwise
minimally toxic to the adult animal. In addition, there may be human data from appropriate studies that
are supportive of no apparent hazard. In the event that a substantial database exists for a given
chemical, but no single study meets current test guidelines, the risk assessor should use scientific
judgment to determine whether the composite database may be viewed as meeting the Sufficient
criteria.
Some important considerations in determining the confidence in the health database are as
follows:
C Data of equivalent quality from human exposures are given more weight than data from
exposures of test species.
C Although a single study of high quality could be sufficient to achieve a relatively high level
of confidence, replication increases the confidence that may be placed in such results.
C Data are available from one or more in vivo studies of acceptable quality with humans or
other mammalian species that are believed to be predictive of human responses.
C Data exhibit a dose-response relationship.
C Results are statistically significant and biologically plausible.
C When multiple studies are available, the results are consistent.
C Sufficient information is available to reconcile discordant data.
C Route, level, duration, and frequency of exposure are appropriate.
C An adequate array of endpoints has been examined.
C The power and statistical treatment of the studies are appropriate.
Any statistically significant deviation from baseline levels for an in vivo effect warrants closer
examination. To determine whether such a deviation constitutes an adverse effect requires an
understanding of its role within a complex system and the determination of whether a true effect has

73
been observed. Application of the above criteria, combined with guidance presented in Section 3.2,
can facilitate such determinations.
The greatest confidence for identification of a reproductive hazard should be placed on
significant adverse effects on sexual behavior, fertility or development, or other endpoints that are
directly related to reproductive function such as menstrual (estrous) cycle normality, sperm evaluations,
reproductive histopathology, reproductive organ weights, and reproductive endocrinology. Agents
producing adverse effects on these endpoints can be assigned to the Sufficient Evidence category if
study quality is adequate.
Less confidence should be placed in results when only measures such as in vitro tests, data from
nonmammals, or structure-activity relationships are available, but positive results may trigger follow-up
studies that extend the preliminary data and determine the extent to which function might be affected.
Results from these types of studies alone, whether or not they demonstrate an effect, may be suggestive,
but should be assigned to the Insufficient Evidence category.
The absence of effects in test species on the endpoints that are evaluated routinely (i.e., fertility,
histopathology, prenatal development, and organ weights) may constitute sufficient evidence to place a
low priority on the potential reproductive toxicity of a chemical. However, in such cases, careful
consideration should be given to the sensitivity of these endpoints and to the quality of the data on these
endpoints. Consideration also should be given to the possibility of adverse effects that may not be
reflected in these routine measures (e.g., germ-cell mutation, alterations in estrous cyclicity or sperm
measures such as motility or morphology, functional effects from developmental exposures).
Judging that the health database indicates a potential reproductive hazard does not mean that
the agent will be a hazard at every exposure level (because of the assumption of a nonlinear dose-
response) or in every situation (e.g., the type and degree of hazard may vary significantly depending on
route and timing of exposure). In the final risk characterization, the summary of the hazard
characterization should always be presented with information on the quantitative dose-response analysis
and, if available, with the human exposure estimates.

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 4. QUANTITATIVE DOSE-RESPONSE ANALYSIS

In quantitative dose-response assessment, a nonlinear dose-response is assumed for noncancer

health effects unless mode of action or pharmacodynamic information indicate otherwise. If sufficient
data are available, a biologically based approach should be used on a chemical-specific basis to predict
the shape of the dose-response curve below the observable range. It is plausible that certain biologic
processes (e.g., Sertoli cell barrier selectivity, metabolic and repair capabilities of the germ cells) may
impede the attainment or maintenance of concentrations of the agent at the target site following
exposure to low-dose levels that would be associated with adverse effects. The assumption of a
nonlinear dose-response suggests that the application of adequate uncertainty factors to a NOAEL,
LOAEL, or benchmark dose will result in an exposure level for all humans that is not attended with
significant risk above background. With a linear dose-response, it is assumed that some risk exists at
any level of exposure, with risk decreasing as exposure decreases.
The NOAEL is the highest dose at which there is no significant increase in the frequency of an
adverse effect in any manifestation of reproductive toxicity compared with the appropriate control
group in a database having sufficient evidence for use in a risk assessment. The LOAEL is the lowest
dose at which there is a significant increase in the frequency of adverse reproductive effects compared
with the appropriate control group in a database having sufficient evidence. A significant increase may
be based on statistical significance or on a biologically significant trend. Evidence for biological
significance may be strengthened by mode of action or other biochemical evidence at lower exposure
levels that supports the causation of such an effect. The existence of a NOAEL in an experimental
animal study does not show the shape of the dose-response below the observable range; it only defines
the highest level of exposure under the conditions of the study that is not associated with a significant
increase in an adverse effect. Alternatively, mathematical modeling of the dose-response relationship
may be performed in the experimental range. This approach can be used to determine a benchmark
dose, which may be used in place of the NOAEL as a point of departure for calculating an RfD, RfC,
MOE, or other exposure estimates.
Several limitations in the use of the NOAEL have been described (Kimmel, C.A. and Gaylor,
1988; U.S. EPA, 1995b): (1) Use of the NOAEL focuses only on the dose that is the NOAEL and
does not incorporate information on the slope of the dose-response curve or the variability in the data;
(2) Because data variability is not taken into account (i.e., confidence limits are not used), the NOAEL
will likely be higher with decreasing sample size or poor study conduct, either of which are usually
associated with increasing variability in the data; (3) The NOAEL is limited to one of the experimental

75
doses; (4) The number and spacing of doses in a study can influence the dose that is chosen for the
NOAEL; and (5) Because the NOAEL is defined as a dose that does not produce an observable
change in adverse responses from control levels and is dependent on the power of the study,
theoretically the risk associated with it may fall anywhere between zero and an incidence just below that
detectable from control levels (usually in the range of 7% to 10% for quantal data). The 95% upper
confidence limit on developmental toxicity risk at the NOAEL has been estimated for several data sets
to be 2% to 6% (Crump, 1984; Gaylor, 1989); similar evaluations have not been conducted on data
for other reproductive effects. Because of the limitations associated with the use of the NOAEL, the
Agency is beginning to use the benchmark dose approach for quantitative dose-response evaluation
when sufficient data are available.
Calculation and use of the benchmark dose are described in the EPA document The Use of
the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995b). The Agency is
currently developing guidance for application of the benchmark dose, including a decision process to
use for the various steps in the analysis (U.S. EPA, 1996c). The benchmark dose is based on a model-
derived estimate of a particular incidence level, such as a 5% or 10% incidence. The BMD/C for a
given endpoint serves as a consistent point of departure for low-dose extrapolation. In some cases,
mode of action data may be sufficient to estimate a BMD/C at levels below the observable range for
the health effect of concern. A benchmark response (BMR) of 5% is usually the lowest level of risk
that can be estimated adequately for binomial endpoints from standard developmental toxicity studies
(Allen et al., 1994a,b). For fetal weight, a continuous endpoint, a 5% change from the control mean
was near the limit of detection for standard prenatal toxicity studies (Kavlock et al., 1995). The
modeling approaches that have been proposed for developmental toxicity (U.S. EPA, 1995b) are, for
the most part, curve-fitting models that have biological plausibility, but do not incorporate mode of
action. Similar approaches can be applied to other reproductive toxicity data to derive dose-response
curves for data in the observed dose range, but may or may not accurately predict risk at low levels of
exposure. Further guidance on the use of the BMD/C is being developed by the Agency (U.S. EPA,
1996c).
The RfD or RfC for reproductive toxicity is an estimate of a daily exposure to the human
population that is assumed to be without appreciable risk of deleterious reproductive effects over a
lifetime of exposure. The RfD or RfC is derived by applying uncertainty factors to the NOAEL, or the
LOAEL if a NOAEL is not available, or to the benchmark dose. Because of the short duration of most
studies of developmental toxicity, a unique value (RfDDT or RfC DT ) is determined for adverse
developmental effects. For adverse reproductive effects on endpoints other than those of

