United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/630/R-96/009
October 1996
Reproductive Toxicity
Risk Assessment
RISK ASSESSMENT FORUM
-------
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
Printed on Recycled Paper
-------
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.
11
-------
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
3.2.5.2. Effects on Puberty 46
3.2.5.3. Adverse Effects 47
111
-------
CONTENTS (continued)
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
Part B: Response to Science Advisory Board and Public Comments
1. Introduction 115
IV
-------
CONTENTS (continued)
2. Response to Science Advisory Board Comments 115
3. Response to Public Comments 117
-------
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
VI
-------
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 Agency's Risk
Assessment Forum and interagency comment by members of subcommittees of the Committee
on the Environment and Natural Resources of the Office of Science and Technology Policy. The
EPA appreciates the efforts of all participants in the process and has tried to address their
recommendations in these Guidelines.
EFFECTIVE DATE: The Guidelines will be effective October 31,1996.
vn
-------
ADDRESSES: The Guidelines will be made available in the following ways:
(1) The electronic version will be accessible on EPA's Office of Research and
Development home page on the Internet at http://www.epa.gov/ORD/WebPubs/repro/.
(2) 3 '/z-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: clegg.eric@epamail.epa.gov.
SUPPLEMENTARY INFORMATION:
A. APPLICATION OF THE GUIDELINES
The EPA is authorized by numerous statutes, including the Toxic Substances Control Act
(TSCA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Clean Air Act, the
Safe Drinking Water Act, and the Clean Water Act, to regulate environmental agents that have
the potential to adversely affect human health, including the reproductive system. These statutes
are implemented through offices within the Agency. The Office of Pesticide Programs and the
Office of Pollution Prevention and Toxics within the Agency have issued testing guidelines (U.S.
EPA, 1982, 1985b, 1996a) that provide protocols designed to determine the potential of a test
substance to produce reproductive (including developmental) toxicity in laboratory animals.
Proposed revisions to these testing guidelines are in the final stages of completion (U.S. EPA,
Vlll
-------
1996a). The Organization for Economic Cooperation and Development (OECD) also has issued
testing guidelines (which are under revision) for reproduction studies (OECD, 1993b).
These Guidelines apply within the framework of policies provided by applicable EPA
statutes and do not alter such policies. They do not imply that one kind of data or another is
prerequisite for action concerning any agent. The Guidelines are not intended, nor can they be
relied upon, to create any rights enforceable by any party in litigation with the United States.
This document is not a regulation and is not intended to substitute for EPA regulations. These
Guidelines set forth current scientific thinking and approaches for conducting reproductive
toxicity risk assessments. EPA will revisit these Guidelines as experience and scientific
consensus evolve.
The procedures outlined here in the Guidelines provide guidance for interpreting,
analyzing, and using the data from studies that follow the above testing guidelines (U.S. EPA
1982, 1985b, 1996a). In addition, the Guidelines provide information for interpretation of other
studies and endpoints (e.g., evaluations of epidemiologic data, measures of sperm production,
reproductive endocrine system function, sexual behavior, female reproductive cycle normality)
that have not been required routinely, but may be required in the future or may be encountered in
reviews of data on particular agents. The Guidelines will promote consistency in the Agency's
assessment of toxic effects on the male and female reproductive systems, including outcomes of
pregnancy and lactation, and inform others of approaches that the Agency will use in assessing
those risks. More specific guidance on developmental effects is provided by the Guidelines for
Developmental Toxicity Risk Assessment (U.S. EPA, 1991). Other health effects guidance is
provided by the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986a, 1996b), the
Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986c), and the Proposed Guidelines
for Neurotoxicity Risk Assessment (U.S. EPA, 1995a). These Guidelines and the four cited above
are complementary.
The Agency has sponsored or participated in several conferences that addressed issues
related to evaluations of reproductive toxicity data which provide some of the scientific bases for
these risk assessment guidelines. Numerous publications from these and other efforts are
available which provide background for these Guidelines (U.S. EPA, 1982,1985b, 1995b;
Galbraith et al., 1983; OECD, 1983; U.S. Congress, 1985, 1988; Kimmel, C.A. et al., 1986;
Francis and Kimmel, 1988; Burger et al., 1989; Sheehan et al., 1989; Seed et al., 1996). Also,
ix
-------
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
x
-------
occurrence of other plausibly related effects such as reduced sperm production, hypospadias, and
cryptorchidism, suggest that an adverse effect may have occurred. However, there is no
definitive evidence that such adverse human health effects have been caused by environmental
chemicals.
Endometriosis is a painful reproductive and immunologic disease in women that is
characterized by aberrant location of uterine endometrial cells, often leading to infertility. It
affects approximately five million women in the United States between 15 and 45 years of age.
Very limited research has suggested a link between dioxin exposure and development of
endometriosis in rhesus monkeys (Rier et al., 1993). Gerhard and Runnebaum (1992) reported
an association in women between occurrence of endometriosis and elevated blood PCB levels,
while a subsequent small clinical study found no significant correlations between disease severity
in women and serum levels of halogenated aromatic hydrocarbons (Boyd et al., 1995).
Even though not all infertile couples seek treatment, and infertility is not the only adverse
reproductive effect, it is estimated that in 1986, Americans spent about $1 billion on medical care
to treat infertility alone (U.S. Congress, 1988). With the increased use of assisted reproduction
techniques in the last 10 years, that amount has increased substantially.
Disorders of the male or female reproductive system may also be manifested as adverse
outcomes of pregnancy. For example, it has been estimated that approximately 50% of human
conceptuses fail to reach term (Hertig, 1967; Kline et al., 1989). Methods that detect pregnancy
as early as eight days after conception have shown that 32%-34% of postimplantation
pregnancies end in embryonic or fetal loss (Wilcox et al., 1988; Zinaman et al., 1996).
Approximately 3% of newborn children have one or more significant congenital malformations
at birth, and by the end of the first 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
xi
-------
reproductive alterations, chemical or physical agents can affect the female and male reproductive
systems at any time in the life cycle, including susceptible periods in development. The
reproductive system begins to form early in gestation, but structural and functional maturation is
not completed until puberty. Exposure to toxicants early in development can lead to alterations
that may affect reproductive function or performance well after the time of initial exposure.
Examples include the actions of estrogens, anti-androgens or dioxin in interfering with male
sexual differentiation (Gill et al., 1979; Gray et al., 1994,1995; Giusti et al., 1995; Gray and
Ostby, 1995). Adverse effects such as reduced fertility in offspring may appear as delayed
consequences of in utero exposure to toxicants. Effects of toxic agents on other parameters such
as sexual behavior, reproductive cycle normality, or gonadal function can also alter fertility
(Chapman, 1983; Dixon and Hall, 1984; Schrag and Dixon, 1985b; U.S. Congress, 1985). For
example, developmental exposure to environmental compounds that possess steroidogenic
(Mattison, 1985) or antisteroidogenic (Schardein, 1993) activity affect the onset of puberty and
reproductive function in adulthood.
Numerous agents have been shown to cause reproductive toxicity in adult male and
female laboratory animals and in humans (Mattison, 1985; Schrag and Dixon, 1985a,b; Waller et
al., 1985; Lewis, 1991). In adult males and females, exposure to agents of abuse, e.g., cocaine,
disrupts normal reproductive function in both test species and humans (Smith, C.G. and Gilbeau,
1985). Numerous chemicals disrupt the ovarian cycle, alter ovulation, and impair fertility in
experimental animals and humans. These include agents with steroidogenic activity, certain
pesticides, and some metals (Thomas, 1981; Mattison, 1985). In males, estrogenic compounds
can be testicular toxicants in rodents and humans (Colborn et al., 1993; Toppari et al., 1995).
Dibromochloropropane (DBCP) impairs spermatogenesis in both experimental animals and
humans by another mechanism. These and other examples of toxicant-induced effects on
reproductive function have been reviewed (Katz and Overstreet, 1981; Working, 1988).
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
xn
-------
be identified. Also, adverse effects on the reproductive systems of the two sexes may not be
detected until a couple attempts to conceive a child.
For successful reproduction, it is critical that the biologic integrity of the human
reproductive system be maintained. For example, the events in the estrous or menstrual cycle are
closely interrelated; changes in one event in the cycle can alter other events. Thus, a short or
inadequate luteal phase of the menstrual cycle is associated with disorders in ovarian follicular
steroidogenesis, gonadotropin secretion, and endometrial integrity (McNatty, 1979; Scommegna
et al., 1980; Smith, S.K. et al., 1984; Sakai and Hodgen, 1987). Toxicants may interfere with
luteal function by altering hypothalamic or pituitary function and by affecting ovarian response
(La Bella etal, 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 x 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
xiii
-------
all stages of risk assessment. These Guidelines adopt an interactive approach by organizing the
process around the components of hazard characterization, the quantitative dose-response
analysis, the exposure assessment, and the risk characterization where hazard characterization
combines hazard identification with qualitative consideration of dose-response relationships,
route, timing, and duration of exposure. This is done because, in practice, hazard identification
for reproductive toxicity and other noncancer health effects 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
xiv
-------
the NOAEL or benchmark dose divided by uncertainty factors to account for interspecies
differences in response, intraspecies variability and deficiencies in the database.
Exposure assessment identifies and describes populations exposed or potentially exposed
to an agent, and presents the type, magnitude, frequency, and duration of such exposures. Those
procedures are considered separately in the Guidelines for Exposure Assessment (U.S. EPA,
1992). However, unique considerations for reproductive toxicity exposure assessments are
detailed in Section 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 Agency's 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
xv
-------
information in this process is discussed. For example, the acceptability of the margin of
exposure (MOE) is a risk management decision, but the scientific bases for generating this value
are discussed here.
Dated: October 15,1996 Signed by EPA Administrator
Carol M. Browner
xvi
-------
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 made. These default assumptions, which are summarized in
1
-------
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 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.
-------
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.
-------
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).
-------
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:
• Identify the strengths and limitations of the database, including all available
epidemiologic and experimental animal studies as well as pharmacokinetic and
mechanistic information.
• Identify and describe key toxicological studies.
• Describe the type(s) of effects.
• Describe the nature of the effects (irreversible, reversible, transient, progressive,
delayed, residual, or latent effects).
• Describe how much is known about how (through what biological mechanism) the
agent produces adverse effects.
• Discuss the other health endpoints of concern.
• Discuss any nonpositive data in humans or experimental animals.
• Discuss the dose-response data (epidemiologic or experimental animal) available for
further dose-response analysis.
• Discuss the route, level, timing, and duration of exposure in studies as compared to
expected human exposures.
• Summarize the hazard characterization, including:
- Major assumptions used,
- Confidence in the conclusions,
- Alternative conclusions also supported by the data,
- Major uncertainties identified, and
- Significant data gaps.
Conduct of a hazard characterization requires knowledge of the protocols in which data
were produced and the endpoints that were evaluated. Sections 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).
-------
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.
-------
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, F, and F2 offspring
are exposed continuously in utero from conception until birth and during the preweaning period.
This allows detection of effects that occur from exposures throughout development, including the
peripubertal and young adult phases. Because the parental and subsequent filial generations have
8
-------
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., F,,
F2). Only the P generation is mated in a single-generation test, while both the P and F,
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 F, male and female offspring
continue to be exposed after weaning (day 21) and through the mating period. Treatment of
mated F, females is continued throughout gestation and lactation. More than one litter may be
produced from either P or F, 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.
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 F, 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
andKimmel, 1988).
A continuous breeding protocol, Fertility (or Reproductive) Assessment by Continuous
Breeding (FACB or RACE), 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 (PvACB), dosing
is extended into the F, generation to make it compatible with the EPA workshop
recommendations for a two-generation design (Francis and Kimmel, 1988). The RACE 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
11
-------
of the test period and their utility has been examined using model compounds in the mouse
(Morrisseyetal., 1989).
Another test method combines the use of multiple endpoints in both sexes of rats with
initiation of treatment at weaning (Gray et ah, 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 F,
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 F,
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 RACE, 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 OECD's
Screening Information Data Set protocol (Scala et al., 1992; Tanaka et al, 1992; OECD, 1993a),
a tripartite protocol developed by the International Conference on Harmonization (International
Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use,
1994; Manson, 1994), and the NTP's Short-Term Reproductive and Developmental Toxicity
Screen (Harris, M.W. et al., 1992). These protocols have been developed for setting priorities for
further testing and should not be considered sufficient by themselves to establish regulatory
exposure levels. Their limited exposure periods do not allow assessment of certain aspects of the
reproductive process, such as developmentally induced effects on the reproductive systems of
offspring, that are covered by the multigeneration reproduction protocols.
The male dominant lethal test was designed to detect mutagenic effects in the male
spermatogenic process that are lethal to the offspring. A female dominant lethal protocol has
also been used to detect equivalent effects on oogenesis (Generoso and Piegorsch, 1993).
A review of the male dominant lethal test has been published as part of the EPA's Gene-
Tox Program (Green et al., 1985). Dominant lethal protocols may use acute dosing (1 to 5 days)
followed by serial matings with one or two females per male per week for the duration of the
12
-------
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 agent's
mutagenic and gametotoxic potential if genotoxicity is to be factored into the risk assessment.
Regardless, significant effects may be observed in a dominant lethal test that are considered
reproductive in nature.
An acute exposure protocol, combined with serial mating, may allow identification of the
spermatogenic cell types that are affected by treatment. However, acute dosing may not produce
adverse effects at levels as low as with subchronic dosing because of factors such as
bioaccumulation. Conversely, if tolerance to an agent is developed with longer exposure, an
effect may be observed after acute dosing that is not detected after longer-term dosing.
Subchronic toxicity tests may have been conducted before a detailed reproduction study is
initiated. In the subchronic toxicity test with rats, exposure usually begins at 6-8 weeks of age
and is continued for 90 days (U.S. EPA, 1982, 1985b). Initiation of exposure at 8 weeks of age
(compared with 6) and exposure for approximately 90 days allows the animals to reach a more
mature stage of sexual development and assures an adequate length of dosing for observation of
effects on the reproductive organs with most agents. The route of administration is often oral or
by gavage but may be dermal or by inhalation. Animals are monitored for clinical signs
throughout the test and are necropsied at the end of dosing.
