EPA/600/FR-91/001

December 1991

Guidelines for
Developmental Toxicity Risk Assessment
Published on December 5, 1991, Federal Register 56(234):63798-63826

Risk Assessment Forum

U.S. Environmental Protection Agency
 Washington, DC

DISCLAIMER

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

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

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 CONTENTS

Lists of Tables and Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Federal Register Preamble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Part A: Guidelines for Developmental Toxicity Risk Assessment

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Definitions and Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Hazard Identification/Dose-Response Evaluation of Agents That Cause Developmental Toxicity 4

3.1. Developmental Toxicity Studies: Endpoints and Their Interpretation . . . . . . . . . . . . . . . . . . 5
3.1.1. Laboratory Animal Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1.1.1 Endpoints of Maternal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.1.2. Endpoints of Developmental Toxicity: Altered Survival,
Growth, and Morphological Development . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1.1.3. Endpoints of Developmental Toxicity: Functional Deficits . . . . . . . . . . . 13
3.1.1.4. Overall Evaluation of Maternal and Developmental Toxicity . . . . . . . . . . 17
3.1.1.5. Short-Term Testing in Developmental Toxicity . . . . . . . . . . . . . . . . . . . 18
3.1.1.6. Statistical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1.2. Human Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.2.1. Epidemiologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1.2.2. Examination of Clusters or Case Reports/Series . . . . . . . . . . . . . . . . . . 31
3.1.3. Other Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1.3.1. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1.3.2. Comparisons of Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2. Dose-Response Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3. Characterization of the Health-Related Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4. Determination of the Reference Dose (RfDDT ) or Reference Concentration (RfC DT )
for Developmental Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

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 CONTENTS (continued)

4. Exposure Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5. Risk Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.2. Integration of the Hazard Identification/Dose-Response Evaluation and Exposure
Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.3. Descriptors of Developmental Toxicity Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.3.1. Estimation of the Number of Individuals Exposed to Levels of Concern . . . . . . . . . 47
5.3.2. Presenting Specific Scenarios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.3.3. Risk Characterization for Highly Exposed Individuals . . . . . . . . . . . . . . . . . . . . . . 47
5.3.4. Risk Characterization for Highly Sensitive or Susceptible Individuals . . . . . . . . . . . 48
5.3.5. Other Risk Descriptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.4. Communicating Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6. Summary and Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Part B: Response to Public and Science Advisory Board Comments

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

2. Intent of the Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

3. Basic Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4. Maternal/Developmental Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

5. Functional Developmental Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

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6. Weight-of-Evidence Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

7. Applicability of the RfDDT Concept and the Benchmark Dose Approach . . . . . . . . . . . . . . . . . . 67

LIST OF TABLES

Table 1. Endpoints of maternal toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Table 2. Endpoints of developmental toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Table 3. Categorization of the health-related database for hazard identification/dose-response

evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

LIST OF FIGURES

Figure 1. Graphical illustration of the benchmark dose approach . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

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 GUIDELINES FOR DEVELOPMENTAL TOXICITY RISK ASSESSMENT
[FRL-4038-3]

AGENCY: U.S. Environmental Protection Agency (EPA).

ACTION: Final Guidelines for Developmental Toxicity Risk Assessment.

SUMMARY: The U.S. Environmental Protection Agency (EPA) is today issuing final amended
guidelines for assessing the risks for developmental toxicity from exposure to environmental agents. As
background information for this guidance, this notice describes the scientific basis for concern about
exposure to agents that cause developmental toxicity, outlines the general process for assessing
potential risk to humans because of environmental contaminants, summarizes the history of these
guidelines, and addresses public and Science Advisory Board comments on the 1989 “Proposed
Amendments to the Guidelines for the Health Assessment of Suspect Developmental Toxicants” [54
FR 9386-9403]. These guidelines, which have been renamed “Guidelines for Developmental Toxicity
Risk Assessment” (hereafter “Guidelines”), outline principles and methods for evaluating data from
animal and human studies, exposure data, and other information to characterize risk to human
development, growth, survival, and function because of exposure prior to conception, prenatally, or to
infants and children. These Guidelines amend and replace EPA’s 1986 “Guidelines for the Health
Assessment of Suspect Developmental Toxicants” [51 FR 34028-34040] by adding new guidance on
the relationship between maternal and developmental toxicity, characterization of the health-related
database for developmental toxicity risk assessment, use of the reference dose or reference
concentration for developmental toxicity (RfDDT or RfC DT ), and use of the benchmark dose approach.
In addition, the Guidelines were reorganized to combine hazard identification and dose-response
evaluation since these are usually done together in assessing risk for human health effects other than
cancer.

EFFECTIVE DATE: The Guidelines will be effective December 5, 1991.

FOR FURTHER INFORMATION, CONTACT: Dr. Carole A. Kimmel, Effects Identification

and Characterization Group, National Center for Environmental Assessment-Washington Division
(8623D), U.S. Environmental Protection Agency, 401 M Street, SW, Washington, DC 20460, TEL:
202-564-3307, FAX: 202-565-0078.

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SUPPLEMENTARY INFORMATION: The Clean Air Act (CAA), the Toxic Substances Control
Act (TSCA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), and other statutes
administered by the EPA authorize the Agency to protect public health against adverse effects from
environmental pollutants. One type of adverse effect of great concern is developmental toxicity, i.e.,
adverse effects produced prior to conception, or during pregnancy and childhood. Exposure to agents
affecting development can result in any one or more of the following manifestations of developmental
toxicity: death, structural abnormality, growth alteration, and/or functional deficit. These manifestations
encompass a wide array of adverse developmental endpoints, such as spontaneous abortions, stillbirths,
malformations, early postnatal mortality, reduced birth weight, mental retardation, sensory loss, and
other adverse functional or physical changes that are manifested postnatally.

The Role of Environmental Agents in Developmental Toxicity

Several environmental agents are established as causing developmental toxicity in humans (e.g.,
lead, polychlorinated biphenyls, methylmercury, ionizing radiation), while many others are suspected of
causing developmental toxicity in humans on the basis of data from experimental animal studies (e.g.,
some pesticides, other heavy metals, glycol ethers, alcohols, and phthalates). Data for several of the
agents identified as causing human developmental toxicity have been compared to the experimental
animal data (Nisbet and Karch, 1983; Kimmel et al., 1984; Hemminki and Vineis, 1985; Kimmel et al.,
1990a). In these comparisons, the agents causing human developmental toxicity in almost all cases
were found to produce effects in experimental animal studies and, in at least one species tested, types
of effects similar to those in humans were generally seen. This information provides a strong basis for
the use of animal data in conducting human health risk assessments. On the other hand, a number of
agents found to cause developmental toxicity in experimental animal studies have not shown clear
evidence of hazard in humans, but the available human data are often too limited to evaluate a cause­
and-effect relationship. The comparison of dose-response relationships is hampered by differences in
route, timing, and duration of exposure. When careful comparisons have been done taking these
factors into account, the minimally effective dose for the most sensitive animal species was generally
higher than that for humans, usually within 10-fold of the human effective dose, but sometimes was 100
times or more higher (e.g., polychlorinated biphenyls [Tilson et al., 1990]). Thus, the experimental
animal data were generally predictive of adverse developmental effects in humans, but in some cases,
the administered dose or exposure level required to achieve these adverse effects was much higher than
the effective dose in humans.

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 In most cases, the toxic effects of an agent on human development have not been fully studied,
even though exposure of humans to that agent may have been established. At the same time, there are
many developmental effects in humans with unknown causes and no clear link with exposure to
environmental agents. The background incidence of human spontaneous abortion, for example, was
estimated by Hertig (1967) to be approximately 50% of all conceptions, and more recently, Wilcox et
al. (1985), using sensitive techniques for detecting pregnancy as early as 9 days postconception,
observed that 35% of postimplantation pregnancies ended in an embryonic or fetal loss. Of those
infants born alive, approximately 7.4% are reduced in weight at birth (i.e., below 2,500 g) (Selevan,
1981), approximately 3% are found to have one or more congenital malformations at birth, and by the
end of the first postnatal year, about 3% more are found to have serious developmental defects
(Shepard, 1986). Of those children born with developmental defects, it has been estimated that 20%
are due to genetic transmission and 10% can be attributed to known exogenous factors (including
drugs, infections, ionizing radiation, and environmental agents), leaving the remaining 70% with
unknown causes (Wilson, 1977). In a recent hospital-based surveillance study (Nelson and Holmes,
1989), 50.7% of congenital malformations were estimated to be due to genetic or multifactorial causes,
while 3.2% were associated with exposure to exogenous agents and 2.9% to twinning or uterine
factors, leaving 43.2% to unknown causes. The proportion of the effects with unknown causes that
may be attributable to environmental agents or to a combination of factors, such as environmental
agents and genetic factors, nutritional deficiencies, alcohol consumption, direct or indirect exposure to
tobacco smoke, use of prescribed and illicit drugs, etc., is unknown.
The social and economic impact of developmental disabilities on the population is extremely
high. Close to one-half of the children in hospital wards are there because of prenatally acquired
malformations (Shepard, 1980). According to the Centers for Disease Control, congenital anomalies,
sudden infant death syndrome, and prematurity combined account for more than 50% of infant mortality
among all races in the United States (National Center for Health Statistics, 1988). In addition, among
the leading causes of estimated years of potential life lost (YPLL) due to death before the age of 65,
congenital anomalies, prematurity, and sudden infant death syndrome combined rank third (Centers for
Disease Control, 1988a,b). The YPLL estimates for developmental defects may actually underestimate
the public health impact because the estimates do not include prenatal deaths, they are based only on
those cases that die before age 65 and do not account for limited quality of life, and pregnancies may
be terminated early due to prenatal diagnosis of developmental defects.
These data provide the basis for a long-standing interest by Federal agencies that deal with
human health to protect against exposures to agents that cause developmental toxicity, and most of

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these regulatory agencies have provisions for considering data on developmental toxicity in protecting
human health. As a step in developing procedures for interpreting toxicity data in the regulatory
context, the National Academy of Sciences/National Research Council, in 1983, published a
framework for the risk assessment process, which EPA uses as the basis for its risk assessment
guidelines and for the assessment of risk due to environmental agents.

The Risk Assessment Process and Its Application to Developmental Toxicity

Risk assessment is the process by which scientific judgments are made concerning the potential
for toxicity to occur in humans. The National Research Council (1983) has defined risk assessment as
including some or all of the following components: hazard identification, dose-response assessment,
exposure assessment, and risk characterization. In general, the process of assessing the risk of human
developmental toxicity may be adapted to this format. In practice, however, hazard identification for
developmental toxicity and other noncancer health effects is usually done in conjunction with an
evaluation of dose-response relationships, since the determination of a hazard is often dependent on
whether a dose-response relationship is present (Kimmel et al., 1990b). One advantage of this
approach is that it reflects hazard within the context of dose, route, and duration and timing of
exposure, all of which are important in comparing the toxicity information available to potential human
exposure scenarios. Second, this approach avoids labeling of chemicals as developmental toxicants on
a purely qualitative basis. For these reasons, the Guidelines combine hazard identification and dose-
response evaluation under one section (Section 3), and characterize both hazard and dose information
as part of the health-related database for risk assessment. If data are considered sufficient for risk
assessment, an oral or dermal reference dose for developmental toxicity (RfD DT ) or an inhalation
reference concentration for developmental toxicity (RfC DT ) is then derived for comparison with human
exposure estimates. A statement of the potential for human risk and the consequences of exposure can
come only from integrating the hazard identification/dose-response evaluation with the human exposure
estimates in the final risk characterization. Combining hazard identification and dose-response
evaluation, as well as development of the RfDDT and RfC DT , are revisions of the 1986 Guidelines.
Hazard identification/dose-response evaluation involves examining all available experimental
animal and human data and the associated doses, routes, and timing and duration of exposures to
determine if an agent causes developmental toxicity and/or maternal or paternal toxicity in that species
and under what exposure conditions. The no-observed-adverse-effect-level (NOAEL) and/or the
lowest-observed-adverse-effect-level (LOAEL) are determined for each study and type of effect.
Based upon the hazard identification/dose-response evaluation and criteria provided in these

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Guidelines, the health-related database can be characterized as sufficient or insufficient for use in risk
assessment (Section 3.3). Because of the limitations associated with the use of the NOAEL, the
Agency is evaluating the use of an additional approach, i.e., the benchmark dose approach (Crump,
1984), for more quantitative dose-response evaluation when sufficient data are available. The
benchmark dose provides an indication of the risk associated with exposures near the NOAEL, taking
into account the variability in the data and the slope of the dose-response curve.
For the determination of the RfDDT or the RfC DT , uncertainty factors are applied to the
NOAEL (or LOAEL, if a NOAEL has not been established) to account for extrapolation from
experimental animals to humans and for variability within the human population. The RfDDT or RfC DT is
generally based on a short duration of exposure as is typically used in developmental toxicity studies in
experimental animals. The use of the terms RfDDT and RfC DT distinguish them from the oral or dermal
reference dose (RfD) and the inhalation reference concentration (RfC), which refer primarily to chronic
exposure situations (U.S. EPA, 1991). Uncertainty factors may also be applied to a benchmark dose
for calculating the RfDDT or RfC DT , but the Agency has little experience with applying this approach
and is currently supporting research efforts to determine the appropriate methods. As more information
becomes available, guidance will be written and published as an addendum to these Guidelines. These
approaches are discussed further in Section 3.4.
The exposure assessment identifies human populations exposed or potentially exposed to an
agent, describes their composition and size, and presents the types, magnitudes, frequencies, and
durations of exposure to the agent. The exposure assessment provides an estimate of human exposure
levels for particular populations from all potential sources.
In risk characterization, the hazard identification/dose-response evaluation and the exposure
assessment for given populations are combined to estimate some measure of the risk for developmental
toxicity. As part of risk characterization, a summary of the strengths and weaknesses in each
component of the risk assessment is discussed along with major assumptions, scientific judgments, and,
to the extent possible, qualitative and quantitative estimates of the uncertainties. Confidence in the
health-related data is always presented in conjunction with information on dose-response and the
RfDDT or RfC DT . If human exposure estimates are available, the exposure basis used for the risk
assessment is clearly described, e.g., highly exposed individuals, or highly sensitive or susceptible
individuals. The NOAEL may be compared to the various estimates of human exposure to calculate
the margin(s) of exposure (MOE). The considerations for determining adequacy of the MOE are
similar to those used in determining the appropriate size of the uncertainty factor for calculating the
RfDDT or RfC DT .

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 Risk assessment is just one component of the regulatory process and defines the potential
adverse health consequences of exposure to a toxic agent. The other component, risk management,
combines risk assessment with statutory directives regarding socioeconomic, technical, political, and
other considerations, to reach decisions about the appropriate regulation of the suspected toxic agents.
Risk management is not dealt with directly in these Guidelines since the basis for decision making goes
beyond scientific consideration alone, but the use of scientific information in this process is discussed in
some cases. For example, the acceptability of the MOE is a risk management decision, but the
scientific bases for establishing this value are discussed here.

History of These Guidelines

In 1984, the Agency published “Proposed Guidelines for the Health Assessment of Suspect
Developmental Toxicants” [49 FR 46324-46331]. Following extensive scientific and public review,
final guidelines were issued on September 24, 1986 [51 FR 34028-34040]. The 1986 Guidelines set
forth principles and procedures to guide EPA scientists in the conduct of Agency risk assessments, to
help promote high scientific quality and Agencywide consistency, and to inform Agency decision
makers and the public about these scientific procedures. In publishing this guidance, EPA emphasized
that one purpose of its risk assessment guidelines was to “encourage research and analysis that will lead
to new risk assessment methods and data,” which in turn would be used to revise and improve the
guidelines, and better guide Agency risk assessors. Thus, the 1986 Guidelines were developed and
published with the understanding that risk assessment is an evolving science and that continued study
could lead to changes.
As expected, Agency experience with the 1986 Guidelines suggested that additional or
alternate approaches should be considered for certain aspects of the guidance. Proposals to amend the
guidelines were considered soon after their publication in September 1986, because of new reviews or
re-evaluations that focused on some of the issues identified for research in the guidelines. Included
were several workshops and symposia cited in the Introduction to these Guidelines. In addition, much
experience had been gained in using the 1986 Guidelines and in instructing others in their use.
Based on this experience, amendments to the 1986 Guidelines were proposed for public
comment in March 1989 [54 FR 9386-9403]. Following receipt and review of the public comments,
they were collated, summarized, and reviewed by scientists within the Agency. On October 27, 1989,
EPA’s Science Advisory Board (SAB) met to review the Proposed Amendments and the summarized
public comments, and to be briefed by Agency scientists concerning proposed responses.