76
developmental toxicity, no special designator is attached. Data on reproductive toxicity (including
developmental toxicity) are considered along with other data on a particular chemical in deriving an RfD
or RfC.
The effect used for determining the NOAEL, LOAEL, or benchmark dose in deriving the RfD
or RfC is the most sensitive adverse reproductive endpoint (i.e., the critical effect) from the most
appropriate or, in the absence of such information, the most sensitive mammalian species (see Sections
2 and 3.2.1). Uncertainty factors for reproductive and other forms of systemic toxicity applied to the
NOAEL or benchmark dose generally include factors of 3 or 10 each for interspecies variation and for
intraspecies variation. Additional factors may be applied to account for other uncertainties that may
exist in the database. In circumstances where only a LOAEL is available, the use of an additional
uncertainty factor of up to 10 may be required, depending on the sensitivity of the endpoints evaluated,
adequacy of dose levels tested, or general confidence in the LOAEL.
Other areas of uncertainty may be identified and modifying factors used depending on the
characterization of the database (e.g., if the only data available are from a one-generation reproductive
effects study; see Section 3.7), data on pharmacokinetics, or other considerations that may alter the
level of confidence in the data (U.S. EPA, 1987). The total size of the uncertainty factor will vary from
agent to agent and requires scientific judgment, taking into account interspecies differences, variability
within species, the slope of the dose-response curve, the types of reproductive effects observed, the
background incidence of the effects, the route of administration, and pharmacokinetic data.
The NOAEL, LOAEL, or the benchmark dose is divided by the total uncertainty factor
selected for the critical effect in the most appropriate or most sensitive mammalian species to determine
the RfD or RfC. If the NOAEL, LOAEL, or benchmark dose for other forms of systemic toxicity is
lower than that for reproductive toxicity, this should be noted in the risk characterization, and this value
should be compared with data from other studies in which adult animals are exposed. Thus,
reproductive toxicity data should be discussed in the context of other toxicity data.
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.

77
 Efforts are underway to develop models that are more biologically based. These models should
provide a more accurate estimation of low-dose risk to humans. The development of biologically
based dose-response models in reproductive toxicology has been impeded by a number of factors,
including limited understanding of the biologic mechanisms underlying reproductive toxicity, intra- and
interspecies differences in the types of reproductive events, lack of appropriate pharmacokinetic data,
and inadequate information on the influence of other types of systemic toxicity on the dose-response
curve. Current research on modes of action in reproductive toxicology is promising and may provide
data that are useful for appropriate modeling in the near future.

4.1. UTILIZATION OF INFORMATION IN RISK CHARACTERIZATION

The hazard characterization and quantitative dose-response evaluations are incorporated into
the final characterization of risk along with information on estimates of human exposure. The analysis
depends on and should describe scientific judgments as to the accuracy and sufficiency of the health-
related data in experimental animals and humans (if available), the biologic relevance of significant
effects, and other considerations important in the interpretation and application of data to humans.
Scientific judgment is always necessary, and in many cases, interaction with scientists in specific
disciplines (e.g., reproductive toxicology, epidemiology, statistics) is recommended.

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 5. EXPOSURE ASSESSMENT

To obtain a quantitative estimate of risk for the human population, an estimate of human
exposure is required. The Guidelines for Exposure Assessment (U.S. EPA, 1992) have been
published separately and will not be discussed in detail here. Rather, issues important to reproductive
toxicity risk assessment are addressed. In general, the exposure assessment describes the magnitude,
duration, schedule, and route of human exposure. Ideally, existing body burden as well as internal
circulating and target organ exposure information for the agent of concern and other synergistic or
antagonistic agents should be described. It should include information on the purpose, scope, level of
detail and approach used, including estimates of exposure and dose by pathway and route for
populations, subpopulations, and individuals in a manner that is appropriate for the intended risk
characterization. It also should provide an evaluation of the overall level of confidence in the estimate(s)
of exposure and dose and the conclusions drawn. This information is usually developed from
monitoring data, from estimates based on modeling of environmental exposures, and from application of
paradigms to exposure data bases. Often quantitative estimates of exposures may not be available
(e.g., workplace or environmental measurements). In such instances, employment or residential
histories also may be used in characterizing exposure in a qualitative sense. The potential use of
biomarkers as indicators of exposure is an area of active interest.
Studies of occupational populations may provide valuable information on the potential
environmental health risks for certain agents. Exposures among environmentally exposed human
populations tend to be lower (but of longer duration) than those in studies of occupationally exposed
populations and therefore may require more observations to assure sufficient statistical power. Also,
reconstruction of exposures is more difficult in an environmental study than in those done in workplace
settings where industrial hygiene monitoring may provide more detailed exposure data.
The nature of the exposure may be defined at a particular point in time or may reflect
cumulative exposure. Each approach makes an assumption about the underlying relationship between
exposure and outcome. For example, a cumulative exposure measure assumes that total exposure is
important, with a greater probability of effect with greater total exposure or body burden. A
dichotomous exposure measure (ever exposed versus never exposed) assumes an irreversible effect of
exposure. Models that define exposure only at a specific time may assume that only the present
exposure is important (Selevan and Lemasters, 1987). The appropriate exposure model depends on
the biologic processes affected and the nature of the chemical under study. Thus, a cumulative or
dichotomous exposure model may be appropriate if injury occurs in cells that cannot be replaced or

