Monday
March 6, 1989
EPA/600/FR-89/001
Part VII
Environmental
Protection Agency
Proposed Amendments to the Guidelines
for the Health Assessment of Suspect
Developmental Toxicants; Request for
Comments; Notice

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Federal Register / Vol. 54, No. 42 / Monday. March 6. 1989 / Notices
ENVIRONMENTAL PROTECTION
AGENCY
IFRL-S533-91
Proposed Amend menu to the
Guideline* lor the Health Assessment
of Suspect Developmental Toxicants
AOlNCr: U.S. Environmental Protection
Agency.
ACTION: Request for comments on the
Proposed Amendments to the Guidelines
for the Health Assessment of Suspect
Developmental Toxicants.	
SuitMAftv. The U.S. Environmental
Protection Agency (EPA] is today
proposing amendments to the
Guidelines for the Health Assessment of
Suspect Developmental Toxicants that
were issued on September 24.1966 (51
FR 34028-34040) (hereafter "current
guidelines").
These proposed amendments are
intended to expand Agency guidance on
the analysis of developmental toxicity
data m accordance with appropriate
scientific standards and with the
policies and procedures established in
the statutes administered by the EPA.
The proposed amendments were
developed as part of an interoffice
guidelines development program under
the auspices of the Agency's Risk
Assessment Forum. The proposed
amendments are based, in part on
recommendations developed in
scientific workshops.
The public is invited to comment and
public comments will be considered in
final Agency decision* on amending the
current guidelines. Cosnnentors are
asked to focus on several special issues,
particularly, (1) a proposed new weight-
cf-evidence scheme and its use. and (2)
the advantages and disadvantages of
using this scheme only for hazard
identification versus using it in
conjunction with dose-response and
exposure assessment information. Also,
comments are invited on the use of the
special term "reference dose for
developmental toxicity (RIDdt)-" The
term RfDt* is used to distinguish the
time-limited reference dose for exposure
dunng development from the reference
dose (RID), which generally refers to
chronic exposure situations.
The proposed amendments are
individually identified and explained in
the Supplementary Information section
of this notice. The full text of the
proposed guidelines is published in the
following section. As used in this notice,
the term "proposed guidelines" refers to
the current guidelines as modified by the
proposed amendments The request for
comment applies only to the proposed
amendments, but EPA will also consider
any important new scientific
information bearing on the proposed
guidelines as a whole
EPA's Science Advisory Board (SAB)
also will review the proposed
amendments at a meeting to be
announced in a future FEDERAL
REGISTER. Agency staff will prepare
summaries of the public and SAB
comments, analyses of major issues
presented by commentors, and Agency
responses to those comments.
Appropriate comments will be
incorporated, and the amended
guidelines will be submitted to the Risk
Assessment Forum and the Risk
Assessment Council for review. The
Risk Assessment Council will consider
comments from the public, the SAB. and
the Risk Assessment Forum in its
recommendations to the EPA
Administrator.
oatc Public comments must be
postmarked by June S. 1989.
ADMKSS: Comments may be mailed or
delivered to: Dr. Carole A. KimmeL
Reproductive and Developmental
Toxicology Branch. Human Health
Assessment Group. Office of Health and
Environmental Assessment (RD-689).
U.S. Environmental Protection Agency,
401M Street SW., Washington, DC
20460.
pon nnrrxen intowmatiom contact.
Dr. Carole A. Kimmel. Telephone: 202-
382-7331.
Inspection and Copies This notice,
references, supporting documents, and
other relevant materials are available
for inspection and copying at the Public
Information Reference Unit, (202) 382-
5926. EPA Headquarters Library, 401 M
Street. SW, Washington. DC between
the hours of &00 aj&. and 4:30 pjn.
SUmiMtMTAHY INFORMATION: In 1984-
85. the Agency proposed nsk
assessment guidelines for
carcinogenicity, exposure assessment,
mutagenicity, developmental toxicity (49
FR 46294-46331), and chemical mixtures
(50 FR 1170-1176). Following extensive
scientific and public review, final
guidelines were issued on September 24,
1986 (51 FR 33992-34054). Each of the
guidelines set forth principles and
procedures to guide EPA scientists In
the conduct of Agency nsk assessments,
to help promote high scientific quality
and Agency-wide 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 nsk
assessment guidelines was to
"encourage research and analysis that
will lead to new nsk assessment
methods and data." which in turn would
be used to revise and improve the
guidelines, and better guide Agency risk
assessors. Thus, each of the 1986 nsk
assessment guidelines was developed
and published with the understand.ng
that nsk assessment is an evolving
science and that continued study could
lead to changes.
As expected. Agency experience with
the current Guidelines for the Health
Assessment of Suspect Developmental
Toxicants suggests that additional or
alternate approaches should be
considered for certain aspects of these
guidelines. Proposals to amend the
current guidelines were considered soon
after their publication in September 1980
because of new reviews or re-
evaluations that focused on some of the
issues identified for research in the
guidelines. These included several
workshops and symposia cited in the
Introduction to the current guidelines In
addition, much expenence has been
gained in using these guidelines and in
instructing others in their use Based on
this expenence, the proposed
amendments are designed to clanfy
certain aspects of the current guidelines,
and the terminology has been updated
to be consistent with that used in other
Agency guidance.
As outlined below, some of the
changes involve substantive revisions to
the current guidelines, while others
simply clanfy or reorganize current
provisions. The remainder of the notice
publishes the full text of the proposed
guidelines, that is. the current guidelines
as modified by the proposed
amendments.
Overview of Proposed Amendments
The major proposed amendments
include stronger statements concerning
guidance on evaluating maternal and
developmental toxicity based on EPA's
1987 workshop on this topic, particularly
about the inter-relationship between
these end points (see Reference 3 in
Section VII of the proposed guidelines).
A major innovation for the proposed
guidelines is a weight-of-evidence
scheme for developmental toxicants
(Section HID) which was developed in a
1987 EPA workshop by experts from
within and outside the Agency.
Lesser changes in the proposed
guidelines include a change in the title
from "Guidelines for the Health
Assessment of Suspect Developmental
Toxicants" to "Proposed Guidelines for
Developmental Toxicity Risk
Assessment." In addition, three other
sections have been revised the Human
Studies section (Section 111 B) was
reonented more towards nsk
assessment than study design, and the
Dose-Response and Risk

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Federal Register / Vol. 54. No. 42 / Monday. March 6. 1989 / Notices
9387
Characterization sections (Sections IV
and VI) were reorganized so that
information on the NOAEL/uncertainty
factor approach and low-dose
extrapolation are contained m the Dose-
Response section (Section IV). and the
margin of exposure (MOE) approach is
contained m the Risk Characterization
section (Secnon VI).
One other proposed change is the
introduction of the term RfDpr for the
reference dose for developmental
toxicity derived from dividing the
NOAEL by an uncertainty factor. This is
to distinguish the developmental toxicity
reference dose (RfDw). which is baaed
cn a short-term exposure as occurs in
most developmental toxicity studies,
from the RfD, which the Agency derives
based on a chronic or sometimes a
subchromc exposure scenario. These
and other proposed changes are
discussed further by section.
Section L Introduction
This section gives the general
background information on
developmental toxicity risk assessment
and the magnitude of the potential for
developmental toxicity problems in the
general population. In the current
guidelines. EPA provide* the general
basis for (be use of data from animal
studies in estimating human nsk. but
docs not describe the assumptions
generally made in this process.
The primary proposed amendment in
this section is a statement of the basic
assumptions made m the nsk
assessment process for developmental
toxicity, e.g., an agent that produces an
adverse developmental effect in
experimental animal studies is assumed
to pose a potential hazard to humans,
and all four possible manifestations of
developmental toxicity (i.e, death,
structural abnormality, growth
alteration, functional deficit) are of
concern for risk assessment The
assumption of a threshold is stated,
although this assumption is currently
being discussed in the Utarature, u
indicated in the proposed amendments.
These assumptions haip to more clearly
identify the basts for die Agency's
approach to nsk assessment described
in the proposed guidelines. In addition,
same background information and
references have been revised.
Section U. Definitions and Terminology
This section sets forth the definitions
of particular terms that are widely used
in the field of developmental toxicology
These include special terms such as
developmental toxicity." "altered
growth." "malformations." and
variations"
The only proposed amendment in this
section is the deletion of the terms
"erabryotoxicity" and "fetotoxicity "
Because ambiguities in these terms have
led to confusion and misuse, they are
not used in the proposed guidelines.
Thus, use of the term "developmental
toxicity," which is a broader tenn, is
encouraged and ambiguities are
eliminated.
Section CD. Hazard Identification of
Developmental Toxicants
This section descnbes the study
designs used in animal studies and the
evaluation and interpretation of end
points. In the current guidelines the title
of this section includes the term
"qualitative assessment." Also, this
section recommends that other EPA risk
assessment guidelines be used when
carcinogenic or mutagenic effects from
developmental exposures are of
concern.	~
The proposed heading for this section
no longer includes the term "qualitative
assessment" since hazard identification
for developmental toxicity also includes
some evaluation of the dose-response
nature of an effect. This change is
proposed because the distinction in the
current guidelines between qualitative
and quantitative assessment has proved
to be unsatisfactory and is not made m
actual practice when using the
guidelines to assess developmental
toxicity data.