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 During this same period, several issues with implications for health effects other than cancer
were under discussion in the Agency and elsewhere. These issues included use of the benchmark dose
(see Section 3.2), exposure descriptors (see Section 5.3), and risk characterization (see Section 5).
Thus, generic discussions on risk assessment issues, along with comments from the public and the SAB,
have influenced the structure and content of these Guidelines.
These revised Guidelines were then reviewed by a number of Agency scientists and official
panels, including the Risk Assessment Forum and the Risk Assessment Council. The revised
Guidelines also were presented to the SAB on March 27, 1991, for final comment. In addition, a
review was conducted by the interagency Working Party on Reproductive Toxicology, Subcommittee
on Risk Assessment of the Federal Coordinating Committee on Science, Engineering and Technology.
Comments of these groups have been considered in the revision of these Guidelines. The full text of the
final “Guidelines for Developmental Toxicity Risk Assessment” is published here.
These Guidelines were developed as part of an interoffice guidelines development program
under the auspices of the Risk Assessment Forum and the Office of Health and Environmental
Assessment (OHEA) in the Agency’s Office of Research and Development. The Agency is continuing
to study risk assessment issues raised in these Guidelines, and will revise them in line with new
information as appropriate.
Following this Preamble are two parts: Part A is the Guidelines and Part B is the Response to
the Public and Science Advisory Board Comments. Part B includes a summary of the issues raised by
the public and the SAB, and the Agency’s responses to those comments.
References, supporting documents, and comments received on the Proposed Amendments, as
well as a copy of the final Guidelines, are available for inspection and copying at the Public Information
Reference Unit Docket (202-260-5926), EPA Headquarters Library, 401 M Street, S.W.,
Washington, DC, between the hours of 8:00 a.m. and 4:30 p.m.

______________________ _______________________________________
Dated: November 26, 1991 Signed by EPA Administrator
William K. Reilly

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PART A: GUIDELINES FOR DEVELOPMENTAL TOXICITY RISK ASSESSMENT

1. INTRODUCTION

These Guidelines describe the procedures that EPA follows in evaluating potential
developmental toxicity associated with human exposure to environmental agents. The Agency has
sponsored or participated in several conferences that addressed issues related to such evaluations and
that provide some of the scientific basis for these Guidelines (U.S. EPA, 1982a; Kimmel et al., 1982b,
1987; Hardin, 1987; Perlin and McCormack, 1988; Kimmel et al., 1989; Kimmel and Francis, 1990;
Kimmel et al., 1990a). The Agency’s authority to regulate substances that have the potential to
interfere with human development is derived from a number of statutes that are implemented through
multiple offices within the EPA. The procedures described herein are intended to promote consistency
in the assessment of developmental toxic effects across program offices within the Agency.
These Guidelines provide a general format for analyzing and organizing the available data for
conducting risk assessments. The Agency previously has issued testing guidelines (U.S. EPA, 1982b,
1985a, 1989a, 1991a) that provide protocols designed to determine the potential of a test substance to
induce structural and/or other adverse effects during development. These risk assessment Guidelines
do not change any prescribed statutory or regulatory standards for the type of data necessary for
regulatory action, but rather provide guidance for the interpretation of studies that follow the testing
guidelines and, in addition, provide limited information for interpretation of other studies (e.g.,
epidemiologic data, functional developmental toxicity studies, and short-term tests) that are not routinely
required, but may be encountered when reviewing data on particular agents.
Since the purpose of risk assessment is to make inferences about potential risks to human
health, the most appropriate data to be used are those deriving from studies of humans. If adequate
human data are not available, then it is necessary to use data obtained from other species. There are a
number of unknowns in the extrapolation of data from animal studies to humans. Therefore, a number
of assumptions must be made on the relevance of effects to potential human risk that are generally
applied in the absence of data. These assumptions provide the inferential basis for the approaches
taken to risk assessment in these Guidelines.
First, it is assumed that an agent that produces an adverse developmental effect in experimental
animal studies will potentially pose a hazard to humans following sufficient exposure during
development. This assumption is based on the comparisons of data for agents known to cause human
developmental toxicity (Nisbet and Karch, 1983; Kimmel et al., 1984; Hemminki and Vineis, 1985;

1
Kimmel et al., 1990a), which indicate that, in almost all cases, experimental animal data are predictive
of a developmental effect in humans.
It is assumed that all of the four manifestations of developmental toxicity (death, structural
abnormalities, growth alterations, and functional deficits) are of concern. In the past, there has been a
tendency to consider only malformations or malformations and death as endpoints of concern. From
the data on agents that are known to cause human developmental toxicity (Nisbet and Karch, 1983;
Kimmel et al., 1984; Hemminki and Vineis, 1985; Kimmel et al., 1990a), there is usually at least one
experimental species that mimics the types of effects seen in humans, but in other species tested, the
type of developmental perturbation may be different. Thus, a biologically significant increase in any of
the four manifestations is considered indicative of an agent’s potential for disrupting development and
producing a developmental hazard.
It is assumed that the types of developmental effects seen in animal studies are not necessarily
the same as those that may be produced in humans. This assumption is made because it is impossible
to determine which will be the most appropriate species in terms of predicting the specific types of
effects seen in humans. The fact that every species may not react in the same way could be due to
species-specific differences in critical periods, differences in timing of exposure, metabolism,
developmental patterns, placentation, or mechanisms of action.
The most appropriate species is used to estimate human risk when data are available (e.g.,
pharmacokinetics). In the absence of such data, it is assumed that the most sensitive species is
appropriate for use, based on observations that humans are as sensitive or more so than the most
sensitive animal species tested for the majority of agents known to cause human developmental toxicity
(Nisbet and Karch, 1983; Kimmel et al., 1984; Hemminki and Vineis, 1985; Kimmel et al., 1990a).
In general, a threshold is assumed for the dose-response curve for agents that produce
developmental toxicity. This is based on the known capacity of the developing organism to compensate
for or to repair a certain amount of damage at the cellular, tissue, or organ level. In addition, because
of the multipotency of cells at certain stages of development, multiple insults at the molecular or cellular
level may be required to produce an effect on the whole organism.

2
 2. DEFINITIONS AND TERMINOLOGY

The Agency recognizes that there are differences in the use of terms in the field of
developmental toxicology. For the purposes of these Guidelines the following definitions will be used.

Developmental toxicology - The study 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.

Altered growth - An alteration in offspring organ or body weight or size. Changes in one endpoint
may or may not be accompanied by other signs of altered growth (e.g., changes in body weight may or
may not be accompanied by changes in crown-rump length and/or skeletal ossification). Altered
growth can be induced at any stage of development, may be reversible, or may result in a permanent
change.

Functional developmental toxicology - The study of alterations or delays in the physiological and/or
biochemical competence of an organism or organ system following exposure to an agent during critical
periods of development pre- and/or postnatally.

Structural abnormalities - Structural alterations in development that include both malformations and
variations.

Malformations and variations - A malformation is usually defined as a permanent structural change

that may adversely affect survival, development, or function. The term teratogenicity is used in these
Guidelines to refer only to malformations. The term variation is used to indicate a divergence beyond
the usual range of structural constitution that may not adversely affect survival or health. Distinguishing
between variations and malformations is difficult since there exists a continuum of responses from the
normal to the extremely deviant. There is no generally accepted classification of malformations and
variations. Other terms that are often used, but no better defined, include anomalies, deformations, and
aberrations.

3
 3. HAZARD IDENTIFICATION/DOSE-RESPONSE EVALUATION OF AGENTS THAT
CAUSE DEVELOPMENTAL TOXICITY

This section discusses the evaluation and interpretation of hazards for a variety of endpoints of
developmental toxicity seen in both human and animal studies, and describes the criteria for
characterizing the sufficiency of the health-related database for conducting a developmental toxicity risk
assessment. It also details the use of dose-response data for determining potential hazards, and
describes the calculation of the RfDDT or RfC DT , a dose or concentration that is assumed to be without
appreciable risk of deleterious developmental effects for a given agent.
Developmental toxicity is expressed as one or more of a number of possible endpoints that may
be used for evaluating the potential of an agent to cause abnormal development. Developmental
toxicity generally occurs in a dose-related manner, may result from short-term exposure (including
single exposure situations) or from longer term low-level exposure, may be produced by various routes
of exposure, and the types of effects may vary depending on the timing of exposure because of a
number of critical periods of development for various organs and functional systems.
The four major manifestations of developmental toxicity are death, structural abnormality,
altered growth, and functional deficit. The relationship among these manifestations may vary with
increasing dose and, especially at higher doses, death of the conceptus may preclude expression of
other manifestations. Of these, all four manifestations have been evaluated in human studies, but only
the first three are traditionally measured in laboratory animals using the conventional developmental
toxicity (also called teratogenicity or Segment II) testing protocol as well as in other study protocols,
such as the multigeneration study or the continuous breeding study. Although functional deficits seldom
have been evaluated in routine testing studies in experimental animals, functional evaluations are
beginning to be required in certain regulatory situations (U.S. EPA, 1986a, 1988a, 1989b, 1991a).
Developmental toxicity can be considered a component of reproductive toxicity, and often it is
difficult to distinguish between effects mediated through the parents versus direct interaction with
developmental processes. For example, developmental toxicity may be influenced by the effects of
toxic agents on the maternal system when exposure occurs during pregnancy or lactation. In addition,
following parental exposure prior to conception, developmental toxicity may result in their offspring and,
potentially, in subsequent generations. Therefore, it is useful to consult the “Proposed Guidelines for
Assessing Male Reproductive Risk” (U.S. EPA, 1988b) and the “Proposed Guidelines for Assessing
Female Reproductive Risk” (U.S. EPA, 1988c) in conjunction with these Guidelines. Mutational
events that occur as a result of exposure to agents that cause developmental toxicity may be difficult to

4
discriminate from other possible mechanisms in standard studies of developmental toxicity. When
mutational events are suspected, the “Guidelines for Mutagenicity Risk Assessment” (U.S. EPA,
1986c), which specifically address the risks of heritable mutation, should be consulted.
Carcinogenic effects have occurred in humans following developmental exposures to
diethylstilbestrol (Herbst et al., 1971). Several additional agents (e.g., direct-acting alkylating agents)
have been shown to cause cancer following developmental exposures in experimental animals, and it
appears from the data collected thus far that agents capable of causing cancer in adults may also cause
transplacental or neonatal carcinogenesis (Anderson et al., 1985). Currently, there is no way to predict
whether the developing offspring or adult will be more sensitive to the carcinogenic effects of an agent.
At present, testing for carcinogenesis following developmental exposure is not routinely required.
However, if this type of effect is reported for an agent, it is considered appropriate to use the
“Guidelines for Carcinogen Risk Assessment” (U.S. EPA, 1986b) for assessing human risk.

3.1. DEVELOPMENTAL TOXICITY STUDIES: ENDPOINTS AND THEIR

INTERPRETATION

3.1.1. Laboratory Animal Studies

This section discusses the endpoints examined in routinely used protocols as well as the use of
other types of studies, including functional studies and short-term tests.
The most commonly used protocol for assessing developmental toxicity in laboratory animals
involves the administration of a test substance to pregnant animals (usually mice, rats, or rabbits) during
the period of major organogenesis, evaluation of maternal responses throughout pregnancy, and
examination of the dam and the uterine contents just prior to term (U.S. EPA, 1982b, 1985a; Food and
Drug Administration [FDA], 1966, 1970; Organization for Economic Cooperation and Development
[OECD], 1981). Some studies may use exposures of one to a few days to investigate periods of
particular sensitivity for induction of abnormalities in specific organs or organ systems. In addition,
developmental toxicity may be evaluated in studies involving exposure to one or both parents prior to
conception, to the conceptus during pregnancy and over several generations, or to offspring during the
prenatal and preweaning periods (U.S. EPA, 1982b, 1985a, 1986a, 1988a, 1991a; FDA, 1966,
1970; OECD, 1981; Lamb, 1985). These Guidelines are intended to provide information for
interpreting developmental effects related to any of these types of exposure.
Appropriate study designs include a number of important factors. For example, test animal
selection is generally based on considerations of species, strain, age, weight, and health status.
Assignment of animals to dose groups by stratified randomization (on the basis of body weight) reduces

5
bias and provides a basis for performing valid statistical tests. At a minimum, a high dose, a low dose,
and one intermediate dose are included. The high dose is selected to produce some minimal maternal
or adult toxicity (i.e., a level that at the least produces marginal but significantly reduced body weight,
reduced weight gain, or specific organ toxicity, and at the most produces no more than 10% mortality).
At doses that cause excessive maternal toxicity (that is, significantly greater than the minimal toxic level),
information on developmental effects may be difficult to interpret and of limited value. The low dose is
generally a NOAEL for adult and offspring effects, although if the low dose produces a biologically or
statistically significant increase in response, it is considered a LOAEL (see Section 3.1.1.6 for a
discussion of biological versus statistical significance). A concurrent control group treated with the
vehicle used for agent administration is a critical component of a well-designed study.
The route of exposure in these studies is usually oral, unless the chemical or physical
characteristics of the test substance or pattern of human exposure suggest a more appropriate route of
administration. In the case of dermal exposure, developmental toxicity studies showing no indication of
maternal or developmental toxicity are considered insufficient for risk assessment unless accompanied
by absorption data (Kimmel and Francis, 1990). Dermal developmental toxicity studies in which skin
irritation is too marked (moderate erythema and/or moderate edema, i.e., raised approximately 1 mm)
also are considered insufficient, since excessive maternal toxicity may be produced from the irritation
rather than from systemic exposure to the agent. Assessment of maternal toxicity is based on signs of
systemic toxicity rather than on local effects such as skin irritation. Absorption data and limited
pharmacokinetic data collected in dermal developmental toxicity studies provide very useful information
in the evaluation of study design and data interpretation (Kimmel and Francis, 1990). Many of these
points also are pertinent to studies by other routes of exposure.
The evaluation of specific endpoints of maternal and developmental toxicity is discussed in the
next several sections. Appropriate historical control data sometimes can be very useful in the
interpretation of these endpoints. Comparison of data from treated animals with concurrent study
controls should always take precedence over comparison with historical control data. The most
appropriate historical control data are those from the same laboratory in which studies were conducted.
Even data from the same laboratory, however, should be used cautiously and examined for subtle
changes over time that may result from genetic alterations in the strain or stock of the species used,
changes in environmental conditions both in the breeding colony of the supplier and in the laboratory,
and changes in personnel conducting studies and collecting data (Kimmel and Price, 1990). Study data
should be compared with recent as well as cumulative historical data. Any change in laboratory

6
procedure that might affect control data should be noted and the data accumulated separately from
previous data.
The next three sections (3.1.1.1, 3.1.1.2, and 3.1.1.3) discuss individual endpoints of maternal
and developmental toxicity as measured in the conventional developmental toxicity study, the
multigeneration study, and, when available, in postnatal studies. Other endpoints specifically related to
reproductive toxicity are covered in the relevant risk assessment guidelines (U.S. EPA, 1988b, 1988c).
The fourth section (3.1.1.4) deals with the integrated evaluation of all data, including the relative effects
of exposure on maternal animals and their offspring, which is important in assessing the level of concern
about a particular agent.