79
repaired (e.g., oocytes); on the other hand, a concurrent exposure model may be appropriate for cells
that are being generated continually (e.g., spermatids).
There are a number of unique considerations regarding the exposure assessment for
reproductive toxicity. Exposure at different stages of male and female development can result in
different outcomes. Such age-dependent variation has been well documented in both experimental
animal and human studies. Prenatal and neonatal treatment can irreversibly alter reproductive function
and other aspects of development in a manner or to an extent that may not be predicted from adult-only
exposure. Moreover, chemicals that alter sexual differentiation in rodents during these periods may
have similar effects in humans, because the mechanisms underlying these developmental processes
appear to be similar in all mammalian species (Gray, 1991).
The susceptibility of elderly males and females to chemical insult has not been well studied.
Although procreative competence may not be a major health concern with elderly individuals, other
biologic functions maintained by the gonads (e.g., hormone production) are of significance (Walker,
1986). An exposure assessment should characterize the likelihood of exposure of these different
subgroups (embryo or fetus, neonate, juvenile, young adult, older adult) and the risk assessment should
factor in the susceptibility of different age groups to the extent possible.
The relationship between time or duration of exposure and observation of male reproductive
effects has particular significance for short-term exposures. Spermatogenesis is a temporally
synchronized process. In humans, germ cells that were spermatozoa, spermatids, spermatocytes, or
spermatogonia at the time of an acute exposure require 1 to 2, 3 to 5, 5 to 8, or 8 to 12 weeks,
respectively, to appear in an ejaculate. That timing may vary somewhat depending on degree of sexual
activity. It is possible that an endpoint may be examined too early or too late to detect an effect if only
a particular cell type was affected during a relatively brief exposure to an agent. The absence of an
effect when observations were made too late suggests either a reversible effect or no effect. However,
an effect that is reversible at lower exposures might become irreversible with higher or longer exposures
or exposure of a more susceptible individual. Thus, the failure to detect transient effects because of
improper timing of observations may be important. If information is available on the type of effect
expected from a class of agents, it may be possible to evaluate whether the timing of endpoint
measurement relative to the timing of the short-term exposure is appropriate. Some information on the
appropriateness of the protocol can be obtained if test animal data are available to identify the most
sensitive cell type or the putative mechanism of action for a given agent.
Compared with acute exposures, the link between exposure and outcome may be more
apparent with relatively constant subchronic or longer exposures that are of sufficient duration to cover

80
all phases of spermatogenesis (Russell et al., 1990). Assessments may be made at any time after this
point as long as exposure remains constant. Time required for the agent or metabolite to attain steady-
state levels should also be considered. Again, application of models of exposure (e.g., dichotomous,
concurrent, or cumulative) depends on the suspected target and chemical mechanism of action.
The reversibility of an adverse effect on the reproductive system can be affected by the degree
and duration of exposure (Clegg, 1995). The degree of stem cell loss is inversely related to the degree
of restoration of sperm production, because repopulation of the germinal epithelium is dependent on the
stem cells (Meistrich, 1982; Foote and Berndtson, 1992). For agents that bioaccumulate, increasing
duration of exposure may also increase the extent of damage to the stem cell population. Damage to
other spermatogenic cell types reduces the number of sperm produced, but recovery should occur
when the toxic agent is removed. Less is known about the effects of toxicity on the Sertoli cells.
Temporary impairment of Sertoli cell function may produce long-lasting effects on spermatogenesis.
Destruction of Sertoli cells or interference with their proliferation before puberty are irreversible effects
because replication ceases after puberty. Sertoli cells are essential for support of the spermatogenic
process and loss of those cells results in a permanent reduction of spermatogenic capability (Foster,
1992).
When recovery is possible, the duration of the recovery period is determined by the time for
regeneration (for stem cells) and repopulation of the affected spermatogenic cell types and appearance
of those cells as sperm in the ejaculate. The time required for these events to occur varies with the
species, the pharmacokinetic properties of the agent, the extent to which the stem cell population has
been destroyed, and the degree of sublethal toxicity inflicted on the stem cells or Sertoli cells. When
the stem cell population has been partially destroyed, humans require more time than mice to reach the
same degree of recovery (Meistrich and Samuels, 1985).
Unique considerations in the assessment of female reproductive toxicity include the duration and
period of exposure as related to the development or stage of reproductive life (e.g., prenatal,
prepubescent, reproductive, or postmenopausal) or considerations of different physiologic states (e.g.,
nonpregnant, pregnant, lactating). For infertility, a cumulative exposure measure assumes destruction of
increasing numbers of primary oocytes with greater lifetime exposure or increasing body burden.
However, humans may be exposed to varying levels of an agent within the study period. Exposures
during certain critical points in the reproductive process may affect the outcomes observed in humans
(Lemasters and Selevan, 1984). In test species, perinatal exposure to androgens or estrogens such as
zearalenone, methoxychlor, and DDT (Bulger and Kupfer, 1985; Gray et al., 1985) have been shown
to advance puberty and masculinize females. Similar effects have been reported in humans (both sexes)

81
exposed neonatally to synthetic estrogens or progestins (Steinberger and Lloyd, 1985; Schardein,
1993). Studies using test species also have shown that exposure to some environmental agents such as
ionizing radiation (Dobson and Felton, 1983) and glycol ethers (Heindel et al., 1989) can deplete the
pool of primordial follicles and thus significantly shorten the females reproductive lifespan.
Furthermore, exposure to compounds at different stages of the ovarian cycle can disrupt or delay
follicular recruitment and development (Armstrong, 1986), ovulation (Everett and Sawyer, 1950;
Terranova, 1980), and ovum transport (Cummings and Perreault, 1990). Compounds that delay
ovulation can lead to significant alterations in egg viability (Peluso et al., 1979), fertilizability of the egg
(Fugo and Butcher, 1966; Butcher and Fugo, 1967; Butcher et al., 1975), and a reduction in litter size
(Fugo and Butcher, 1966). After ovulation, single exposures to microtubule poisons such as
carbendazim may impair the completion of meiosis in the fertilized oocyte with adverse developmental
consequences (Perreault et al., 1992; Zuelke and Perreault, 1995). Thus, knowledge of when acute
exposures occur relative to the females lifespan and reproductive cycle can provide insight into how an
agent disrupts reproductive function.
DES is a classic example of an agent causing different effects on the reproductive system in the
developing organism compared with those in adults (McLachlan, 1980). DES, as well as other agents
with estrogenic or anti-androgenic activity, interferes with the development of the Mullerian and
Wolffian duct systems and thereby causes irreversible structural and functional damage to the
developing reproductive system. In adults, the reproductive effects that are caused by the estrogenic
activity of DES do not necessarily result in permanent damage.
Unique considerations for developmental effects are duration and period of exposure as related
to stage of development (i.e., critical periods) and the possibility that even a single exposure may be
sufficient to produce adverse developmental effects. Repeated exposure is not a necessary prerequisite
for developmental toxicity to be manifested, although it should be considered in cases where there is
evidence of cumulative exposure or where the half-life of the agent is long enough to produce an
increasing body burden over time. For these reasons, it is assumed that, in most cases, a single
exposure at the critical time in development is sufficient to produce an adverse developmental effect.
Therefore, the human exposure estimates used to calculate the MOE for an adverse developmental
effect or to compare to the RfD or RfC are usually based on a single daily dose that is not adjusted for
duration or pattern (e.g., continuous or intermittent) of exposure. For example, it would be
inappropriate to use time-weighted averages or adjustment of exposure over a different time frame than
that actually encountered (such as the adjustment of a 6-hour inhalation exposure to account for a 24-
hour exposure scenario) unless pharmacokinetic data were available to indicate an accumulation with