The discussion of potential
carcinogenic effects following
development exposure is proposed to be
expanded somewhat, as are the
statements on potential mutational
events. These changes would emphasize
the importance of considering potential
carcinogenic and mutagenic effects
resulting from developmental exposures.
More extensive information on
conducting nsk assessments for these
types of effects is provided in the
Guidelines for Carcinogen Risk
Assessment (51 FR 33892) or the
Guidelines for Mutagenicity Risk
Assessment (51 FR 34006).
A. Laboratory Animal Studies of
Developmental Toxicity: End Points and
Their Interpretation
This section provides general
information on the protocols typically
used to assess developmental toxicity.
There are no proposed amendments to
this section.
A1 End Points of Maternal Toxicity.
This section descnbes the types of
maternal end points evaluated m
developmental toxicity studies and
provides guidance for the hazard
assessment.
The proposed amendments to this
section include the addition of support
from adverse histopathology findings to
the use of alterations in organ weights
as a sign of maternal toxicity This
change would indicate more eleariy the
basis fof the use of maternal organ
changes as signs of maternal toxicity
A-Z End Points of Developmental
Toxicity.
This section descnbes the types of
developmental end points evaluated in
developmental toxicity studies and
provides guidance for the hazard
assessment
There are no proposed amendments to
this section.
A.3. Functional Developmental
Toxicology.
This section provides information on
the state-of-the-art in the evaluation of
functional effects resulting from
developmental exposures.
Developmental neurotoxicity is briefly
reviewed, along with other areas of
functional evaluation. Since the
publication of the current guidelines in
1988. specific testing in this area has
been proposed or required by the
Agency for certain agents. ~ .
The proposed amendments to tSis
section reflect the current regulatory
status for developmental neurotoxicity
testing in the Agency. The Office cf
Toxic Substances (OTS) recently
proposed developmental neurotoxicity
testing guidelines and finalized at least
one test rule requiring such testing (see
Reference 28 in Section VII of the
proposed guidelines). In addiuon. the
Science Advisory Panel for the Office of
Pesticide Programs (OPP) has approved
the development of testing guidelines for
developmental neurotoxicity. The
proposed amendments note these
activities and identify the proposed
bases for OPP and OTS requirement* for
such testing.
A 4. Overall Evaluation of Maternal
and Developmental Toxicity.
This section discusses the relationship
of maternal and developmental toxicity
and the evaluation of developmental
toxicity data in the presence of ma'ernal
toxicity. In the current guidelines, the
statement is made that developmental
effects at maternally toxic doses should
not be discounted as being secondary to
maternal toxicity.
A stronger statement is proposed in
this section concerning the finding of
developmental toxicity in the presence
of maternal toxicity, i e„ 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

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Federal Register / Vol. 54. No. 42 / Monday, March 6. 1989 / Notices
Also, it is proposed that information be
added on the importance of evaluating
both maternal and developmental
toxicity for the final characterisation of
risk as suggested by participants at the
EPA-sponsored workshop on "The
Evaluation of Maternal and
Developmental Toxicity." This would
indicate that maternal toxicity (even in
the absence of developmental toxicity)
is an important end point to evaluate in
the context of all available toxicity data.
A.3. Short-Term Testing in
Developmental Toxicity.
This section summarizes in vivo and
in vitro approaches to short-term testing
for developmental toxicity. In the
current guidelines, the ChernoB/
Kavlock assay is described, but more
recent work, including a NIOSH-
sponsored conference on this testing
procedure, has appeared in the
literature
The proposed amendment would
update the section to include recent
information on the Chemoff/Kavlock
assay, in particular, that from the
NIOSH-sponaored workshop on
"Evaluation of the Chemoff/Kavlock
Test for Developmental Toxicity."
A.6. Statistical Considerations.
This section describes approaches to
the statistical evaluation of data from
animal developmental toxicity studies
and includes important issues of study
design that affect interpretation of data.
There are no proposed amendments to
this section.
B.	Hunan Studies
This section describes the evaluation
of human data for developmental toxic
effects. In the current guidelines, this
section discusses important
considerations of study design and
evaluation, but does not provide much
guidance to the nsk assessor on the
relative importance of various types of
human data.
The proposed amendments would
reorganize and modify this section to
give more specific Information
concerning the use of human data in risk
assessment (e.g„ greatest weight should
be given to carefully designed
epidemiologic studies with more precise
measures of exposure; studies with a
low probability of biased data should
cany more weight in a risk asseument).
These revisions would make this section
consistenrwith similar sections m the
Proposed Guidelines for Assessing Male
Reproductive Risk and Female
Reproductive Risk.
C.	Other Considerations
This section discusses the importance
of pharmacokinetic data and structure-
activity considersUons. if available, in
the nsk assessment of developmental
toxicants.
There are no proposed amendments to
this section.
D. Weight-of-Evidence Determination
This section describes the important
considerations in determining the
relative weight of various kinds of
experimental and/or human evidence in
estimating the nsk of development
toxicity m humans. In the current
idelines. vanous factors are listed as
ing important, but there is no
systematic procedure for categorizing
the level of confidence in the available
data.
A weight-of-evidence scheme is
proposed that defines three levels of
confidence for data used to identify
developmental hazards and to assess
the nsk of human developmental
toxicity. The language used in the
scheme is intentionally broad to allow
for scientific judgment in classifying
data using this scheme, and
classification of agents using this
scheme would require experience with
developmental toxicity data. The intent
of the discussion is that the scheme
would not be used in isolation, but
would be the first step that must be
combined with information on dose-
response and exposure for the final
characterization of nsk.
IV. Dose-Response Assessment
This section describes the evaluation
of the dose-response data from
developmental toxicity studies In the
current guidelines, certain terminology
(e.g.. NOEL LOEL) is used in a way that
is no longer consistent with its usage in
other Agency guidance In addition,
certain topics (e^.. the margin of safety,
now termed the margin of exposure) that
are discussed as dose-response issues in
the current guidelines are treated as risk
charactenzation issues in other Agency
guidance.
The proposed heading for this section
no longer includes the term
"'quantitative assessment." since s sharp
separation between qualitative and
quantitative assessment in the current
guidelines is not made in practice. Dose-
Response Assessment is Section IVA. in
the current guidelines
The proposed amendments to this
section incorporate terminology (e.g.,
NOAEL LOAEL. RfD) that would make
the proposed guidelines consistent with
other Agency guidance The section
discusses the identification of the
NOAEL/LOAEL. the factors used in
establishing the appropnate uncertainty
factor, and the calculation of the RfDpr.
These proposed changes would also be
consistent with the way in which
chronic RfDs are calculated However,
in the proposed guidelines, the term
RfDoT- based on short-term exposure, is
introduced to distinguish it from the
general RfD. An updated discussion of
the status of mathematical approaches
for dose-response modeling and low-
dose extrapolation for developmental
toxicity is also included.
V. Exposure Assessment
This section describes the issues of
concern for developmental toxicity in
the estimation of the human exposure
levels. In the current guidelines, this
section includes information related to
human exposure-effect relationships
that is actually more closely related to
determining dose-effect relationship in
humans.
The proposed amendments to this
section. Section IV.B. in the current
guidelines, include transfemng some
guidance from the section on
determining human exposure-effect
relationships to Section IV (Dose-
Response Assessment) since this
discussion is more involved with dose-
response assessment in humans. The
remaining information in this section
focuses primarily on the special
considerations concerning exposure
assessment for developmental toxicity
Another proposed change in this section
would more clearly indicate that since a
single exposure at the critical time in
development is sufficient to produce an
adverse developmental effect, the
human exposure estimate used to
calculate the margin of exposure is
usually based on a single dose that is
not adjusted for duration of exposure,
and the number of exposures is not
considered important unless there is
evidence for a cumulative effect
VL Risk Characterization
This section desenbes the
summarization of all the toxicology and
exposure data in the final stage of the
nsk asseument process. In the current
guidelines, this section also includes a
discussion of mathematical approaches
to quantitative nsk assessment.
The proposed amendments to the nsk
charactenzation section. Section IV C in
the current guidelines, include a
discussion of the Margin of Exposure
approach. The discussion of dose-
response models and nsk extrapolation
procedures has been moved to Section
IV. Dose-Response Assessment in the
proposed guidelines
YH. References
This section includes a full list of
references for the proposed guidelines
and is Section V in the current

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Federal Register / Vol. 54. No. 42 / Monday. March 0. 1989 / Notices
9389
guidelines. Appropriate reference
changes and additions have been made
to conform to the proposed
amendments.
Dale February 23.1983.
John A. Moot*.