3.1.1.1. Endpoints of Maternal Toxicity

A number of endpoints that may be observed as possible indicators of maternal toxicity are
listed in Table 1. Maternal mortality is an obvious endpoint of toxicity; however, a number of other
endpoints can be observed that may give an indication of the more subtle adverse effects of an agent.
For example, in well-conducted studies, the mating and fertility indices provide information on the
general fertility rate of the animal stock used and are important indicators of toxic effects to adults if
treatment begins prior to mating or implantation. Changes in gestation length may indicate effects on the
process of parturition.
Body weight and change in body weight are viewed collectively as indicators of maternal
toxicity for most species, although these endpoints may not be as useful in rabbits, because body weight
changes are usually more variable (Kimmel and Price, 1990) and, in some strains of rabbits, body
weight is not a good indicator of pregnancy status. Body weight changes may provide more
information than a daily body weight measured during treatment or during gestation. Changes in weight
gain during treatment could occur that would not be reflected in the total weight change throughout
gestation, because of compensatory weight gain that may occur following treatment but before sacrifice.
For this reason, changes in weight gain during treatment can be examined as another indicator of
maternal toxicity.
Changes in maternal body weight corrected for gravid uterine weight at sacrifice may indicate
whether the effect is primarily maternal or intrauterine. For example, a significant reduction in weight
gain throughout gestation and in gravid uterine weight without any change in corrected maternal weight
gain generally would indicate an intrauterine effect. Conversely, a change in corrected weight gain and
no change in gravid uterine weight generally would suggest maternal toxicity and little or no intrauterine
effect. An alternate estimate of maternal weight change during gestation can be obtained by subtracting

7
the sum of the weights of the fetuses. However, this weight does not include the uterine or placental
tissue, or the amniotic fluid.

8
 Table 1. Endpoints of maternal toxicity

Mortality

Mating index [(no. with seminal plugs or sperm/no. mated) × 100]

Fertility index [(no. with implants/no. of matings) × 100]

Gestation length (useful when animals are allowed to deliver pups)

Body weight

Day 0
During gestation
Day of necropsy

Body weight change

Throughout gestation
During treatment (including increments of time within treatment period)
Post-treatment to sacrifice
Corrected maternal (body weight change throughout gestation minus gravid
uterine weight or litter weight at sacrifice)

Organ weights (in cases of suspected target organ toxicity and especially when
supported by adverse histopathology findings)

Absolute
Relative to body weight
Relative to brain weight

Food and water consumption (where relevant)

Clinical evaluations

Types, incidence, degree, and duration of clinical signs

Enzyme markers
Clinical chemistries

Gross necropsy and histopathology

9
 Changes in other endpoints may also be important. For example, changes in relative and
absolute organ weights may be signs of a maternal effect, especially when an agent is suspected of
causing specific organ toxicity and when such findings are supported by adverse histopathologic findings
in those organs. Food and water consumption data are useful, especially if the agent is administered in
the diet or drinking water. The amount ingested (total and relative to body weight) and the dose of the
agent (relative to body weight) can then be calculated, and changes in food and water consumption
related to treatment can be evaluated along with changes in body weight and body weight gain. Data
on food and water consumption also are useful when an agent is suspected of affecting appetite, water
intake, or excretory function.
Clinical evaluations of toxicity also may be used as indicators of maternal toxicity. Daily clinical
observations may be useful in describing the profile of maternal toxicity and alterations in general
homeostasis. Enzyme markers and clinical chemistries may be useful indicators of exposure but must
be interpreted carefully as to whether or not a change constitutes toxicity. Gross necropsy and
histopathology data (when specified in the protocol) may aid in determining toxic dose levels. The
minimum amount of information considered useful for evaluating maternal toxicity [as noted in the
“Proceedings of the Workshop on the Evaluation of Maternal and Developmental Toxicity” (Kimmel et
al., 1987)], includes morbidity or mortality, maternal body weight and body weight gain, clinical signs of
toxicity, food and water consumption (especially if dosing is via food or water), and necropsy for gross
evidence of organ toxicity. In a well-designed study, maternal toxicity is determined in the pregnant
and/or lactating animal over an appropriate part of gestation and/or the neonatal period, and is not
assumed or extrapolated from other adult toxicity studies.

3.1.1.2. Endpoints of Developmental Toxicity: Altered Survival, Growth, and Morphological

Development
Because the maternal animal, and not the conceptus, is the individual treated during gestation,
data generally are calculated as incidence per litter or as number and percent of litters with particular
endpoints. Table 2 indicates the ways in which offspring and litter endpoints may be expressed.
When treatment of females begins prior to implantation, an increase in preimplantation loss
could indicate an adverse effect on gamete transport, the fertilization process, uterine toxicity, the
developing blastocyst, or on the process of implantation itself. If treatment begins around the time of
implantation (i.e., day 6 of gestation in the mouse, rat, or rabbit), an increase in preimplantation loss
probably reflects variability that is not treatment-related in the animals being used, but the data should
be examined carefully to determine if there is a dose-response

10
 Table 2. Endpoints of developmental toxicity

Litters with implants

No. implantation sites/dam
No. corpora lutea (CL)/dama
Percent preimplantation loss
(CL - implantations) × 100a
CL
No. and percent live offspringb/litter
No. and percent resorptions/litter
No. and percent litters with resorptions
No. and percent late fetal deaths/litter
No. and percent nonlive (late fetal deaths + resorptions) implants/litter
No. and percent litters with nonlive implants
No. and percent affected (nonlive + malformed) implants/litter
No. and percent litters with affected implants
No. and percent litters with total resorptions
No. and percent stillbirths/litter
No. and percent litters with live offspring

Litters with live offspring

No. and percent live offspring/litter
Viability of offspringc
Sex ratio/litter
Mean offspring body weight/litterc
Mean male or female body weight/litterc
No. and percent offspring with external, visceral, or skeletal malformations/litter
No. and percent malformed offspring/litter
No. and percent litters with malformed offspring
No. and percent malformed males or females/litter
No. and percent offspring with external, visceral, or skeletal variations/litter
No. and percent offspring with variations/litter
No. and percent litters having offspring with variations
Types and incidence of individual malformations
Types and incidence of individual variations
Individual offspring and their malformations and variations
(grouped according to litter and dose)
Clinical signs (type, incidence, duration, and degree)
Gross necropsy and histopathology

a
Important when treatment begins prior to implantation. May be difficult to assess in mice.
b
Offspring refers both to fetuses observed prior to term and to pups following birth. The endpoints examined
depend on the protocol used for each study.
c
Measured at selected intervals until termination of the study.

11
relationship. If preimplantation loss is related to dose, further studies would be necessary to determine
the mechanism and extent of such effects.
The number and percent of live offspring per litter, based on all litters, may include litters that
have no live implants. The number and percent of resorptions and late fetal deaths give some indication
of when the conceptus died, and the number and percent of nonlive implants per litter (postimplantation
loss) is a combination of these two measures. Expression of data as the number and percent of litters
showing an increased incidence for these endpoints may be less useful than incidence per litter because,
in the former case, a litter is counted whether one or all implants were resorbed, dead, or nonlive.
If a significant increase in postimplantation loss is found after exposure to an agent, the data
may be compared not only with concurrent controls, but also with recent historical control data
(preferably from the same laboratory), since there is considerable interlitter variability in the incidence of
postimplantation loss (Kimmel and Price, 1990). If a given study control group exhibits an unusually
high or low incidence of postimplantation loss compared to historical controls, then scientific judgment
must be used to determine the adequacy of the study for risk assessment purposes.
The endpoint “affected implants” (i.e., the combination of nonlive and malformed conceptuses)
sometimes reflects a better dose-response relationship than does the incidence of nonlive or malformed
offspring taken individually. This is especially true at the high end of the dose-response curve in cases
when the incidence of nonlive implants per litter is greatly increased. In such cases, the malformation
rate may appear to decrease because only unaffected offspring have survived. If the incidence of
prenatal deaths or malformations is unchanged, then the incidence of affected implants will not provide
any additional dose-response information. In studies where maternal animals are allowed to deliver
pups normally, the number of stillbirths per litter should also be noted.
The number of live offspring per litter, based on those litters that have one or more live
offspring, may be unchanged even though the incidence of nonlive in all litters is increased. This could
occur either because of an increase in the number of litters with no live offspring, or an increase in the
number of implants per litter. A decrease in the number of live offspring per litter is generally
accompanied by an increase in the incidence of nonlive implants per litter unless the implant numbers
differ among dose groups. In postnatal studies, the viability of live-born offspring should be determined
at selected intervals until termination of the study.
The sex ratio per litter, as well as the body weights of males and females, can be examined to
determine whether or not one sex is preferentially affected by the agent. However, this is an unusual
occurrence.

12
 A change in offspring body weight is a sensitive indicator of developmental toxicity, in part
because it is a continuous variable. In some cases, offspring weight reduction may be the only indicator
of developmental toxicity. While there is always a question as to whether weight reduction is a
permanent or transitory effect, little is known about the long-term consequences of short-term fetal or
neonatal weight changes. Therefore, when significant weight reduction effects are noted, they are used
as a basis to establish the NOAEL. Several other factors should be considered in the evaluation of
fetal or neonatal weight changes; for example, in polytocous animals, fetal and neonatal weights are
usually inversely correlated with litter size, and the upper end of the dose-response curve may be
affected by smaller litters and increased fetal or neonatal weight. Additionally, the average body weight
of males is greater than that of females in the more commonly used laboratory animals.
Live offspring are generally examined for external, visceral, and skeletal malformations and
variations. If only a portion of the litter is examined for one or more endpoints, then random selection
of those pups examined introduces less bias in the data. An increase in the incidence of malformed
offspring may be indicated by a change in one or more of the following endpoints: the incidence of
malformed offspring per litter, the number and percent of litters with malformed offspring, or the number
of offspring or litters with a particular malformation that appears to increase with dose (as indicated by
the incidence of individual types of malformations).
Other ways of examining the data include determining the incidence of external, visceral, and
skeletal malformations and variations that may indicate the organs or organ systems affected. A listing
of individual offspring with their malformations and variations may give an indication of the pattern of
developmental deviations. All of these methods of expressing and examining the data are valid for
determining the effects of an agent on structural development. However, care must be taken to avoid
counting offspring more than once in the evaluation of any single endpoint based on number or percent
of offspring or litters. The incidence of individual types of malformations and variations may indicate
significant changes that are masked if the data on all malformations and/or variations are pooled.
Appropriate historical control data can be especially helpful in the interpretation of malformations and
variations, particularly those that normally occur at a low incidence and may or may not be related to
dose in an individual study.
Although a dose-related increase in malformations is interpreted as an adverse developmental
effect of exposure to an agent, the biological significance of an altered incidence of anatomical variations
is more difficult to assess, and must take into account what is known about developmental stage (e.g.,
with skeletal ossification), background incidence of certain variations (e.g., 12 or 13 pairs of ribs in
rabbits), or other strain- or species-specific factors. However, if variations are significantly increased in

13
a dose-related manner, these should also be evaluated as a possible indication of developmental
toxicity.
In addition, although some investigators have considered certain of these effects to simply be
associated with manifestations of maternal toxicity noted at similar dose levels (Khera, 1984, 1985,
1987), such effects are still toxic manifestations and as such are generally considered a reasonable basis
for Agency regulation and/or risk assessment. On a somewhat similar note, the conclusion of
participants in a “Workshop on Reproductive Toxicity Risk Assessment” (Kimmel et al., 1986) was
that dose-related increases in defects that may occur spontaneously are as relevant as dose-related
increases in any other developmental toxicity endpoints.

3.1.1.3. Endpoints of Developmental Toxicity: Functional Deficits

Developmental effects that are induced by exogenous agents are not limited to death, structural
abnormalities, and altered growth. Rather, it has been demonstrated in a number of instances that
alterations in the functional competence of an organ or a variety of organ systems may result from
exposure during critical developmental periods that may occur between conception and sexual
maturation. Sometimes, these functional defects are observed at dose levels below those at which
other indicators of developmental toxicity are evident (Rodier, 1978). Such effects may be transient or
reversible in nature, but generally are considered adverse. Testing for functional developmental toxicity
has not been required routinely by regulatory agencies in the United States, but studies in developmental
neurotoxicity are beginning to be required by the EPA when other information indicates the potential for
adverse functional developmental effects (U.S. EPA, 1986a, 1988a, 1989b, 1991a). Data from
postnatal studies, when available, are considered very useful for further assessment of the relative
importance and severity of findings in the fetus and neonate. Often, the long-term consequences of
adverse developmental outcomes noted at birth are unknown, and further data on postnatal
development and function are necessary to determine the full spectrum of potential developmental
effects. Useful data can also be derived from well-conducted multigeneration studies, although the dose
levels used in these studies may be much lower than in studies with shorter-term exposure.
Much of the early work in functional developmental toxicology was related to behavioral
evaluations, and the term “behavioral teratology” became prominent in the mid-1970s. Recent
advances in this area have been reviewed in several publications (Riley and Vorhees, 1986; Kimmel,
1988; Kimmel et al., 1990a). Several expert groups have focused on the functions that should be
included in a behavioral testing battery (World Health Organization [WHO], 1984; Buelke-Sam et al.,
1985; Leukroth, 1986). These include: sensory systems, neuromotor development, locomotor activity,

14
learning and memory, reactivity and/or habituation, and reproductive behavior. No testing battery has
fully addressed all of these functions, but it is important to include as many as possible, and several
testing batteries have been developed and evaluated for use in testing (Buelke-Sam et al., 1985;
Tanimura, 1986; Elsner et al., 1986).
The Agency recently has developed a “generic” developmental neurotoxicity test guideline that
can be used for both pesticides and industrial chemicals (U.S. EPA, 1991a). Because of its design, the
developmental neurotoxicity testing protocol may be conducted as a separate study, concurrently with
or as a follow-up to a developmental toxicity (Segment II) study, or be folded into a multigeneration
study in the second generation. Testing is generally conducted in the rat. In the protocol for the
separate study, the test agent is administered orally (other routes may be used on a case-by-case basis)
to at least three treated groups and one concurrent control group of animals on day 6 of gestation
through day 10 postnatally. The highest dose level is selected to induce some overt signs of maternal
toxicity, but not result in more than a 20% reduction in weight gain during gestation and lactation. This
dose also is selected to avoid in utero or neonatal death or malformations sufficient to preclude a
meaningful evaluation of developmental neurotoxicity. At least 20 litters are required per treatment
group. For behavioral tests, one female and one male pup per litter are randomly selected and assigned
to one of the following tests: motor activity, auditory startle, and learning and memory in animals at
weaning and as adults. Neuropathological evaluation and determination of brain weights are conducted
on selected pups at postnatal day 11 and at termination of the study.
Several criteria for selecting agents for developmental neurotoxicity testing have been suggested
(Buelke-Sam et al., 1985; Levine and Butcher, 1990), including: agents that cause central nervous
system malformations, psychoactive drugs and chemicals, agents that cause adult neurotoxicity,
hormonally active agents, and chemicals that are structurally related to others that cause developmental
neurotoxicity or for which widespread exposure and/or release is expected. Data from developmental
neurotoxicity studies should be evaluated in light of the data that may have triggered such testing as well
as all other toxicity data available.
Less work has been done on other developing functional systems, but the assessment of
postnatal renal morphological and functional development may serve as a model for the use of postnatal
evaluations in the risk assessment process. As an example, standard morphological analyses of the
kidneys of fetal rodents have detected treatment-related changes in the relative growth of the renal
papilla versus the renal cortex, an effect considered in some cases to be a malformation
(hydronephrosis), while in other cases a variation (apparent hydronephrosis, enlarged or dilated renal
pelvis). While some investigators (Woo and Hoar, 1972) have provided data suggesting that the

15
morphological effect represents a transient developmental delay, others have shown that it can persist
well into postnatal life and that physiological function is compromised in the affected individuals
(Kavlock et al., 1987a, 1988; Daston et al., 1988; Couture, 1990). Thus, the biological interpretation
of this effect on the basis of fetal examinations alone is tenuous (U.S. EPA, 1985b). In addition, the
critical period for inducing renal morphological abnormalities extends into the postnatal period
(Couture, 1990), and studies on perinatally induced renal growth retardation (Kavlock et al., 1986,
1987b; Slotkin et al., 1988; Gray et al., 1989; Gray and Kavlock, 1991) have shown that renal
function is generally altered in such conditions, but that manifestation of the dysfunction is not readily
predictable. Thus, both morphological and functional assessment of the kidneys after birth can provide
useful and complementary information on the persistence and biological significance of expressions of
developmental toxicity.
Although not as well studied, data indicate that the cardiovascular, respiratory, immune,
endocrine, reproductive, and digestive systems also are subject to alterations in functional competence
(Kavlock and Grabowski, 1983; Fujii and Adams, 1987) following exposure during development.
Currently, there are no standard testing procedures for these functional systems; however, when data
are encountered on a chemical under review, they are considered in the risk assessment process.
Direct extrapolation of functional developmental effects to humans is limited in the same way as
for other endpoints of developmental toxicity, i.e., by the lack of knowledge about underlying
toxicological mechanisms and their significance. In evaluations of a limited number of agents known to
cause developmental neurotoxic effects in humans, Adams (1986) concluded that these agents produce
similar developmental neurotoxic effects in animals and humans. This conclusion was strongly
supported by the results of a recent “Workshop on the Qualitative and Quantitative Comparability of
Human and Animal Developmental Neurotoxicity,” sponsored by EPA and the National Institute on
Drug Abuse (NIDA), at which participants critically evaluated and compared the effects of agents
known to cause human developmental neurotoxicity with the effects seen in experimental animal studies
(Kimmel et al., 1990a). The high degree of qualitative correlation between human and experimental
animal data for the agents evaluated lends strong support for the use of experimental animals in
assessing the potential risk for developmental neurotoxicity in humans. Thus, as for other endpoints of
developmental toxicity, the assumption can be made that functional effects in animal studies indicate the
potential for altered development in humans, although the types of developmental effects seen in
experimental animal studies will not necessarily be the same as those that may be produced in humans.
Thus, when data from functional developmental toxicity studies are encountered for particular agents,
they should be considered in the risk assessment process.