The endpoints that are usually evaluated for the male reproductive system include visual
examination of the reproductive organs, plus weights and histopathology for the testes,
epididymides, and accessory sex glands. For the females, endpoints may include visual
13
-------
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.
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
14
-------
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 Agency's 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.
Some of the endpoints identified above are used to calculate ratios or indices (NRC1,
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.
15
-------
Table 2. Couple-mediated endpoints of reproductive toxicity
Multigeneration studies
Mating rate, time to mating (time to pregnancy*)
Pregnancy rate*
Delivery rate*
Gestation length*
Litter size (total and live)
Number of live and dead offspring (fetal death rate*)
Offspring gender* (sex ratio)
Birth weight*
Postnatal weights*
Offspring survival*
External malformations and variations*
Offspring reproduction*
Other reproductive endpoints
Ovulation rate
Fertilization rate
Preimplantation loss
Implantation number
Postimplantation loss*
Internal malformations and
variations*
Postnatal structural and
functional development*
*Endpoints that can be obtained with humans.
16
-------
Table 3. Selected indices that may be calculated from endpoints of reproductive toxicity in
test species
MATING INDEX
Number of males or females mating x 100
Number of males or females cohabited
Note: Mating is used to indicate that evidence of copulation (observation or other
evidence of ejaculation such as vaginal plug or sperm in vaginal smear) was obtained.
FERTILITY INDEX
Number of cohabited females becoming pregnant x 100
Number of nonpregnant couples cohabited
Note: Because both sexes are often exposed to an agent, distinction between sexes 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 x 100
Number of females with evidence of pregnancy
LIVE BIRTH INDEX
Number of live offspring x 100
Number of offspring delivered
SEX RATIO
Number of male offspring
Number of female offspring
4-DAY SURVIVAL INDEX (VIABILITY INDEX)
Number of live offspring at lactation day 4 x 100
Number of live offspring delivered
Note: This definition assumes that no standardization of litter size is done until after the
day 4 determination is completed.
17
-------
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 x 100
Number of live offspring born
Note: If litters were standardized to equalize numbers of offspring per litter, number of
offspring after standardization'should be used instead of number born alive. When no
standardization is done, measure is called weaning index. When standardization is done,
measure is called lactation index.
PREWEANING INDEX
Number of live offspring born -
Number of offspring weaned x 100
Number of live offspring born
Note: If litters were standardized to equalize numbers of offspring per litter, then number
of offspring remaining after standardization should be used instead of number born.
18
-------
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.
19
-------
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 NO AEL,
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 NO AEL, 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.
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
20
-------
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 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
21
-------
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
fo& 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 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.
22
-------
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.
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.
23
-------
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 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
24
-------
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.
25
-------
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
Ley dig cell hyperplasia. Blockage of the efferent ducts by cells sloughed from the germinal
epithelium or the efferent ducts themselves can lead to an increase in testis weight due to fluid
accumulation (Hess et al., 1991; Nakai et al., 1993), an effect that could offset the effect of
depletion of the germinal epithelium on testis weight. Thus, while testis weight measurements
may not reflect certain adverse testicular effects and do not indicate the nature of an effect, a
significant increase or decrease is indicative of an adverse effect.
Pituitary gland weight can provide valuable insight into the reproductive status of the
animal. However, the pituitary contains cell types that are responsible for the regulation of a
variety of physiologic functions including some that are separate from reproduction. Thus,
changes in pituitary weight may not necessarily reflect reproductive impairment. If weight
changes are observed, gonadotroph-specific histopathologic evaluations may be useful in
identifying the affected cell types. This information may then be used to judge whether the
observed effect on the pituitary is related to reproductive system function and therefore an
adverse reproductive effect.
Prostate and seminal vesicle weights are androgen-dependent and may reflect changes in
the animal's endocrine status or testicular function. Separation of the seminal vesicles and
coagulating gland (dorsal prostate) is difficult in rodents. However, the seminal vesicle and
prostate can be separated and results may be reported for these glands separately or together, with
or without their secretory fluids. Differential loss of secretory fluids prior to weighing could
produce artifactual weights. Because the seminal vesicles and prostate may respond differently
to an agent (endocrine dependency and developmental susceptibility differ), more information
may be gained if the weights were examined separately.
26
-------
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, mice, and dogs. Included is guidance for identification and
27
-------
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 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
28
-------
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 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.
29
-------
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
andZenick, 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 agent's mutagenicity and its ability to induce abnormal sperm, sperm
head morphology has been a frequently reported sperm variable in toxicologic studies on test
species (Wyrobek et al., 1983b). The tendency has been to conclude that increased incidence of
sperm head malformations reflects germ-cell mutagenicity. However, not every mutagen induces
sperm head abnormalities, and other nonmutagenic chemicals may alter sperm head morphology.
For example, microtubule poisons may cause increases in abnormal sperm head incidence,
presumably by interfering with spermiogenesis, a microtubule-dependent process (Russell et al.,
30
-------
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
etal., 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 functional characteristics, i.e., progressive motility and the potential to
fertilize. With chemical exposures, perturbation of this balance may occur, producing alterations
31
-------
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., 199la;
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 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
32
-------
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 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
33
-------
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.
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
34
-------
may affect the endpoints used for evaluation. Therefore, knowledge of the reproductive status of
the female at necropsy, including the stage of the estrous cycle, can facilitate detection and
interpretation of effects with endpoints such as uterine weight and histopathology of the ovary
and uterus. Necropsy of all test animals at the same stage of the estrous cycle can reduce the
variance of test results with such measures.
A variety of measures to evaluate the integrity of the female reproductive system has been
used in toxicity studies. With appropriate measures, a comprehensive evaluation of the
reproductive process can be achieved, including identification of target organs and possible
elucidation of the mechanisms involved in the agent's effect(s). Areas that may be examined in
evaluations of the female reproductive system are listed in Table 5.
Reproductive function in the female is controlled through complex interactions involving
the central nervous system (particularly the hypothalamus), pituitary, ovaries, the reproductive
tract, and the secondary sexual organs. Other nongonadotrophic components of the endocrine
system may also modulate reproductive system function. Because it is difficult to measure
certain important aspects of female reproductive function (e.g., increased rate of follicular atresia,
ovulation failure), assessment of the endocrine status may provide needed insight that is not
otherwise available.
To understand the significance of effects on the reproductive endpoints, it is critical that
the relationships between the various reproductive hormones and the female reproductive organs
be understood. Although certain effects may be identified routinely as adverse, all of the results
should be considered in the context of the known biology.
The format used below for presentation of the female reproductive endpoints is altered
from that used for the male to allow examination of events that are linked and that fluctuate with
the changing endocrine status. Particularly, the organ weight, gross morphology, and histology
are combined for each organ. Endpoints and endocrine factors for the individual female
reproductive organs are discussed, with emphasis on the nonpregnant animal. This is followed
by examination of measures of cyclicity and their interpretation. Then, considerations relevant to
prepubertal, pregnant, lactating, and aging females are presented.
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
35
-------
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.
36
-------
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-I mice when body weights were 90%, 80%, or 70% of controls.
Female rats were resistant to effects on reproductive function at 80% of control weight whereas
mice showed adverse effects at 80% 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 stages of maturation (Plowchalk et al., 1993). These techniques
37
-------
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:
• Increase or decrease in ovarian weight
• Increased incidence of follicular atresia
• Decreased number of primary follicles
• Decreased number or lifespan of corpora lutea
• Evidence of abnormal folliculogenesis or luteinization, including cystic follicles,
luteinized follicles, and failure of ovulation
• Evidence of altered puberty or premature reproductive senescence
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 animal's estrous cycle stage. A number of environmental compounds (e.g.,
pesticides such as methoxychlor and chlordecone, mycotoxins, polychlorinated biphenyls,
alkylphenols, and phytoestrogens) possess varying degrees of estrogenic activity and have the
potential to stimulate the female reproductive tract (Barlow and Sullivan, 1982; Bulger and
Kupfer, 1985; Hughes, 1988).
When pregnant or postpartum animals are examined, the numbers of implantation sites or
implantation scars should be counted. This information, along with corpus luteum counts, can be
used to calculate pre- and postimplantation losses.
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 progestogens (Warren et al., 1967), and extended treatment with these compounds
leads to hypertrophy and hyperplasia. Conversely, inhibition of ovarian activity and reduced
38
-------
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.
• Infantile or malformed uterus or cervix
• Decreased or increased uterine weight
• Endometrial hyperplasia, hypoplasia, or aplasia
• 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 behavior. Other histologic alterations that may be observed include aplasia,
hypoplasia, and hyperplasia of the vaginal epithelial cell lining.
39
-------
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 uremral 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:
• Increases or decreases in weight
• Infantile or malformed vagina or vulva, including masculinized vulva or increased
ano-genital distance
• Vaginal hypoplasia or aplasia
• Altered timing of vaginal opening
• Abnormal vaginal smear cytology pattern
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 accompanied by hyperprolactinemia and constant vaginal estrus.
Decreased pituitary weight is less common but may result from decreased estrogenic stimulation
(Cooper et al., 1989).
40
-------
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 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
41
-------
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 (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
42
-------
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
. Typically, in the adult, if the vaginal epithelium becomes cornified and remains so in
riisfi to toxicant exposure, it is the result of the agent's estrogenic properties (i.e., DES or
methoxychJ.or), 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, t he 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
t
altered gonadatropin secretion will contain several large follicles and no corpora lutea. Females
in con stant 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
rnay 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,
43
-------
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; Sonawanse,
1995). Reduced growth of young could be caused by reduced milk availability, palatabilitv, w
quality, by ingestion of a toxic agent secreted into the milk, or by other factors unrelateed to
lactational ability (e.g., deficient suckling ability or deficient maternal behavior). Perinatal
exposure to steroid hormones and other chemicals can alter mammary gland morp hology and
tumor potential in adulthood. Because of the tendency for mobilization of lipids .from adipose
tissue and secretion of those lipids into milk by lactating females, milk may contain. Hpophilic
agents at concentrations equal to or higher than those present in the blood or organs of the a'am.
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
44
-------
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 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.
45
-------
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
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
46
-------
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 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
47
-------
to reduction in sperm production or altered sexual behavior. Chemicals with estrogenic or anti-
androgenic activity have been identified that are capable, with sufficient exposure levels, of
causing effects of these types in males (Gray et al., 1994; Harrison et al., 1995; Kelce et al.,
1995). While sensitivity may differ, it is likely that mechanisms of action for these endocrine
disrupting agents will be consistent across mammalian species. Chemicals with the ability to
interact with the Ah receptor (e.g., dioxin or PCBs) may also disrupt reproductive system
development or function (Brouwer et al., 1995; Safe, 1995). Several of the effects seen with
exposure of male and female rats and hamsters differ from those caused by estrogens, indicating
a different mechanism of action.
The developing nervous system can be a target of chemicals. In rats, sexual
differentiation of the CNS can be modified by hormonal treatments or exposure to environmental
agents that mimic or interfere with the action of certain hormones. Prior to gender
differentiation, the brain is inherently female or at least bipotential (Gorski, 1986). Thus, the
functional and structural sex differences in the CNS are not due directly to sex differences in
neuronal genomic expression, but rather are imprinted by the gonadal steroid environment during
development.
Chemicals with endocrine activity have been shown to masculinize the CNS of female
rats. Examples include chlordecone (Gellert, 1978), DDT (Bulger and Kupfer, 1985), and
methoxychlor (Gray et al., 1989). Exposure of newborn female rats to these agents during the
critical period of sexual differentiation can alter the timing of puberty and perturb subsequent
reproductive function, presumably by altering the development of the neural mechanisms that
regulate gonadotropin secretion.
In females, the situation is more complex than in males due to the female cycle, the
fertilization process, gestation and lactation. All of the functions of the female reproductive
system are under endocrine control, and therefore can be susceptible to disruption by effects on
the reproductive endocrine system.
As with males, disturbance of the normal endocrine patterns during development can
result in abnormal development of the female reproductive tract at exposure levels that tend to be
lower than those affecting adult females (Gellert, 1978; Brouwer et al., 1995). Consistent with
the differentiation mechanism described above, exposure of genotypic females to androgens
causes formation of pseudohermaphrodite reproductive tracts with varying degrees of severity as
well as alteration of brain imprinting. However, exposure to estrogenic substances during
development also results in adverse effects on anatomy and function including, in rats,
malformations of the genitalia. Exposure of human females to diethylstilbestrol in utero has
been shown to cause an increased incidence of vaginal clear cell adenoma (Giusti et al., 1995).
48
-------
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; 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
49
-------
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 Ley dig 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 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).
50
-------
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.
"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
51
-------
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.
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).
52
-------
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 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 woman's 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).
53
-------
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, 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
54
-------
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 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 couple's 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
55
-------
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 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
56
-------
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 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.
57
-------
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 individual's body burden
and consequently the actual dose during the critical period. The probability of misclassification
of exposure status may affect the ability to 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; a = 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.
58
-------
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 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
individual's 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
59
-------
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 (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 individual's self-report of his/her
occupational exposures and health characteristics more reliable than his/her mate's 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
60
-------
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.
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
61
-------
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 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. EPA's Office of Prevention, Pesticides, and Toxic Substances
has published protocols for metabolism studies that may be adapted to provide information
useful in reproductive toxicity risk assessment for a suspect agent. Pharmacokinetic studies in
62
-------
reproductive toxicology are most useful if the data are obtained with animals that are at the same
reproductive status and stage of life (e.g., pregnant, nonpregnant, embryo or fetus, neonate,
prepubertal, adult) at which reproductive insults are expected to occur in humans.
Specific guidance regarding both the development and application of pharmacokinetic
data was agreed on by the participants of the Workshop on Dermal Developmental Toxicity
Studies (Kimmel, C.A. 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.,
63
-------
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 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
64
-------
examined. Because 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.
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
65
-------
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, the risk assessor must consider each study's 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,
66
-------
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 EPA's 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 more definitive data are developed. Identification of the increased uncertainty
and justification for the adjustment of the uncertainty factor should be stated clearly.
67
-------
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 EPA's 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.
68
-------
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:
• Data of equivalent quality from human exposures are given more weight than data
from exposures of test species.