Chairman Risk Astessmem Council
Coalaou
I. Introduction
A General
B Background
U. Definition* and Terminology
111. Hazard Identification of Developmental
Toxicant*
A. Laboratory Animal Studies of
Developmental Toxicity. End Point* and
Their Interpretation
1 End Point* of Maternal Toxicity
2.	End Point* of Developmental Toxicity
3.	Functional Developmental Toxicology
4 Overall Evaluation of Maternal and
Developmental Toxicity
S. Short-tens Testing m Developmental
Toxicity
a. In Vivo Mammalian Developmental
Toxicity Screen
b In Vitro Developmental Toxicity Screens
8 Statistical Consideration*
B Human Studies
1 Examination of Ousters. Case Reports.
or Case Series
Z. Epidemiologic Studies
a General Design Considerations
b Selection of Outcomes for Study
c. Reproductive History Studies
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Federal Register / Vol. 54. No. 42 I Monday. March 6. 1989 / Notices
B. Background
The background incidence of
developmental defects in the human
population is quite large For example.
Hertig (11) estimated that approximately
50% of human conceptuses fail to reach
term: Wilcox (12). using biochemical
techniques for detecting pregnancy as
early as B days postconception.
observed that 35% of pregnancies ended
in an embryonic or fetal loss.
Approximately 3% of newborn children
are found to have one or more
significant congenita) malformations at
birth, and by the end of the first
postnatal year, about 3% more are found
to have serious developmental defects
(13). Of these, it is estimated that 20%
are of known genetic transmission. 10%
are attributable to known environmental
factors, and the remainder result from
unknown causes (14). Also,
approximately 7.4% of children are
reduced in weight at birth (i.e.. below
2500 g) (15).
Gose to one-half of the children in
hospital wards are there because of
prenatally acquired malformations (16).
The Centers for Disease Control recently
evaluated the enormity of the problem
of developmental disabilities in the
United States. Among all races,
congenital anomalies, sudden infant
death syndrome, and prematurity
combined account for more than 50% of
Infant mortality in the United StateB
(17).	In addition, among the leading
causes of estimated years of potential
life lost (YPLL) before the age of 65.
congenital anomalies ranks fifth,
prematurity ranks sixth, and sudden
infant death syndrome ranks seventh
(18)	The YPLL estimates may actually
underestimate the public health Impact
of congenital anomalies because
statistics on the following may not be
represented (19): (1) Anomalies is
infants who die shortly after birth may
not be diagnosed and death may not be
attributed to congenital anomalies: (2)
YPLL estimates are based only on live
births and therefore do not take into
account the number of fetuses with
anomalies that were spontaneously
aborted or infants that were stillborn: (3)
with prenatal diagnoses of chromosomal
abnormalities and neural tube defects,
pregnancies may be terminated and thus
these statistics are not represented in
the YPLL estimates.
Exposure to agents affecting
development can result in any one or
more of four possible manifestations
(death, structural abnormality, growth
alteration, and/or functional deficit)
Therefore, assessment efforts should
encompass a wide arTay of adverse
developmental end points, such as
spontaneous abortions, stillbirths,
malformations, early postnatal
mortality, reduced birth weight and
other adverse functional or physical
changes that are manifested postnatally
Numerous agents have been shown to
be developmental toxicants in animal
test systems (IB). Several of them have
also been shown to be the cause of
adverse developmental effects in
humans, including alcohol aminoptenn.
busulfan. chlorobiphenyls.
diethylstilbestrol. isotretinoin, lead
organic mercury, thalidomide, and
valproic acid (13, 20.21). Although a
number of agents found to be
developmental toxicants in
experimental animal studies have not
shown clear evidence of hazard in
humans, the available human data are
Inadequate to determine a cause and
effect relationship. Comparisons of
human and experimental animal data
have been mude for a limited number of
agents that are human developmental
toxicants (22-24). In these comparisons,
there was almost always qualitative
concordance of effects between humans
and at least one species tested. bIso. the
minimally effective dose (MED) for the
most sensitive animal species was
approximately OJ to 100 tunes the
human MED. not accounting for
differences in the incidence of effect at
the MED. Thus, there is some basis for
estimating the nsk of exposure to human
development based on data from animal
studies.
However, there are a number of
unknowns in the extrapolation of data
from animal studies to humans.
Therefore, a number of assumptions
must be made which are generally
applied. These assumptions are the
bases for the approaches taken to nsk
assessment in these Guidelines.
First an agent that produces an
adverse developmental effect in
experimental animal studies is assumed
to pose a potential hazard to humans
following exposure dunng development.
This assumption is based on the
comparisons of data for known human
developmental toxicants (22-24) In
almost all cases, the experimental
animal data would have predicted 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 end points of concern.
From the data on agents that are known
human developmental toxicants (22-24).
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, the appearance of 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
is probably due to species—specific
differences in critical periods,
metabolism, developmental patterns, or
mechanisms of action.
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. In the absence of such
data, the most sensitive species is used,
based on the fact that for the maionty of
known human developmental toxicants,
humans are as sensitive or more so than
the most sensitive animal species (22-
24).
In general, a threshold is assumed for
the dose-response curve for most
developmental toxicants. 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. There are uncertainties
concerning this assumption that are
being discussed currently in the
literature (25,28).
11. 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 and terminology
will be used.
Developmental toxicology. The study
of adverse effects on the developing
organism that may result from exposure
prior to conception (either parent),
dunng prenatal development, or
postnatally to the time of sexual
maturation Adverse developmental
effects may be detected at any point in
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Federal Register / Vol. 54. No. 42 / Monday. March 8. 1989 / Notices
93$
the life span 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 m
offspring organ or body weight or si2e.
Changes is one end point 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
functional competence of the organism
or organ system following exposure to
an agent during critical periods of
development pre- and/or postnatally.
Malformations and Variations. A
malformation is usually defined as a
permanent structural change that may
adversely affect survival development
or function. The term teratogenicity,
which is used to describe these types of
structural abnormalities, will be used in
these Guidelines to refer only to
structural defects. A 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 smce there
exists a continuum of responses from
the normal to the extreme deviant
There is no generally accepted
classification of malformations and
variations. Other terminology that is
often used, but no better defined,
includes anomalies, deformation*, and
aberrations.
Ill Hazard Identification of
Developmental Toxicants
Developmental toxicity is expressed
as one or more of a number of possible
end points that may be used for
evaluating the potential of an agent to
cause abnormal development The four
types of effects on th« coaceptus that
may be produced by developmental
exposure to toxicants ladude death,
structural abnormality, altered growth,
and functional defidta. Of these, all four
types of effects 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 Q) testing
protocol as well as in other study
protocols, such as the multigeneration
study Although functional deficits have
been shown to occur subsequent to
developmental exposures in humans.
such effects seldom have been
evaluated in routine testing studies in
experimental animals. However,
functional evaluations are beginning to
be examined under certain regulatory
situations (27. 28).
Carcinogenic effects of developmental
exposures have occurred in humans
resulting from the use of
diethylatilbestrol for the maintenance of
pregnancy (29). Several agents have
been shown to cause cancer following
developmental exposures in
experimental animals, and it appears
from the data collected thus far that
agents which are capable of causing
cancer in adults may also cause
transplacental or neonatal
carcinogenesis (30). There is no way to
predict whether adults or developing
animals mil 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 (31) for assessing human
nsk. Mutational events also may occur
as a result of exposure to developmental
toxicants but may be difficult to
discriminate from other possible
mechanisms in standard studies of
developmental toxicity. When
mutational events are suspected from
further experiments, the Guidelines for
Mutagenicity Risk Assessment (32)
should be consulted: however these
guidelines specifically address heritable
and not somatic mutational nsk.
A. Laboratory Animal Studies of
Developmental Toxicity: End Points and
Their Interpretation
This section will discuss the end
points examined in routinely-used
protocols as well as the use of other
types of studies, induding 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) dunng the period of major
organogenesis, evaluation of maternal
responses throughout pregnancy, and
examination of the dam and the uterine
contents just prior to term (7.8. 33-35).
Other protocols may use exposures of
one to a few days to investigate periods
of particular sensitivity for induction of
anomalies in specific organs or organ
systems [38) In addition, developmental
toxicity may be evaluated in studies
involving exposure of one or both
parents prior to conception, of the
conceptus dunng pregnancy and over
several generations, or of offspring
dunng the late prenatal and early
postnatal penods (7.8, 27. 28. 33-33. 37)
These Guidelines are intended to
provide information for interpreting
developmental effects related to any of
these types of exposure. Since many of
the end points evaluated also are
related to effects on the parental
reproductive systems, these Guidelines
should be used in conjunction with
those published on assessing male and
female reproductive risk |3& 39).
Study designs should tndude. at a
minimum, a high dose, a low dose, and
one intermediate dose. The high dose
should produce some maternal or adult
toxidty (i.e., a level which at the least
produces marginal but significantly
reduced body weight weight gam. or
specific organ toxicity, and at the most
produces no more than 10% mortality).
The low dose should demonstrate a
NOAEL for adult and offspnng effects.