16
 Some guidance is provided here concerning important general concepts of study design and
evaluation for functional developmental toxicity studies.
C Several aspects of study design are similar to those important in standard developmental
toxicity studies (e.g., a dose-response approach with the highest dose producing minimal
overt maternal or perinatal toxicity, number of litters large enough for adequate statistical
power, randomization of animals to dose groups and test groups, litter generally considered
the statistical unit, etc.).
C A replicate study design provides added confidence in the interpretation of data.
C A pharmacological/physiological challenge may be valuable in evaluating function and
“unmasking” effects not otherwise detectable, particularly in the case of organ systems that
are endowed with a reasonable degree of functional reserve capacity.
C Functional tests with a moderate degree of background variability may be more sensitive to
the effects of an agent on behavioral endpoints than are tests with low variability that may
be impossible to disrupt without being life-threatening (Butcher et al., 1980).
C A battery of functional tests, in contrast to a single test, is usually needed to evaluate the full
complement of organ function in an animal; tests conducted at several ages may provide
more information about maturational changes and their persistence.
C Critical periods for the disruption of functional competence include both the prenatal and
the postnatal periods to the time of sexual maturation, and the effect is likely to vary
depending on the time and degree of exposure.
C Interpretation of data from studies in which postnatal exposure is included should take into
account possible interaction of the agent with maternal behavior, milk composition, pup
suckling behavior, possible direct exposure of pups via dosed feed or water, etc.
Although interpretation of functional data may be limited at present, it is clear that functional
effects must be evaluated in light of other toxicity data, including other forms of developmental toxicity
(e.g., structural abnormalities, perinatal death, and growth retardation). The level of confidence in an
adverse effect may be as important as the type of change seen, and confidence may be increased by
such factors as replicability of the effect either in another study of the same function or by convergence
of data from tests that purport to measure similar functions. A dose-response relationship is considered
an important measure of chemical effect; in the case of functional effects, both monotonic and biphasic
dose-response curves are likely, depending on the function being tested.
Finally, there are at least three general ways in which the data from these studies may be useful
for risk assessment purposes: (1) to help elucidate the long-term consequences of fetal and neonatal

17
effects; (2) to indicate the potential for an agent to cause functional alterations and the effective doses
relative to those that produce other forms of toxicity; and (3) for existing environmental agents, to
suggest organ systems to be evaluated in exposed human populations.

3.1.1.4. Overall Evaluation of Maternal and Developmental Toxicity

As discussed previously, individual endpoints of maternal and developmental toxicity are
evaluated in developmental toxicity studies. In order to interpret the data fully, an integrated evaluation
must be performed considering all maternal and developmental endpoints.
Agents that produce developmental toxicity at a dose that is not toxic to the maternal animal are
especially of concern because the developing organism is affected but toxicity is not apparent in the
adult. However, the more common situation is when adverse developmental effects are produced only
at doses that cause minimal maternal toxicity; in these cases, the developmental effects are still
considered to represent developmental toxicity and should not be discounted as being secondary to
maternal toxicity. At doses causing excessive maternal toxicity (that is, significantly greater than the
minimal toxic dose), information on developmental effects may be difficult to interpret and of limited
value. Current information is inadequate to assume that developmental effects at maternally toxic doses
result only from maternal toxicity; rather, when the LOAEL is the same for the adult and developing
organisms, it may simply indicate that both are sensitive to that dose level. Moreover, whether
developmental effects are secondary to maternal toxicity or not, the maternal effects may be reversible
while effects on the offspring may be permanent. These are important considerations for agents to
which humans may be exposed at minimally toxic levels either voluntarily or involuntarily, since several
agents are known to produce adverse developmental effects at minimally toxic doses in adult humans
(e.g., smoking, alcohol, isotretinoin).
Since the final risk assessment not only takes into account the potential hazard of an agent, but
also the nature of the dose-response relationship, it is important that the relationship of maternal and
developmental toxicity be evaluated and described. Then, information from the exposure assessment is
used to determine the likelihood of exposure to levels near the maternally toxic dose for each agent and
the risk for developmental toxicity in humans.
Although the evaluation of developmental toxicity is the primary objective of standard studies
within this area, maternal effects seen within the context of developmental toxicity studies should be
evaluated as part of the overall toxicity profile for a given chemical. Maternal toxicity may be seen in
the absence of or at dose levels lower than those producing developmental toxicity. If the maternal
effect level is lower than that in other evaluations of adult toxicity, this implies that the pregnant female is

18
likely to be more sensitive than the nonpregnant female. Data from reproductive and developmental
toxicity studies on the pregnant female should be used in the overall assessment of risk.
Approaches for ranking agents according to their relative maternal and developmental toxicity
have been proposed; Schardein (1983) has reviewed several of these. Several approaches involve the
calculation of ratios relating an adult toxic dose to a developmentally toxic dose (Johnson, 1981; Fabro
et al., 1982; Johnson and Gabel, 1983; Brown and Freeman, 1984). Such ratios may describe in a
qualitative and roughly quantitative fashion the relationship of maternal (adult) and developmental
toxicity. However, at the U.S. EPA-sponsored “Workshop on the Evaluation of Maternal and
Developmental Toxicity” (Kimmel et al., 1987), there was no agreement as to the validity or utility of
these approaches in other aspects of the risk assessment process. This is due in part to uncertainty
about factors that can affect the ratios. For example, the number and spacing of dose levels,
differences in study design (e.g., route and/or timing of exposure), the relative thoroughness in the
assessment of maternal and developmental endpoints examined, species differences in response, and
differences in the slope of the dose-response curves for maternal and developmental toxicity can all
influence the maternal and developmental effects observed and the resulting ratios (Kimmel et al., 1987;
U.S. EPA, 1985b). Also, maternal and developmental endpoints used in the ratios need to be better
defined to permit cross-species comparison. Until such information is available, the applicability of
these approaches in risk assessment is not justified.

3.1.1.5. Short-Term Testing in Developmental Toxicity

The need for short-term tests for developmental toxicity has arisen from the need to establish
testing priorities for the large number of agents in or entering the environment, the interest in reducing
the number of animals used for routine testing, and the expense of testing. These approaches may be
useful in making preliminary evaluations of potential developmental toxicity, for evaluating structure-
activity relationships, and for assigning priorities for further, more extensive testing. Furthermore, as the
risk assessment process begins to incorporate more pharmacokinetic and mechanistic data, short-term
tests should be particularly useful. Kimmel (1990) has recently discussed the potential application of in
vitro systems in risk assessment in a context that is broader than chemical screening. However, the
Agency currently considers a short-term test as “insufficient” by itself to carry out a risk assessment
(see Section 3.3).
Although short-term tests for developmental toxicity are not routinely required, such data are
encountered in the review of chemicals. Two approaches are considered here in terms of their
contribution to the overall testing process: an in vivo mammalian screen and in vitro test systems.

19
3.1.1.5.1. In vivo mammalian developmental toxicity tests. The most widely studied in vivo
short-term approach is that developed by Chernoff and Kavlock (1982). This approach is based on
the hypothesis that a prenatal injury, which results in altered development, will be manifested postnatally
as reduced viability and/or impaired growth. When originally proposed, the test substance was
administered to mice over the period of major organogenesis at a single dose level that would elicit
some degree of maternal toxicity. At the NIOSH “Workshop on the Evaluation of the
Chernoff/Kavlock Test for Developmental Toxicity” (Hardin, 1987), use of a second lower dose level
was encouraged to potentially reduce the chances of false positive results, and the recording of
implantation sites was recommended to provide a more precise estimate of postimplantation loss
(Kavlock et al., 1987c). In this approach, the pups are counted and weighed shortly after birth, and
again after 3-4 days. Endpoints that are considered in the evaluation include: general maternal toxicity
(including survival and weight gain), litter size, pup viability and weight, and gross malformations in the
offspring. Several schemes have been proposed for ranking the results as a means of prioritizing agents
for further testing (Chernoff and Kavlock, 1982; Brown, 1984; Schuler et al., 1984).
The mouse was chosen originally for this test because of its low cost, but the procedure has
been applied to the rat as well (Wickramaratne, 1987). The test can predict the potential for
developmental toxicity of an agent in the species used while extrapolation of risk to other species,
including humans, has the same limitations as for other testing protocols. The EPA Office of Toxic
Substances has developed testing guidelines for this procedure (U.S. EPA, 1985c), and the Office of
Pesticide Programs has applied similar protocols on a case-by-case basis (U.S. EPA, 1985b). The
National Toxicology Program also has developed a protocol that incorporates aspects of a range-
finding study, with the intent of providing information on appropriate exposure levels should a standard
developmental toxicity study be required (Morrissey et al., 1989). Although testing guidelines are
available, such procedures are required on a case-by-case basis. Application of this procedure in the
risk assessment process within the Office of Toxic Substances has been described (Francis and
Farland, 1987), and the experiences of a number of laboratories are detailed in the proceedings of a
NIOSH-sponsored workshop (Hardin, 1987).
Recently, the OECD developed a screening protocol to be used for prioritizing existing
chemicals for further testing (draft as of March 22, 1990). This protocol is similar to the design of the
Chernoff-Kavlock test except that it involves exposure of male and female rats 2 weeks prior to
mating, throughout mating and gestation, and postnatally to day 4. Male animals are exposed following
mating for a period corresponding to that of the females. Adult animals are evaluated for general
toxicity and effects on reproductive organs. Pups are counted, weighed, and examined for any gross

20
physical or behavioral abnormalities at birth and on postnatal day 4. This protocol permits evaluation of
reproductive and developmental toxicity following repeated dosing with an agent, provides an indication
for the need to conduct additional studies, and provides guidance in the design of further studies.
Currently, this study design is insufficient by itself to make an estimate of human risk without further
studies to confirm and extend the observations.

3.1.1.5.2. In vitro developmental toxicity screens. Test systems that fall under the general heading
of “in vitro” developmental toxicity screens include any system that employs a test subject other than the
intact pregnant mammal. Examples of such systems include isolated whole mammalian embryos in
culture, tissue/organ culture, cell culture, and developing nonmammalian organisms. These systems
have long been used to assess events associated with normal and abnormal development, but more
recently they have been considered for their potential as screens in testing (Wilson, 1978; Kimmel et
al., 1982b; Brown and Fabro, 1982). Many of these systems are now being evaluated for their ability
to predict the developmental toxicity of various agents in intact mammalian systems. This validation
process requires certain considerations in study design, including defined endpoints for toxicity and an
understanding of the system’s ability to handle various test agents (Kimmel et al., 1982a; Kimmel,
1985; FDA, 1987; Brown, 1987).
While in vitro test systems can provide significant information, they are considered insufficient,
by themselves, for carrying out a risk assessment (see Section 3.3). In part, this is due to limitations in
the application of the data to the whole-animal situation. But it is also due to the lack of assays that
have been fully validated, as has been noted in several reviews of available in vitro systems (FDA,
1987; Brown, 1987; Faustman, 1988) and at a recent workshop on in vitro teratology (Morrissey et
al., 1991).

3.1.1.6. Statistical Considerations

In the assessment of developmental toxicity data, statistical considerations require special
attention. Since the litter is generally considered the experimental unit in most developmental toxicity
studies, and fetuses or pups within litters do not respond independently, the statistical analyses are
generally designed to analyze the relevant databased on incidence per litter or on the number of litters
with a particular endpoint. The analytical procedures used and the results, as well as an indication of
the variance in each endpoint, should be evaluated carefully when reviewing data for risk assessment
purposes. Analysis of variance (ANOVA) techniques, with litter nested within dose in the model, take
the litter variable into account while allowing use of individual offspring data and an evaluation of both

21
within and between litter variance as well as dose effects. Nonparametric and categorical procedures
have also been widely used for binomial or incidence data. In addition, tests for dose-response trends
can be applied. Although a single statistical approach has not been agreed upon, a number of factors
important in the analysis of developmental toxicity data have been discussed (Haseman and Kupper,
1979; Kimmel et al., 1986).
Studies that employ a replicate experimental design (e.g., two or three replicates with 10 litters
per dose per replicate rather than a single experiment with 20 to 30 litters per dose group) allow
broader interpretation of study results since the variability between replicates can be accounted for
using ANOVA techniques. Replication of effects due to a given agent within a study, as well as among
studies or laboratories, provides added strength in the use of data for the estimation of risk.
An important factor to consider in evaluating data is the power of a study (i.e., the probability
that a study will demonstrate a true effect), which is limited by the sample size used in the study, the
background incidence of the endpoint observed, the variability in the incidence of the endpoint, and the
analysis method. As an example, Nelson and Holson (1978) have shown that the number of litters
needed to detect a 5% or 10% change was dramatically lower for fetal weight (a continuous variable
with low variability) than for resorptions (a binomial response with high variability). With the current
recommendation in testing protocols being 20 rodents per dose group (U.S. EPA, 1982b, 1985a), the
minimum change detectable is an increased incidence of malformations 5 to 12 times above control
levels, an increase 3 to 6 times the in utero death rate, and a decrease 0.15 to 0.25 times the fetal
weight. Thus, even within the same study, the ability to detect a change in fetal weight is much greater
than for the other endpoints measured. Consequently, for statistical reasons only, changes in fetal
weight are often observed at doses below those producing other signs of developmental toxicity. Any
risk assessment should present the detection sensitivity for the study design used and for the endpoint(s)
evaluated.
Although statistical analyses are important in determining the effects of a particular agent, the
biological significance of data is most relevant. It is important to be aware that with the number of
endpoints that can be observed in standard protocols for developmental toxicity studies, a few
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. This may be true especially for the incidence of malformations or in utero death because of the
low power of standard study designs in which a relatively large difference is required to be statistically
significant. It should be apparent from this discussion that a great deal of scientific judgment, based on

22
experience with developmental toxicity data and with principles of experimental design and statistical
analysis, may be required to adequately evaluate such data.

3.1.2. Human Studies

In principle, human data are preferred for risk assessment. However, the complexities of
obtaining sufficient human data are such that these data are not available for many potential toxicants.
The following describes the methods of generation of human data, their evaluation, and the weight they
should be given in risk assessments.
The category of “human studies” includes both epidemiologic studies and other reports of
individual cases or clusters of events. Greatest weight should be given to carefully designed
epidemiologic studies with more precise measures of exposure, since they can best evaluate exposure-
response relationships (see Section 4). Epidemiologic studies in which exposure is presumed based on
occupational title or residence (e.g., some case-referent and all ecologic studies) may contribute data to
qualitative risk assessments, but are of limited use for quantitative risk assessments because of the
generally broad categorical groupings. Reports of individual cases or clusters of events may generate
hypotheses of exposure-outcome associations, but require further confirmation with well-designed
epidemiologic or laboratory studies. These reports of cases or clusters may give added support to
associations suggested by other human or animal data, but cannot stand by themselves in risk
assessments. Risk assessors should seek the assistance of professionals trained in epidemiology when
conducting a detailed analysis.