82
continuous exposure. In the case of intermittent exposures, examination of the peak exposures as well
as the average exposure over the time of exposure would be important.
It should be recognized that, based on the definitions used in these Guidelines, almost any
segment of the human population may be at risk for a reproductive effect. Although the reproductive
effects of exposures may be manifested while the exposure is occurring (e.g., menstrual disorder,
decreased sperm count, spontaneous abortion) some effects may not be detectable until later in life
(e.g., endocrine disruption of reproductive tract development, premature reproductive senescence due
to oocyte depletion), long after exposure has ceased.

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 6. RISK CHARACTERIZATION

6.1. OVERVIEW
A risk characterization is an essential part of any Agency report on risk whether the report is a
preliminary one prepared to support allocation of resources toward further study, a site-specific
assessment, or a comprehensive one prepared to support regulatory decisions. A risk characterization
should be prepared in a manner that is clear, reasonable, and consistent with other risk
characterizations of similar scope prepared across programs in the Agency. It should identify and
discuss all the major issues associated with determining the nature and extent of the risk and provide
commentary on any constraints limiting more complete exposition. The key aspects of risk
characterization are: (1) bridging risk assessment and risk management, (2) discussing confidence and
uncertainties, and (3) presenting several types of risk information. In this final step of a risk assessment,
the risk characterization involves integration of toxicity information from the hazard characterization and
quantitative dose-response analysis with the human exposure estimates and provides an evaluation of
the overall quality of the assessment, describes risk in terms of the nature and extent of harm, and
communicates results of the risk assessment to a risk manager. A risk manager can then use the risk
assessment, along with other risk management elements, to make public health decisions. The
information should also assist others outside the Agency in understanding the scientific basis for
regulatory decisions.
Risk characterization is intended to summarize key aspects of the following components of the
risk assessment:
C The nature, reliability, and consistency of the data used.
C The reasons for selection of the key study(ies) and the critical effect(s) and their relevance
to human outcomes.
C The qualitative and quantitative descriptors of the results of the risk assessment.
C The limitations of the available data, the assumptions used to bridge knowledge gaps in
working with those data, and implications of using alternative assumptions.
C The strengths and weaknesses of the risk assessment and the level of scientific confidence
in the assessment.
C The areas of uncertainty, additional data/research needs to improve confidence in the risk
assessment, and the potential impacts of the new research.

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 The risk characterization should be limited to the most significant and relevant data, conclusions,
and uncertainties. When special circumstances exist that preclude full assessment, those circumstances
should be explained and the related limitations identified.
The following sections describe these aspects of the risk characterization in more detail, but do
not attempt to provide a full discussion of risk characterization. Rather, these Guidelines point out
issues that are important to risk characterization for reproductive toxicity. Comprehensive general
guidance for risk characterization is provided by Habicht (1992) and Browner (1995).

6.2. INTEGRATION OF HAZARD CHARACTERIZATION, QUANTITATIVE DOSE-

RESPONSE, AND EXPOSURE ASSESSMENTS
In developing each component of the risk assessment, risk assessors must make judgments
concerning human relevance of the toxicity data, including the appropriateness of the various test animal
models for which data are available, and the route, timing, and duration of exposure relative to the
expected human exposure. These judgments should be summarized at each stage of the risk
assessment process. When data are not available to make such judgments, as is often the case, the
background information and assumptions discussed in the Overview (Section 1) provide default
positions. The default positions used and the rationale behind the use of each default position should be
clearly stated. In integrating the parts of the assessment, risk assessors must determine if some of these
judgments have implications for other portions of the assessment, and whether the various components
of the assessment are compatible.
The description of the relevant data should convey the major strengths and weaknesses of the
assessment that arise from availability and quality of data and the current limits of understanding of the
mechanisms of toxicity. Confidence in the results of a risk assessment is a function of confidence in the
results of these analyses. Each section (hazard characterization, quantitative dose-response analysis,
and exposure assessment) should have its own summary, and these summaries should be integrated into
the overall risk characterization. Interpretation of data should be explained, and risk managers should
be given a clear picture of consensus or lack of consensus that exists about significant aspects of the
assessment. When more than one interpretation is supported by the data, the alternative plausible
approaches should be presented along with the strengths, weaknesses, and impacts of those options. If
one interpretation or option has been selected over another, the rationale should be given; if not, then
both should be presented as plausible alternatives.

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 The risk characterization should not only examine the judgments, but also should explain the
constraints of available data and the state of knowledge about the phenomena studied in making them,
including:
C The qualitative conclusions about the likelihood that the chemical may pose a specific
hazard to human health, the nature of the observed effects, under what conditions (route,
dose levels, time, and duration) of exposure these effects occur, and whether the health-
related data are sufficient and relevant to use in a risk assessment.
C A discussion of the dose-response patterns for the critical effect(s) and their relationships
to the occurrence of other toxicity data, such as the shapes and slopes of the dose-
response curves for the various other endpoints; the rationale behind the determination of
the NOAEL, LOAEL, and/or benchmark dose; and the assumptions underlying the
estimation of the RfD, RfC, or other exposure estimate.
C Descriptions of the estimates of the range of human exposure (e.g., central tendency, high
end), the route, duration, and pattern of the exposure, relevant pharmacokinetics, and the
size and characteristics of the various populations that might be exposed.
C The risk characterization of an agent being assessed for reproductive toxicity should be
based on data from the most appropriate species or, if such information is not available, on
the most sensitive species tested. It also should be based on the most sensitive indicator of
an adverse reproductive effect, whether in the male, the female (nonpregnant or pregnant),
or the developing organism, and should be considered in relation to other forms of toxicity.
The relevance of this indicator to human reproductive outcomes should be described. The
rationale for those decisions should be presented.
If data to be used in a risk characterization are from a route of exposure other than the
expected human exposure, then pharmacokinetic data should be used, if available, to extrapolate
across routes of exposure. If such data are not available, the Agency makes certain assumptions
concerning the amount of absorption likely or the applicability of the data from one route to another
(U.S. EPA, 1985a, 1986b). Discussion of some of these issues may be found in the Proceedings of
the Workshop on Acceptability and Interpretation of Dermal Developmental Toxicity Studies
(Kimmel, C.A. and Francis, 1990) and Principles of Route-to-Route Extrapolation for Risk
Assessment (Gerrity et al., 1990). The risk characterization should identify the methods used to
extrapolate across exposure routes and discuss the strengths and limitations of the approach.
The level of confidence in the hazard characterization and quantitative dose-response evaluation
should be stated to the extent possible, including placement of the agent into the appropriate category