A concurrent control group treated with
the vehide used for agent
administration should be included. The
route of exposure is usually oral
although data from other routes may
sometimes be useful especially if
supported by pharmacokinetic
information. Test animals should be
selected based on considerations of
species, strain, age. weight and health
status, and should be randomized to
dose groups in order to reduce bias and
provide a basis for performing valid
statistical tests.
The next three sections discuss
individual end points of maternal and
developmental toxicity as measured in
the conventional developmental toxicity
study and the multigeneration study,
and. on occasion. In postnatal studies.
Other end points specifically related to
reproductive toxicity are covered in the
relevant risk assessment guidelines (38,
39). The fourth section deals with the
integrated evaluation of all data,
including the relative effects of exposure
on maternal animals and their offspnng.
which is important in assessing the level
of concern about a particular agent It,
should be noted that appropnate
historical control data can be helpful m
the interpretation of end points of
maternal and developmental toxicity
1. End Points of Maternal Toxicity A
number of end points that may be
observed as possible indicators of
maternal toxicity are listed in Table 1.
Maternal mortality is an obvious end
point of toxicity; however, a number uf
other end points can be observed whn.h
may give an indication of the subtle
effects of an agent For examcle. in weil-
conducted studies, the fertility and
gestation indices provide informanon on
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the general fertility rate of the animal
stock used and are important indicators
of toxic effects to adults if treatment
begins pnor to mating or implantation
Changes in gestation length may
indicate effects an the process of
parturition.
Table 1. End Points of Maternal Toxicity
Mortality
Fertility Index (no. with seminal plugs or
sperm/no. mated)
Gestation Index (no. with implants/no.
with seminal plugs or sperm)
Gestation Length (when allowed to
deliver pops)
Body Weight
Day 0
During gestation
Sacrifice day
Body Weight Change
Throughout gestation
During treatment (including
increments of time within treatment
penod)
Post-treatment of sacrifice
Corrected maternal (body weight
change throughout gestation minus
gravid uterine weight or litter
weight at sacrifice)
Organ Weights (in cases of inspected
specific organ toxicity and when
supported by advene
histopathology findings)
Absolute
Relative to body weight
Food and Water Consumption (where
relevant)
Clinical Evaluations
Types, incidence and duration of
signs
Enzyme markers
Clinical chemistries
Gross Necropsy and Histopathology
Body weight and the change m body
weight are viewed collectively as
indicators of maternal toxicity for most
species, althought these end points may
not be as useful in rabbits, because
body weight changes in some strains of
rabbits are not good indicators 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 dunng treatment could
occur that would not be reflected in the
total weight change throughout
gestation, because of compensatory
weight jam that may occur following
treatment but before sacrifice. For this
reason, changes in weight gain dunng
treatment can be examined as another
Indicator or maternal toxicity.
Changes in maternal body weight
corrected for gravid uterine weight at
sacnfice may indicate whether the effect
is pnmanly maternal or fetal. For
example, there may be a significant
reduction in weight gain throughout
gestation and in gravid uterine weight,
but no change in corrected maternal
weight gam which would generally
indicate an intrauterine effect.
Conversely, a change in corrected
weight gain and no change in gravid
uterine weight generally suggests
maternal toxicity and little or not
intrauterine effect An alternate estimate
of maternal weight change during
gestation can be obtained by subtracting
the sum of the weights of the fetuses.
However, this weight does not include
the uterine tissue, placental tissue, or
the amniotic fluid.
Changes in other end points may also
be important. For example, changes in
relative and absolute organ weights may
be signs of b maternal effect when an
agent is suspected or 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 are also useful when as
agent is suspected of affecting appetite,
water intake, or excretory function.
Clinical evaluations of toxicity may also
be used as indicator* of maternal
toxicity. Daily clinical observations may
be useful describing the profile or
maternal toxicity. 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/data
considered useful for evaluating
maternal toxicity Jas noted in the
Proceedings of the Workshop on the
Evaluation of Maternal and
Developmental Toxicity (3)). includes:
morbidity or mortality, maternal body
weight and body weight gain: clinical
signs of toxicity: food (and water, if
dosing is via drinking water)
consumption, and necropsy for gross
evidence of organ toxicity. Maternal
toxicity should be determined in the
pregnant and/or lactating animal over
an appropnate part of gestauon and/or
the neonatal penod. and should not be
assumed or extrapolated from other
adult toxicity studies.
2. End Points of Developmental
Toxicity. Because the maternal animal,
and not the conceptus. is the individual
treated dunng gestation, data generally
should be calculated as incidence per
Utter or as number and percent of litters
with particular end points. Table 2
indicates the way in which offspring and
litter end points may be expressed.
Table 2. End Points of Developmental
Toxidty
Litters with implants
No. implantation sites/dam
No. corpora lutea (CL)/dam'
Percent preimplantatioa loss
(CL-implantations) X IDO'/CL
No. and percent live offspring '/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/
Utter
No. and percent liters with nonlive
implants
No. and percent affected (nonlive •+¦
malformed) implants/litter
No. and percent litters with affected
Implants
No. and per cant litters with total
resorptions
No. and percent stillhirths/litter
Litters with Jive offspring
No. and percent litters with live
offspring
No. and percent live offspring/litter
Viability of offspring*
Sex ratio/litter
Mean offspring body weight/litter'
Mean male body weight/litter*
Mean female body weight/litter'
No. and percent externally malformed
offsprmg/litter
No. and percent viscerally malformed
offspring/litter
No. and percent skeletally malformed
offsprmg/litter
No. and percent malformed offspring/
Utter
No. and percent Utters with
malformed offspring
No. and percent malformed males/
litter
No. and percent malformed females/
Utter
No. and percent offspring with
vanations/Utter
No. and percent Utters having
offspring with variations
Types and incidence of individual
malformations
Types and incidence of individual
venations
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Individual offspring and their
malformations and variations
(grouped according to litter and
dose)
Clinical signs
Gross necropsy and histopathology
' Important when treatment begins prior to
implantation. May be difficult to assess in
mica
• Offspring refers both to fetuses observed
pnor to tern or to pups following birth. The
end points examined depend on the protocol
used for each study
' Measured at selected intervals until
termination of the study
When treatment begins pnor to
implantation, an increase in
preimpiantation loss could indicate an
adverse effect on the fertilization
process, ovum transport, utenne
toxicitv. the developing blastocyst, or on
the process of implantation itself. If
treatment begins around the tune of
implantation (i.e.. day 6 of gestation in
the mouse, rat. or rabbit), an increase in
preimpiantation loss probably reflects
normal variability in the animals being
used, but the data should be examined
carefully to determine whether or not
the effect is dose related. U
preimpiantation loss is related to dose m
either case, 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. Resorptions and late fetal
deaths give some indication of when the
conceptus died, and the number and
percent nonlive implants per litter (post-
implantation loss) is a combination of
resorptions and late fetal deaths. The
number and percent of litters showing
an increased incidence for these end
points is generally useful but may be
less useful than incidence per litter
because, in the former case, a litter is
counted whether it has one or all
resorbed. dead, or nonlive implants.
If a significant increase in
postimplantation lot* is found after
exposure to an agrat. the data may be
compared not only with concurrent
controls, but also with recent historical
control data, since there is considerable
intarhtter variability in the incidence of
post-implantation loss (40) 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 end point for affected implants
(i e. the combination of nonlive and
malformed conceptuses) gives an
indication of the total intrauterine
response to an agent and some'imes
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
unplanta per Utter is greatly increased.
In such cases, the malformation rate
may appear to decrease because only
unaffected offspring have survived. If
the incidence of prenatal death or
malformation 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 m the number of implants
per litter. A decrease in the number of
live offspring per litter should be
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 annual
occurrence.
A change in offspring body weight is a
sensitive indicator of developmental
toxicity, in part because it is a
continuous vanable In some cases,
offspnng weight reduction may be the
only indicator of developmental toxicity
While there is always a question
remaining 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,
weight reduction should be used to
establish the NOAEL There are other
factors that 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 confounded by
smaller litters and increased fetal or
neonatal weight Additionally, the
average body weight of males :s greater
than that of females in the more
commonly used laboratory animals
Live offspring should be examined fa
external, visceral, and skeletal
malformations. If only a portion of the
litter is examined, then it is preferable
that those examined be randomly
selected from each litter. An increase in
the incidence of malformed offspnng
may be indicated by a change in one or
more of the following end points: the
incidence of malformed offspnng per
litter, the number and percent of litters
with malformed offspnng. or the numbei
of offspnng or Utters 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 the incidence of external,
visceral, and skeletal malformations
which may indicate the general systems
affected. A listing of individual offspnng
with their malformations and venations
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. eare*must be
taken to avoid counting offspnng more
than once in evaluating any single end
point based on number or percent of
offspnng or litters. The incidence of
individual types of malformations and
vanations should be examined for
significant changes which may be
masked if the data cn all malformations
and vanations are pooled. Appropriate
historical control data are helpful in the
interpretation of malformations and
vanations. especially those that
normally occur at a low incidence and
may or may not be related to dose in 
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Federal Register / Vol. 54, No. 42 / Monday, March 6. 1989 / Notices
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 subtle
alterations in the functional competence
of an organ or a variety of organ
systems may mult from exposure
during critical developmental periods
that may occur between conception and
sexual maturation. Often, these
functional defects are observed at dose
levels below those at which gross
malformations are evident (42). Such
testing has not been routinely required
in the United States, but studies are
beginning to be required when other
information indicates the potential for
adverse functional effects (27.20). Data
from postnatal studies, when available,
are considered very useful for the
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 needed to
determine the full spectrum of potential
developmental effects. In some cases,
useful data can be derived from well-
executed multigeneration studies.