• Although a single study of high quality could be sufficient to achieve a relatively
high level of confidence, replication increases the confidence that may be placed in
such results.
• Data are available from one or more in vivo studies of acceptable quality with
humans or other mammalian species that are believed to be predictive of human
responses.
• Data exhibit a dose-response relationship.
• Results are statistically significant and biologically plausible.
• When multiple studies are available, the results are consistent.
• Sufficient information is available to reconcile discordant data.
• Route, level, duration, and frequency of exposure are appropriate.
• An adequate array of endpoints has been examined.
• The power and statistical treatment of the studies are appropriate.
Any statistically significant deviation from baseline levels for an in vivo effect warrants
closer examination. To determine whether such a deviation constitutes an adverse effect requires
an understanding of its role within a complex system and the determination of whether a "true
effect" has been observed. Application of the above criteria, combined with guidance presented
in Section 3.2, can facilitate such determinations.
69
-------
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.
70
-------
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 pharmacodynaniic 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 doses; (4) The number and spacing of doses in a
71
-------
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 RfCDT) is
determined for adverse developmental effects. For adverse reproductive effects on endpoints
other than those of developmental toxicity, no special designator is attached. Data on
72
-------
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.
73
-------
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.
74
-------
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
75
-------
cells that cannot be replaced or 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
76
-------
cover all phases of spermatogenesis (Russell et al., 1990). Assessments may be made at any time
after this point as long as exposure remains constant. Time required for 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 stern 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) exposed
neonatally to synthetic estrogens or progestins (Steinberger and Lloyd, 1985; Schardein, 1993).
77
-------
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 female's reproductive lifespan.
Furthermore, exposure to compounds at different stages of the ovarian cycle can disrupt or delay
follicular recruitment and development (Armstrong, 1986), ovulation (Everett 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 female's lifespan and reproductive
cycle can provide insight into how an agent disrupts reproductive function.
DBS 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). DBS, 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 DBS 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 MOB for an adverse developmental effect or to compare to the RfD or RfC are
usually based on a single daily dose that is not adjusted for duration or pattern (e.g., continuous
or intermittent) of exposure. For example, it would be inappropriate to use time-weighted
averages or adjustment of exposure over a different time frame than that actually encountered
(such as the adjustment of a 6-hour inhalation exposure to account for a 24-hour exposure
scenario) unless pharmacokinetic data were available to indicate an accumulation with
continuous exposure. In the case of intermittent exposures, examination of the peak exposures as
well as the average exposure over the time of exposure would be important.
78
-------
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.
79
-------
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:
• The nature, reliability, and consistency of the data used.
• The reasons for selection of the key study(ies) and the critical effect(s) and their
relevance to human outcomes.
• The qualitative and quantitative descriptors of the results of the risk assessment.
• The limitations of the available data, the assumptions used to bridge knowledge gaps
in working with those data, and implications of using alternative assumptions.
• The strengths and weaknesses of the risk assessment and the level of scientific
confidence in the assessment.
• The areas of uncertainty, additional data/research needs to improve confidence in the
risk assessment, and the potential impacts of the new research.
The risk characterization should be limited to the most significant and relevant data,
conclusions, and uncertainties. When special circumstances exist that preclude full assessment,
those circumstances should be explained and the related limitations identified.
The following sections describe these aspects of the risk characterization in more detail,
but do not attempt to provide a full discussion of risk characterization. Rather, these Guidelines
80
-------
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.
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:
• 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.
81
-------
• 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.
• Descriptions of the estimates of the range of human exposure (e.g., central tendency,
high end), the route, duration, and pattern of the exposure, relevant
pharmacokinetics, and the size and characteristics of the various populations that
might be exposed.
• The risk characterization of an agent being assessed for reproductive toxicity should
be based on data from the most appropriate species or, if such information is not
available, on the most sensitive species tested. It 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 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.
82
-------
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.
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.
83
-------
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?
• How good is the key study?
• Are the data from laboratory or field studies? In a single or multiple species?
• What adverse reproductive endpoints were observed, and what is the basis for
the critical effect?
• Describe other studies that support this finding.
• 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:
• What types of data were used (e.g., human ecologic, case-control or cohort
studies, or case reports or series)?
• Describe the degree to which exposures were described.
• Describe the degree to which confounding factors were considered.
• 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?
• Discuss relevant studies of mechanisms of action or metabolism.
• Does this information aid in the interpretation of the toxicity data?
• 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:
• Confidence in conclusions
• Alternative conclusions that are also supported by the data
• Significant data gaps
• Highlights of major assumptions
84
-------
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?
• If laboratory animal data were used:
Which species were used?
Most sensitive, average of all species, or other?
Were any studies excluded? Why?
• 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?
• How was the RfD/RfC (or the acceptable range) calculated?
• What assumptions and uncertainty factors were used?
• What is the confidence in the estimates?
C. Discuss the route, level, and duration of exposure observed, as compared to expected
human exposures.
• 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?
• 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.
Ill. 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?
85
-------
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:
• Results of different approaches, i.e., modeling, monitoring, probability
distributions;
• Limitations of each, and the range of most reasonable values;
• 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)?
86
-------
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?
87
-------
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 Agency's 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 descriptors) 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 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.
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
89
-------
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.
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 Agency's current methods for developing
reference doses and reference concentrations (RfDs and RfCs) are designed to protect sensitive
90
-------
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:
• What are the reproductive risks if a pesticide applicator applies this pesticide without
using protective equipment?
• What are the reproductive risks if this site becomes residential in the future?
• What are the reproductive risks if we set the standard at 100 ppb?
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
91
-------
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 assessor's 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
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.
92
-------
7. REFERENCES
Aafjes, J.H., Vels, J.M., Schenck, E. (1980) Fertility of rats with artificial oligozoospermia. J. Reprod. Fertil.
58:345-351.
Adler, NT., Toner, J.P. (1986) The effect of copulatory behavior on sperm transport and fertility in rats. In:
Komisaruk, B.R., Siegel, H.I., Chang, M.F., Feder, H.H. Reproduction: Behavioral and Neuroendocrine
Perspective. New York Academy of Science, New York. pp. 21-32.
Allen, B.C., Kavlock, R.J., Kimmel, C.A., Faustman, E.M. (1994a) Dose-response assessment for developmental
toxicity: II. Comparison of generic benchmark dose estimates with NOAELs. Fundam. Appl. Toxicol. 23:487-495.
Allen, B.C., Kavlock, R.J., Kimmel, C.A., Faustman, E.M. (1994b) Dose-response assessment for developmental
toxicity: HI. Statistical models. Fundam. Appl. Toxicol. 23:496-509.
Amann, R.P. (1981) A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J.
Androl. 2:37-58.
American Academy of Pediatrics Committee on Drugs. (1994) The transfer of drugs and other chemicals into human
milk. Pediatrics 93:137-150.
Armstrong, D.L. (1986) Environmental stress and ovarian function. Biol. Reprod. 34:29-39.
Atterwill, C.K., Flack, J.D. (1992) Endocrine Toxicology. Cambridge University Press, Cambridge.
Auger, J., Kunstman, J.M., Czyglik, F., Jouannet, P. (1995) Decline in semen quality among fertile men in Paris
during the past 20 years. N. Engl. J. Med. 332:281-285.
Axelson, O. (1985) Epidemiologic methods in the study of spontaneous abortions: sources of data, methods, and
sources of error. In: Hemminki, K., Sorsa, M., Vaino, H. Occupational Hazards and Reproduction. Hemisphere,
Washington, pp. 231-236.
Baird, D.D., Wilcox, AJ. (1985) Cigarette smoking associated with delayed conception. JAMA 253:2979-2983.
Baird, D.D., Wilcox, A.J., Weinberg, C.R. (1986) Using time to pregnancy to study environmental exposures. Am.
J. Epidemiol. 124:470-480.
Baird, S.J.S., Cohen, J.T., Graham, J.D., Shlyakhter, A.I., Evans, J.S. (1996) Noncancer risk assessment: a
probabilistic alternative to current practice. Human Ecol. Risk Assess. 2:79-102.
Barlow, S.M., Sullivan, P.M. (1982) Reproductive Hazards of Industrial Chemicals. Academic Press, London.
Barsotti, D.A., Abrahamson, L.J., Allen, J.R. (1979) Hormonal alterations in female rhesus monkeys fed a diet
containing 2,3,7,8-TCDD. Bull. Environ. Contam. Toxicol. 21:463-469.
Beach, F.A. (1979) Animal models for human sexuality. In: Ciba Foundation Symposium No. 62, Sex, Hormones
and Behavior. Elsevier-North Holland, London, pp. 113-143.
Bemdtson, W.E. (1977) Methods for quantifying mammalian spermatogenesis: a review. J. Anim. Sci. 44:818-833.
Bernstein, M.E. (1984) Agents affecting the male reproductive system: effects of structure on activity. Drug Metab.
Rev. 15:941-996.
Biava, C.G., Smuckler, E.A., Whorton, D. (1978) The testicular morphology of individuals exposed to
dibromochloropropane. Exp. Mol. Pathol. 29:448-458.
93
-------
Blazak, W.F., Ernst, T.L., Stewart, B.E. (1985) Potential indicators of reproductive toxicity, testicular sperm
production and epididymal sperm number, transit time and motility in Fischer 344 rats. Fundam. Appl. Toxicol.
5:1097-1103.
Blazak, W.F., Treinen, K.A., Juniewicz, P.E. (1993) Application of testicular sperm head counts in the assessment of
male reproductive toxicity. In: Chapin, R.E. and Heindel, J.J. Methods in Toxicology: Male Reproductive
Toxicology. Academic Press, San Diego, pp. 86-94.
Bloom, A.D. (1981) Guidelines for reproductive studies in exposed human populations. Guideline for studies of
human populations exposed to mutagenic and reproductive hazards. Report of Pane! II. March of Dimes Birth
Defects Foundation, White Plains, NY, pp. 37-110.
Boyd, J.A., Clark, G.C., Walmer, O.K., Patterson, D.G., Needham, L.L., Lucier, G.W. (1995) Endometriosis and the
environment: biomarkers of toxin exposure. Abstract from Endometriosis 2000 Workshop, May 15-17.
Boyers, S.P., Davis, R.O., Katz, D.F. (1989) Automated semen analysis. Curr. Probi. Obstet. Gynecol. Fertil.
12:173-200.
Brawer, J.R., Finch, C.E. (1983) Normal and experimentally altered aging processes in the rodent hypothalamus and
pituitary. In: Walker, R.F., Cooper. R.L. Experimental and Clinical Interventions in Aging. Marcel Dekker, New
York. pp. 45-65.
Brouwer, A., Ahlborg, U.G., Vandenberg, M., Birnbaum, L.S., Boersma, E.R., Bosveld, B., Denison, M.S., Gray,
L.E., Hagmar, L., Holene, E., Huisman, M., Jacobson, S.W., Jacobson, J.L., Koopmanesseboom, C., Koppe, J.G.,
Kulig, B.M., Morse, D.C., Muckle, G., Peterson, R.E., Sauer, P.J.J., Seegal, R.F., Smitsvanprooije, A.E., Touwen,
B.C.L., Weisglaskuperus, N., Winneke, G. (1995) Functional aspects of developmental toxicity of polyhalogenated
aromatic hydrocarbons in experimental animals and human infants. Eur. J. Pharmacol. 293:1-40.
Browner, C.M. (1995) EPA risk characterization program. U.S. EPA Memorandum, March 21, 1995. Available
from the EPA Air docket.
Brown-Grant, K., Davidson, J.M., Grieg, F. (1973) Induced ovulation in albino rats exposed to constant light. J,
Endocrinol. 57:7-22.
Bujan, L., Mansat, A., Pontonnier, F., Mieusset, R. (1996) Time series analysis of sperm concentration in fertile men
in Toulouse, France between 1977 and 1992. Br. Med. J. 312:471-472.
Bulger, W.H., Kupfer, D. (1985) Estrogenic activity of pesticides and other xenobiotics on the uterus and male
reproductive tract. In: Thomas, J.A., Korach, K.S., McLachlan, J.A. Endocrine Toxicology. Raven Press, New
York. pp. 1-33.
Burch, T.K., Macisco, J.J., Parker, M.P. (1967) Some methodologic problems in the analysis of menstrual data. Int.
J. Fertil. 12:67-76.
Burger, E.J., Tardiff, R.G., Scialli, A.R., Zenick, H. (1989) Sperm Measures and Reproductive Success. Alan R.
Liss, New York.
Butcher, R.L., Fugo, N.W. (1967) Overripeness and the mammalian ova. II. Delayed ovulation and chromosome
anomalies. Fertil. Steril. 18:297-302.
Butcher, R.L., Blue, J.D., Fugo, N.W. (1969) Overripeness and the mammalian ova. III. Fetal development at
midgestation and at term. Fertil. Steril. 20:223-231.
Butcher, R.L., Collins, W.E., Fugo, N.W. (1975) Altered secretion of gonadotropins and steroids resulting from
delayed ovulation in the rat. Endocrinology 96:576-586.
94
-------
Byskov, A.G., Hoyer, P.E. (1994) Embryology of mammalian gonads and ducts. In: Knobil, E., Neill, J.D. The
Physiology of Reproduction. Raven Press, New York. pp. 487-540.
Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E. (1992) Evidence for decreasing quality of semen during
past 50 years. Br. Med. J. 305:609-613.
Cassidy, S.L., Dix, K.M., Jenkins, T. (1983) Evaluation of a testicular sperm head counting technique using rats
exposed to dimethoxyethyl phthalate (DMEP), glycerol alpha-monochlorohydrin (GMCH), epichlorohydrin (ECH),
formaldehyde (FA), or methyl methanesulphonate (MMS). Arch. Toxicol. 53:71-78.
Chapin, R.E. (1988) Morphologic evaluation of seminiferous epithelium of the testis. In: Lamb, J.C., Foster, P.M.D.
Physiology and Toxicology of Male Reproduction. Academic Press, New York. pp. 155-177.
Chapin, R.E., Heindel, J.J. (1993) Methods in Toxicology: Male Reproductive Toxicology. Academic Press, San
Diego.