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regarding the sufficiency of the health-related data (see Section 3.7). A comprehensive risk assessment
ideally includes information on a variety of endpoints that provide insight into the full spectrum of
potential reproductive responses. A profile that integrates both human and test species data and
incorporates both sensitive endpoints (e.g., properly performed and fully evaluated histopathology) and
functional correlates (e.g., fertility) allows more confidence in a risk assessment for a given agent.
Descriptions of the nature of potential human exposures are important for prediction of specific
outcomes and the likelihood of persistence or reversibility of the effect in different exposure situations
with different subpopulations (U.S. EPA, 1992; Clegg, 1995).
In the risk assessment process, risk is estimated as a function of exposure, with the risk of
adverse effects increasing as exposure increases. Information on the levels of exposure experienced by
different members of the population is key to understanding the range of risks that may occur. Where
possible, several descriptors of exposure such as the nature and range of populations and their various
exposure conditions, central tendencies, and high-end exposure estimates should be presented.
Differences among individuals in absorption rates, metabolism, or other factors mean that individuals or
subpopulations with the same level and pattern of exposure may have differing susceptibility. For
example, the consequences of exposure can differ markedly between developing individuals, young
adults and aged adults, including whether the effects are permanent or transient. Other considerations
relative to human exposures might include pregnancy or lactation, potential for exposures to other
agents, concurrent disease, nutritional status, lifestyle, ethnic background and genetic polymorphism,
and the possible consequences. Knowledge of the molecular events leading to induction of adverse
effects may be of use in determining the range of susceptibility in sensitive populations.
An outline to serve as a guide and formatting aid for developing reproductive risk
characterizations for chemical-specific risk assessments can be found in Table 7. A common format
will assist risk managers in evaluating and using reproductive risk characterization. The outline has two
parts. The first part tracks the reproductive risk assessment to bring forward its major conclusions.
The second part pulls the information together to characterize the reproductive risk.

6.3. DESCRIPTORS OF REPRODUCTIVE RISK

Descriptors of reproductive risk convey information and answer questions about risk, with each
descriptor providing different information and insights. There are a number of ways to describe risk.
Details on how to use these descriptors can be obtained from the guidance on risk characterization
(Browner, 1995) from which some of the information below has been extracted.

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 In most cases, the state of the science is not yet adequate to define distributions of factors such
as population susceptibility. The guidance principles below discuss a variety of risk descriptors that
primarily reflect differences in estimated exposure. If a full description of the range of susceptibility in
the population cannot be presented, an effort should be made to identify subgroups that, for various
reasons, may be particularly susceptible.

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Table 7. Guide for developing chemical-specific risk characterizations for reproductive
effects

PART ONE
Summarizing Major Conclusions in Risk Characterization

I. Hazard Characterization
A. What is (are) the key toxicological study (or studies) that provides the basis for health
concerns for reproductive effects?
C How good is the key study?
C Are the data from laboratory or field studies? In a single or multiple species?
C What adverse reproductive endpoints were observed, and what is the basis for the
critical effect?
C Describe other studies that support this finding.
C Discuss any valid studies which conflict with this finding.

B. Besides the reproductive effect observed in the key study, are there other health endpoints
of concern? What are the significant data gaps?

C. Discuss available epidemiological or clinical data. For epidemiological studies:

C What types of data were used (e.g., human ecologic, case-control or cohort studies,
or case reports or series)?
C Describe the degree to which exposures were described.
C Describe the degree to which confounding factors were considered.
C Describe the degree to which other causal factors were excluded.

D. How much is known about how (through what biological mechanism) the chemical
produces adverse reproductive effects?
C Discuss relevant studies of mechanisms of action or metabolism.
C Does this information aid in the interpretation of the toxicity data?
C What are the implications for potential adverse reproductive effects?

E. Comment on any nonpositive data in animals or people, and whether these data were
considered in the hazard characterization.

F. If adverse health effects have been observed in wildlife species, characterize such effects
by discussing the relevant issues as in A through E above.

G. Summarize the hazard characterization and discuss the significance of each of the
following:
C Confidence in conclusions
C Alternative conclusions that are also supported by the data
C Significant data gaps

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C Highlights of major assumptions

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Table 7. Guide for developing chemical-specific risk characterizations for reproductive
effects (continued)

II. Characterization of Dose-Response

A. What data were used to develop the dose-response curve? Would the result have been
significantly different if based on a different data set?
C If laboratory animal data were used:
Which species were used?
Most sensitive, average of all species, or other?
Were any studies excluded? Why?
C If epidemiological data were used:
Which studies were used?
Only positive studies, all studies, or some other combination?
Were any studies excluded? Why?
Was a meta-analysis performed to combine the epidemiological studies? What
approach was used?
Were studies excluded? Why?

B. Was a model used to develop the dose-response curve and, if so, which one? What
rationale supports this choice? Is chemical-specific information available to support this
approach?
C How was the RfD/RfC (or the acceptable range) calculated?
C What assumptions and uncertainty factors were used?
C What is the confidence in the estimates?

C. Discuss the route, level, and duration of exposure observed, as compared to expected
human exposures.
C Are the available data from the same route of exposure as the expected human
exposures? If not, are pharmacokinetic data available to extrapolate across route of
exposure?
C How far does one need to extrapolate from the observed data to environmental
exposures? One to two orders of magnitude? Multiple orders of magnitude? What
is the impact of such an extrapolation?

D. If adverse health effects have been observed in wildlife species, characterize

dose-response information using the process outlined in A through C above.

III. Characterization of Exposure

A. What are the most significant sources of environmental exposure?
Are there data on sources of exposure from different media?
What is the relative contribution of different sources of exposure?
What are the most significant environmental pathways for exposure?

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92
Table 7. Guide for developing chemical-specific risk characterizations for reproductive
effects (continued)

B. Describe the populations that were assessed, including the general population, highly
exposed groups, and highly susceptible groups.

C. Describe the basis for the exposure assessment, including any monitoring, modeling, or
other analyses of exposure distributions such as Monte Carlo or krieging.

D. What are the key descriptors of exposure?

Describe the (range of) exposures to: average individuals, high-end individuals,
general population, high exposure group(s), children, susceptible populations, males,
females (nonpregnant, pregnant, lactating).
How was the central tendency estimate developed?
What factors and/or methods were used in developing this estimate?
How was the high-end estimate developed?
Is there information on highly exposed subgroups?
Who are they?
What are their levels of exposure?
How are they accounted for in the assessment?

E. Is there reason to be concerned about cumulative or multiple exposures because of

biological, ethnic, racial, or socioeconomic reasons?