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 (43.44).
Several expert groups have focused on
the functions that should be included in
a behavioral testing battery (45-47), and
these include: sensory systems,
neuromotor development, locomotor
activity, learning and memory, reactivity
and/or habituation, and reproductive
behavior. No tesung battery has
adequately addressed all of these
functions, but it is important to include
as many as possible. Several testing
batteries have been developed and
evaluated (46.48.49). The US. EPA
Office of Toxic Substances (OTS) has
developed a guideline for developmental
neurotoxicity testing (28) that includes
some evaluation of all the categories
hated above except for reproductive
behavior, and also includes
requirements for brain weights and
neuropathology. Several criteria for
selecting agents for developmental
neurotoxicity testing have been
suggested (40). including- agents that
cause central nervous system
malformations, pyschosctive drugs and
chemicals, adult neurotoxicants.
hormonally-active agents, and chemicals
that are structurally related to other
developmental neurotoxicants. 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 data
have accumulated to indicate that the
cardiopulmonary, immune, endocrine,
digestive, and unnary systems, as well
as the central nervous system are
subject to alterations in functional
competence (50.51) 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 and
evaluated in the nsk assessment
process.
Extrapolation of functional
developmental effects to humans is
limited by the lack of knowledge about
underlying toxicological mechanisms
and their significance as is true for other
end points of developmental toxicity. In
comparisons made on a limited number
of agents known to cause developmental
neurotoxic effects in humans (52). these
agents also have been shown to produce
developmental neurotoxic effects in
animal species. As for other end points
of developmental toxicity, the
assumption is made that functional
effects in ammal studies indicate the
potential for altered development in
humans. When data from functional
developmental toxicity studies ire
encountered for particular agents, they
should be evaluated and included in the
nsk assessment prooess.
Same guidance is provided here
concerning important general concepts
of study design and evaluation for
functional developmental toxicity
studies.
•	Several aspects of study design are
similar to those important in standard
developmental toxicity studies (e^. 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
animal* to dose groups and test groups,
btter generally considered the statistical
unit etc.),
•	A replicate study design provides
added confidence in the interpretation
of data.
•	Use of a pharmacological challenge
may be valuable in evaluating function
and "unmasking" effects not othewise
detectable, particularly in the case of
organ systems that are endowed with a
reasonable degree of functional reserve
capacity.
•	Use of functional tests with a
moderate degree of background
variability may be more sensitive to the
effects of an agent than are tests with
low variability that may be impossible
to disrupt without being life-threatening.
Butcher et al. (S3) discussed this with
relation to behavioral end points.
•	A battery of functional tests, in
contrast to a single test, usually
provides a more thorough evaluation of
the functioanl competence of an ammal:
tests conducted at several ages may
provide more information about
maturabonal changes and their
persiatence.
•	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.
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^. structural abnormalities,
perinatal death, and growth
retardation). The level of confidence in
an adverse effect may be more
important than the type of change seen,
and confidence may be increased by
such factors as rephcability of the effect
either in another study of die 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, and
both may be appropriate depending on
the function being tested. Finally, there
are at least three general ways m which
the data from these studies may be
useful for nsk assessment purposes: (1)
To help elucidate the long-term
consequences of fetal and neonatal
findings; (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.
4. Overall Evaluation of Maternal and
Developmental Toxicity.
As discussed previously, individual
end points 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 ell maternal and
developmental end points.
Those agents that produce
developmental toxicity at a dose that is
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9395
not toxic to the maternal animal are of
greatest concern because the developing
organism appears to be more sensitive
than the adult. However, when adverse
developmental effects are produced only
at minimal maternally toxic doses, they
are still considered to represent
developmental tonaty and should not
be discounted as being secondary to
maternal toxicity Current information is
inadequate to assume that
developmental effects at maternally
toxic doses result only from maternal
toxicity: rather, when the lowest
observed advene effect level (LOAEL)
is the same for the adult and developing
organisms, it may simply indicate that
both are sensitive to that dose level.
Moreover, 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 minimal!?
toxic levels either voluntarily or in the
workplace, since several agents are
known to produce adverse
developmental effects at minimally toxic
doses in adult humans (e.g., smoking,
alcohol).
Since the final nsk assessment not
only takes into account the potential
b izard 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 nsk for developmental toxicity
in humans.
If. on the ether hand, maternal toxicity
is seen in the absence of or at dose
levels lower than those producing
developmental toxicity, and if the effect
level is lower than that in evaluations of
other types of adult toxicity, this implies
that the pregnant female is likely to be
more sensitive than the nonpregnant
female and the data from the pregnant
female should be used to assess nsk.
Although the evaluation of
developmental toxicity la the primary
obiective of standard studies within this
area, maternal effects seen within the
context of developmental toxicity
studies should be evalnated as pert of
the overall toxicity profile for a given
chemical.
Approaches for ranking agents
according to their relative maternal and
developmental toxicity have been
proposed: Schardein (20) has reviewed
several of these. Several approaches
involve the calculation of ratios relating
an adult toxic dose to a developmental^
toxic dose (54-57) Such ratios may
desenbe 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
(3). there was no agreement as to the
validity or utility of these approaches ut
other aspects of the nsk assessment
process. This is in part due 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 an/or timing of
exposure), and species differences in
response (3. 56), can influence the
maternal and developmental effects and
the resulting ratios. Also, the end points
used in the ratios need to be better
defined to permit cross-species
comparison. (Jntil such information is
available, the applicability of these
approaches in risk assessment is not
justified.
5. Short-term Testing w
Developmental Toxicity. The need for
short-term tests for developmental
toxicity has ansea from the need to
establish testing pnonties 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. Two
approaches are considered here in terms
of their contnbotion to the overall
testing process: 1) an m vivo mammalian
screen, and 2) a variety of in vitro
systems. Currently, neither approach is
considered as a replacement for routine
in vivo development toxicity testing in
experimental animals, and should not be
used to make the final decision as to an
agent's developmental toxicity. Rather,
such tests may be useful in making
preliminary evaluations of
developmental toxicity, for evaluating
structure-activity relationships, and for
assigning pnonties for further, more
extensive testing. Although such short-
term tests are not routinely required,
data sometimes are encountered in the
review of chemicals: the comments are
provided here for guidance In the
evaluation of such data.
o. In vivo mamahan developmental
toxicity screen. The most widely studied
in vivo short-term approach is that
developed by Chemoff and Kavlock
(59). This approach is based on the
hypothesis that ajrgnaiaLinjary. which
results in altered development wdl be
manifested postnatally as reduced
viability and/or impaired growth. When
originally proposed, the test substance
was administered to mice over the
penod of maior organogenesis at a
single dose level that would elicit some
degree of maternal toxicity At the
MOSH Workshop on the Evaluation of
the Chemoff/Kavlock Test for
Developmental Toxicity (4). 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 preuse
estimate of postimplantation loss (80).
In this approach, the pups are counted
and weighed shortly after birth, and
again after 3-4 days. End points that are
considered m the evaluation include,
general maternal toxicity (including
survival and weight gain), litter size, and
viability, weight and gross
malformations m the offspnng. Basic
pnonty-settmg categories for more
extensive testing have been suggested,
l) agents that induce perinatal death
should receive highest pnority, 2) agents
that induce pennatal weight changes
should be ranked lower in pnonty. and
3) agents that induce no effect should
receive the lowest pnonty (59). Another
scheme that has been proposed applies
a numerical ranking to the results as a
means of pnontmng agents for further
testing (01.62).
The mouse was chosen originally for
this test because of its low cost, but the
procedure has been applied to the rat as
well (63). The test will predict the
potential for developmental toxicity of
an agent in the species used while
extrapolation of nsk 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 (64). Although the testing
guidelines are available, such
procedures are required on a case-by-
case basis. Application of this procedure
in the nsk assessment process within
the Office of Toxic Substances has been
desenbed (65). and the expenences of a
number of laboratones are detailed in
the proceedings of the NIOSH workshop
(i).
b. 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 nonmammahan
organisms. These systems have long
been used to assess events associated
with normal and abnormal development,
but only recently have they been
considered for this potential as screens
in testing (66-66) Many of these systems
are now being evaluated for their ability
to predict the developmental toxicity of
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various agents In Intact mammalian
systems. This validation process
requires certain considerations in study
design, including defined end points for
toxicity and an understanding of the
system's ability to handle various test
agents (67,69-71).