Chapin, R.E., Filler, R.S., Gulati, D., Heindel, J.J., Katz, D.F., Mebus, C.A., Obasaju, F., Perreault, S.D., Russell,
S.R., Schrader, S., Slott, V., Sokol, R.Z., Toth, G. (1992) Methods for assessing rat sperm motility. Reprod.
Toxicol. 6:267-273.
Chapin, R.E., Gulati, D.K., Barnes, L.H., Teague, J.L. (1993a) The effects of feed restriction on reproductive
function in Sprague-Dawley rats. Fundam. Appl. Toxicol. 20:23-29.
Chapin, R.E., Gulati, O.K., Fail, P.A., Hope, E., Russell, S.R., Heindel, J.J., George, J.D., Grizzle, T.B., Teague,
J.L. (1993b) The effects of feed restriction on reproductive function in Swiss CD-I mice. Fundam. Appl. Toxicol.
20:15-22.
Chapman, R.M. (1983) Gonadal injury resulting from chemotherapy. In: Mattison, D.R. Reproductive Toxicology.
Alan R. Liss, New York. pp. 149-161.
Clegg, E.D. (1995) Reversibility of effects: overview and reproductive systems. Inhal. Toxicol. 7:881-889.
Colborn, T., vom Saal, F.S., Soto, A.M. (1993) Developmental effects of endocrine-disrupting chemicals in wildlife
and humans. Environ. Health Perspect. 101:378-384.
Colie, C.F. (1993) Male mediated teratogenesis. Reprod. Toxicol. 7:3-9.
Collins, T.F.X. (1978) Multigeneration reproduction studies. In: Wilson, J.G., Fraser, F.C. Handbook of Teratology.
Plenum Press, New York, pp. 191 -214.
Cooper, R.L., Walker, R.F. (1979) Potential therapeutic consequences of age-dependent changes in brain
physiology. Interdis. Topics Gerontol. 15:54-76.
Cooper, R.L., Conn, P.M., Walker, R.F. (1980) Characterization of the LH surge in middle-aged female rats. Biol.
Reprod. 23:611-615.
Cooper, R.L., Chadwick, R.W., Rehnberg, G.L., Goldman, J.M., Booth, K.C., Hein, J.F., McElroy, W.K. (1989)
Effect of lindane on hormonal control of reproductive function in the female rat. Toxicol. Appl. Pharmacol. 99:384-
394.
Cooper, R.L., Goldman, J.M., Vandenbergh, J.G. (1993) Monitoring of the estrous cycle in the laboratory rodent by
vaginal lavage. In: Heindel, J.J., Chapin, R.E. Methods in Toxicology: Female Reproductive Toxicology.
Academic Press, San Diego, pp. 45-56.
95
-------
Cooper, R.L., Barrett, M.A., Goldman, J.M., Rehnberg, G.R., McElroy, W.K., Stoker, I.E. (1994) Pregnancy
alterations following xenobiotic-induced delays in ovulation in the female rat. Fundam. Appl. Toxicol. 22:474-480.
Cooper, R.L., Stoker, I.E., Goldman, J.M., Parrish, M.B., Tyrey, L. (1996) Effect of atrazine on ovarian function in
the rat. Reprod. Toxicol. 10(4):257-264.
Crisp, T.M. (1992) Organization of the ovarian follicle and events in its biology: oogenesis, ovulation or atresia.
Mutat. Res. 296:89-106.
Crump, K.S. (1984) A new method for determining allowable daily intakes. Fundam. Appl. Toxicol. 4:854-871.
Csapo, A.I., Pulkkinen, M. (1978) Indispensability of the human corpus luteum in the maintenance of early
pregnancy: lutectomy evidence. Obstet. Gynecol. Surv. 33:69.
Cummings, A.M., Gray, L.E. (1987) Methoxychlor affects the decidual cell response of the uterus but not other
progestational parameters in female rats. Toxicol. Appl. Pharmacol. 90:330-336.
Cummings, A.M., Perreault, S.D. (1990) Methoxychlor accelerates embryo transport through the rat reproductive
tract. Toxicol. Appl. Pharmacol. 102:110-116.
Darney, S.P. (1991) In vitro assessment of gamete integrity. In: Goldberg, A.M. In Vitro Toxicology: Mechanisms
and New Technology. Mary Ann Liebert, Inc., New York. pp. 63-75.
Davis, D.L., Friedler, G., Mattison, D., Morris, R. (1992) Male-mediated teratogenesis and other reproductive
effects: biologic and epidemiologic findings and a plea for clinical research. Reprod. Toxicol. 6:289-292.
de Boer, P., van der Hoeven, F.A., Chardon, J.A.P. (1976) The production, morphology, karyotypes and transport of
spermatozoa from tertiary trisomic mice and the consequences for egg fertilization. J. Reprod. Fertil. 48:249-256.
Dixon, R.L., Hall, J.L. (1984) Reproductive toxicology. In: Hayes, A.W. Principles and Methods of Toxicology.
Raven Press, New York. pp. 107-140.
Dobbins, J.G., Eifler, C.W., Buffler, P.A. (1978) The use of parity survivorship analysis in the study of reproductive
outcomes. Presented at the Society for Epidemiologic Research Conference, Seattle, WA: June, 1978.
Dobson, R.L., Felton, J.S. (1983) Female germ cell loss from radiation and chemical exposure. Am. J. Ind. Med.
4:175-190.
Drouva, S.V., Laplante, E., Kordon, C. (1982) Alpha 1-adrenergic receptor involvement in the LH surge in
ovariectomized estrogen-primed rats. Eur. J. Pharmacol. 81:341-344.
Egeland, G.M., Sweeney, M.H., Fingerhut, M.A., Wille, K.K., Schnorr, T.M., Halperin, W.E. (1994) Total serum
testosterone and gonadotropins in workers exposed to dioxin. Am. J. Epidemiol. 139:272-281.
Egnatz, D.G., Ott, M.G., Townsend, J.C., Olson, R.D., Johns, D.B. (1980) DBCP and testicular effects in chemical
workers; an epidemiological survey in Midland. J. Occup. Med. 22:727-732.
Epidemiology Workgroup for the Interagency Regulatory Liaison Group. (1981) Guidelines for documentation of
epidemiologic studies. Am. J. Epidemiol. 114:609-613.
Everett, J.W., Sawyer, C.H. (1950) A 24-hour periodicity in the "LH-release apparatus" of female rats disclosed by
barbiturate sedation. Endocrinology 47:198-218.
Everson, R.B., Sandier, D.P., Wilcox, A.J., Schreinemachers, D., Shore, D.L., Weinberg, C. (1986) Effect of passive
exposure to smoking on age at natural menopause. Br. Med. J. 293:792.
96
-------
Fabia, J., Thuy, T.D. (1974) Occupation of father at time of children dying of malignant disease. Br. J. Prev. Soc.
Med. 28:98-100.
Fawcett, D.W. (1986) Bloom and Fawcett: A Textbook of Histology. W. B. Saunders, Philadelphia, PA.
Filler, R. (1993) Methods for evaluation of rat epididymal sperm morphology. In: Chapin, R.E., Heindel, J.J.
Methods in Toxicology: Male Reproductive Toxicology. Academic Press, San Diego, pp. 334-343.
Finch, C.E., Felicio, L.S., Mobbs, C.V. (1984) Ovarian and steroidal influences on neuroendocrine aging processes
in female rodents. Endocrinol. Rev. 5:467-497.
Fink, G. (1988) Gonadotropin secretion and its control. In: Knobil, E., Neill, J.D. The Physiology of Reproduction.
Raven Press, New York. pp. 1349-1377.
Fisch, H., Goluboff, E.T., Olson, J.H., Feldshuh, J., Broder, S.J., Barad, D.H. (1996) Semen analyses in 1,283 men
from the United States over a 25-year period: no decline in fertility. Fertil. Steril. 65:1009-1014.
Foote, R.H., Berndtson, W.E. (1992) The Germinal Cells. In: Scialli, A.R., Clegg, E.D. Reversibility in Testicular
Toxicity Assessment. CRC Press, Boca Raton, pp. 1-55.
Foote, R.H., Schermerhorn, B.C., Simkin, M.E. (1986) Measurement of semen quality, fertility, and reproductive
hormones to assess dibromochloropropane (DBCP) effects in live rabbits. Fundam. Appl. Toxicol. 6:628-637.
Forsberg, J.G. (1981) Permanent changes induced by DES at critical stages in human and model systems. Biol. Res.
Pregnancy 2:168-175.
Foster, P.M.D. (1992) The Sertoli cell. In: Scialli, A.R., Clegg, E.D. Reversibility in Testicular Toxicity
Assessment. CRC Press, Boca Raton, pp. 57-86.
Francis, E.Z., Kimmel, G.L. (1988) Proceedings of the workshop on one- vs two-generation reproductive effects
studies. J. Am. Coll. Toxicol. 7:911-925.
Franken, D.R., Burkman, L.J.; Coddington, C.C., Oehninger, S., Hodgen, G.D. (1990) Human hemizona attachment
assay. In: Acosta, A.A., Swanson, R.J., Ackerman, S.B., Kruger, T.F., VanZyl, J.A., Menkveld, R. Human
Spermatozoa in Assisted Reproduction. Williams and Wilkins, Baltimore, pp. 355-371.
Fugo, N.W., Butcher, R.L. (1966) Overripeness and the mammalian ova. I. Overripeness and early embryonic
development. Fertil. Steril. 17:804-814.
Gaffey, W.R. (1976) A critique of the standard mortality ratio. J. Occup. Med. 18:157-160.
Galbraith, W.M., Voytek, P., Ryon, M.S. (1983) Assessment of risks to human reproduction and development of the
human conceptus from exposure to environmental substances. In: Christian, M.S., Galbraith, W.M., Voytek, P.,
Mehlman, M.A. Advances in Modern Environmental Toxicology. Princeton Scientific Publ., Princeton, pp. 41-153.
Galletti, F., Klopper, A. (1964) The effect of progesterone on the quantity and distribution of body fat in the female
rat. Acta Endocrinol. 46:379-386.
Gardner, M.J., Hall, A.J., Snee, M.P., Downes, S., Powell, C.A., Terrell, J.D. (1990a) Methods and basic data of
case-control study of leukaemia and lymphoma among young people near Sellafield nuclear plant in West Cumbria.
Br. Med. J. 300:429-434.
Gardner, M.J., Snee, M.P., Hall, A.J., Powell, C.A., Downes, S., Terrell, J.D. (1990b) Results of case-control study
of leukaemia and lymphoma among young people near Sellafield nuclear plant in West Cumbria. Br. Med. J.
300:423-429.
97
-------
Gaylor, D.W. (1989) Quantitative risk analysis for quanta! reproductive and developmental effects. Environ. Health
79:243-246.
Gellert, R.J. (1978) Kepone, mirex, dieldrin, and aldrin: estrogenic activity and the induction of persistent vaginal
estrus and anovulation in rats following neonatal treatment. Environ. Res. 16:131-138.
Generoso, W.M., Piegorsch, W.W. (1993) Dominant lethal tests in male and female mice. In: Chapin, R.E.,
Heindel, J.J. Methods in Toxicology: Male Reproductive Toxicology. Academic Press, San Diego, pp. 124-139.
Generoso, W.M., Rutledge, J.C., Cain, K.T., Hughes, L.A., Braden, P.W. (1987) Exposure of female mice to
ethylene oxide within hours after mating leads to fetal malformation and death. Mutat. Res. 176:269-274.
George, F.W., Wilson, J.D. (1994) Sex determination and differentiation. In: Knobil, E., Neill, J.D. The Physiology
of Reproduction. Raven Press, New York. pp. 3-28.
Gerhard, I., Runnebaum, B. (1992) Grenzen der hormonsubstitution bei Schadstoffbelastung und fertilitatsstorungen.
Zentralbl. Gynakol. 114:593-602.
Gerrity, T.R., Henry, C.J., Bronaugh, R., et al. (1990) Summary report of the workshops on principles of route-to-
route extrapolation for risk assessment. In: Gerrity, T.R., Henry, CJ. Principles of Route-To-Route Extrapolation
for Risk Assessment. Elsevier Science Publ. Co., New York. pp. 1-12.
Gill, W.B., Schumacher, F.B., Bibbo, M., Straus, F.H., Schoenberg, H.W. (1979) Association of diethylstilbestrol
exposure in utero with cryptorchidism, testicular hypoplasia and semen abnormalities. J. Urol. 122:36-39.
Ginsburg, J., Okolo, S., Prelevic, G., Hardiman, P. (1994) Residence in London area and sperm density. Lancet
343:230.
Giusti, R.M., Iwamoto, K., Hatch, E.E. (1995) Diethylstilbestrol revisited: a review of the long-term health effects.
Ann. Intern. Med. 122:778-788.
Giwercman, A., Carlsen, E., Keiding, N., Skakkebaek, N.E. (1993) Evidence for increasing incidence of
abnormalities of the human testis: A review. Environ. Health Perspect. 101:65-71.
Goldman, J.M., Cooper, R.L., Laws, S.C., Rehnberg, G.L., Edwards, T.L., McElroy, W.K., Hein, J.F. (1990)
Chlordimeform-induced alterations in endocrine regulation within the male rat reproductive system. Toxicol. Appl.
Pharmacol. 104:25-35.
Goldman, J.M., Cooper, R.L., Edwards, T.L., Rehnberg, G.L., McElroy, W.K., Hein, J.F. (1991) Suppression of the
luteinizing hormone surge by chlordimeform in ovariectomized, steroid-primed female rats. Pharmacol. Toxicol.
68:131-136.
Gorski, R.A. (1979) The neuroendocrinology of reproduction: an overview. Biol. Reprod. 20:111-127.
Gorski, R.A. (1986) Sexual differentiation of the brain: a model for drug-induced alterations of the reproductive
system. Environ. Health Perspect. 70:163-175.
Gray, L.E. (1991) Delayed effects on reproduction following exposure to toxic chemicals during critical periods of
development. In: Cooper, R.L., Goldman, J.M., Harbin, T.J. Aging and Environmental Toxicology: Biological and
Behavioral Perspectives. Johns Hopkins University Press, Baltimore, pp. 183-210.