F. If adverse reproductive effects have been observed in wildlife species, characterize wildlife
exposure by discussing the relevant issues as in A through E above.

G. Summarize exposure conclusions and discuss the following:

C Results of different approaches, i.e., modeling, monitoring, probability distributions;
C Limitations of each, and the range of most reasonable values;
C Confidence in the results obtained, and the limitations to the results

PART TWO
Risk Conclusions and Comparisons

IV. Risk Conclusions

A. What is the overall picture of risk, based on the hazard, quantitative dose-response, and
exposure characterizations?
B. What are the major conclusions and strengths of the assessment in each of the three main
analyses (i.e., hazard characterization, quantitative dose-response, and exposure
assessment)?

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Table 7. Guide for developing chemical-specific risk characterizations for reproductive
effects (continued)

C. What are the major limitations and uncertainties in the three main analyses?

D. What are the science policy options in each of the three major analyses?
What are the alternative approaches evaluated?
What are the reasons for the choices made?

V. Risk Context
A. What are the qualitative characteristics of the reproductive hazard (e.g., voluntary vs.
involuntary, technological vs. natural, etc.)? Comment on findings, if any, from studies of
risk perception that relate to this hazard or similar hazards.

B. What are the alternatives to this reproductive hazard? How do the risks compare?

C. How does this reproductive risk compare to other risks?

How does this risk compare to other risks in this regulatory program, or other similar risks
that the EPA has made decisions about?
Where appropriate, can this risk be compared with past Agency decisions, decisions by
other federal or state agencies, or common risks with which people may be familiar?
Describe the limitations of making these comparisons.

D. Comment on significant community concerns which influence public perception of risk.

VI. Existing Risk Information

Comment on other reproductive risk assessments that have been done on this chemical by
EPA, other federal agencies, or other organizations. Are there significantly different conclusions
that merit discussion?

VII. Other Information

Is there other information that would be useful to the risk manager or the public in this situation
that has not been described above?

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6.3.1. Distribution of Individual Exposures
Risk managers are interested generally in answers to questions such as: (1) Who are the
people at the highest risk and why? (2) What is the average risk or distribution of risks for individuals
in the population of interest? and (3) What are they doing, where do they live, etc., that might be putting
them at this higher risk?
Exposure and reproductive risk descriptors for individuals are intended to provide answers to
these questions. To describe the range of risks, both high-end and central tendency descriptors are
used to convey the distribution in risk levels experienced by different individuals in the population. For
the Agencys purposes, high-end risk descriptors are plausible estimates of the individual risk for those
persons at the upper end of the risk distribution. Given limitations in current understanding of variability
in individuals sensitivity to agents that cause reproductive toxicity, high-end descriptors will usually
address high-end exposure or dose. Conceptually, high-end exposure means exposure above
approximately the 90th percentile of the population distribution, but not higher than the individual in the
population who has the highest exposure. Central tendency descriptors generally reflect central
estimates of exposure or dose. The descriptor addressing central tendency may be based on either the
arithmetic mean exposure (average estimate) or the median exposure (median estimate), either of which
should be clearly labeled. The selection of which descriptor(s) to present in the risk characterization
will depend on the available data and the goals of the assessment.

6.3.2. Population Exposure

Population risk refers to assessment of the extent of harm for the population as a whole. In
theory, it can be calculated by summing the individual risks for all individuals within the subject
population. That task requires more information than is usually available. Questions addressed by
descriptors of population risk for reproductive effects would include: What portion of the population is
within a specified range of some reference level, e.g., exceeds the RfD (a dose), the RfC (a
concentration), or other health concern level?
For reproductive effects, risk assessment techniques have not been developed generally to the
point of knowing how to add risk probabilities, although Hattis and Silver (1994) have proposed
approaches for certain case-specific situations. Therefore, the following descriptor is usually
appropriate: An estimate of the percentage of the population, or the number of persons, above a
specified level of risk or within a specified range of some reference level (e.g., exceeds the RfD, RfC,
LOAEL, or other specific level of interest). The RfD or RfC is assumed to be a level below which no
significant risk occurs. Therefore, information from the exposure assessment on the populations below

95
the RfD or RfC (not likely to be at risk) and above the RfD or RfC (may be at risk) may be useful
information for risk managers. Estimating the number of persons potentially removed from the may be
at risk category after a contemplated action is taken may be particularly useful to a risk manager
considering possible actions to ameliorate risk for a population. This descriptor must be obtained
through measuring or simulating the population distribution.

6.3.3. Margin of Exposure

In the risk characterization, dose-response information and the human exposure estimates may
be combined either by comparing the RfD or RfC and the human exposure estimate or by calculating
the margin of exposure (MOE). The MOE is the ratio of the NOAEL or benchmark dose from the
most appropriate or sensitive species to the estimated human exposure level from all potential sources
(U.S. EPA, 1985a). If a NOAEL is not available, a LOAEL may be used in the calculation of the
MOE, but consideration for the acceptability would be different than when a NOAEL is used.
Considerations for the acceptability of the MOE are similar to those for the selection of uncertainty
factors applied to the NOAEL, LOAEL, or the benchmark dose for the derivation of an RfD. The
MOE is presented along with the characterization of the database, including the strengths and
weaknesses of the toxicity and exposure data, the number of species affected, and the information on
dose-response, route, timing, and duration. The RfD or RfC comparison with the human exposure
estimate and the calculation of the MOE are conceptually similar, but may be used in different
regulatory situations.
The choice of approach is dependent on several factors, including the statute involved, the
situation being addressed, the database used, and the needs of the decisionmaker. The RfD, RfC, or
MOE are considered along with other risk assessment and risk management issues in making risk
management decisions, but the scientific issues that should be taken into account in establishing them
have been addressed here.

6.3.4. Distribution of Exposure and Risk for Different Subgroups

A risk manager might also ask questions about the distribution of the risk burden among various
segments of the subject population such as the following: How do exposure and reproductive risk
impact various subgroups? and What is the population risk of a particular subgroup? Questions about
the distribution of exposure and reproductive risk among such population segments require additional
risk descriptors.

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6.3.4.1. Highly Exposed
The purpose of this measure is to describe the upper end of the exposure distribution, allowing
risk managers to evaluate whether certain individuals are at disproportionately high or unacceptably high
risk. The objective is to look at the upper end of the exposure distribution to derive a realistic estimate
of relatively highly exposed individual(s). The high end of the risk distribution has been defined
(Habicht, 1992; Browner, 1995) as above the 90th percentile of the actual (either measured or
estimated) distribution. Whenever possible, it is important to express the number or proportion of
individuals who comprise the selected highly exposed group and, if data are available, discuss the
potential for exposure at still higher levels.
Highly exposed subgroups can be identified and, where possible, characterized, and the
magnitude of risk quantified. This descriptor is useful when there is (or is expected to be) a subgroup
experiencing significantly different exposures or doses from those of the larger population. These
subpopulations may be identified by age, sex, lifestyle, economic factors, or other demographic
variables. For example, toddlers who play in contaminated soil and consumers of large amounts of fish
represent subpopulations that may have greater exposures to certain agents.
If population data are absent, it will often be possible to describe a scenario representing high-
end exposures using upper percentile or judgment-based values for exposure variables. In these
instances, caution should be taken not to overestimate the high-end values if a reasonable exposure
estimate is to be achieved.