8. Statistical Considerations. In the
assessment of developmental toxicity
data, statistical considerations require
•pedal attention. Since the litter is
generally considered the experimental
unit in most developmental toxicity
studies, the statistical analyses should
be designed to analyze the relevant data
based on Incidence per Utter or on the
number of litters with a particular end
point The analytical procedures used
and the results, as well as an indication
of the variance in each end point should
be clearly indicated in the presentation
of data. 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 within and between
litter variance as well as dose effects.
Nonparametnc and categorical
iirocedures have also been widely used
or 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 (41,72).
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 for 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 determine in
evaluating data is the power of a study
(i.e~ the probability thhl a study will
demonstrate a true effect), which u
limited by the sample size used ui the
study, the background incidence of the
end point observed, the variability in the
incidence of the end point and the
analysis method. As an example. Nelson
and Holson (73) have shown that the
number of litters needed to detect a 5%
or 10% change was dramatically lower
for felal 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 (7. B), it
is possible to detect an increased
incidence of malformations in the range
of 5 to 12 times above control levels, an
increase of 3 to B times the in utero
death rate, and a decrease of 0.15 to 0-25
tunes 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 end points measured.
Consequently, for statistical reasons
only, changes in fetal weight are often
observable 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 end
pointfs) evaluated.
Although statistical analyses are
important in determining the effects of a
particular agent the biological
significance of data should not be
overlooked. For example, with the
number of end points that can be
observed in 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 statistical analyses do not
indicate a significant effect This may be
true especially for the incidence of
malformations or in utero death where 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 experience with developmental
toxicity data and with principles of
experimental design and statistical
analysis, may be required to adequately
evaluate such data.
B. Human Studies
The category of "human studies"
includes both epidemiologic studies and
other reports of individual cases or
clusters of events. 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,
liese 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. 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 IV)
Epidemiologic studies in which exposure
is presumed based on occupational title
or residence (e g.. some case-control and
all ecologic studies) may contribute data
to qualitative nsk assessments, but are
of limited use fur quantitative risk
assessments because of the generally
broad categorical groupings. Risk
assessors should seek the assistance or
professionals trained in epidemiology
when conducting a detailed analysis
1.	Examination of Clusters. Case
Reports, or Case Series. The
identification of cases or clusters of
adverse developmental effects is
generally limited to those identified by
the women involved, or clinically by
their physicians. Examples of outcomes
more easily identified include fetal loss
in mid to late pregnancy or congenital
malformations. Identification of other
effects, such as embryonic loss may be
difficult to separate from subfertility/
infertility. Identification of such "non-
events" (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 may have
Importance in the recognition of
developmental toxicants, they may be of
greatest use in suggesting topics for
further investigation (74).
2.	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 evalution do not
exist The following is a discussion of
factors that affect the relative weight
assigned a particular study in a nsk
assessment
a. General design considerations.
Factors that affect a study's usefulness
for risk assessment include the power of
the study, potential bias in data
collection, control of potential nsk
factors, effect modifiers and
confoundera. and statistical factors (41.
75-80):
(1) The power of the study: The
power, or ability of a study to detect a
true effect is dependent on the size of
the study group, the frequency of the
outcome in the general population, and
the level of excess nsk to be identified
In a cohort study, common outcomes,
such as recognized embryo/fetal loss,
require hundreds of pregnancies in order
to have a high probability of detecting a
modest increase in risk (e.g.. 133 in both
exposed and unexposed groups to delect
a twofold increase: alpha <0.05. power
— 80%). while less common outcomes,
such as the total of all malformations
recognized at birth, require thousands of
pregnancies to have the same
probability (e.g.. more than 1200 in both
exposed and unexposed groups) (15. 75.
76. 81. G2). In case-control studies, study
sizes are dependent upon the frequency
of exposure within the source
population
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9397
A poster on determination of power
of the actual study is useful in
cvaluatmg negative findings. Negative
findings in a study of low power would
be given considerably less weight than
either a pcmuve study, or a negative
study with high power.
(2) Potential hia* ui data collection:
Sources of bias may indude selection
bias aad information bias (S3). Selection
bias may occur when an individual's
willingness to participate vanes with
certain characteristics relating to the
exposure statu* or health status of that
individual In addition, selection bias
may operate in the identification o!
subjects for study. For example, for
studies of very early loaa. use of hospital
records to identify embryonic or early
fetal losa will underescertain events,
because women are not always
hospitalized for these outcomes. More
weight would be given m a risk
assessment to a study in which a mora
complete list of pregnancies is obtained
by. for example, either interviewing the
women in the study or. in a prospective
study, collecting biological data (e.g..
human chorionic gonadotropin
measurements) of pregnancy status from
study members. 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. Thus, a study using hospital
records to identify congenital
malformations would be given more
weight in a nsk assessment.
Information bias may result from
misclaniTication 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-control studies) ia
which the respondent belongs. Use of
highly structured questionnaires and/or
"blinding" of the interviewer will reduce
the likelihood of sueb bias. Studies with
lower likelihood of such types of bias
should carry more weight is a nsk
assessment
When data are collected by interview
or questionnaire, the appropnate
respondent depends upon 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 cwnts to be considerably more
complete and valid than those of the
husbands (78). Studies based on
interview data from the appropnate
respondent (e g_ the woman when
examining her pregnancy history) would
carry more weight than those from
proxy respondents (e.g, the man when
examining his partner's pregnancy
history).
Data from any source may be prone to
errors or bias. Validation with an
independent data source (e g., vital or
hospital records), or use of btomarkers
of exposure or outcome, where possible,
may indicate the presence or absence of
bias and increase confidence m the
results of the study. Those studies with
a low probability of biased data should
carry more weight (81,84).
(3)	Control of potential risk factors,
effect modifiers, and confounder*:
Potential nsk factors may include
smoking, alcohol coosumptioa drug use.
past reproductive history, and
environmental and occupational
exposure. Such characteristics should be
examined, when appropnate. for the
outcome uader study, and should be
controlfed for in the study design and/or
analysis.
The potential for characteristics of the
subiects to be effect modifiers and/or
confouaden should also be considered.
An effect modifier is a factor that
produces different exposure-response
relationships at different levels of the
effect modifier. For example, maternal
age would be an effect modifier if the
nsk associated with a given exposure
increased with the mother's age. A
confounder is associated with both the
exposure and outcome, and these
interrelationships conld distort both the
magnitude and direction of the measure
of association between the exposure of
interest and the outcome. For example,
smoking might be a confounder in a
study of the association of
socioeconomic status and low birth
weight, since smoking has been
associated with both.
Both effect modifiers and confounder*
need to be controlled in the analysis to
improve the estimate of the effects of
exposure (85). A more in-depth
discussion may be found elsewhere (83,
86). The statistical techniques used to
control for these factors require careful
consideration in their application and
interpretation (83,85). Studies that fail
to account for these important factors
should be given less weight in a risk
assessment.
(4)	Statistical factors. As in animal
studies, pregnancies expenenccd by the
same woman are not independent
events. In animal studies, the litter is
generally used as the unit of measure to
deal with nomndependence of events.
This approach is difficult in humans
since the pregnancies are sequential,
with the nsk factors changing for
different pregnancies (15. 41.81.96). If
more than one pregnancy per woman is
included, as is often necessary due to
small study groups, the use of
nomndependent observations
overestimates the true size of the
population at nsk and artificially
increases the significance level (87).
Some approaches to deal with these
issues have been suggested (81.88). At
this point in time, a generally accepted
solution to this problem has not been
developed.
b. Selection of outcomes for study. As
already discussed, a number of end
points can be considered in the
evaluation of advene developmental
effects. However, some of the outcomes
are not easily observed in humans.
These include early embryonic loss and
reproductive capacity of the offspnng.
Currently, the most feasible end points
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 subfertihty/infertility which
in some cases might be evidence of very
early embryonic loss. Factors requiring
control in the design or analysis (such as
jther nsk factors, 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 since differing spectra of
outcomes may occur at different
exposure levels, the size and
domnqmphic characteristics of the
population, and the ability to observe
the reproductive outcome in humans.
Improved methods for identifying soir.e
outrumes such as embryonic or very
cjr'v fetal loss using new human
chorionic gonadotropin (hCG) assays
may change the spectrum of outcomes
available for study (12).
Demographic characteristics of the
population, such as mantal stutus. age
distribution, education, and pnor
reproductive history are associated with
the probability of whether couples will
attempt to have children. There may
also be differences m the use of birth
control, which would affect the number
of outcomes available for study.
Additionally, workers may move in and
out of areas with differing levels and
types of exposures, affecting the number
of exposed and companson pregnancies
for study Larger populations are usually
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Federal Register / Vol. 54. No. 42 / Monday, March 6. 1989 / Notices
necessary in environmental settings,
since the exposures in environmental
settings are generally much lower than
in occupational settings.
a Reproductive history studies.