Gray, L.E., Ostby, J.S. (1995) In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology
and function in female rat offspring. Toxicol. Appl. Pharmacol. 133:285-294.
98
-------
Gray, L.E., Ferrell, J.M., Ostby, J.S. (1985) Alteration of behavioral sex differentiation by exposure to estrogenic
compounds during a critical neonatal period: effects of zearalenone, methoxychlor, and estradiol in hamster.
Toxicol. Appl. Pharmacol. 80:127-136.
Gray, L.E., Ostby, J., Sigmon, R., Ferrell, J., Linder, R., Cooper, R., Goldman, J., Laskey, J. (1988) The
development of a protocol to assess reproductive effects of toxicants in the rat. Reprod. Toxicol. 2:281-287.
Gray, L.E., Ostby, J., Ferrell, J., Rehnberg, G, Linder, R., Cooper, R., Goldman, J., Slott, V., Laskey, J. (1989) A
dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat.
Fundam. Appl. Toxicol. 12:92-108.
Gray, L.E., Ostby, J., Linder, R., Goldman, J., Rehnberg, G., Cooper, R. (1990) Carbendazim-induced alterations of
reproductive development and function in the rat and hamster. Fundam. Appl. Toxicol. 15:281-297.
Gray, L.E., Ostby, J.S., Kelce, W.R. (1994) Developmental effects of an environmental antiandrogen: the fungicide
vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129:46-52.
Gray, L.E., Kelce, W.R., Monosson, E., Ostby, J.S., Birnbaum, L.S. (1995) Exposure to TCDD during development
permanently alters reproductive function in male Long Evans rats and hamsters: reduced ejaculated and epididymal
sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol. Appl.
Pharmacol. 131:108-118.
Green, S., Auletta, A., Fabricant, R., Kapp, M., Sheu, C., Springer, J., Whitfield, B. (1985) Current status of
bioassays in genetic toxicology: the dominant lethal test. Mutat. Res. 154:49-67.
Greenland, S. (1987) Quantitative methods in the review of epidemiologic literature. Epidemiol. Rev. 9:1-30.
Gulati, O.K., Hope, E., Teague, J., Chapin, R.E. (1991) Reproductive toxicity assessment by continuous breeding in
Sprague-Dawley rats: a comparison of two study designs. Fundam. Appl. Toxicol. 17:270-279.
Gustafsson, J.-A., Mode, A., Norstedt, G., Hokfelt, T., Sonnenschein, C., Eneroth, P., Skett, P. (1980) The
hypothalamo-pituitary-liver axis: a new hormonal system in control of hepatic steroid and drug metabolism.
Biochem. Act. Hormones 14:47-89.
Habicht, F.H. (1992) Guidance on risk characterization for risk managers and risk assessors. U.S. EPA,
Memorandum to Assistant Administrators and Regional Administrators, February 26, 1992. Available from the EPA
Air docket.
Hales, B., Crosman, K., Robaire, B. (1992) Increased post-implantation loss and malformations among the F2
progeny of male rats chronically treated with cyclophosphamide. Teratology 45:671-678.
Harris, M.W., Chapin, R.E., Lockhart, A.C., Jokinen, M.P., Allen, J.D., Haskins, E.A. (1992) Assessment of a short-
term reproductive and developmental toxicity screen. Fundam. Appl. Toxicol. 19:186-196.
Harris, R.Z., Benet, L.Z., Schwartz, J.B. (1995) Gender effects in pharmacokinetics and pharmacodynamics. Drugs
50:222-239.
Harrison, P.T.C., Humfrey, C.D.N., Litchfield, M., Peakall, D., Shuker, L.K. (1995) IEH Assessment on
Environmental Oestrogens: Consequences to Human Health and Wildlife. MRC Institute for Environment and
Health. Leicester, UK.
Haschek, W.M., Rousseaux, C.G. (1991) Handbook of Toxicologic Pathology. Academic Press, New York.
Hatch, M., Kline, J. (1981) Spontaneous abortion and exposure to the herbicide 2,4,5-T: a pilot study. U.S.
Environmental Protection Agency, Washington, D.C. EPA-560/6-81-006.
99
-------
Hattis, D., Silver, K. (1994) Human interindividual variability: a major source of uncertainty in assessing risks for
noncancer health effects. Risk Analysis 14:421-431.
Heindel, J.J., Chapin, R.E. (1993) Methods in Toxicology: Female Reproductive Toxicology. Academic Press, San
Diego.
Heindel, J.J., Thomford, P.J., Mattison, D.R. (1989) Histological assessment of ovarian follicle number in mice as a
screen of ovarian toxicity. In: Hirshfield, A.N. Growth Factors and the Ovary. Plenum Press, New York. pp. 421-
426.
Hemminki, K., Vineis, P. (1985) Extrapolation of the evidence on teratogenicity of chemicals between humans and
experimental animals: chemicals other than drugs. Teratogenesis Carcinog. Mutagen. 5:251-318.
Hemminki, K., Mutanen, P., Luoma, K., Saloniemi, I. (1980) Congenital malformations by the parental occupation
in Finland. Int. Arch. Occup. Environ. Health 46:93-98.
Hemminki, K., Saloniemi, I., Salonen, T. (1981) Childhood cancer and paternal occupation in Finland. J.
Epidemiol. Community Health 35:11-15.
Hertig, A.T. (1967) The overall problem in man. In: Benirschke, K. Comparative Aspects of Reproductive Failure.
Springer-Verlag, New York. pp. 11-41.
Hervey, E., Hervey, G.R. (1967) The effects of progesterone on body weight and composition in the rat. J.
Endocrinol. 37:361-384.
Hess, R.A. (1990) Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat
seminiferous epithelium: light microscopic observations of perfusion-flxed and plastic-embedded testes. Biol.
Reprod. 43:525-542.
Hess, R.A., Moore, B.J. (1993) Histological methods for evaluation of the testis. In: Chapin, R.E., Heindel, J.J.
Methods in Toxicology: Male Reproductive Toxicology. Academic Press, San Diego, pp. 52-85.
Hess, R.A., Moore, B.J., Forrer, J., Linder, R.E., Abuel-Atta, A.A. (1991) The fungicide Benomyl (methyl 1-
(butylcarbamoyl)-2-benzimidazolecarbamate) causes testicular dysfunction by inducing the sloughing of germ cells
and occlusion of efferent ductules. Fundam. Appl. Toxicol. 17:733-745.
Heywood, R., James, R.W. (1985) Current laboratory approaches for assessing male reproductive toxicity. In:
Dixon, R.L. Reproductive Toxicology. Raven Press, New York. pp. 147-160.
Hogue, C.J.R. (1984) Reducing misclassification errors through questionnaire design. In: Lockey, J.E., Lemasters,
O.K., Keye, W.R. Reproduction: the new frontier in occupational and environmental health research. Alan R. Liss,
Inc., New York. pp. 81-97.
Holloway, A.J., Moore, H.D.M., Foster, P.M.D. (1990a) The use of in vitro fertilization to detect reductions in the
fertility of male rats exposed to 1,3-dinitrobenzene. Fundam. Appl. Toxicol. 14:113-122.
Holloway, A.J., Moore, H.D.M., Foster, P.M.D. (1990b) The use of rat in vitro fertilization to detect reductions in
the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether. Reprod. Toxicol. 4:21-27.
Holmes, R.L., Ball, J.N. (1974) The Pituitary Gland: A Comparative Account. Cambridge University Press,
Cambridge.
Huang, H.H., Meites, J. (1975) Reproductive capacity of aging female rats. Neuroendocrinology 17:289-295.
100
-------
Hugenholtz, A.P., Bruce, W.R. (1983) Radiation induction of mutations affecting sperm morphology in mice.
Mutat. Res. 107:177-185.
Hughes, C.L. (1988) Phytochemical mimicry of reproductive hormones and modulation of herbivore fertility by
phytoestrogens. Environ Health Perspect. 78:171-175.
Hurtt, M.E., Zenick, H. (1986) Decreasing epididymal sperm reserves enhances the detection of ethoxyethanol-
induced spermatotoxicity. Fundam. Appl. Toxicol. 7:348-353.
Imagawa, W., Yang, 1, Guzman, R., Nandi, S. (1994) Control of mammary gland development. In: Knobil, E.,
O'Neill, J.D. The Physiology of Reproduction. Raven Press, New York. pp. 1033-1063.
International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human UseTt994)
ICH harmonized tripartite guideline, Detection of Toxicity to Reproduction for Medicinal Products. Federal/
Register 59(1831):48746-48752.
Irvine, S., Cawood, E., Richardson, D., MacDonald, E., Aitken, J. (1996) Evidence of deteriorating semen quality in
the United Kingdom: birth cohort study in 577 men in Scotland over 11 years. Br. Med. J. 312:467-471.
Joffe, M. (1985) Biases in research on reproduction and women's work. Int. J. Epidemiol. 14:118-123.
Jones, T.C., Mohr, U, Hunt, R.D. (1987) Genital System. Springer-Verlag, New York.
Katz, D.F., Overstreet, J.W. (1981) Sperm motility assessment by videomicrography. Fertil. Steril. 35:188-193.
Katz, D.F., Diel, L., Overstreet, J.W. (1982) Differences in the movement of morphologically normal and abnormal
human seminal spermatozoa. Biol. Reprod. 26:566-570.
Kavlock, R.J., Allen, B.C., Kimmel, C.A., Faustman, E.M. (1995) Dose-response assessment for developmental
toxicology: benchmark doses for fetal weight changes. Fundam. Appl. Toxicol. 26:211-222.
Kelce, W.R., Stone, C.R., Laws, S.C., Gray, L.E., Kemppainen, J.A., Wilson, E.M. (1995) Persistent DDT
metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature 375:581-585.
Kesner, J.S., Wright, D.M., Schrader, S.M., Chin, N.W, Krieg, E.F. (1992) Methods of monitoring menstrual
function in field studies: Efficacy of methods. Reprod. Toxicol. 6:385-400.
Kimmel, C.A., Francis, E.Z. (1990) Proceedings of the workshop on the acceptability and interpretation of dermal
developmental toxicity studies. Fundam. Appl. Toxicol. 14:386-398.
Kimmel, C.A., Gaylor, D.W. (1988) Issues in qualitative and quantitative risk analysis for developmental toxicology.
Risk Analysis 8:15-20.
Kimmel, C.A., Holson, J.F., Hogue, C.J., Carlo, G.L. (1984) Reliability of experimental studies for predicting
hazards to human development. National Center for Toxicological Research, Jefferson, AR. NCTR Technical
Report for Experiment No. 6015.
Kimmel, C.A., Kimmel, G.L., Frankos, V. (1986) Interagency Regulatory Liaison Group workshop on reproductive
toxicity risk assessment. Environ. Health 66:193-221.
Kimmel, C.A., Rees, D.C., Francis, E.Z. (1990) Proceedings of the workshop on the qualitative and quantitative
comparability of human and animal developmental neurotoxicity. Neurotoxicol. Teratol. 12:173-292.
Kimmel, G.L., Clegg, E.D., Crisp, T.M. (1995) Reproductive toxicity testing: a risk assessment perspective. In:
Witorsch, R.J. Reproductive Toxicology. Raven Press, New York. pp. 75-98.
101
-------
Kissling, G. (1981) A generalized model for analysis of nonindependent observations. Dissertation. University of
North Carolina.
Kleinbaum, D.G., Kupper, L.L., Morgenstern, H. (1982) Epidemiologic Research: Principle and Quantitative
Methods. Lifetime Learning Publications, London.
Kline, J., Stein, Z., Susser, M. (1989) Conception to Birth: Epidemiology of Prenatal Development. Oxford
University Press, New York.
Klinefelter, G.R., Laskey, J.W., Kelce, W.R., Ferrell, J., Roberts, N.L., Suarez, J.D., Slott, V. (1994a)
Chloroethylmethanesulfonate-induced effects on the epididymis seem unrelated to altered Leydig cell function. Biol.
Reprod. 51:82-91.
Klinefelter, G.R., Laskey, J.W., Perreault, S.D., Ferrell, J, Jeffay, S., Suarez, J., Roberts, N. (1994b) The ethane
dimethanesulfonate-induced decrease in the fertilizing ability of cauda epididymal sperm is independent of the testis.
J. Androl. 15:318-327.
Knobil, E., Neill, J.D., Greenwald, G.S., Markert, C.L., Pfaff, D.W. (1994) The Physiology of Reproduction. Raven
Press, New York.
Ku, W.W., Chapin, R.E., Wine, R.N., Gladen, B.C. (1993) Testicular toxicity of boric acid (BA): relationship of
dose to lesion development and recovery in the F344 rat. Reprod. Toxicol. 7:305-319.
Kupfer, D. (1987) Critical evaluation of methods for detection and assessment of estrogenic compounds in
mammals: strengths and limitations for application to risk assessment. Reprod. Toxicol. 2:147-153.
Kurman, R., Norris, H.J. (1978) Germ cell tumors of the ovary. Pathol. Annu. 13:291.
Kwa, S.L., Fine, L.J. (1980) The association between parental occupation and childhood malignancy. J. Occup.
Med. 22:792-794.
La Bella, F.S., Dular, R., Lemons, P., Vivian, S., Queen, M. (1973a) Prolactin secretion is specifically inhibited by
nickel. Nature 245:330-332.
La Bella, F.S., Dular, R., Vivian, S., Queen, G. (1973b) Pituitary hormone releasing activity of metal ions present in
hypothalamic extracts. Biochem. Biophys. Res. Commun. 52:786-791.
Lamb, J.C. (1985) Reproductive toxicity testing: evaluating and developing new testing systems. J. Am. Coll.
Toxicol. 4:163-171.
Lamb, J.C., Chapin, R.E. (1985) Experimental models of male reproductive toxicology. In: Thomas, J.A., Korach,
K.S., McLachlan, J.A. Endocrine Toxicology. Raven Press, New York. pp. 85-115.
Lamb, J.C., Foster, P.M.D. (1988) Physiology and Toxicology of Male Reproduction. Academic Press, New York.
Lamb, J.C., Jameson, C.W., Choudhury, H., Gulati, D.K. (1985) Fertility assessment by continuous breeding:
evaluation of diethylstilbestroi and a comparision of results from two laboratories. J. Am. Coll. Toxicol. 4:173-183.