6.3.4.2. Highly Susceptible

Highly susceptible subgroups also can be identified and, if possible, characterized, and the
magnitude of risk quantified. This descriptor is useful when the sensitivity or susceptibility to the effect
for specific subgroups is (or is expected to be) significantly different from that of the larger population.
Therefore, the purpose of this measure is to quantify exposure of identified sensitive or susceptible
populations to the agent of concern. Sensitive or susceptible individuals are those within the exposed
population at increased risk of expressing the adverse effect. Examples might be pregnant or lactating
women, women with reduced oocyte numbers, men with borderline sperm counts, or infants. To
calculate risk for these subgroups, it will be necessary sometimes to use a different dose-response
relationship; e.g., upon exposure to a chemical, pregnant or lactating women, elderly people, children of
varying ages, and people with certain illnesses may each be more sensitive than the population as a
whole.

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 In general, not enough is understood about the mechanisms of toxicity to identify sensitive
subgroups for most agents, although factors such as age, nutrition, personal habits (e.g., smoking,
consumption of alcohol, and abuse of drugs), existing disease (e.g., diabetes or sexually transmitted
diseases), or genetic polymorphisms may predispose some individuals to be more sensitive to the
reproductive effects of various agents.
It is important to consider, however, that the Agencys current methods for developing
reference doses and reference concentrations (RfDs and RfCs) are designed to protect sensitive
populations. If data on sensitive human populations are available (and there is confidence in the quality
of the data), then the RfD is based on the dose level at which no adverse effects are observed in the
sensitive population. If no such data are available (for example, if the RfD is developed using data from
humans of average or unknown sensitivity), then an additional 3- to 10-fold factor may be used to
account for variability between the average human response and the response of more sensitive
individuals (see Section 4).
Generally, selection of the population segments to consider for high susceptibility is a matter of
either a priori interest in the subgroup (e.g., environmental justice considerations), in which case the
risk assessor and risk manager can jointly agree on which subgroups to highlight, or a matter of
discovery of a sensitive or highly exposed subgroup during the assessment process. In either case,
once identified, the subgroup can be treated as a population in itself and characterized in the same way
as the larger population using the descriptors for population and individual risk.

6.3.5. Situation-Specific Information

Presenting situation-specific scenarios for important exposure situations and subpopulations in
the form of what if? questions may be particularly useful to give perspective to risk managers on
possible future events. The question being asked in these cases is, for any given exposure level, what
would be the resulting number or proportion of individuals who may be exposed to levels above that
value?
What if ...? questions, such as those that follow, can be used to examine candidate risk
management options:
C What are the reproductive risks if a pesticide applicator applies this pesticide without using
protective equipment?
C What are the reproductive risks if this site becomes residential in the future?
C What are the reproductive risks if we set the standard at 100 ppb?

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 Answering such what if? questions involves a calculation of risk based on specific
combinations of factors postulated within the assessment. The answers to these what if? questions do
not, by themselves, give information about how likely the combination of values might be in the actual
population or about how many (if any) persons might be subjected to the potential future reproductive
risk. However, information on the likelihood of the postulated scenario would be desirable to include in
the assessment.
When addressing projected changes for a population (either expected future developments or
consideration of different regulatory options), it usually is appropriate to calculate and consider all the
reproductive risk descriptors discussed above. When central tendency or high-end estimates are
developed for a scenario, these descriptors should reflect reasonable expectations about future
activities. For example, in site-specific risk assessments, future scenarios should be evaluated when
they are supported by realistic forecasts of future land use, and the reproductive risk descriptors should
be developed within that context.

6.3.6. Evaluation of the Uncertainty in the Risk Descriptors

Reproductive risk descriptors are intended to address variability of risk within the population
and the overall adverse impact on the population. In particular, differences between high-end and
central tendency estimates reflect variability in the population but not the scientific uncertainty inherent in
the risk estimates. As discussed above there will be uncertainty in all estimates of reproductive risk.
These uncertainties can include measurement uncertainties, modeling uncertainties, and assumptions to
fill data gaps. Risk assessors should address the impact of each of these factors on the confidence in
the estimated reproductive risk values.
Both qualitative and quantitative evaluations of uncertainty provide useful information to users of
the assessment. The techniques of quantitative uncertainty analysis are evolving rapidly and both the
SAB (Loehr and Matanoski, 1993) and the NRC (1994) have urged the Agency to incorporate these
techniques into its risk analyses. However, it should be noted that a probabilistic assessment that uses
only the assessors best estimates for distributions of population variables addresses variability, but not
uncertainty. Uncertainties in the estimated risk distribution need to be evaluated separately. An
approach has been proposed for estimating distribution of uncertainty in noncancer risk assessments
(Baird et al., 1996).

6.4. SUMMARY AND RESEARCH NEEDS

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 These Guidelines summarize the procedures that the EPA will follow in evaluating the potential
for agents to cause reproductive toxicity. They discuss the assumptions that must be made in risk
assessment for reproductive toxicity because of gaps in our knowledge about underlying biologic
processes and how these compare across species. Research to improve the interpretation of data and
interspecies extrapolation is needed. This research includes studies that: (1) more completely
characterize and define female and male reproductive endpoints, (2) more completely characterize the
types of developmental toxicity possible, (3) evaluate the interrelationships among endpoints, (4)
examine quantitative extrapolation between endpoints (e.g., sperm count) and function (e.g., fertility),
(5) provide a better understanding of the relationships between reproductive toxicity and other forms of
toxicity, (6) explore pharmacokinetic disposition of the target, and (7) examine mechanistic phenomena
related to pharmacokinetic disposition. These types of studies, along with further evaluation of a
nonlinear dose-response for susceptible populations, should provide methods to more precisely assess
risk.

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Barlow, S.M., Sullivan, F.M. (1982) Reproductive Hazards of Industrial Chemicals. Academic Press, London.

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Beach, F.A. (1979) Animal models for human sexuality. In: Ciba Foundation Symposium No. 62, Sex, Hormones and
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Berndtson, W.E. (1977) Methods for quantifying mammalian spermatogenesis: a review. J. Anim. Sci. 44:818-833.

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Zenick, H., Clegg, E.D., Perreault, S.D., Klinefelter, G.R., Gray, L.E. (1994) Assessment of male reproductive toxicity: a
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937-988.