3.1.2.1. Epidemiologic Studies

Good epidemiologic studies provide the most relevant information for assessing human risk. As
there are many different designs for epidemiologic studies, simple rules for their evaluation do not exist.

3.1.2.1.1. 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; U.S. EPA, 1981; Wilcox, 1983; Sever and Hessol, 1984; Axelson, 1985; Tilley et al., 1985;
Kimmel et al., 1986). Some of the more prominent factors are as follows:
(a) 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 in order to have a high probability of detecting a modest increase

23
in risk (e.g., 133 in both exposed and unexposed groups to detect a doubling of background; alpha =
0.05, power = 80%), while less common outcomes, such as the total of all malformations recognized at
birth, require thousands of pregnancies to have the same probability (e.g., more than 1,200 in both
exposed and unexposed groups) (Bloom, 1981; Selevan, 1981; Sever and Hessol, 1984; Selevan,
1985; Stein et al., 1985; Kimmel et al., 1986). In case-referent studies, study sizes are dependent on
the frequency of exposure within the source population. The confidence one has in the results of a
study without positive findings is related to the power of the study to detect meaningful differences in the
endpoints studied.
Power may be enhanced by combining populations from several studies using a meta-analysis
(Greenland, 1987). The combined analysis would increase confidence in the absence of risk for agents
with negative findings. However, care must be exercised in the combination of potentially dissimilar
study groups.
A posteriori determination of power of the actual study may be useful in evaluating
contradictory studies in risk assessment. Absence of positive findings in a study of low power would be
given less weight than either a positive study or a null study (one with no significant differences) with
high power. Positive findings from very small studies are open to question due to the instability of the
risk estimates and the potential for highly selected study groups.
(b) Potential bias in data collection: Sources of bias may include selection bias and information
bias (Rothman, 1986). Selection bias may occur when an individual’s willingness to participate varies
with certain characteristics relating to the exposure status or health status of that individual. In addition,
selection bias may operate in the identification of subjects for study. For example, in studies of
embryonic loss, use of hospital records to identify embryonic or early fetal loss will underascertain
events, because women are not always hospitalized for these outcomes. More weight might be given in
a risk assessment to a study in which a more complete list of pregnancies is obtained by, for example,
collecting biological data [e.g., human chorionic gonadotropin (hCG) measurements] on pregnancy
status from study members. These studies may also be affected by bias. 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; however, this strategy carries with it the potential for recall bias, discussed in further
detail below. A second example of different levels of ascertainment of events is the use of hospital
records to study congenital malformations. Hospital records contain more complete data on
malformations than do birth certificates (Mackeprang et al., 1972). Consequently, birth defects
registries that are based on searches of hospital records are more complete than those based on vital

24
records (Selevan, 1986). Thus, a study using hospital records to identify congenital malformations
would be given more emphasis in a risk assessment than one using birth certificates.
Studies of working women present the potential for additional bias since some factors that
influence employment status may also be associated with 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).
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 will reduce the likelihood of such bias.
Studies with lower likelihood of the above-listed biases 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 considerably more
complete and valid than those of the husbands (Selevan, 1980). A more recent study (Schnatter,
1990) found small, nonsignificant improvements in reporting of birth weights by mothers compared to
fathers, and that males who provide early fetal loss data with the aid of their wives give better data
(borderline significance). Studies based on interview data from the appropriate respondent(s) would
carry more weight than those from proxy respondents (e.g., the specific individual when examining
exposure history and the woman or both partners when examining pregnancy history).
Data from any source may be prone to errors or bias. All types of bias are difficult to assess;
however, validation with an independent data source (e.g., vital or hospital records) or use of
biomarkers of exposure or outcome, where possible, may indicate the degree of bias present and
increase confidence in the results of the study. Those studies with a low probability of biased data
should carry more weight (Axelson, 1985; Stein and Hatch, 1987).
Differential misclassification, i.e., when certain subgroups are more likely to have misclassified
data than others, may either raise or lower the risk estimate. Nondifferential misclassification will bias
the results toward a finding of “no effect” (Rothman, 1986).
(c) Collection of data on other risk factors, effect modifiers, and confounders: Risk factors for
reproductive and developmental toxicity include such characteristics as age, smoking, alcohol

25
consumption, drug use, and past reproductive history. Additionally, occupational and environmental
exposures are potential risk factors for reproductive and developmental effects. Known and potential
risk factors should be examined to identify those that may be effect modifiers or confounders. An effect
modifier is a factor that produces different exposure-response relationships at different levels of that
factor. For example, maternal age would be an effect modifier if the risk associated with a given
exposure increased with the mother’s age. A confounder is a variable that is a risk factor for the
disease under study and is associated with the exposure under study, but is not a consequence of the
exposure. A confounder may distort both the magnitude and direction of the measure of association
between the exposure of interest and the outcome. For example, socioeconomic status might be a
confounder in a study of the association of smoking and fertility, since socioeconomic status may be
associated with both.
Studies that fail to account for effect modifiers and confounders should be given less weight in a
risk assessment. Both of these important factors 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, 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).
(d) Statistical factors: As in animal studies, pregnancies experienced by the same woman are
not independent events (Kissling, 1981; Selevan, 1985). Women who have had embryo/fetal loss are
reported to be more likely to have subsequent losses (Leridon, 1977). In animal studies, the litter is
generally used as the unit of measure to deal with nonindependence of events. In studies of humans,
pregnancies are sequential with the risk factors changing for different pregnancies, making analyses
considering nonindependence of events very difficult (Epidemiology Workgroup, 1981; Kissling,
1981). If more than one pregnancy per woman is included, as is often necessary due to small study
groups, the use of nonindependent observations overestimates the true size of the groups being
compared, thus artificially increasing the probability of reaching statistical significance (Stiratelli et al.,
1984). Biased estimates of risk might also result if family size confounds the relationship between
exposure and outcome. Some approaches to deal with these issues have been suggested (Kissling,
1981; Stiratelli et al., 1984; Selevan, 1985). At this point in time, a generally accepted solution to this
problem has not been developed.

3.1.2.1.2. Selection of outcomes for study. As already discussed, a number of endpoints can be
considered in the evaluation of adverse developmental effects. However, some of the outcomes are not

26
easily observed in humans, such as early embryonic loss and reproductive capacity of the offspring.
Currently, the most feasible endpoints for epidemiologic studies are reproductive history studies of
some pregnancy outcomes (e.g., embryo/fetal loss, birth weight, sex ratio, congenital malformations,
postnatal function, and neonatal growth and survival) and measures of fertility/infertility, which would
include indirect evaluations of very early embryonic loss. Postnatal outcomes for examination could
include physical growth and development, organ or system function, and behavioral effects of exposure.
Factors requiring control in the design or analysis (such as effect modifiers and confounders) may vary
depending on the specific outcomes selected for study.
The developmental outcomes available for epidemiologic examination are limited by a number
of factors, including the relative magnitude of the exposure, because differing spectra of outcomes may
occur at different exposure levels, different size and demographic characteristics of the population, and
different ability to observe the developmental outcome in humans. Improved methods for identifying
some outcomes such as very early embryonic loss using new hCG assays may change the spectrum of
outcomes available for study (Wilcox et al., 1985; Sweeney et al., 1988).
Demographic characteristics of the population, such as marital status, age distribution,
education, socioeconomic status (SES), and prior reproductive history are associated with the
probability of whether couples will attempt to have children. Differences in the use of birth control
would also affect the number of outcomes available for study. In addition, women with live births are
more likely to terminate employment than are those with other outcomes, such as infertility or early
embryonic loss. Thus, retrospective studies of female exposure that do not include terminated women
workers may be of limited use in risk assessment because the level of risk for these outcomes is likely
to be overestimated (Lemasters and Pinney, 1989).
In addition to the above-mentioned factors, developmental endpoints may be envisioned as
effects recognized at various points in a continuum, starting at conception through death of the offspring.
Thus, a malformed stillbirth would not be included in a study of defects observed at live birth, even
though the etiology could be identical (Stein et al., 1975; Bloom, 1981). A shift in the patterns of
outcomes could result from differences in timing or in level of exposure (Selevan and LeMasters,
1987).

3.1.2.1.3. Reproductive history studies. (a) Measures of fertility: Normally, studies of sub- or
infertility would not be included in an evaluation of developmental effects. However, in humans it is
difficult to identify very early embryonic loss, and distinguish it from sub- or infertility. Thus, studies that
examine sub- or infertility indirectly examine loss very early in the gestational period. Infertility or

27
subfertility may be thought of as a nonevent: a couple is unable to have children within a specific time
frame. Therefore, the epidemiologic measurement of reduced fertility is typically indirect, and is
accomplished by comparing birth rates or time intervals between births or pregnancies. In these evalu­
ations, the couple’s joint ability to procreate is estimated. One method, the Standardized Birth Ratio
(SBR; also referred to as the Standardized Fertility Ratio), compares the number of births observed to
those expected based on the person-years of observation stratified by factors such as time period, age,
race, marital status, parity, contraceptive use, etc. (Wong et al., 1979; Levine et al., 1980, 1981;
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).
Analysis of the time period between recognized pregnancies or live births has been suggested
as another indirect measure of fertility (Dobbins et al., 1978; Baird et al., 1986; Weinberg and Gladen,
1986). Because the time interval 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.
Fertility may also be affected by alterations in sexual behavior. However, limited data are
available linking toxic exposures to these alterations in humans. Moreover, such data are not easily
obtained in epidemiology studies. More information on this subject is available in the proposed male
and female reproductive risk assessment guidelines (U.S. EPA, 1988b, 1988c).
(b) Pregnancy outcomes: Pregnancy outcomes examined in human studies of parental
exposures may include embryo/fetal loss, congenital malformations, birth weight, sex ratio at birth, and
postnatal effects (e.g., physical growth and development, organ or system function, and behavioral
effects of exposure). Postnatal effects are discussed in more detail in the next section. As mentioned
previously, epidemiologic studies that focus on only one type of pregnancy outcome may miss a true
effect of exposure because of the continuum of outcomes. Examination of individual outcomes could
mask a true effect due to reduced power resulting from fewer events for study. Studies that examine
multiple endpoints could yield more information, but the results may be difficult to interpret.
Evidence of a dose-response relationship is usually an important criterion in the assessment of a
toxic exposure. However, traditional dose-response relationships may not always be observed for
some endpoints. For example, with increasing dose, a pregnancy might end in a 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

28
description, see Section 3.1.2.1.5). Therefore, a risk assessment should, when possible, attempt to
look at the interrelationship of different reproductive endpoints and patterns of exposure.
(c) Postnatal developmental effects: These effects may include changes in growth, behavior,
organ or system function, or cancer. Studies of neurological and reproductive function are discussed
here as examples. Postnatal behavioral and functional effects in humans have been examined for a small
number of environmental and occupational agents (e.g., lead, PCBs, methyl mercury, alcohol). For
some agents (e.g., lead and PCBs), subtle changes have been observed in groups of children at lower
exposures than for other developmental effects (e.g., Bellinger et al., 1987; Needleman, 1988; Davis et
al., 1990; Tilson et al., 1990). This may not be true for all toxic agents. These subtle differences would
be difficult to identify in individuals, but could result in an overall shifting of mean values when
comparing groups of exposed and unexposed children. Some postnatal studies have examined infants
or young children using standard developmental scales (e.g., Brazelton Neonatal Behavioral
Assessment Scale, Bayley Scales of Infant Development, Stanford Binet IV, and Wechsler Scales) and
some biologic measure of exposure (e.g., blood lead levels). These tests are designed to examine
certain endpoints and have been developed to cover certain age ranges. Certain tests examine specific
aspects of development. For example, the Bayley Scales look at motor and language development, but
do not examine sensory function. Batteries of tests are important for a proper evaluation because of the
possibility of interrelated effects, e.g., hearing deficits and language development. Thus, batteries of
tests will give a clearer indication of direct effects of exposure resulting in postnatal developmental
deficits.
Factors that may influence the examination of these effects include parental education, SES,
obstetrical history, and health characteristics independent of exposure that may affect functional
measurement (e.g., injuries and infections). Many social and lifestyle factors may also affect scoring on
these scales (e.g., neonatal-maternal interactions, SES, home environment).
Studies of premature infants carry special problems. For proper comparisons, tests keyed to
age in very young children (less than 2.5 years of age) need to “correct” the age for premature infants to
the age they would have been had they been born at term. In addition, premature infants or those with
low birth weight for their gestational age may have problems resulting from the birth process not directly
related to exposure (e.g., intraventricular hemorrhage in the brain which can then cause developmental
problems). Thus, the developmental effects resulting from exposure may have their own sequelae.
Other studies may examine effects occurring at a later age (e.g., in utero exposure and cancer in
young women). This long time interval typically carries with it the need for retrospective studies, with
the inherent limitations in accurate determination of exposure, effect modifiers, and confounders. Risk

29
assessment methods for cancer are described in the “Guidelines for Carcinogen Risk Assessment”
(U.S. EPA, 1986b).
Reproductive effects may result from developmental exposures. For example, environmental
exposures may result in oocyte toxicity, in which a loss of primordial oocytes irreversibly affects a
woman’s fertility. The exposures of importance may occur during both the prenatal period and after
birth. 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). Risk assessment methods for female reproductive effects are
described in the “Proposed Guidelines for Assessing Female Reproductive Risk” (U.S. EPA, 1988c).
Developmental exposures to males could affect their reproductive function (e.g., deplete stem
or Sertoli cells potentially affecting sperm production) (Zenick and Clegg, 1989). If stem cell death
occurs with exposure at any age, recovery is possible as long as some stem cells survive. The same is
true for Sertoli cells, except that they cease multiplication before puberty. Thus, cell replication cannot
compensate for Sertoli cell death after puberty. Human studies of stem and Sertoli cells would be
difficult due to the invasiveness of the measure. Less direct measures, e.g., sperm count, morphology,
and motility, could be evaluated, but this would not indicate what cells or stage of spermatogenesis had
been affected. Risk assessment methods for male reproductive effects are described in the “Proposed
Guidelines for Assessing Male Reproductive Risk” (U.S. EPA, 1988b).
In addition to the above effects, genetic damage to germ cells may result from developmental
exposures. Outcomes resulting from germ-cell mutations could include reduced probability of
conception as well as increased probability of embryo/fetal loss and other developmental effects.
These endpoints could be studied using the approaches described above. However, a human germ-cell
mutagen has not yet been demonstrated (U.S. EPA, 1986c). Based on animal studies, critical
exposures are to germ cells or early zygotes. Germ-cell mutagenicity could also be expressed as
genetic diseases in future generations. Unfortunately, these studies would be very difficult to conduct in
human populations because of the long time lag between exposure and outcome. For more
information, refer to the “Guidelines for Mutagenicity Risk Assessment” (U.S. EPA, 1986c).

3.1.2.1.4. Community studies/surveillance programs. Epidemiologic studies may also be based

on broad populations such as a community, a nationwide probability sample, or surveillance programs
(such as birth defects registries). Other studies have examined environmental exposures, such as toxic
agents in the water system, and adverse pregnancy outcome (Swan et al., 1989; Deane et al., 1989).
Unfortunately, in these studies maternally mediated effects may be difficult to distinguish from paternally

30
mediated effects. In addition, the presumably lower exposure levels (compared to industrial settings)
may require very large groups for study. A number of case-referent studies have examined the
relationship between broad classes of parental occupation in certain communities or countries and
embryo/fetal loss (Silverman et al., 1985), birth defects (Hemminki et al., 1980; Kwa and Fine, 1980;
Papier, 1985), and childhood cancer (Kwa and Fine, 1980; Zack et al., 1980; Hemminki et al., 1981;
Peters et al., 1981). In these reports, jobs are typically classified into broad categories based on the
probability of exposure to certain classes or levels of exposure (e.g., Kwa and Fine, 1980). 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, such studies are not typically useful for risk assessment
of a particular agent.
Surveillance programs may also exist in occupational settings. In this case, reproductive
histories and/or clinical evaluations could be followed to monitor for reproductive effects of exposures.
Both could yield very useful data for risk assessment; however, a clinical evaluation program would be
costly to maintain, and there are numerous impediments to the collection of reliable and valid
information in the workplace. These might include concerns similar to those previously discussed plus
potentially low participation rates due to employee sensitivities and confidentiality concerns.