Langley, F.A., Fox, H. (1987) Ovarian tumors. Classification, histogenesis, etiology. In: Fox, H. Haines and
Taylor's Obstetrical and Gynaecologic Pathology. Churchill Livingstone, Edinburgh, pp. 542-555.
Lantz, G.D., Cunningham, G.R., Huckins, C, Lipshultz, L.I. (1981) Recovery from severe oligospermia after
exposure to dibromochloropropane. Fertil. Steril. 35:46-53.
102
-------
LeFevre, J., McClintock, M.K. (1988) Reproductive senescence in female rats: a longitudinal study of individual
differences in estrous cycles and behavior. Biol. Reprod. 38:780-789.
Lemasters, O.K. (1992) Occupational exposures and effects on male and female reproduction. In: Rom, W.N.
Environmental and Occupational Medicine. Little, Brown, Boston, MA. pp. 147-170.
Lemasters, O.K., Pinney, S.M. (1989) Employment status as a confounder when assessing occupational exposures
and spontaneous abortion. J. Clin. Epidemiol. 42:975-981.
Lemasters, O.K., Selevan, S.G. (1984) Use of exposure data in occupational reproductive studies. Scan. J. Work.
Environ. Health 10:1-6.
Lemasters, G.K., Selevan, S.G. (1993) Toxic exposures and reproduction: a view of epidemiology and surveillance.
In: Scialli, A.R., Zinaman, M.J. Reproductive Toxicology and Infertility. McGraw-Hill, New York. pp. 307-321.
Leridon, H. (1977) Human Fertility: The Basic Components. The University of Chicago Press, Chicago.
Le Vier, R.R., Jankowiak, M.E. (1972) The hormonal and antifertility activity of 2,6-cis-
diphenylhexamethylcyclotetra-siloxane in the female rat. Biol. Reprod. 7:260-266.
Levine, R.J. (1983) Methods for detecting occupational causes of male infertility: reproductive history versus semen
analysis. Scand. J. Work Environ. Health 9:371-376.
Levine, R.J., Symons, M.J., Balogh, S.A., Arndt, D.M., Kaswandik, N.R., Gentile, J.W. (1980) A method for
monitoring the fertility of workers: I. Method and pilot studies. J. Occup. Med. 22:781-791.
Levine, R.J., Symons, M.J., Balogh, S.A., Milby, T.H., Whorton, M.D. (1981) A method for monitoring the fertility
of workers: II. Validation of the method among workers exposed to dibromochloropropane. J. Occup. Med. 23:183-
188.
Levine, R.J., Blunden, P.B., DalCorso, R.D., Starr, T.B., Ross, C.E. (1983) Superiority of reproductive histories to
sperm counts in detecting infertility at a dibromochloropropane manufacturing plant. J. Occup. Med. 25:591-597.
Lewis, J.R. (1991) Reproductively Active Chemicals: A Reference Guide. Van Nostrand Reinhold, New York.
Lindbohm, M.L., Hemminki, K., Bonhomme, M.G., Anttila, A., Rantala, K., Keikkila, P., Rosenberg, M.J. (1991)
Effects of paternal occupational exposure on spontaneous abortions. Am. J. Public Health 81:1029-1033.
Linder, R.E., Hess, R.A., Strader, L.F. (1986) Testicular toxicity and infertility in male rats treated with 1,3-
dinitrobenzene. J. Toxicol. Environ. Health 19:477-489.
Linder, R.E., Strader, L.F., Barbee, R.R., Rehnberg, G.L., Perreault, S.D. (1990) Reproductive toxicity of a single
dose of 1,3-dinitrobenzene in two ages of young adult male rats. Fundam. Appl. Toxicol. 14:284-298.
Linder, R.E., Strader, L.F., Slott, V.L., Suarez, J.D. (1992) Endpoints of spermatotoxicity in the rat after short
duration exposures to fourteen reproductive toxicants. Reprod. Toxicol. 6:491-505.
Lipshultz, L.I., Ross, C.E., Whorton, D., Thomas, M., Smith, R., Joyner, R.E. (1980) Dibromochloropropane and its
effect on testicular function in man. J. Urol. 124:464-468.
Liu, D.Y., Baker, H.W.G. (1992) Tests of human sperm function and fertilization in vitro. Fertil. Steril. 58:465-483.
Loehr, R.A., Matanoski, G.M. (1993) Letter to Carol M. Browner, EPA Administrator, re: quantitative uncertainty
analysis for radiological assessments. U.S. EPA Science Advisory Board, July 23, 1993 (EPA-SAB-RAC-COM-93-
006).
103
-------
Long, J.A., Evans, H.M. (1922) The oestrous cycle in the rat and its associated phenomena. Mem. Univ. Calif. 6:1-
111.
Mackeprang, ML, Hay, S., Lunde, A.S. (1972) Completeness and accuracy of reporting of malformations on birth
certificates. HSMHA Health Reports 84:43-49.
Manson, J.M. (1994) Testing of pharmaceutical agents for reproductive toxicity. In: Kimmel, C.A., Buelke-Sam, J.
Developmental Toxicology. Raven Press, New York. p. 379.
Manson, J.M., Kang, Y.J. (1994) Test methods for assessing female reproductive and developmental toxicology. In:
Hayes, A.W. Principles and Methods of Toxicology. Raven Press, New York. pp. 989-1037.
Mason, H.J. (1990) Occupational cadmium exposure and testicular endocrine function. Hum. Exp. Toxicol. 9:91-94.
Martison, D.R. (1985) Clinical manifestations of ovarian toxicity. In: Dixon, R.L. Reproductive Toxicology. Raven
Press, New York. pp. 109-130.
Martison, D.R., Nightingale, M.R. (1980) The biochemical and genetic characteristics of murine ovarian aryl
hydrocarbon (benzo(a)pyrene) hydroxylase activity and its relationship to primary oocyte destruction by polycyclic
aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 56:399-408.
Martison, D.R., Thomford, P.J. (1989) The mechanisms of action of reproductive toxicants. Toxicol. Pathol. 17:364-
376.
Martison, D.R., Thorgeirsson, S.S. (1978) Gonadal aryl hydrocarbon hydroxylase in rats and mice. Cancer Res.
38:1368-1373.
McDonald, A.D., McDonald, J.C., Armstrong, B., Cherry, N.M., Nolin, A.D., Robert, D. (1989) Father's occupation
and pregnancy outcome. Br. J, Ind. Med. 46:329-333.
McGregor, A.J., Mason, H.J. (1991) Occupational mercury vapour exposure and testicular, pituitary and thyroid
endocrine function. Hum. Exp. Toxicol. 10:199-203.
McKinney, J.D., Waller, C.L. (1994) Polychlorinated biphenyls as hormonally active structural analogues. Environ.
Health Perspect. 102:290-297.
McLachlan, J.A. (1980) Estrogens in the Environment. Elsevier North Holland, New York.
McMichael, A.J. (1976) Standardized mortality ratios and the healthy worker effect: scratching beneath the surface.
J. Occup. Med. 18:165-168.
McNatty, K.P. (1979) Follicular determinants of corpus luteum function in the human ovary. Adv. Exp. Med. Biol.
112:465-481.
Meistrich, M.L. (1982) Quantitative correlation between testicular stem cell survival, sperm production, and fertility
in the mouse after treatment with different cytotoxic agents. J. Androl. 3:58-68.
Meistrich, M.L. (1986) Critical components of testicular function and sensitivity to disruption. Biol. Reprod. 34:17-
28.
Meistrich, M.L., Brown, C.C. (1983) Estimation of the increased risk of human infertility from alterations in semen
characteristics. Fertil. Steril. 40:220-230.
Meistrich, M.L., Samuels, R.C. (1985) Reduction in sperm levels after testicular irradiation of the mouse: a
comparison with man. Radial. Res. 102:138-147.
104
-------
Meistrich, M.L., van Beek, M.E.A.B. (1993) Spermatogonial stem cells: assessing their survival and ability to
produce differentiated cells. In: Chapin, R.E., Heindel, J.J. Methods in Toxicology: Male Reproductive
Toxicology. Academic Press, San Diego, pp. 106-123.
Meyer, C.R. (1981) Semen quality in workers exposed to carbon disulfide compared to a control group from the
same plant J. Occup. Med. 23:435-439.
Milby, T.H., Whorton, D. (1980) Epidemiological assessment of occupationally related chemically induced sperm
count suppression. J. Occup. Med. 22:77-82.
Milby, T.H., Whorton, M.D., Stubbs, H.A., Ross, C.E., Joyner, R.E., Lipshultz, L.I. (1981) Testicular function
among epichlorohydrin workers. Br. J. Ind. Med. 38:372-377.
Morris, I.D., Bardin, C.W., Gunsalus, G., Ward, J.A. (1990) Prolonged suppression of spermatogenesis by oestrogen
does not preserve the seminiferous epithelium in procarbazine-treated rats. Int. J. Androl. 13:180-189.
Morrissey, R.E., Lamb, J.C., Schwetz, B.A., Teague, J.L., Morris, R.W. (1988a) Association of sperm, vaginal
cytology, and reproductive organ weight data with results of continuous breeding reproduction studies in Swiss (CD-
1) mice. Fundam. Appl. Toxicol. 11:359-371.
Morrissey, R.E., Schwetz, B.A., Lamb, J'.C., Ross, M.D., Teague, J.L., Morris, R.W. (1988b) Evaluation of rodent
sperm, vaginal cytology, and reproductive organ weight data from National Toxicology Program 13-week studies.
Fundam. Appl. Toxicol. 11:343-358.
Morrissey, R.E., Lamb, J.C., Morris, R.W., Chapin, R.E., Gulati, O.K., Heindel, J.J. (1989) Results and evaluations
of 48 continuous breeding reproduction studies conducted in mice. Fundam. Appl. Toxicol. 13:747-777.
Mosher, W.D., Pratt, W.F. (1990) Fecundity and infertility in the United States, 1965-88. Report 192, National
Center for Health Statistics, Hyattsville, MD.
Mukhtar, H., Philpot, R.M., Lee, I.P., Bend, J.R. (1978) Developmental aspects of epoxide-metabolizing enzyme
activities in adrenals, ovaries, and testes of the rat. In: Mahlum, D.D., Sikov, M.R., Hackett, P.L., Andrew, F.D.
Developmental Toxicology of Energy Related Pollutants. Technical Information Center, U.S. Department of
Energy, Springfield, VA. pp. 89-104.
Na, J.Y., Garza, F., Terranova, P.P. (1985) Alterations in follicular fluid steroids and follicular hCG and FSH
binding during atresia in hamsters. Proc. Soc. Exp. Biol. Med. 179:123-127.
Nakai, M., Moore, B.J., Hess, R.A. (1993) Epithelial reorganization and irregular growth following carbendazim-
induced injury of the efferent ductules of the rat testis. Anat. Rec. 235:51-60.
National Research Council (1977) Reproduction and teratogenicity tests. In: Principles and Procedures for
Evaluating the Toxicity of Household Substances. National Academy Press, Washington, DC.
National Research Council. (1983) Risk Assessment in the Federal Government: Managing the Process. National
Academy Press, Washington, DC.
National Research Council. (1989) Biologic Markers in Reproductive Toxicity. National Academy Press,
Washington, DC.
National Research Council. (1994) Science and Judgment in Risk Assessment. National Academy Press,
Washington, DC.
Nestor, A., Handel, M.A. (1984) The transport of morphologically abnormal sperm in the female reproductive tract
ofmice. Gamete Res. 10:119-125.
105
-------
Nett, T.M. (1989) Hormonal evaluation of testicular function: species variation. J. Am. Coll. Toxicol. 8:539-549.
Nisbet, I.C.T., Karen, N.J. (1983) Chemical hazards to human reproduction, Park Ridge, N.J., Noyes Data Corp.
Oberlander, G., Yeung, C.H., Cooper, T.G. (1994) Induction of reversible infertility in male rats by oral ornidazole
and its effects on sperm motility and epididymal secretions. J. Reprod. Fertil. 100:551-559.
Organization for Economic Cooperation and Development. (1983) First addendum to OECD guideline 415 for
testing of chemicals, "One-Generation Rreproduction Toxicity." OECD, Paris, pp. 1-8.
Organization for Economic Cooperation and Development. (1993a) Draft guidelines for testing chemicals: combined
repeated dose toxicity study with the reproduction/developmental toxicity screening test. #422. OECD, Paris.
Organization for Economic Cooperation and Development. (1993b) First amendment to OECD guidelines 416,
"Two Generation Reproduction Toxicity". OECD, Paris, pp. 1-8.
Oskarsson, A., Hallen, I.P., Sundberg, J. (1995) Exposure to toxic elements via breast milk. Analyst 120:765-770.
Pang, C.N., Zimmerman, E., Sawyer, C.H. (1977) Morphine inhibition of preovulatory surges of plasma luteinizing
hormone and follicle stimulating hormone in the rat. Endocrinology 101:1726-1732.
Papier, C.M. (1985) Parental occupation and congenital malformations in a series of 35,000 births in Israel. Prog.
Clin. Biol. Res. 163:291-294.
Paul, M. (1993) Occupational and Environmental Reproductive Hazards. Williams and Wilkins, Baltimore.
Paulsen, C.A., Berman, N.G., Wang, C. (1996) Data from men in greater Seattle area reveals no downward trend in
semen quality: further evidence that deterioration of semen quality is not geographically uniform. Fertil. Steril.
65:1015-1020.
Peluso, J.J., Bolender, D.L., Perri, A. (1979) Temporal changes associated with the degeneration of the rat oocyte.
Biol. Reprod. 20:423-430.
Perreault, S.D. (1989) Impaired gamete function: implications for reproductive toxicology. In: Working, P.K.
Toxicology of the Male and Female Reproductive Systems. Hemisphere, New York. pp. 217-229.
Perreault, S.D., Jeffay, S.C. (1993) Strategies and methods for the functional evaluation of oocytes and zygotes. In:
Heindel, J.J., Chapin, R.E. Methods in Toxicology: Female Reproductive Toxicology. Academic Press, San Diego.
pp. 92-109.