(1)	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 possibly postnatal survival,
growth, and function. Epidemiologic
studies that focus on only one type of
pregnancy outcome may miss a true
effect of exposure. As mentioned above,
some reproductive end points can be
thought of as a continuum of adverse
effects; for example, a malformed
stillbirth would not be included in a
study of defects observed at live birth,
even though the etiology could be
identical (75,89). Studies that examine
multiple end points could yield more
information, but the results may be
difficult to interpret. Evidence of a dose-
response relationship is usually an
inportant criterion in the assessment of
a toxic exposure. However, traditional
dose-response relationships may not
always be observed for some end
points. For example, with increasing
dose, a pregnancy might end in an
embryo/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 (90.91). Therefore, a nsk
assessment should when possible,
attempt to look at the interrelationship
of different reproductive end points and
patterns of exposure.
(2)	Measures of fertility: Normally,
studies of subfertiljty/infertility would
not be included in an evaluation of
developmental effects. However, in
humans it is difficult to identify very
early embryonic loss, and to distinguish
it from subfertility/infertility. Thus,
studies that examine subfertility or
infertility indirectly examine loss very
early in the gestational penod. Studies
of subfertility may be thought of as the
study of non-events: 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 evaluations, 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 penod. age. race, mantal status.
parity, contraceptive use. etc. (92-94)
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 (87.95) and
in usefulness for risk assessment
Analysis of the time period between
recognized pregnancies or live births
has been suggested as another indirect
measure of fertility (96). Because the
time interval between births increases
with increasing parity (97). comparisons
within birth order (parity) are more
appropriate. A statistical method (Cox
regression) con 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 subiect is
available in the Proposed Guidelines for
Assessing Male Reproductive Risk (38)
and the Proposed Guidelines for
Assessing Female Reproductive Risk
(39).
d. Community studies/surveillance
programs. Epidemiologic studies may
also be based upon broad populations
such as a community, a nationwide
probability sample, or surveillance
programs (such as birth defects
registries) A number of case-control
studies have examined the relationship
between broad classes of parental
occupation in certain communities or
countries, and embryo/fetal loss (98).
birth defects (99-101). and childhood
cancer (100.102-104). 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.. 100). Such studies are
most helpful in the Identification of
topics for additional study However,
because of the broad groupings of types
of levels of exposure, such studies are
not typically useful for nsk assessment
of a particular agent.
Surveillance programs may also exist
in occupational settings. In this case,
reproductive histories and/or cluneal
evaluation could monitor for
reproductive effects of exposures. Both
could yield very useful data for nsk
assessment; however, a clinical
evaluation program would be costly to
maintain.
C. Other Considerations
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,
placental metabolism and transfer,
comparative metabolism, and
concentrations of the parent compound
and metabolites in the maternal animal
and eonceptus 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, including that of
humans (105.106). 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 pregnant 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 (107).
2. Comparisons of Molecular
Structure. Comparisons of the chemical
or physical properties of an agent with
those of known developmental toxicants
may provide some indication of 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
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.
D. Weight-of-Evidence Determination
Information from all available Btudies.
whether indicative of potential concern
or not must be evaluated and factored
into a weight-of-evidence judgment as to
the likelihood that an agent may pose a
nsk for developmental toxicity in
humans. The primary considerations are
the human data (which are seldom
available) and the experimental animal
data. The qualitative assessment for
developmental toxicity should consider
quality of the data, resolving power of
the studies, number and types of end
points examined, relevance of route and
timing of exposure, appropnateness of
the dose selection, replication of effects,
number of species examined, and
availability of human case reports or
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9399
series, and/or epidemiologic study data.
In addition, pharmacokinetic data and
structure-activity considerations, as well
as other factora that may affect the
strength 0f the evidence, should be
taken into account Therefore, all data
pertinent to developmental toxicity
should be examined in the evaluation of
a chemical's potential to cause
developmental toxicity in humans, and
sound scientific judgment should be
exercised in interpreting the data in
terms of the risk for adverse human
developmental health effects.
A categorization scheme for the
weight of evidence hat been developed.
It contains several broad categories that
reflect the accumulated data base on
agents and serves as an indicator of
whether exposure to the substance may
cause developmental toxicity in humans.
It represents one important step ui the
evaluation of agents. However, the nsk
of any given exposure to an agent can
only be derived from an appreciation of
its intrinsic biological activity and the
nature of the anticipated exposure
conditions. These important aspects are
developed in subsequent sections of this
Guideline.
Placing an agent in a particular
weight-of-evidence category such as
"adequate evidence for human
developmental toxicity" does not mean
that it will be a developmental toxicant
at every dose (because of the
assumption of a threshold) or in every
situation (e.g. hazard may vary
significantly depending on route and
timing of exposure). Thus, in the final
characterization of nsk. the weight-of-
evidence determination should always
be presented in conjunction with
information on dose-response (NOAEL
and/or LOAEL), and. if available, with
the human exposure estimate.
The weight-of-evidence scheme
(outlined in Table 3) defines three levels
of confidence for data used to identify
developmental hazards and to assess
the nsk of human developmental
toxicity- definitive evidence, adequate
evidence, and inadequate evidence.
Within the definitive evidence and
adequate evidence atafories. there are
subcategories for evidence indicating
adverse effects and for evidence
indicating no apparent effects. In both
categories, the evidence required to
classify an agent as demonstrating no
adverse effects is greater than that
required to demonstrate an adverse
effect and must include evaluations of a
variety of potential manifestations of
developmental toxicity Greater
evidence is required because it is much
more difficult both biologically and
statistically lo support a finding of no
apparent adverse effect than one of an
adverse effect. Most agents meeting
current testing requirements would be
expected to fall within the adequate
evidence category, while many for
which little or no informahon is
available would be classified in the
inadequate category. Few agents would
be expected to fall into the definitive
evidence category because the human
data necessary to meet the criteria for
this category would be difficult to
obtain.
TABLE 3. WEIGHT OF EVIDENCE
SCHEME FOR DEVELOPMENTAL
TOXICITY
Definitive Evidence for
—Human Developmental Toxicity
—No Apparent Human Developmental
Toxicity
Adequate Evidence for
—Potential Human Developmental Toxicity
—No Apparent Potential Human
Developmental Toxicity
Inadequate Evidence for Determining
Potential Human Developmental Toxicity
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 experimental design
and statistical analysts, may be required
to adequately evaluate the data base. To
allow for this, the language used in the
scheme is intentionally broad.
Definitive Evidence for
—Human Developmental Toxicity
This category includes agents for
which there is sufficient evidence from
epidemiologic studies for the scientific
community to judge that a cause and
effect relationship exists. Case reports
in conjunction with other supporting
evidence may also be used.
—No Apparent Human
Developmental Toxicity
Agents in this category have not been
associated with developmental toxicity
in well-executed epidemiologic studies
(e.g.. case control and cohort) with
adequate power A variety of potential
manifestations of developmental
toxicity have been studied. Supporting
animal data may or may not be
available.
Adequate Evidence for
—Potential Human Developmental
Toxicity
This category includes agents for
which sufficient evidence exists for
them to be considered potential human
developmental toxicants The minimum
evidence necessary for considering an
agent a potential human developmental
toxicant would include data from an
appropriate, well-executed study m a
single experimental animal species that
demonstrates developmental toxicity,
and/or strong suggestive evidence from
adequate cluucal/epidemiologic studies.
Evidence may be modified by further
data, such as studies in additional
species or by other routes of exposure,
and replication of the findings.
Development of pharmacokinetic or
mechanistic information may reduce
uncertainties in extrapolation to the
human. The strength of the evidence
increases as it approaches the definition
for definitive human developmental
toxicity.
—No Apparent Potential Human
Developmental Toxicity
This category includes agents with
data from appropriate well-executed
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
animal. In addition, there may be human
data from adequate studies supportive
of no advene effects. _ .
Inadequate Evidence for Determining
Potential Human Developmental
Toxicity
This category includes agents for
which there is less than the minimum
sufficient evidence necessary for
assessing human nsk. However, data on
agents that fall into this category may be
used to determine the need for
additional testing or information that
would then, if adequate, move the agent
into the adequate evidence category.
This category includes a variety of
types of information such as the lack of
any data on the developmental toxicity
potential of an agent, data from an
appropnate well-executed study in a
single species showing no
developmental toxicity, data from
poorly-conducted studies in animals
(e.g., small numbers of animals,
inappropnate dose selection, other
confounding factors) or inadequate dati
in humans. Additionally, data on
structure/activity relationships, short-
term test data, pharmacokinetic data, or
data on metabolic precursors of the
agent of interest could be used to call for
further testing but would be considered
insufficient by themselves to assess
human nsk.
IV. Dose-Response Assessment
When quantitative human dose-effect
data are available and with sufficient
range of exposure, dose-response
relationships may be examined Data on
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Federal Register / Vol. 54. No. 42 / Monday. March 6. 1989 / Notices
exposure from human studies are
usually qualitative, such as employment
or residence histories; quantitative or
dose data are frequently not available.