3.1.2.1.5. Identification of exposures important for developmental effects. For all examinations
of the relationship between developmental effects and potentially toxic exposures, the identification of
the appropriate exposure is crucial. Preconceptional exposures to either parent and in utero exposures
have been associated with the more commonly examined outcomes (e.g., fetal loss, malformations, birth
weight, and measures of infertility). These exposures, plus postnatal exposure from breast milk, food,
and the general environment, may be associated with postnatal developmental effects (e.g., changes in
behavioral and cognitive function, or growth). The magnitude of exposure may affect the spectrum of
outcomes observed. This issue is discussed in more detail in Sections 3.1.1.2 and 3.2.
Infants and young children may receive disproportionate levels of exposure due to their
tendency to “put everything” in their mouths (pica) and the greater time they spend on the floor.
Carpets may serve as a reservoir for toxic agents (e.g., pesticides and lead dust), and the air nearer the
floor may have greater levels of certain airborne toxicants (e.g., mercury from latex paints).
Exposures in environmental settings are frequently lower than in industrial and agricultural
settings. However, this relationship may change as exposures are reduced in workplaces, and as more
is learned about environmental exposures (e.g., indoor air exposures, pesticides usage). Larger
populations are necessary in settings with lower exposures (Lemasters and Selevan, 1984). Other

31
factors affect the identification of reproductive or developmental events with various levels of exposure.
Exposed individuals may move in and out of areas with differing levels and types of exposures, affecting
the number of exposed and comparison events for study. Thus, exposures can be short-term or
chronic.
Data on exposure from human studies are frequently qualitative, such as employment or
residence histories. More quantitative data may be difficult to obtain due to the nature of certain study
designs (e.g., retrospective studies) and historical limitations in exposure measurements. Many
developmental outcomes result from exposures during certain critical times. The appropriate exposure
classification depends on the outcome(s) studied, the biologic mechanism affected by exposure, and the
biologic half-life of the agent. The biologic half-life, in combination with the patterns of exposure (e.g.,
continuous or intermittent) affect the individual’s body burden and consequently the “true” dose during
the critical period. The probability of misclassification of exposure status may affect the ability to
recognize a true effect in a study (Selevan, 1981; Hogue, 1984; Lemasters and Selevan, 1984; Sever
and Hessol, 1984; Kimmel et al., 1986). As more prospective studies are done, better estimates of
exposure will be developed.

3.1.2.2. Examination of Clusters or Case Reports/Series

The identification of cases or clusters of adverse pregnancy outcomes is generally limited to
those identified by the women involved, or clinically by their physicians. Examples of outcomes more
easily identified include mid-to-late fetal loss or congenital malformations. Identification of other effects,
such as very early embryonic loss, may be difficult to separate from the study of sub- or infertility. Such
“nonevents” (e.g., lack of pregnancies or children) are much harder to recognize than are
developmental effects such as malformations resulting from in utero exposure. While case reports have
been important in the recognition of some agents that cause developmental toxicity, they may be of
greatest use in suggesting topics for further investigation (Hogue, 1985). 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 animals also occur in humans. Previous discussion of the use of human data should
be taken into account wherever possible.

3.1.3. Other Considerations

Several other types of information may be considered in the evaluation and interpretation of
human and animal data. Information on pharmacokinetics and structure-activity relationships may be
very useful, but is often lacking for developmental toxicity risk assessments.

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3.1.3.1. Pharmacokinetics
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 and/or peak plasma concentrations, placental
metabolism and transfer, excretion in breast milk, comparative metabolism, and concentrations of the
parent compound and metabolites may be useful in predicting risk for developmental toxicity. Such
data may also be helpful in defining the dose-response curve, developing a more accurate comparison
of species sensitivity (Wilson et al., 1975, 1977), determining dosimetry at target sites, and comparing
pharmacokinetic profiles for various dosing regimens or routes of exposure. Pharmacokinetic studies in
developmental toxicology are most useful if conducted in animals at the stage when developmental
insults occur. The correlation of pharmacokinetic parameters and developmental toxicity data may be
useful in determining the contribution of specific pharmacokinetic parameters to the effects observed
(Kimmel and Young, 1983).
While human pharmacokinetic data are often lacking, absorption data in laboratory animals for
studies conducted by any relevant route of exposure may assist in the interpretation of the
developmental toxicity studies in the animal models for the purposes of risk assessment. Specific
guidance regarding both the development and application of pharmacokinetic data was agreed upon by
the participants at the “Workshop on the Acceptability and Interpretation of Dermal Developmental
Toxicity Studies” (Kimmel and Francis, 1990). It was concluded that absorption data are needed both
when a dermal developmental toxicity study shows no developmental effects and when developmental
effects are seen. The results of a dermal developmental toxicity study showing no adverse
developmental effects and without blood level data (as evidence of dermal absorption) are potentially
misleading and would be insufficient for risk assessment, especially if interpreted as a “negative” study.
In studies where developmental toxicity is detected, regardless of the route of exposure, absorption
data can be used to establish the internal dose in maternal animals for risk extrapolation purposes.

3.1.3.2. Comparisons of Molecular Structure

Comparisons of the chemical or physical properties of an agent with those known to cause
developmental toxicity may indicate a potential for developmental toxicity. Such information may be
helpful in setting priorities for testing of agents or for evaluation of potential toxicity when only minimal
data are available. Structure-activity relationships have not been well studied in developmental
toxicology, although data are available that suggest structure-activity relationships for certain classes of

33
chemicals (e.g., glycol ethers, steroids, retinoids). Under certain circumstances (e.g., in the case of new
chemicals), this is one of several procedures used to evaluate the potential for toxicity when little or no
data are available.

3.2. DOSE-RESPONSE EVALUATION

The evaluation of dose-response relationships for developmental toxicity includes the evaluation
of data from both human and animal studies. When quantitative dose-response data are available in
humans and with sufficient range of exposure, dose-response relationships may be examined. Since
data on human dose-response relationships have been available infrequently, the dose-response
evaluation is usually based on the assessment of data from tests performed in laboratory animals.
Evidence for a dose-response relationship is an important criterion in the assessment of
developmental toxicity, which is usually based on limited data from standard studies using three dose
groups and a control group. Most agents causing developmental toxicity in humans alter development
at doses within a narrow range near the lowest maternally toxic dose (Kimmel et al., 1984). Therefore,
for most agents, the exposure situations of concern will be those that are potentially near the maternally
toxic dose range. For those few agents that produce developmental effects at much lower levels than
maternal effects, the potential for exposing the conceptus to damaging doses is much greater than when
the maternal and developmental toxic doses are similar. As mentioned previously (Section 3.1.1.2),
however, traditional dose-response relationships may not always be observed for some endpoints. For
example, as exposure increases, embryolethal levels may be reached, resulting in an observed decrease
in malformations with increasing dose (Wilson, 1973; Selevan and LeMasters, 1987). The potential for
this response pattern indicates that dose-response relationships of individual endpoints as well as
combinations of endpoints (e.g., dead and malformed combined) must be carefully examined and
interpreted.
The evaluation of dose-response relationships includes the identification of effective dose levels
as well as doses that are associated with no increased incidence of adverse effects when compared
with controls. Much of the focus is on the identification of the critical effect(s) (i.e., the adverse
effect(s) observed at the lowest dose level) and the LOAEL and NOAEL associated with that
developmental effect, which may be any of the four manifestations of developmental toxicity. The
NOAEL is defined as the highest dose at which there is no statistically or biologically significant
increase in the frequency of an adverse effect in any of the possible manifestations of developmental
toxicity when compared with the appropriate control group in a data base characterized as having
sufficient evidence for use in a risk assessment (see Section 3.3). The LOAEL is the lowest dose at

34
which there is a statistically or biologically significant increase in the frequency of adverse developmental
effects when compared with the appropriate control group in a database characterized as having
sufficient evidence. Although a threshold is assumed for developmental effects, the existence of a
NOAEL in an animal study does not prove or disprove the existence or level of a biological threshold;
it only defines the highest level of exposure under the conditions of the study that is not associated with
a significant increase in adverse effects.
Several limitations in the use of the NOAEL have been described (Gaylor, 1983; Crump,
1984; Kimmel and Gaylor, 1988; Gaylor, 1989; Brown and Erdreich, 1989, Kimmel, 1990): (1) Use
of the NOAEL focuses only on the dose that is the NOAEL, and does not incorporate information on
the slope of the dose-response curve or the variability in the data. (2) Since 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 is 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 study influence the dose chosen for the NOAEL. (5) Since the NOAEL is defined as a dose
that does not produce an observed increase 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). Crump (1984) and Gaylor (1989) have estimated the upper confidence limit on risk at
the NOAEL to be 2% to 6% for specific developmental endpoints from several data sets.
Because of the limitations associated with the use of the NOAEL (Kimmel and Gaylor, 1988;
Gaylor, 1989; Kimmel, 1990), the Agency is evaluating the use of an additional approach for more
quantitative dose-response evaluation when sufficient data are available, i.e., the benchmark dose
(Crump, 1984). The benchmark dose is based on a model-derived estimate of a particular incidence
level, such as 10% incidence. More specifically, the benchmark dose (BD) is derived by modeling the
data in the observed range, selecting an incidence level within or near the observed range (e.g., the
effective dose to produce a 10% increased incidence of response, the ED10), and determining the upper
confidence limit on the model. The upper confidence value corresponding to, for example, a 10%
excess in response is used to derive the BD, which is the lower confidence limit on dose for that level of
excess response, in this case the LED10 (see Figure 1).
Various mathematical approaches have been proposed for deriving the benchmark dose for
developmental toxicity data (e.g., Crump, 1984; Rai and Van Ryzin, 1985; Kimmel and Gaylor, 1988;
Faustman et al., 1989; Chen and Kodell, 1989; Kodell et al., 1991). Such models may be used to
calculate the benchmark dose, and the particular model used may be less critical since estimation of the

35
benchmark dose is limited to the observed dose range. Since the model is only used to fit the observed
data, the assumptions about the existence or nonexistence of a threshold are not as pertinent. Thus,
models that fit the empirical data may well provide a reasonable estimate of the benchmark dose,
although biological factors known to influence data should be incorporated into the model [e.g.,
intralitter correlations, correlations among endpoints (Ryan et al., 1991)]. The Agency is currently
conducting studies to evaluate the application of several models to actual data sets for calculating the
benchmark dose, to determine the minimum data required for modeling, and to develop methods for
application to continuous data. In addition, information from these studies will be used to develop
guidance for application of the benchmark dose approach to the calculation of the RfD DT or the RfC DT ,
since the Agency has limited experience with this approach (see Section 3.4 for a discussion of the
RfDDT and RfC DT ).
Using the benchmark dose approach, an LED10 can be calculated for each effect of an agent
for which there is a database with sufficient evidence to conduct a risk assessment. In some cases, the
data may be sufficient to also estimate the ED05 or ED01, which should be closer

36
 35

Figure 1. This graphical illustration of the benchmark dose approach is based on Crump (1984) and Kimmel and Gaylor (1988). The
benchmark dose (BD) is derived by modeling the data in the observed range, selecting an incidence level within or near the observed range
(e.g., the effective dose to produce a 10% increased incidence of response, the ED10), and determining the upper confidence limit on the
model. The upper confidence value corresponding to, for example, a 10% excess in response is used to derive the BD, which is the lower
confidence limit on dose for that level of excess response, in this case the LED10. The RfDDT or RfC DT estimated by applying uncertainty
factors (UF) to the BD would be greater than or equal to the BD/UF.
to a true no-effect dose. A level between the ED01 and the ED10 usually corresponds to the lowest
level of risk that can be estimated for binomial endpoints from standard developmental toxicity studies
Certain principles are especially applicable for determining the NOAEL, LOAEL, and
benchmark dose for developmental toxicity studies. First, the NOAEL, LOAEL, or benchmark dose
are identified for both developmental and maternal or adult toxicity, based on the information available
from studies in which developmental toxicity has been evaluated. The NOAEL, LOAEL, or
benchmark dose for maternal or adult toxicity should be compared with the corresponding values from
other adult toxicity data to determine if the pregnant or lactating female or the paternal animal (if
exposure is prior to mating) may be more sensitive to an agent than adult males or nonpregnant females
in other toxicity studies that generally involve longer exposure times.
Second, for developmental toxic effects, a primary assumption is that a single exposure at a
critical time in development may produce an adverse developmental effect, i.e., repeated exposure is
not a necessary prerequisite for developmental toxicity to be manifested. In most cases, however, the
data available for developmental toxicity risk assessment are from studies using exposures over several
days of development, and the NOAEL, LOAEL, and/or benchmark dose is most often based on a
daily dose, e.g., mg/kg-day. Usually, the daily dose is not adjusted for duration of exposure because
appropriate pharmacokinetic data are not available. In cases where such data are available,
adjustments may be made to provide an estimate of equal average concentration at the site of action for
the human exposure scenario of concern. For example, inhalation studies often use 6 hr/day exposures
during development. If the human exposure scenario is continuous and pharmacokinetic data indicate
an accumulation with continuous exposure, appropriate adjustments can be made. If, on the other
hand, the human exposure scenario of concern is very brief or intermittent, pharmacokinetic data
indicating a long half-life may also require adjustment of dose. When quantitative absorption data by
any route of exposure are available, the NOAEL may be adjusted accordingly; e.g., absorption of 50%
of administered dose could result in a 50% reduction in the NOAEL. If absorption in the experimental
species has been determined, but human absorption is not known, human absorption is generally
assumed to be the same as that for the species with the greatest degree of absorption. NOAELs from
inhalation exposure studies are adjusted to derive a human equivalent concentration (HEC) by taking
into account known anatomical and physiological species differences (e.g., minute volume, respiratory
rate, etc.) (U.S. EPA, 1991b).
In summary, the dose-response evaluation identifies the NOAEL, LOAEL, or benchmark
dose, defines the range of doses for a given agent that are effective in producing developmental and
maternal toxicity; the route, timing, and duration of exposure; species specificity of effects, and any

38
pharmacokinetic or other considerations that might influence the comparison with human exposure
scenarios. This information should always accompany the characterization of the health-related
database (discussed in the next section).

3.3. CHARACTERIZATION OF THE HEALTH-RELATED DATABASE

This section describes the process for evaluating the health-related database as a whole on a
particular agent and provides criteria for characterizing the evidence for judging a potential
developmental hazard in humans within the context of expected exposure or dose. This determination
provides the basis for judging whether or not there are sufficient data for proceeding further in the risk
assessment process. This section does not address the nature and magnitude of human health risks,
which are discussed as part of the final characterization of risk along with estimates of potential human
exposure and the relevancy of available data for estimating human risk. Characterization of hazard
potential within the context of exposure or dose should assist the risk assessor in clarifying the strengths
and uncertainties associated with a particular database. Because a complex interrelationship exists
among study design, statistical analysis, and biological significance of the data, a great deal of scientific
judgment, based on experience with developmental toxicity data and with the principles of study design
and statistical analysis, may be required to adequately evaluate the database. Scientific judgment is
always necessary, and in many cases, interaction with scientists in specific disciplines (e.g.,
developmental toxicology, epidemiology, statistics) is recommended.
A categorization scheme for characterizing the evidence for developmental toxicity is presented
in Table 3. The categorization scheme contains two broad categories, sufficient evidence and
insufficient evidence, which are defined in the table. Data from all available studies, whether indicative
of potential hazard or not, must be evaluated and factored into a judgment as to the strength of evidence
available to support a complete risk assessment for developmental toxicity. The primary considerations
are the human data, if available, and the experimental animal data. The judgment of whether the data
are sufficient or insufficient should consider quality of the data, power of the studies, number and types
of endpoints examined, replication of effects, relevance of the test species to humans, relevance of route
and timing of exposure for both human and animal studies, appropriateness of the dose selection in
animal studies, and number of species examined. In addition, pharmacokinetic data and structure-
activity considerations, data from other toxicity studies, as well as other factors that may affect the
strength of the evidence, should be taken into account.
In general, the categorization is based on criteria that define the minimum evidence necessary to
conduct a hazard identification/dose-response evaluation. Establishing the

39
 Table 3. Categorization of the health-related database for hazard identification/dose­
response evaluation

SUFFICIENT EVIDENCE

The sufficient evidence category includes data that collectively provide enough information to
judge whether or not a human developmental hazard could exist within the context of dose, duration,
timing, and route of exposure. This category includes both human and experimental animal evidence.