Perreault, S.D., Jeffay, S., Poss, P., Laskey, J.W. (1992) Use of the fungicide carbendazim as a model compound to
determine the impact of acute chemical exposure during oocyte maturation and fertilization on pregnancy outcome in
the hamster. Toxicol. Appl. Pharmacol. 114:225-231.
Peters, J.M., Preston-Martin, S., Yu, M.C. (1981) Brain tumors in children and occupational exposure of the parents.
Science 213:235-237.
Plowchalk, D.R., Smith, B.J., Mattison, D.R. (1993) Assessment of toxicity to the ovary using follicle quantitation
and morphometrics. In: Heindel, J.J., Chapin, R.E. Methods in Toxicology: Female Reproductive Toxicology.
Academic Press, San Diego, pp. 57-68.
Qiu, J., Hales, B.F., Robaire, B. (1995) Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure.
Biol. Reprod. 53:1465-1473.
106
-------
Ratcliffe, J.M., Clapp, D.E., Schrader, S.M., Turner, T.W., Oser, J., Tanaka, S., Hornung, R.W., Halperin, W.E.
(1986) Semen quality in 2-ethoxyethanol-exposed workers. Health Hazard evaluation report, HETA 84-415-1688.
Department of Health and Human Services, National Institute for Occupational Safety and Health, Cincinnati, Ohio.
Ratcliffe, J.M., Schrader, S.M., Steenland, K., Clapp, D.E., Turner, T., Hornung, R.W. (1987) Semen quality in
papaya workers with long term exposure to ethylene dibromide. Br. J. Ind. Med. 44:317-326.
Ratcliffe, J.M., Schrader, S.M., Clapp, D.E., Halperin, W.E., Turner, T.W., Horning, R.W. (1989) Semen quality in
workers exposed to 2-ethoxyethanol. Br. J. Ind. Med. 46:399-406.
Redi, C.A., Garagna, S., Pellicciari, C., Manfredi-Romanini, M.G., Capanna, E., Winking, H., Gropp, A. (1984)
Spermatozoa of chromosomally heterozygous mice and their fate in male and female genital tracts. Gamete Res.
9:273-286.
Rier, S.E., Martin, D.C., Bowman, R.E., Dmowski, W.P., Becker, J.L. (1993) Endometriosis in rhesus monkeys
(Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxicol. 21:
433-441.
Robaire, B., Smith, S., Hales, B.F. (1984) Suppression of spermatogenesis by testosterone in adult male rats: effect
on fertility, pregnancy outcome and progeny. Biol. Reprod. 31:221-230.
Rosenberg, M.J., Wyrobeck, A.J., Ratcliffe, J., Gordon, L.A., Watchmaker, G., Fox, S.H., Moore, D.H. (1985)
Sperm as an indicator of reproductive risk among petroleum refinery workers. Br. J. Ind. Med. 42:123-127.
Rothman, K.J. (1986) Modern epidemiology. Little, Brown, Boston.
Rowland, A.S., Baird, D.D., Weinberg, C.R., Shore, D.L., Shy, C.M., Wilcox, A.J. (1992) Reduced fertility among
women employed as dental assistants exposed to high levels of nitrous oxide. N. Engl. J. Med. 327:993-997.
Rubin, H.B., Henson, D.E. (1979) Effects of drugs on male sexual function. In: Advances in Behavioral
Pharmacology. Academic Press, New York. pp. 65-86.
Russell, L.D. (1983) Normal testicular structure and methods of evaluation under experimental and disruptive
conditions. In: Clarkson, T.W., Nordberg, G.F., Sager, P.R. Reproductive and Developmental Toxicity of Metals.
Plenum Publishing Co., New York. pp. 227-252.
Russell, L.D., Malone, J.P., McCurdy, D.S. (1981) Effect of microtubule disrupting agents, colchicine and
vinblastine, on seminiferous tubule structure in the rat. Tissue Cell 13:349-367.
Russell, L.D., Ettlin, R., Sinha Hikim, A.P., Clegg, E.D. (1990) Histological and Histopathological Evaluation of the
Testis. Cache River Press, Clearwater, FL.
Safe, S.H. (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-
dioxin and related compounds. Pharmacol. Ther. 67:247-281.
Sakai, C.N., Hodgen, G.D. (1987) Use of primate folliculogenesis models in understanding human reproductive
biology and applicablity to toxicology. Reprod. Toxicol. 1:207-222.
Samuels, S.J. (1988) Lessons from a surveillance program of semen quality. Reprod. Toxicol. 2:229-231.
Savitz, D.A., Harlow, S.D. (1991) Selection of reproductive health end points for environmental risk assessment.
Environ. Health 90:159-164.
Savitz, D.A., Sonnenfeld, N.L., Olshan, A.F. (1994) Review of epidemiologic studies of paternal occupational
exposure and spontaneous abortion. Am. J. Ind. Med. 25:361-383.
107
-------
Scala, R.A., Bevan, C., Beyer, B.K. (1992) An abbreviated repeat dose and reproductive/developmental toxicity test
for high production volume chemicals. Regul. Toxicol. Pharmacol. 16:73-80.
Schardein, J.L. (1993) Chemically Induced Birth Defects. Marcel Dekker, New York.
Schrader, S.M., Chapin, R.E., Clegg, E.D., Davis, R.O., Fourcroy, J.L., Katz, D.F., Rothmann, S.A., Toth, G.,
Turner, T.W., Zinaman, M. (1992) Laboratory methods for assessing human semen in epidemiologic studies: a
consensus report. Reprod. Toxicol. 6:275-279.
Schrag, S.D., Dixon, R.L. (1985a) Occupational exposures associated with male reproductive dysfunction. Ann.
Rev. Pharmacol. Toxicol. 25:567-592.
Schrag, S.D., Dixon, R.L. (1985b) Reproductive effects of chemical agents. In: Dixon, R.L. Reproductive
Toxicology. Raven Press, New York. pp. 301-319.
Schwetz, B.A., Rao, K.S., Park, C.N. (1980) Insensitivity of tests for reproductive problems. J. Environ. Pathol.
Toxicol. 3:81-98.
Scialli, A.R., Clegg, E.D. (1992) Reversibility in Testicular Toxicity Assessment. CRC Press, Boca Raton.
Scommegna, A., Vorys, N., Givens, J.R. (1980) Menstrual dysfunction. In: Gold, J.J., Josimovich, J.B.
Gynecologic Endocrinology. Harper and Row, Hagerstown, MD.
Seed, J., Chapin, R.E., Clegg, E.D., Darney, S.P., Dostal, L., Foote, R.H., Hurtt, M.E., Klinefelter, G.R., Makris,
S.L., Schrader, S., Seyler, D., Sprando, R., Treinen, K.A., Veeranachaneni, R., Wise, L.D. (1996) Methods for
assessing sperm motility, morphology, and counts in the rat, rabbit and dog: a consensus report. Reprod. Toxicol.
10:237-244.
Selevan, S.G. (1980) Evaluation of data sources for occupational pregnancy outcome studies. Thesis. University of
Cincinnati.
Selevan, S.G. (1981) Design considerations in pregnancy outcome studies of occupational populations. Scand. J.
Work Environ. Health 7:76-82.
Selevan, S.G. (1985) Design of pregnancy outcome studies of industrial exposure. In: Hemminki, K., Sorsa, M.,
Vainio, H. Occupational Hazards and Reproduction. Hemisphere, Washington, DC. pp. 219-229.
Selevan, S.G. (1991) Environmental exposures and reproduction. In: Keily, M. Reproductive and Perinatal
Epidemiology. CRC Press, Boca Raton, pp. 115-130.
Selevan, S.G., Lemasters, O.K. (1987) The dose response fallacy in human reproductive studies of toxic exposure. J.
Occup. Med. 29:451-454.
Selevan, S.G., Edwards, B., Samuels, S. (1982) Interview data from both parents on pregnancies and occupational
exposures. How do they compare? Am. J. Epidemiol. 116:583.
Sever, L.E., Hessol, N.A. (1984) Overall design considerations in male and female occupational reproductive
studies. In: Lockey, I.E., Lemasters, G.K., Keye, W.R. Reproduction: The New Frontier in Occupational and
Environmental Research. Alan R. Liss, Inc., New York. pp. 15-48.
Sharpe, R.M. (1994) Regulation of spermatogenesis. In: Knobil, E., Neill, J.D. The Physiology of Reproduction.
Raven Press, New York. pp. 1363-1434.
108
-------
Sheehan, D.M., Young, J.F., Slikker, W., Gaylor, D.W., Mattison, D.R. (1989) Workshop on risk assessment in
reproductive and developmental toxicology: addressing the assumptions and identifying the research needs. Regul.
Toxicol. Pharmacol. 10:110-122.
Shepard, T.H. (1986) Human teratogenicity. Adv. Pediatrics 33:225-268.
Silverman, J., Kline, J., Hutzler, M. (1985) Maternal employment and the chromosomal characteristics of
spontaneously aborted conceptions. J. Occup. Med. 27:427-438.
Skett, P. (1988) Biochemical basis of sex differences in drug metabolism. Pharmacol. Then 38:269-304.
Slott, V.L., Perreault, S.D. (1993) Computer-assisted sperm analysis of rodent epididymal sperm motility using the
Hamilton-Thorne motility analyzer. In: Chapin, R.E., Heindel, J.J. Methods in Toxicology: Male Reproductive
Toxicology. Academic Press, San Diego, pp. 319-333.
Slott, V.L., Suarez, J.D., Simmons, J.E., Perreault, S.D. (1990) Acute inhalation exposure to epichlorohydrin
transiently decreases rat sperm velocity. Fundam. Appl. Toxicol. 15:597-606.
Slott, V.L., Suarez, J.D., Perreault, S.D. (1991) Rat sperm motility analysis: methodologic considerations. Reprod.
Toxicol. 5:449-458.
Slott, V.L., Jeffay, S.C., Suarez, J.D., Barbee, R.R., Perreault, S.D. (1995) Synchronous assessment of sperm
motility and fertilizing ability in the hamster following treatment with alpha-chlorohydrin. J. Androl. 16:523-535.
Smith, C.G. (1983) Reproductive toxicity: hypothalamic-pituitary mechanisms. Am. J. Ind. Med. 4:107-112.
Smith, C.G., Gilbeau, P.M. (1985) Drug abuse effects on reproductive hormones. In: Thomas, J.A., Korach, K.S.,
McLachlan, J.A. Endocrine Toxicology. Raven Press, New York. pp. 249-267.
Smith, E.R., Davidson, J.M. (1974) Luteinizing hormone releasing factor in rats exposed to constant light: effects of
mating. Neuroendocrinology 14:129-138.
Smith, S.K., Lenton, E.A., Landgren, B.M., Cooke, I.D. (1984) The short luteal phase and infertility. Br. J. Obstet.
Gynaecol. 91:1120-1122.
Snell, L.M., Little, B.B., Knoll, K.A., Johnston, W.L., et al. (1992) Reliability of birth certificate reporting of
congenital anomalies. Am. J. Perinatol. 9:219-222.
Sonawane, B.R. (1995) Chemical contaminants in human milk: an overview. Environ. Health. Perspect. 103:197-
205.
Sonawane, B.R., Yaffe, S.J. (1983) Delayed effects of drug exposure during pregnancy: reproductive function. Biol.
Res. Pregnancy 4:48-55.
Starr, T.B., Dalcorso, R.D., Levine, R.J. (1986) Fertility of workers: a comparision of logistic regression and indirect
standardization. Am. J. Epidemiol. 123:490-498.
Stein, A. and Hatch, M. (1987) Biological markers in reproductive epidemiology: prospects and precautions.
Environ. Health 74:67-75.
Stein, Z., Kline, J., Shrout, P. (1985) Power in surveillance. In: Hemminki, K., Sorsa, M., Vainio, H. Occupational
hazards and reproduction. Hemisphere, Washington, DC. pp. 203-208.
Steinberger, E., Lloyd, J.A. (1985) Chemicals affecting the development of reproductive capacity. In: Dixon, R.L.
Reproductive Toxicology. Raven Press, New York.
109
-------
Stevens, K.R., Gallo, M.A. (1989) Practical considerations in the conduct of chronic toxicity studies. In: Hayes,
A.W. Principles and Methods of Toxicology. Raven Press, New York. pp. 237-250.
Stiratelli, R., Laird, N., Ware, J.H. (1984) Random-effects models for serial observations with binary responses.
Biometrics 40:961-971.
Stoker, T.E., Goldman, J.M., Cooper, R.L. (1993) The dithiocarbamate fungicide thiram disrupts the hormonal
control of ovulation in the female rat. Reprod. Toxicol. 7:211-218.
Sweeney, A.M., Meyer, M.R., Aarons, J.H., Mills, J.L., LaPorte, R.E. (1988) Evaluation of methods for the
prospective identification of early fetal losses in environmental epidemiology studies. Am. J. Epidemiol. 127:843-
850.
Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., Imaida, K., Takahashi, M., Hayashi, Y.,
Kurokawa, Y., Tobe, M. (1992) Combined repeat dose and reproductive/developmental toxicity screening test
(OECD): familiarization using cyclophosphamide. Fundam. Appl. Toxicol. 18:89-95.
Terranova, P.P. (1980) Effects of phenobarbital-induced ovulatory delay on the follicular population and serum
levels of steroids and gonadotropins in the hamster: a model for atresia. Biol. Reprod. 23:92-99.
Thomas, J.A. (1981) Reproductive hazards and environmental chemicals: a review. Toxic Subst. J. 2:318-348.
Thomas, J.A. (1991) Toxic responses of the reproductive system. In: Amdur, M.O., Doull, J., Klaassen, C.D.
Casarett and Doull's Toxicology. Pergamon Press, New York. pp. 484-520.
Tilley, B.C., Barnes, A.B., Bergstrahl, E., Labarthe, D., Noller, K.L., Colton, T., Adam, E. (1985) A comparision of
pregnancy history recall and medical records: implications for retrospective studies. Am. J. Epidemiol. 121:269-
281.