In human studies, es pea ally
retrospective ones, linking of specific
time periods and specific exposures,
even on a qualitative leveL may be
difficult due to errors of recall or
recordkeeping (where records are
available). The appropriate exposure
depends on the outcome(s) studied, the
biologic mechanism affected by
exposure, and the half-life of the
exposure. The probability of
mis classification of exposure status may
affect the ability of a study to recognize
a true effect (15.41.76.108.109).
Since data on human dose-effect
relationships are rarely available, the
dose-response assessment is usually
based on the evaluation of tests
performed in laboratory animals.
Evidence for a dose-response
relationship is an important criterion in
the assessment of developmental
toxicity, although this may be based on
limited data from standard studies using
three dose groups aad a control group.
Moat human developmental toxicants
that have been studied alter
development at doses within a narrow
range near the lowest maternally toxic
dose 1221. Therefore, for most chemicals,
the exposure situations of concern will
be those that are potentially within this
range. For those few chemicals where
developmental effects occur at much
lower levels than maternal effects, the
potential for exposing the conceptus to
damaging doses is much greater. As
mentioned previously (section IQ-A^.),
however, traditional dose-response
relationships may not always be
observed for some end points. For
example, as the exposure level nses.
embryo/fetolethal levels may be
reached, resulting in an observed
decrease in malformations with
increasing dose (8t 90). The potential
for this response pattern indicates that
dose-response relationships of
individual end points as well as
combinations of end points (eg* dead
and malformed combined) must be
carefully examined and Interpreted.
Identification of a NOAEL and/or
LOAEL is based on the lowest dose at
which an advene effect is detected from
any adequate developmental toxicity
study. Adequacy of the data to be used
for determination must be fudged using
the weight-of-evidence approach
discussed in section 1ILD. NOAELa and
applied uncertainty factors may be used
to determine a reference dose for
developmental toxicity (RtDpr) that is
assumed to be below the threshold for
an increase in advene developmental
effects. The RIDn is based on a short
duration of exposure as is typically used
in developmental toxicity studies. The
term RfDor is used to distinguish from
the RfD which refers to chronic
exposure situations (lO).iJncertainty
factors for developmental toxicity
generally include a 10-fold factor for
interspecies variation and a 10-fold
factor for intraspedes variation. In
general, an additional uncertainty factor
is not applied to account for duration of
exposure. Additional factors may be
applied due to a variety of uncertainties
that exist In the data base. For example,
the standard study design for a
developmental toidaty study calls for a
low dose that demonstrates a NOAEL
but there may be circumstances where a
nsk assessment must be based on the
results of a study in which a NOAEL for
developmental toxicity was not
identified. Rather, the lowest dose
administered caused significant effect(s)
and was identified as the LOAEL In
circumstances where only a LOAEL is
available, questions relative to the
sensitivity of end points reported,
adequacy of dose levels tested, or
confidence in the LOAEL reported may
require the use of an additional
uncertainty factor of 10 (10). The total
uncertainty factor selected is then
divided into the NOAS./LOAEL for the
most sensitive end point from the most
appropriate and/or sensitive
mammalian species to determine the
RfDw
Although the Agency currently uses
the NOAEL/uncertainty factor approach
to establish an RfDg* discussions of risk
extrapolation procedures have noted
that improved mathematical tools are
needed for developing estimates of
potential human developmental nsk (45.
110). Gaylor (111) suggested an
approach for estimating nsk that
combines the use of mathematical
models for low-dose estimation of nsk
with the application of an uncertainty
factor based on a preselected level of
risk. This approach is similar to
approaches proposed for carcinogenesis,
but does not preclude the possibility of a
threshold, and may provide a more
quantitative approach to estimating nsk
Another approach proposed by Rai and
Van Ryzin (112) and recently applied by
Faustman et al. (113). uses a simple two-
component developmental model in
which the first component represents a
dose-related nsk to the litter
environment and the second component
expresses the nsk to an individual
offspring conditional upon a
predisposing nsk to the Utter These
approaches and others have been
summarized recently (5). In addition,
other methods for expressing nsk are
being sought and will be applied, if
considered appropnate.
The development of biologically-
based dose-response models in
developmental toxicology is 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, and
the influence of maternal effects on the
dose-response curve. A biological
threshold is assumed for moBt
developmental effects based on known
homeoatatic. compensatory, or adaptive
mechanisms that must be overcome
before a toxic end point is manifested,
and on the rationale that the embryo is
known to have some capacity for repair
of damage or insult (90). In addition,
most developmental deviations are
probably multifactorial in nature (114).
Although a threshold is assumed for
developmental effects, the existence of a
NOAEL in an study does not
prove or disprove the existence or level
of a true threshold: it only defines the
highest level of exposure under the
conditions of the study that is not
associated with a significant increase in
effect The uncertainties concerning this
assumption are being discussed
currently in the literature (25.28)
In conclusion, dose-response findings
in developmental toxicity studies are
used as pari of the risk characterization
This use is dependent upon scientific
Judgment as to the accuracy and
adequacy of the data, bi addition, the
slope of the dose-response curve should
be considered in conjunction with a
determination as to the adequacy of the
"exposure levels tested, the sensitivity of
the end points reported, and the
appropnatenesi of the experimental
design to determine a level of
confidence m the data and the resultant
confidenoe in the LOAEL NOAEL and
the uncertainty factors applied to obtain
theRfDsr-
V. Exposure Assessment
In order to obtain a quantitative
estimate of risk for the human
population, an estimate of human
exposure is required. The Guidelines for
Estimating Exposures have been
published separately (115) and will not
be diacuased in detail here. In general,
the exposure assessment descnbes the
magnitude, duration, schedule, and route
of exposure. This information is
developed from monitoring data and
from estimates based on modeling of
environmental exposures. Unique
considerations for developmental
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Federal Register / Vol. 54. No. 42 / Monday. March 6. 1989 / Notices
94
toxicity are duration and period of
exposure aa related to stage of
development (i.e., critical periods), and
the 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). For these reasons, it is
assumed that a single exposure at the
critical time in development is sufficient
to produce an adverse developmental
effect. Therefore, the human exposure
estimate used to calculate the margin of
exposure is usually based on a single
dose that is not ad)usted for duration of
exposure, and the number of exposures
is not considered important unless there
is evidence for a cumulative effect. It
should be recognized also that exposure
of almost any segment of the human
population (i.e., fertile men and womea
the conceptus. and the child up to the
age of sexual maturation) may lead to
risk to the developing organism.
VI. Risk Characterization
Many uncertainties described in these
Guidelines are associated with the
toxicological and exposure components
of risk assessments in developmental
toxicology In the past these
uncertainties have often not been
readily apparent or consistently
presented. The presentation of any nsk
assessment for developmental toxicity
should be accompanied by statements
concerning the weight of the evidence,
dose-response relationships and
assumptions underlying the estimation
of the RfDor. esumates of human
exposure, and any factors that affect the
quality and precision of the assessment.
The nsk characterization of an agent
should be based on data from the most
appropnate 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 and should be
considered in relationship to other forms
of toxicity.
In the nsk chanctvfcution. the dose-
response and the exposure
estimate may be conMaed either by
companng the RIDdt and the human
exposure estimate or by calculating the
margin of exposure (MOE). The MOE is
the ratio of the NOAEL from the most
appropnate or sensitive species to the
estimated human exposure level from all
potential sources (53) If a NOAEL is not
available, a LOAEL may be used in the
calculation of the MOE. In this case, the
NOAEL may be estimated from the
LOAEL by applying an uncerta.nty
factor (10-fold) to assess the impact on
the MOE (53) The MOE is presented
along with a discussion of the weight of
evidence, including the nature and
quality of the hazard and exposure data,
the number of species affected, and the
dose-response information.
The RfDor companson with the
human exposure estimate and the
calculation of the MOE are conceptually
similar but are used in different
regulatory situations. The choice of
approach is dependent upon several
factors, including the statute involved,
the situation being addressed, the data
base used, and the needs of the decision
maker. The RIDdt and/or the MOE are
considered along with other risk
assessment and nsk management issues
in making nsk management decisions,
but the scientific issues that must be
taken into account in establishing them
have been addressed here.
These Guidelines summarize the
procedures that the U S. Environmental
Protection Agency will follow 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. Further studies that: (l) Delineate
the mechanisms of developmental
toxicity and pathogenesis. (2) provide
comparative pharmacokinetic data, and
(3) elucidate the functional modalities
that may be altered by exposure to toxic
agents, will aid in the interpretation of
data and interspecies extrapolation.
These types of studies, along with
further evaluation of the relationship
between maternal and developmental
toxicity and the concept of a threshold,
will provide for the development of
improved mathematical models to more
precisely assess nsk.
VH Reference*
1 U S. Environmental Protection Agency
Assessment of nsks 10 human reproduction
and to development of the human conceptus
from exposure to environmental substances,
pp 89-110 Available from- MTIS. Springfield,
VA DEB2-007897
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198& Sept 24 Guidelines for estimating
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34054
[FR Doc 89-4068 Tiled 3-3-89: 8 45 am|
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