Sufficient Human Evidence: This category includes data from epidemiologic studies (e.g., case control
and cohort) that provide convincing evidence for the scientific community to judge that a causal
relationship is or is not supported. A case series in conjunction with strong supporting evidence may
also be used. Supporting animal data may or may not be available.

Sufficient Experimental Animal Evidence/Limited Human Data: This category includes data from
experimental animal studies and/or limited human data that provide convincing evidence for the scientific
community to judge if the potential for developmental toxicity exists. The minimum evidence necessary
to judge that a potential hazard exists generally would be data demonstrating an adverse developmental
effect in a single, appropriate, well-conducted study in a single experimental animal species. The
minimum evidence needed to judge that a potential hazard does not exist would include data from
appropriate, well-conducted laboratory animal studies in several species (at least two) which evaluated
a variety of the potential manifestations of developmental toxicity and showed no developmental effects
at doses that were minimally toxic to the adult.

INSUFFICIENT EVIDENCE

This category includes situations for which there is less than the minimum sufficient evidence
necessary for assessing the potential for developmental toxicity, such as when no data are available on
developmental toxicity, as well as for databases from studies in animals or humans that have a limited
study design (e.g., small numbers, inappropriate dose selection/exposure information, other
uncontrolled factors), or data from a single species reported to have no adverse developmental effects,
or databases limited to information on structure/activity relationships, short-term tests,
pharmacokinetics, or metabolic precursors.

40
minimum sufficient human evidence necessary to do a hazard identification/dose-response evaluation is
difficult, since there are often 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, there are currently few agents that can be classified in this category. In the
case of animal data, agents that have been tested adequately in laboratory animals according to current
test guidelines generally would be included in the “Sufficient Experimental Animal Evidence/Limited
Human Data” category. The strength of evidence for a database increases with replication of the
findings and with additional animal species tested. Information on pharmacokinetics or mechanisms, or
on more than one route of exposure may reduce uncertainties in extrapolation to the human.
More evidence is necessary to judge that an agent is unlikely to pose a hazard for
developmental toxicity than that required to judge a potential hazard. This is because it is more difficult,
both biologically and statistically, to support a finding of no apparent adverse effect than a finding of an
adverse effect. For example, to judge that a hazard for developmental toxicity could exist for a given
agent, the minimum evidence necessary would be data from a single, appropriate, well-executed study
in a single experimental animal species that demonstrate developmental toxicity, and/or suggestive
evidence from adequately conducted clinical/epidemiologic studies. On the other hand, to judge that an
agent is unlikely to pose a hazard for developmental toxicity, the minimum evidence would include data
from appropriate, well-executed laboratory animal studies in several species (at least two) which
evaluated a variety of the potential manifestations of developmental toxicity and showed no adverse
developmental effects at doses that were minimally toxic to the adult animal. In addition, there may be
human data from appropriate studies supportive of no adverse developmental effects.
If a database on a particular agent includes less than the minimum sufficient evidence (as defined
in the “Insufficient Evidence” category) necessary for a risk assessment, but some data are available,
this information could be used to determine the need for additional testing. 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 Evidence” criteria. 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 in an attempt to define the strength of evidence of the database for
assessing the potential for developmental toxicity.
Judging that the health-related database is sufficient to indicate a potential developmental hazard
does not mean that the agent will be a hazard at every exposure level (because of the assumption of a

41
threshold) or in every situation (e.g., hazard may vary significantly depending on route and timing of
exposure). In the final risk characterization, the characterization of the health-related database should
always be presented with information on the dose-response evaluation (e.g., LOAEL, NOAEL, and/or
benchmark dose), exposure route, timing and duration of exposure, and with the human exposure
estimate.

3.4. DETERMINATION OF THE REFERENCE DOSE (RfD DT ) OR REFERENCE

CONCENTRATION (RfCDT ) FOR DEVELOPMENTAL TOXICITY
The RfDDT or RfC DT is an estimate of a daily exposure to the human population that is assumed
to be without appreciable risk of deleterious developmental effects. The use of the subscript DT is
intended to distinguish these terms from the reference dose (RfD) for oral or dermal exposure or the
reference concentration (RfC) for inhalation exposure, terms that refer primarily to chronic exposure
situations (U.S. EPA, 1991b). The RfD DT or RfC DT is derived by applying uncertainty factors to the
NOAEL (or the LOAEL, if a NOAEL is not available), or the benchmark dose. To date, the Agency
has applied uncertainty factors only to the NOAEL or LOAEL to derive an RfD DT or RfC DT . The
Agency is planning eventually to use the benchmark dose approach as the basis for derivation of the
RfDDT or RfC DT and will develop guidance as information is acquired and analyzed from ongoing
Agency studies.
The most sensitive developmental effect (i.e., the critical effect) from the most appropriate
and/or sensitive mammalian species is used for determining the NOAEL, LOAEL, or the benchmark
dose in deriving the RfDDT or RfC DT (Section 3.2). Uncertainty factors (UFs) for developmental and
maternal toxicity applied to the NOAEL generally include a 10-fold factor for interspecies variation and
a 10-fold factor for intraspecies variation. In general, an uncertainty factor is not applied to account for
duration of exposure.
Additional factors may be applied to account for other uncertainties or additional information
that may exist in the database. For example, the standard study design for a developmental toxicity
study calls for a low dose that demonstrates a NOAEL, but in some cases, the lowest dose
administered may cause significant adverse effect(s), and thus be identified as the LOAEL. 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. In addition, if a benchmark dose has been calculated, it
may be used to help interpret how close the LOAEL is to a level that would not be detectable from
controls (equivalent to the NOAEL), and thus the size of the uncertainty factor to be applied. Other

42
modifying factors (MFs) may be used depending on the characterization of the database (Section 3.3),
data on pharmacokinetics, or other considerations that may alter the level of confidence in the data
(U.S. EPA, 1991b). The total size of the uncertainty factor will vary from agent to agent and will
require the exercise of scientific judgment, taking into account interspecies differences, variability within
species, the slope of the dose-response curve, the background incidence of the effects, the route of
administration, and pharmacokinetic data.
As stated above, there is little experience with the application of uncertainty factors to the
benchmark dose approach for calculating the RfDDT or RfC DT , and there are several issues that must
be addressed prior to its use for this purpose. For example, which benchmark dose (e.g., LED01,
LED05, LED10) should be used for calculating the RfDDT or RfC DT , and what are the appropriate
uncertainty factors that should be applied to the benchmark dose for deriving the RfDDT or RfC DT ?
That is, should the uncertainty factor applied to an LED10 be similar to that applied to a LOAEL, or
should the uncertainty factor applied to an LED01 be equal to or less than that applied to a NOAEL?
These and other questions are being addressed in ongoing Agency studies on the calculation of the
RfDDT or RfC DT using the benchmark dose approach. As results become available, and as further
guidance is developed, this information will be published as a supplement to these Guidelines.
The total uncertainty factor selected is divided into the NOAEL or LOAEL (or the benchmark
dose) for the critical effect in the most appropriate and/or sensitive mammalian species to determine the
RfDDT or RfC DT . If the NOAEL, LOAEL, or benchmark dose for maternal toxicity is lower than that
for developmental toxicity, this should be noted in the risk characterization, and this value compared
with data from other studies in which adult animals are exposed.
The modeling approaches that have been proposed for developmental toxicity are, for the most
part, statistical probability models that do not take into account underlying biological processes or
mechanisms (e.g., Crump, 1984; Rai and Van Ryzin, 1985; Kimmel and Gaylor, 1988; Faustman et al.,
1989; Chen and Kodell, 1989; Kodell et al., 1991). These models can be applied to derive dose-
response curves for data in the observed dose range, but may or may not accurately predict risk at low
levels of exposure. It has generally been assumed that there is a biological threshold for developmental
toxicity; however, a threshold for a population of individuals may or may not exist because of other
endogenous or exogenous factors that may increase the sensitivity of some individuals in the population.
Thus, the addition of a toxicant may result in an increased risk for the population, but not necessarily for
all individuals in the population.
Models that are more biologically based should provide a more accurate estimation of low-
dose risk to humans. The development of biologically based dose-response models in developmental

43
toxicology has been limited by a number of factors, including a lack of understanding of the biological
mechanisms underlying developmental toxicity, intra/interspecies differences in the types of
developmental events, appropriate pharmacokinetic data, and the influence of maternal effects on the
dose-response curve. The Agency is currently supporting several major research efforts to develop
biologically based dose-response models for developmental toxicity risk assessment that include the
consideration of threshold under its Research to Improve Health Risk Assessment program.

3.5. SUMMARY
In summary, the hazard identification/dose-response evaluation of developmental toxicity data is
used as part of the final characterization of risk along with information on estimates of human exposure.
This analysis depends on scientific judgment as to the accuracy and sufficiency of the health-related
data, biological relevance of significant effects, the conditions of human exposure, and other
considerations important in the extrapolation of data from animals to humans. Scientific judgment is
always necessary, and in many cases, interaction with scientists in specific disciplines (e.g.,
developmental toxicology, epidemiology, statistics) is recommended.

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 4. EXPOSURE ASSESSMENT

In order to obtain quantitative estimates of risk for human populations, estimates of human
exposure are required. This discussion is not intended to provide definitive guidance on exposure
assessment; the “Guidelines for Estimating Exposures” have been published separately (U.S. EPA,
1986d) and will not be discussed in detail here. Rather, the issues important to developmental toxicity
risk assessment are addressed. In general, the exposure assessment describes the magnitude, duration,
frequency, and route(s) of exposure. This information is usually developed from monitoring data and
from estimates based on various scenarios of environmental exposures.
There are several exposure considerations that are unique for developmental toxicity. For
example, exposure to developing individuals is often secondary via placental transfer or through breast
milk. Thus, exposure to the embryo/fetus or child may not be the same as for the pregnant or lactating
mother, and measurements of an agent in maternal or cord blood and in breast milk may provide a
better estimate of developmental exposure. Direct exposure of neonates and children may also occur
via environmental media such as water, air and soil, and thus may require estimates of exposure from
multiple sources. Duration and period of exposure also must be related to stage of development, if
possible (e.g., first, second, or third trimester of pregnancy, infancy, early, middle, and late childhood,
adolescence, etc.). These stages of development may have different sensitivities to agents, and
exposure estimates should be derived for as many as possible. In addition, exposure to either parent
prior to conception must be considered in relation to adverse developmental effects.
There is also a possibility that a single exposure may be sufficient to produce adverse
developmental effects (i.e., 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 sufficiently long to produce an increasing body burden
over time). Therefore, it is assumed that, in most cases, a single exposure at any of several
developmental stages may be sufficient to produce an adverse developmental effect. Most of the data
available for risk assessment involve exposures over several days of development. Thus, human
exposure estimates used to calculate margins of exposure (MOE, see following section) or to compare
with the RfDDT or RfC DT are usually based on a daily dose that is not adjusted for duration or pattern of
exposure. For example, it would be inappropriate in developmental toxicity risk assessments 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-hr inhalation exposure to account for a 24-hr exposure
scenario), unless pharmacokinetic data were available to indicate an accumulation with continuous

45
exposure. In the case of intermittent exposures, examination of the peak exposure(s), as well as the
average exposure over the time period of exposure, would be important.
It should be recognized that, based on the definition used in these Guidelines for developmental
toxicity, exposure of almost any segment of the human population may lead to risk to the developing
organism. This would include fertile men and women, the developing embryo and fetus, and children up
to the age of sexual maturation. Although some effects of developmental exposures may be manifested
while the exposure is occurring (e.g., spontaneous abortion, structural abnormality present at birth,
childhood mental retardation), some effects may not be detectable until later in life, long after exposure
has ceased (e.g., perinatally induced carcinogenesis, impaired reproductive function, shortened
lifespan).

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 5. RISK CHARACTERIZATION

5.1. OVERVIEW
Risk characterization is the culmination of the risk assessment process. In this final step, risk
characterization involves integration of the toxicity information from the hazard identification/dose­
response evaluation with the human exposure estimates and provides an evaluation of the overall quality
of the assessment, describes risk in terms of the nature and extent of harm, and communicates the
results of the risk assessment to a risk manager. The risk manager can then use the risk assessment,
along with other risk management elements, to make public health decisions. The following sections
describe these three aspects of the risk characterization in more detail, but do not attempt to provide a
full discussion of risk characterization. Rather, these Guidelines point out issues that are important to
risk characterization for developmental toxicity.

5.2. INTEGRATION OF THE HAZARD IDENTIFICATION/DOSE-RESPONSE

EVALUATION AND EXPOSURE ASSESSMENT
In developing the hazard identification/dose-response and exposure portions of the risk
assessment, the risk assessor makes many judgments concerning human relevance of the toxicity data,
including the appropriateness of the various animal models for which data are available, the route,
timing, and duration of exposure relative to expected human exposure, etc. These judgments should be
summarized at each stage of the risk assessment process (e.g., the biological relevance of anatomical
variations may be made in the hazard identification process, or species differences in metabolic patterns
in the dose-response evaluation). When data are not available to make such judgments, as is often the
case, the background information and assumptions discussed in the Introduction (Section 1) provide a
default position. The risk assessor 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 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 the
analysis of these elements. Each of these elements should have its own characterization as a part of it.
Interpretation of data should be explained, and the risk manager should be given a clear picture of
consensus or lack of consensus that exists about significant aspects of the assessment. Whenever more
than one view is supported by the data and choosing between them is difficult, both views should be

47
presented. If one has been selected over another, the rationale should be given; if not, then both should
be presented as plausible alternative results.
The risk characterization should not only examine the judgments, but also explain the constraints
of available data and the state of knowledge about the phenomena studied in making them, including:
C the qualitative conclusions about the likelihood that the agent 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 to use in a risk assessment;
C a discussion of the dose-response patterns for the critical effect(s), data such as the shapes
and slopes of the dose-response curves for the various endpoints, the rationale behind the
determination of the NOAEL, LOAEL, and/or calculation of the benchmark dose, and the
assumptions underlying the estimation of the RfDDT or RfC DT ; and
C the estimates of the magnitude of human exposure, the route, duration, and pattern of the
exposure, relevant pharmacokinetics, and the size and characteristics of the populations
exposed.
The risk characterization of an agent should be based on data from the most appropriate
species, or, if such information is not available, on the most sensitive species tested. It should also be
based on the most sensitive indicator of toxicity, whether maternal, paternal, or developmental, when
such data are available, and should be considered in relationship to other forms of toxicity.
If data used in characterizing risk 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, 1984, 1985b).
The level of confidence in the hazard identification/dose-response evaluation should be stated to
the extent possible, including determination of the appropriate category regarding sufficiency of the
health-related data. A comprehensive risk assessment ideally includes information on a variety of
endpoints that provide insight into the full spectrum of developmental responses. A profile that
integrates both human and test species data and incorporates a broad range of developmental effects
provides more confidence in a risk assessment for a given agent.
The ability to describe the nature of human exposure is important for prediction of specific
outcomes and the likelihood of permanence or reversibility of the effect. An important part of this effort
is a description of the nature of the exposed populations. For example, the consequences of exposure
to the developing individual versus the adult can differ markedly and again can influence whether the

48
effects are transient or permanent. Other considerations relative to human exposures might include
potential synergistic effects, increased susceptibility resulting from concurrent exposures to other agents,
concurrent disease, and nutritional status.