Toppari, J., Larsen, J.C., Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L.J., Jegou, B., Jensen, T.K.,
Jouannet, P., Keiding, N., Leffers, H., McLachlan, J.A., Meyer, O., Muller, J., Rajpert-De Meyts, E., Scheike, T.,
Sharpe, R., Sumpter, J., Skakkebaek, N. (1995) Male Reproductive Health and Environmental Chemicals with
Estrogenic Effects. Miljoprojekt nr. 290. Danish Environmental Protection Agency.
Toth, G.P, Stober, J.A., Read, E.J., Zenick, H., Smith, M.K. (1989a) The automated analysis of rat sperm motility
following subchronic epichlorohydrin administration: methodologic and statistical considerations. J. Androl.
10:401-415.
Toth, G.P., Zenick, H., Smith, M.K. (1989b) Effects of epichlorohydrin on male and female reproduction in Long-
Evans rats. Fundam. Appl. Toxicol. 13:16-25.
Toth, G.P., Stober, J.A., George, E.L., Read, E.J., Smith, M.K. (1991a) Sources of variation in the computer-assisted
motion analysis of rat epididymal sperm. Reprod. Toxicol. 5:487-495.
Toth, G.P., Stober, J.A., Zenick, H., Read, E.J., Christ, S.A., Smith, M.K. (1991b) Correlation of sperm motion
parameters with fertility in rats treated subchronically with epichlorohydrin. J. Androl. 12:54-61.
Toth, G.P., Wang, S.R., McCarthy, H., Tocco, D.R., Smith, M.K. (1992) Effects of three male reproductive
toxicants on rat cauda epididymal sperm motion. Reprod. Toxicol. 6:507-515.
Treloar, A.E., Boynton, R.E., Borghild, G.B., Brown, B.W. (1967) Variation in the human menstrual cycle through
reproductive life. Int. J. Fertil. 12:77-126.
Tsai, S.P., Wen, C.P. (1986) A review of methodological issues of the standardized mortality ratio (SMR) in
occupational cohort studies. Int. J. Epidemiol. 15:8-21.
110
-------
Tucker, H.A. (1994) Lactation and its hormonal control. In: Knobil, E., O'Neill, J.D. The Physiology of
Reproduction. Raven Press, New York. pp. 1065-1098.
Tyl, R.W. (1987) Developmental toxicity in toxicologic research and testing. In: Ballantyne, B. Perspectives in
Basic and Applied Toxicology. John Wright, Bristol, pp. 203-208.
U.S. Congress. (1985) Reproductive Health Hazards in the Workplace. Office of Technology Assessment, OTA-
BA-266, U.S. Government Printing Office, Washington, DC.
U.S. Congress. (1988) Infertility: Medical and Social Choices. Office of Technology Assessment, OTA-BA-358,
U.S. Government Printing Office, Washington, DC.
U.S. Environmental Protection Agency. (1982) Reproductive and Fertility Effects. Pesticide Assessment Guidelines,
Subdivision F. Hazard Evaluation: Human and Domestic Animals. Office of Pesticides and Toxic Substances,
Washington, D.C. EPA-540/9-82-025.
U.S. Environmental Protection Agency. (1985a) Hazard Evaluation Division Standard Evaluation Procedure.
Teratology Studies. Office of Pesticide Programs, Washington, DC. pp. 22-23.
U.S. Environmental Protection Agency. (1985b) Toxic Substances Control Act Test Guidelines: Final Rules.
Federal Register 50 (188):39426-39436.
U.S. Environmental Protection Agency. (1986a) Guidelines for Carcinogen Risk Assessment. Federal Register.
51(185):33992-34003.
U.S. Environmental Protection Agency. (1986b) Guidelines for Estimating Exposures. Federal Register
51(185):34042-34054.
U.S. Environmental Protection Agency. (1986c) Guidelines for Mutagenicity Risk Assessment. Federal Register
51(185):34006-34012.
U.S. Environmental Protection Agency. (1987) Reference Dose (RfD): Description and Use in Health Risk
Assessments. Integrated Risk Information System (IRIS): Appendix A. Integrated Risk Information System
Documentation, Vol. 1. EPA/600/8-66/032a.
U.S. Environmental Protection Agency. (1991) Guidelines for Developmental Toxicity Risk Assessment. Federal
Register 56(234):63798-63826.
U.S. Environmental Protection Agency. (1992) Guidelines for Exposure Assessment. Federal Register
57(104):22888-22938.
U.S. Environmental Protection Agency. (1995a) Proposed Guidelines for Neurotoxicity Risk Assessment. Federal
Register 60(192):52032-52056.
U.S. Environmental Protection Agency. (1995b) The Use of the Benchmark Dose Approach in Health Risk
Assessment. EPA/630/R-94/007.
U.S. Environmental Protection Agency. (1996a) Health Effects Test Guidelines OPPTS 870.3800: Reproduction and
Fertility Effects (Draft). Federal Register 61(43):8282-8283.
U.S. Environmental Protection Agency. (1996b) Proposed Guidelines for Carcinogen Risk Assessment. Federal
Register 61 (79): 17960-18011.
U.S. Environmental Protection Agency. (1996c) Benchmark Dose Technical Guidance Document. EPA/600/P-
96/002A.
Ill
-------
Van Waeleghem, K., De Clerq, N., Vermeulen, L., Schoonjans, F., Comhaire, F. (1996) Deterioration of sperm
quality in young healthy Belgian men. Hum. Reprod. 11:325-329.
Vierula, M., Niemi, M, Keiski, A., Saarikoski, M., Suominen, J. (1996) High and unchanged sperm counts of
Finnish men. Int. J. Androl. 19:11-17.
Wade, G.N. (1972) Gonadal hormones and behavioral regulation of body weight. Physiol. Behav. 8:523-534.
Walker, R.F. (1986) Age factors potentiating drug toxicity in the reproductive axis. Environ. Health 70:185-191.
Walker, R.F., Schwartz, L.W., Manson, J.M. (1988) Ovarian effects of an anti-infiammatory-immunomodulatory
drug in the rat. Toxicol. Appl. Pharmacol. 94:266-275.
Waller, D.P., Killinger, J.M., Zaneveld, L.J.D. (1985) Physiology and toxicology of the male reproductive tract. In:
Thomas, J.A., Korach, K.S., McLachlan, J.A. Endocrine Toxicology. Raven Press, New York. pp. 269-333.
Wang, G.H. (1923) The relation between the "spontaneous" activity and the oestrous cycle in the white rat. Comp.
Psychol. Monographs 2:1-27.
Warren, J.C., Cheatum, S.G., Greenwald, G.S., Barker, K.L. (1967) Cyclic variation of uterine metabolic activity in
the golden hamster. Endocrinology. 80:714-718.
Weinberg, G.R., Gladen, B.C. (1986) The beta-geometric distribution applied to comparative fecundability studies.
Biometrics 42:547-560.
Weinberg, C.R., Baird, D.D., Wilcox, A.J. (1994) Sources of bias in studies of time to pregnancy. Stat. Med.
13:671-681.
Weir, P.J., Rumberger, D. (1995) Isolation of rat sperm from the vas deferens for sperm motion analysis. Reprod.
Toxicol. 9:327-330.
Welch, L.S., Schrader, S.M., Turner, T.W., Cullen, M.R. (1988) Effects of exposure to ethylene glycol ethers on
shipyard painters: II. Male reproduction. Am. J. Ind. Med. 14:509-526.
Welch, L.S., Plotkin, E., Schrader, S. (1991) Indirect fertility analysis in painters exposed to ethylene glycol ethers:
sensitivity and specificity. Am. J. Ind. Med. 20:229-240.
Whorton, D., Milby, T.H. (1980) Recovery of testicular function among DBCP workers. J. Occup. Med. 22:177-
179.
Whorton, D., Krauss, R.M., Marshall, S., Milby, T.H. (1977) Infertility in male pesticide workers. Preliminary
communication. Lancet 2(8051): 1259-1261.
Whorton, D., Milby, T.H., Krauss, R.M., Stubbs, H.A. (1979) Testicular function in DBCP exposed pesticide
workers. J. Occup. Med. 21:161-166.
Wilcox, A.J. (1983) Surveillance of pregnancy loss in human populations. Am. J. Ind. Med. 4:285-291.
Wilcox, A.J., Weinburg, C.R., Wehmann, R.E., Armstrong, E.G., Canfield, R.E., Nisula, B.C. (1985) Measuring
early pregnancy loss: laboratory and field methods. Fertil. Steril. 44:366-374.
Wilcox, A.J., Weinberg, C.R., O'Connor, J.F., Baird, D.D., Schlatterer, J.P., Canfield, R.E., Armstrong, E.G.,
Nisula, B.C. (1988) Incidence of early pregnancy loss. N. Engl. J. Med. 319:189-194.
112
-------
Williams, J., Gladen, B.C., Schrader, S.M., Turner, T.W., Phelps, J.L., Chapin, R.E. (1990) Semen analysis and
fertility assessment in rabbits: statistical power and design considerations for toxicology studies. Fundam. Appl.
Toxicol. 15:651-665.
Wilson, J.G. (1973) Environment and Birth Defects. Academic Press, New York.
Wilson, J.G. (1977) Embryotoxicity of drugs in man. In: Wilson, J.G., Fraser, F.C. Handbook of Teratology.
Plenum Press, New York. pp. 309-355.
Wilson, J.G., Scott, W.J., Ritter, E.J., Fradkin, R. (1975) Comparative distribution and embryotoxicity of
hydroxyurea in pregnant rats and rhesus monkeys. Teratology 11:169-178.
Wilson, J.G., Ritter, E.J., Scott, W.J., Fradkin, R. (1977) Comparative distribution and embryotoxicity of
acetylsalicylic acid in pregnant rats and rhesus monkeys. Toxicol. Appl. Pharmacol. 41:67-78.
Witorsch, R.J. (1995) Reproductive Toxicology. Raven Press, New York.
Wolff, M.S. (1993) Lactation. In: Paul, M. Occupational and Environmental Reproductive Hazards. Williams and
Wilkins, Baltimore, pp. 60-75.
Wong, O., Utidjian, H.M.D., Karten, V.S. (1979) Retrospective evaluation of reproductive performance of workers
exposed to ethylene dibromide. J. Occup. Med. 21:98-102.
Working, P.K. (1988) Male reproductive toxicity: comparison of the human to animal models. Environ. Health
77:37-44.
Working, P.K. (1989) Toxicology of the Male and Female Reproductive Systems. Hemisphere, New York.
World Health Organization. (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-
Cervical Mucus Interaction. Third edition. Cambridge University Press, Cambridge.
Wright, D.M., Kesner, J.M., Schrader, S.M., Chin, N.W., Wells, V.E., Krieg, E.F. (1992) Methods of monitoring
menstrual function in field studies: attitudes of working women. Reprod. Toxicol. 6:401-409.
Wyrobek, A.J. (1982) Sperm assays as indicators of chemically-induced germ cell damage in man. In: Mutagenicity:
New Horizons in Genetic Toxicology. Academic Press, New York. pp. 337-349.
Wyrobek, A.J. (1984) Identifying agents that damage human spermatogenesis: abnormalities in sperm concentration
and morphology. In: Monitoring human exposure to carcinogenic and mutagenic agents. Proceedings of a joint
symposium held in Espoo, Finland. Dec. 12-15, 1983. International Agency for Research on Cancer, Lyon, France.
Wyrobek, A.J., Bruce, W.R. (1978) The induction of sperm-shape abnormalities in mice and humans. In: Hollander,
A., de Serres, F.J. Chemical Mutagens: Principles and Methods for Their Detection. Plenum Press, New York.
Wyrobek, A.J., Gordon, L.A., Burkhart, J.G., Francis, M.W., Kapp, R.W., Letz, G., Mailing, H.V., Topham, J.C.,
Whorton, D.M. (1983a) An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman
mammals. Mutat. Res. 115:1-72.
Wyrobek, A.J., Gordon, L.A., Burkhart, J.G., Francis, M.W., Kapp, R.W., Jr., Letz, G., Mailing, H., V, Topham,
J.C., Whorton, D.M. (1983b) An evaluation of human sperm as indicators of chemically induced alterations of
spermatogenic function. Mutat. Res. 115:73-148.
113
-------
Wyrobek, A.J., Watchmaker, G., Gordon, L. (1984) An evaluation of sperm tests as indicators of germ-cell damage
in men exposed to chemical or physical agents. In: Lockey, J.E., Lemasters, O.K., Keye, W.R. Reproduction: The
New Frontier in Occupational and Environmental Health Research. Alan R. Liss, New York, ppi 385-407.
Yeung, C.H., Oberlander, G., Cooper, T.G. (1992) Characterization of the motility of maturing rat spermatozoa by
computer-aided objective measurement. J. Reprod. Fertil. 96:427-441.
Zeger, S.L., Liang, K.Y. (1986) Longitudinal data analysis for discrete and continuous outcomes. Biometrics 42:121-
130.
Zenick, H., Blackburn, K., Hope, E., Baldwin, D.J. (1984) Evaluating male reproductive toxicity in rodents: a new
animal model. Teratogenesis Carcinog. Mutagen. 4:109-128.
Zenick, H., Clegg, E.D., Perreault, S.D., Klinefelter, G.R., Gray, L.E. (1994) Assessment of male reproductive
toxicity: a risk assessment approach. In: Hayes, A.W. Principles and Methods of Toxicology. Raven Press, New
York. pp. 937-988.
Zinaman, M.J., Clegg, E.D., Brown, C.C., O'Connor, J., Selevan, S.G. (1996) Estimates of human fertility and
pregnancy loss. Fertil. Steril. 65:503-509.
Zuelke, K.A., Perreault, S.D. (1995) Carbendazim (MBC) disrupts oocyte spindle function and induces aneuploidy
in hamsters exposed during fertilization (meiosis II). Mol. Reprod. Dev. 42:200-209.
114
-------
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 health effects data in
Table 5. The Agency's intent for Table 5 is not to characterize the available data, but rather to
115
-------
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 SAB's
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 available, the Agency will continue to apply a
default uncertainty factor to account for potential differences in susceptibility.
116
-------
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 Agency's 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 EPA's 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 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
117
-------
sufficiently. The risk characterization section has been expanded substantially to reflect the
recent guidance provided within EPA for application in all risk assessments.
AU.S. GOVERNMENT PRINTING OFHCE;1999-750-101/00016
118
------- |