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Zuelke, K.A., Perreault, S.D. (1995) Carbendazim (MBC) disrupts oocyte spindle function and induces aneuploidy in
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PART B: RESPONSE TO SCIENCE ADVISORY BOARD AND PUBLIC COMMENTS

1. INTRODUCTION

A notice of availability for public comment of these Guidelines was published in the Federal
Register (FR) in February 1994. Seven responses were received. These Guidelines were presented
to the Environmental Health Committee of the Science Advisory Board (SAB) on July 19, 1994. The
report of the SAB was provided to the Agency in May 1995, with further communication from the
SAB Executive Committee provided in December 1995.
The SAB and public comments were diverse and represented varying perspectives. Many of
the comments were favorable and expressed agreement with positions taken in the proposed guidelines.
A number of the comments addressed items that were more pertinent to testing guidance than risk
assessment guidance or were otherwise beyond the scope of these Guidelines. Some of those were
generic issues that are not system specific. Others were topics that have not been developed
sufficiently and should be viewed as research issues. There were conflicting views about the need to
provide additional detailed guidance about decision-making in the evaluation process as opposed to
promoting extensive use of scientific judgment. Also, comments provided specific suggestions for
clarification of details.

2. RESPONSE TO SCIENCE ADVISORY BOARD COMMENTS

In general, the SAB found the overall scientific foundations of the draft guidelines positions to
be generally sound. However, recommendations were made to improve specific areas.
The SAB recommended that EPA retain separate sections for identification and dose-response
assessment in the draft guidelines. In subsequent meetings involving the SAB Executive Committee,
members of the Clean Air Scientific Advisory Committee, and the Environmental Health Committee,
this issue was explored further. After discussion, the SAB agreed with expanding the hazard
identification to include certain components of the dose-response assessment. The resulting hazard
characterization provides an evaluation of hazard within the context of the dose, route, timing, and
duration of exposure. The next step, the dose-response analysis, quantitatively evaluates the
relationship between dose or exposure and severity or probability of effect in humans. EPA has revised
these Guidelines to reflect that position which is consistent also with the 1994 NRC report, Science
and Judgment in Risk Assessment. The SAB suggested an alternative scheme for characterizing

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health effects data in Table 5. The Agencys intent for Table 5 is not to characterize the available data,
but rather to judge whether the database is sufficient to proceed further in the risk assessment process.
The text has been modified to clarify the intended use of this table and to ensure that it is consistent with
the reorganization of the Guidelines into separate hazard characterization and quantitative dose-
response analysis sections.
The SAB supported the concept of using a gender neutral default assumption, but indicated that
more discussion to support this assumption was needed. In particular, the Committee indicated that a
fuller discussion is needed on information to the contrary (to obviate the need for making this default
assumption), as well as additional guidance for using this and other default assumptions in risk
characterization. The Agency agrees with this recommendation and provides further guidance on the
use of the gender neutral default assumption. In keeping with recent Agency guidance on risk
characterization, discussion on the use of default assumptions has been expanded in the risk
characterization section of these Guidelines.
The SAB in its reviews of the reproductive toxicity and neurotoxicity risk assessment guidelines
discussed assumptions about the behavior of the dose-response curve. The SABs advice has been
that the Agency examine available data first, and only use nonlinear behavior as a default if available
data do not define the dose-response curve. The SAB also recommended that the benchmark dose
method be considered as a possible alternative to the NOAEL/LOAEL approach. The Agency
agrees.
The SAB recommended that more discussion be devoted to the issue of disruption of endocrine
systems by environmental agents. The section on Endocrine Evaluations has been expanded to include
endocrine disruption of the reproductive system during development in addition to effects on adults.
The SAB supported the principle in the Guidelines that more than one negative study is
necessary to judge that a chemical is unlikely to pose a reproductive hazard. That principle has been
retained and, as recommended by the SAB, an explicit statement included that data from a second
species are necessary to determine that sufficient information is available to indicate that an agent is
unlikely to pose a hazard.
The SAB recommended that the topic of susceptible populations be expanded and that the
Guidelines should indicate that relevant information be incorporated into risk assessments when
possible. To address this issue, the Agency has emphasized potential differences in risks in children at
different stages of development, females (including pregnant and lactating females), and males, and
indicated that relevant information on differential risks for susceptible populations should be included in
the risk characterization section when available. When specific information on differential risks is not

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available, the Agency will continue to apply a default uncertainty factor to account for potential
differences in susceptibility.
The SAB recommended that the Agency provide more specific guidance for exposure
assessment issues that arise when characterizing exposure for reproductive toxicants. The Agency
agrees and has indicated that an exposure assessment: include a statement of purpose, scope, level of
detail, and approach used; present the estimate of exposure and dose by pathway and route for
individuals, population segments, and populations in a manner appropriate for the intended risk
characterization; and provide an evaluation of the overall level of confidence (including consideration of
uncertainty factors) in the estimate of exposure and dose and the conclusions drawn. The SAB
recommended that the MOE discussion be modified to address specific circumstances where the
administered dose and the effective dose are known to be different. The discussion has been
modified to emphasize that pharmacokinetic data, when available, be utilized to address such instances.
The SAB recommended that the Agency expand substantially the discussion of overall strategy
to evaluate exposure from mixtures, exposures to multiple single agents, and exposures to the same
agent via different routes. It is anticipated that this type of information will be addressed in the
Agencys upcoming revisions to the chemical mixture guidelines.

3. RESPONSE TO PUBLIC COMMENTS

In addition to numerous supportive statements, several issues were indicated although each
issue was raised by a very limited number of submissions. Use of the benchmark dose was supported
along with the suggestion that the amount of text could be reduced on that subject. The text has been
reduced and reference made to the report, The Use of the Benchmark Dose Approach in Health
Risk Assessment (U.S. EPA, 1995b). A request was made for increased emphasis on paternally
mediated effects on offspring. The text in that section has been expanded to provide additional
discussion and references. Concern was expressed about the existence of constraints on the use of
professional judgment in the risk assessment process, particularly in determining the relevance and
sufficiency of the database, in evaluating biological plausibility of statistically different effects, and in the
determination of uncertainty factors. Requests also have been made to provide additional criteria for
when and under what conditions the risk assessment process will be used. These Guidelines emphasize
the importance of using scientific judgment throughout the risk assessment process. They provide
flexibility to permit EPAs offices and regions to develop specific guidance suited to their particular
needs. The comment was made that the exposure assessment and risk characterization sections were

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not developed as well as the rest of the document. In 1992, EPA published Guidelines for Exposure
Assessment (U.S. EPA, 1992) that were intended to apply generically to noncancer risk assessments.
These Guidelines only address aspects of exposure that are specific to reproduction and have been
developed sufficiently. The risk characterization section has been expanded substantially to reflect the
recent guidance provided within EPA for application in all risk assessments.

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