5.3. DESCRIPTORS OF DEVELOPMENTAL TOXICITY RISK

There are a number of ways to describe risks. These include:

5.3.1. Estimation of the Number of Individuals Exposed to Levels of Concern

The RfDDT or RfC DT is assumed to be a level at or below which no significant risk occurs.
Therefore, information on the populations at or below the RfDDT or RfC DT (“not likely to be at risk”)
and above the RfDDT or RfC DT (“may be at risk”) may be useful information for risk managers.
This method is particularly useful to a risk manager considering possible actions to ameliorate
risk for a population. If the number of persons in the “at risk” category can be estimated, then the
number of persons potentially removed from the “at risk” category after a contemplated action is taken
can be used as an indication of the efficacy of that action.

5.3.2. Presenting Specific Scenarios

Presenting specific scenarios in the form of “what if?” questions is particularly useful to give
perspective to the risk manager, especially where criteria, tolerance limits, or media quality limits are
being set. The question being asked in these cases is, “At this proposed limit, what would be the
resulting risk for developmental toxicity above the RfDDT ?”

5.3.3. Risk Characterization for Highly Exposed Individuals

This measure and the next are examples of specific scenarios. The purpose of this measure is
to describe the upper end of the exposure distribution. This allows risk managers to evaluate whether
certain individuals are at disproportionately high or unacceptably high risk.
The objective of looking at the upper end of the exposure distribution is to derive a realistic
estimate of a relatively highly exposed individual(s), for example, by identifying a specified upper
percentile of exposure in the population and/or by estimating the exposure of the most highly exposed
individual(s). Whenever possible, it is important to express the number of individuals who comprise the
highly exposed group and discuss the potential for exposure at still higher levels.
If population data are absent, it will often be possible to describe a scenario representing high-
end exposures using upper percentile or judgment-based values for exposure variables. In these

49
instances, caution should be taken not to overestimate the high-end values if a “reasonable” exposure
estimate is to be achieved.

5.3.4. Risk Characterization for Highly Sensitive or Susceptible Individuals

The purpose of this measure is to quantify exposure to identified sensitive or susceptible
populations to the effect of concern. Sensitive or susceptible individuals are those within the exposed
population at increased risk of expressing the adverse effect. All stages of development might be
considered highly sensitive or susceptible, but certain subpopulations can sometimes be identified
because of critical periods for exposure; for example, pregnant or lactating women, infants, children,
adolescents.
In general, not enough is understood about the mechanisms of toxicity to identify sensitive
subgroups for all agents, although factors such as nutrition, personal habits (e.g., smoking, alcohol
consumption, illicit drug abuse), or pre-existing disease (e.g., diabetes) may predispose some
individuals to be more sensitive to the developmental effects of various agents.

5.3.5. Other Risk Descriptors

In risk characterization, dose-response information and the human exposure estimates may be
combined either by comparing the RfDDT or RfC DT and the human exposure estimate or by calculating
the margin of exposure (MOE). The MOE is the ratio of the NOAEL from the most appropriate or
sensitive species to the estimated human exposure level from all potential sources (U.S. EPA, 1985b).
If a NOAEL is not available, a LOAEL may be used in the calculation of the MOE, but considerations
for the acceptability would be different from those when a NOAEL is used. Considerations for the
acceptability of the MOE are similar to that for the uncertainty factor applied to the LOAEL, NOAEL,
or the benchmark dose. 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 dose-response, route, timing, and duration information. The RfD DT or RfC DT
comparison with the human exposure estimate and the calculation of the MOE are conceptually similar
but are used in different regulatory situations. If the MOE is equal to or more than the uncertainty
factor used as a basis for an RfD DT or RfC DT , then the need for regulatory concern is likely to be
reduced.
The choice of approach is dependent upon several factors, including the statute involved, the
situation being addressed, the database used, and the needs of the decision maker. While these
methods of describing risk do not actually estimate risks per se, they give the risk manager some sense

50
of how close the exposures are to levels of concern. The RfD DT , RfC DT , and/or the MOE are
considered along with other risk assessment and risk management issues in making risk management
decisions, and the scientific issues that must be taken into account in establishing them have been
addressed here.

5.4. COMMUNICATING RESULTS

Once the risk characterization is completed, the focus turns to communicating results to the risk
manager. The risk manager uses the results of the risk characterization, other technological factors, and
nontechnological social and economic considerations in reaching a regulatory decision. Because of the
way in which these risk management factors may impact different cases, consistent but not necessarily
identical risk management decisions must be made on a case-by-case basis. Consequently, it is entirely
possible and appropriate that an agent with a specific risk characterization may be regulated differently
under different statutes. These Guidelines are not intended to give guidance on the nonscientific aspects
of risk management decisions.

51
 6. SUMMARY AND RESEARCH NEEDS

These Guidelines summarize the procedures that the U.S. Environmental Protection Agency
uses in evaluating the potential for agents to cause developmental toxicity. While these are the first
amendments to the developmental toxicity guidelines issued in 1986, further revisions and updates will
be made as advances occur in the field. These Guidelines discuss the assumptions that should be made
in risk assessment for developmental toxicity because of gaps in our knowledge about underlying
biological processes and how these compare across species.
Research to improve the risk assessment process is needed in a number of areas. For example,
research is needed to delineate the mechanisms of developmental toxicity and pathogenesis, provide
comparative pharmacokinetic data, examine the validity of short-term in vivo and in vitro tests, elucidate
possible functional alterations and their critical periods of exposure to toxic agents, develop improved
animal models to examine the developmental effects of exposure during the premating and early
postmating periods and in neonates, further evaluate the relationship between maternal and
developmental toxicity, provide insight into the concept of threshold, develop approaches for improved
mathematical modeling of adverse developmental effects, and improve animal models for examining the
effects of agents given by various routes of exposure. Epidemiologic studies with quantitative measures
of exposure are also strongly encouraged. Such research will aid in the evaluation and interpretation of
data on developmental toxicity, and should provide methods to more precisely assess risk.

52
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PART B: RESPONSE TO PUBLIC AND SCIENCE ADVISORY BOARD COMMENTS

1. INTRODUCTION

This section summarizes the major issues raised in the public and Science Advisory Board
(SAB) comments on the Proposed Amendments to the Guidelines for the Health Assessment of
Suspect Developmental Toxicants published March 6, 1989 [54 FR 9386-9403]. Comments were
received from 25 individuals or organizations. The Agency’s initial summary of the public comments
and proposed responses were presented to the Environmental Health Committee of the SAB on
October 27, 1989. The report of the SAB Committee was provided to the Agency on April 23, 1990.
The SAB and public comments were diverse and addressed issues from a variety of
perspectives. The majority of the comments were favorable and in support of the Proposed
Amendments to the Guidelines. Many praised the Agency’s efforts as being timely and well-justified.
Most commentors also gave specific comments or criticisms for further consideration, clarification, or
re-evaluation. For example, there was concern expressed about the Guidelines imposing further testing
requirements, particularly functional testing, and many commentors felt that the Proposed Amendments
discounted the role of maternal toxicity in developmental toxicity. In addition, there was concern that
the proposed weight-of-evidence scheme would promote labeling of agents as causing developmental
toxicity before the entire risk assessment process was completed.
The SAB Committee also indicated that the proposed revisions were adequately founded in
developmental toxicology and represented a step forward for the Agency. They suggested that the
Agency revisit the weight-of-evidence scheme to avoid confusion with more commonly applied uses of
such classifications, and to develop a more powerful conceptual approach. Further, the SAB
Committee urged that the Agency begin to move away from the current use of the no-observed­
adverse-effect level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) basis for
calculating the reference dose for developmental toxicity to a benchmark dose and confidence limit
approach tied to empirical models of dose-response relationships.
In response to the comments, the Agency has modified or clarified many sections of the
Guidelines. For the purposes of this discussion, the major issues reflected by the public and SAB
comments are discussed. Several minor recommendations, which are not discussed specifically here,
also were considered by the Agency in the revision of these Guidelines.

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 2. INTENT OF THE GUIDELINES

Many of the public comments indicated some misunderstanding of the intent of the Guidelines,
apparently assuming that the risk assessment guidelines impose testing requirements. In particular,
some commentors suggested that because the Agency was providing guidance on the interpretation of
tests not required in the EPA testing guidelines, the Agency was suggesting that these tests be required
in the future.
The 1986 Guidelines and the 1989 Proposed Amendments clearly state that these guidelines
are not Agency testing guidelines, but rather are intended to ensure uniform interpretation of all existing,
relevant data. However, to avoid any confusion, the discussion of study designs has been changed to
avoid the impression that these Guidelines set testing requirements. In the evaluation of data on an
agent for risk assessment, relevant data are often encountered that have been generated from
nontraditional tests. In such cases, it is imperative that the Agency provide guidance so that all data
considered to be relevant are included in the risk assessment and are interpreted uniformly.

3. BASIC ASSUMPTIONS

In the 1986 Guidelines, several assumptions were implicit in the approach to risk assessment,
but were not explicitly stated. These assumptions were detailed in the 1989 Proposed Amendments.
Comments received from the public and the SAB favored presentation of these assumptions and
generally agreed with the wording, except for the fourth assumption, which concerns the use of the most
relevant or most sensitive species. The 1989 Proposed Amendments stated that “it is assumed that the
most sensitive species should be used to estimate human risk. When data are available (e.g.,
pharmacokinetic, metabolic) to suggest the most appropriate species, that species will be used for
extrapolation.” The SAB recommended that, for this assumption, the basic position of the Agency
should be to use data from the most relevant species, and that use of data from the most sensitive
species should be the default position. In addition, the SAB recommended that the threshold
assumption be considered carefully in the dose-response assessment of any agent, and that the Agency
develop more comprehensive approaches to risk assessment as discussed further in the following
sections.
Changes have been made in the statement of the basic assumptions in line with the SAB and
public comments that clarify, but do not alter, the intent of the assumptions.

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 4. MATERNAL/DEVELOPMENTAL TOXICITY

The 1989 Proposed Amendments stated that “when adverse developmental effects are
produced only at maternally toxic doses, they are still considered to represent developmental toxicity
and should not be discounted as being secondary to maternal toxicity.” This statement and others
concerning the interpretation of developmental toxicity in the presence of maternal toxicity were the
subject of a considerable number of public comments and were also addressed by the SAB. In
general, commentors were divided in their opinions on whether they supported the Agency’s statements
or felt that they discounted the role of maternal toxicity in developmental toxicity, but in general, the
recommended changes did not significantly alter the intent of the statements. The SAB endorsed the
proposed revision, and suggested that the Agency retain the statement that was made in the Proposed
Amendments.
In these Guidelines, the position is further clarified by indicating that when maternal toxicity is
significantly greater than the minimal maternally toxic dose, developmental effects at that dose may be
difficult to interpret. This statement is added to clarify, but not to change, the intent or meaning of the
statements regarding the relationship between maternal and developmental toxicity. From a risk
assessment point of view, whether a developmental effect is or is not secondary to maternal toxicity
does not impact on the selection of the NOAEL or other dose-response methodology.

5. FUNCTIONAL DEVELOPMENTAL TOXICITY

The 1989 Proposed Amendments provided information on the state-of-the-art in the evaluation
of functional effects resulting from developmental exposures. Several commentors voiced strong
objection to this section because they perceived it as indicating an imminent requirement for testing.
Several indicated there are no standard methods for functional testing, some felt that functional
endpoints should not be used to establish the NOAEL, and others voiced concern about the problems
with using postnatal exposures in animal studies.
The final Guidelines further update this section to include a discussion of the latest changes in
the requirements for functional developmental toxicity testing by the Agency, and reflect the current
approach to interpretation of such data, with incorporation of information from the EPA/NIDA­
sponsored “Workshop on the Qualitative and Quantitative Comparability of Human and Animal
Developmental Neurotoxicity” (1990). The intent of these Guidelines, as stated above, is not to change
testing requirements but to give guidance when these types of data are encountered in the risk

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assessment process. The Guidelines also indicate that functional developmental toxicity endpoints will
be used for establishing the NOAEL when they are found to be the adverse effect occurring at the
lowest dose in appropriate, well-conducted studies. Interpretation of postnatal exposure data is a
concern, and must take into consideration effects on the mother, her offspring, and possible
interactions; a statement to this effect has been added. Further interpretation of data will be discussed
in the guidance being developed by the Agency on neurotoxicity risk assessment.

6. WEIGHT-OF-EVIDENCE SCHEME

The 1989 Proposed Amendments described important considerations in determining the

relative weight of various kinds of data in estimating the risk of developmental toxicity in humans. The
intent of the proposed weight-of-evidence (WOE) scheme was that it not be used in isolation, but be
used as the first step in the risk assessment process, to be integrated with dose-response information
and the exposure assessment.
The WOE scheme was the subject of a considerable number of public comments, and was one
of the major concerns of the SAB. The concern of public commentors was that the reference to human
developmental toxicity in this scheme suggested that a chemical could be prematurely designated, and
perhaps labeled, as causing developmental toxicity in humans prior to the completion of the risk
assessment process. The SAB suggested that the intended use of this scheme was not consistent with
the use of the term “weight of evidence” in other contexts, since WOE is usually thought of as an
evaluation of the total composite of information available to make a judgment about risk. In addition,
the SAB Committee proposed that the Agency consider development of a more conceptual approach
using decision analytical techniques to predict the relationships among various outcomes.
In the final Guidelines, the terminology used in the WOE scheme has been completely changed
and retitled “Characterization of the Health-Related Database.” The intended purpose of the scheme is
to provide a framework and criteria for making a decision on whether or not sufficient data are
available to conduct a risk assessment. This decision is based on the available data, whether animal or
human, and does not necessarily imply human hazard. This decision process is part of, but not the
complete, WOE evaluation, which also takes into account the RfDDT or RfC DT and the human
exposure information, culminating in risk characterization.
The final Guidelines also place strong emphasis on the integration of the dose-response
evaluation with hazard information in characterizing the sufficiency of the health-related database. In
line with this approach, the Guidelines have been reorganized to combine hazard identification and

69
dose-response evaluation. Finally, the SAB comments on developing a conceptual matrix provide an
interesting challenge, but current data indicate that the relationships among endpoints of developmental
toxicity are not consistent across chemicals or species. The Agency is currently supporting modeling
efforts to further explore the relationship among various development toxicity endpoints and the
development of biologically based dose-response models that consider multiple effects.

7. APPLICABILITY OF THE RfD DT CONCEPT AND

THE BENCHMARK DOSE APPROACH

The 1989 Proposed Amendments introduced the term “reference dose for developmental
toxicity - RfDDT ,” based on short-term exposure, to distinguish it from the reference dose (RfD), which
is used for chronic exposure situations. The public comments received generally supported the RfDDT
approach. The SAB also agreed with the concept of the RfD DT for developmental toxicity risk
assessment, based on short-term exposure. In addition, the SAB urged the Agency to consider
strengthening the RfD approach by moving to more quantitative alternatives to the NOAEL. In
particular, the use of a benchmark dose approach to replace the NOAEL was strongly suggested.
The final Guidelines have incorporated many of the SAB Committee’s suggestions concerning
the development of more quantitative approaches to the RfD, and state that the Agency is beginning to
use the benchmark dose approach for comparison with and interpretation of the NOAEL. That is,
benchmark dose calculations may allow better interpretation of dose-response data and, in particular,
what level of risk may be associated with the NOAEL. The Agency also has developed the concept of
an inhalation reference concentration (RfC), and the RfC DT is being calculated for inhalation
concentrations based on developmental toxicity. Guidance for use of the benchmark dose in the
calculation of the RfDDT or RfC DT is not included in the final Guidelines, because of the limited
experience of the Agency with this approach. There are several issues that must be addressed prior to
its use for this purpose; for example, which benchmark dose (e.g., LED01, LED05, LED10) should be
used for calculating the RfDDT or RfC DT , and what are the appropriate uncertainty factors that should
be applied to the benchmark dose for deriving the RfD DT or RfC DT ? Should the uncertainty factor
applied to an LED10 be similar to that applied to a LOAEL, or should the uncertainty factor applied to
an LED01 be equal to or less than that applied to a NOAEL? These and other questions are being
addressed in ongoing Agency studies on the calculation of the RfDDT or RfC DT using the benchmark
dose approach. As results become available, and as further guidance is developed, this information will
be published as a supplement to these Guidelines.

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