t -J
&EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/600/8-87/045
August 1987
Research and Development
The Risk Assessment
Guidelines of 1986
SFUND RECORDS CTR
99750
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EPA/600/8-87/045
August 1987
THE RISK ASSESSMENT
GUIDELINES OF 1986
i
I U.S. Environmental Protection Agency
i Washington, B.C.
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DISCLAIMER
This document has been reviewed in accordance wilh U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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CONTENTS
Preface iv
Abstract v
GUIDELINES FOR CARCINOGEN RISK ASSESSMENT 1-1
GUIDELINES FOR MUTAGENICITY RISK ASSESSMENT 2-1
GUIDELINES FOR THE HEALTH RISK ASSESSMENT
OF CHEMICAL MIXTURES 3-1
GUIDELINES FOR THE HEALTH ASSESSMENT
OF SUSPECT DEVELOPMENTAL TOXICANTS 4-1
GUIDELINES FOR ESTIMATING EXPOSURES 5-1
111
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PREFACE
On September 24, 1986, the U.S. Environmental Protection Agency (EPA) issued risk assessment
guidelines relating to five areas: carcinogenicity, mutagenicity, chemical mixtures, suspect developmental
toxicants, and estimating exposures (51 FR 33992-34054). The guidelines were developed to promote high
technical quality and Agencywide consistency in the risk assessment process.
The guidelines were developed partly in response to a 1983 National Academy of Sciences publication
entitled "Risk Assessment in the Federal Government: Managing the Process," which recommended that
Federal regulatory agencies establish risk assessment guidelines. An EPA task force, convened by then
Administrator William D. Ruekelshaus to study ways to improve the scientific foundation for Agency
regulatory decisions, accepted the recommendation, and work on the guidelines began early in 1984.
The guidelines are products of a two-year Agency development and review process which included many
scientists from the larger scientific community. They were developed as part of an interoffice guidelines
development program under the auspices of the Office of Health and Environmental Assessment in the
Agency's Office of Research and Development. The scientists involved were skilled in each topic, and early
drafts were peer-reviewed by experts from academia, industry, public interest groups, and other
governmental agencies. Subsequently, proposed guidelines were published in the Federal Register, reviewed
by special panels of EPA's Science Advisory Board (SAB), and revised to take into account public and SAB
comments. After final EPA review and Office of Management and Budget review, the guidelines were signed
by EPA Administrator Lee M. Thomas on August 22, 1986, and published in the Federal Register on
September 24,1986.
Each of the five guidelines provides both technical information and science policy guidance relating to the
conduct of EPA risk assessments and presentation of risk assessment information. The guidelines are
sufficiently flexible to allow skilled scientists to make appropriate technical judgments on a case-by-case
basis, giving full consideration to all relevant scientific information. The guidelines also stress that risk
assessments should include a discussion of the strengths and weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well as the scientific basis and rationale for each assessment.
They require risk assessors to inform Agency decisionmakers and the public about the assumptions used in
and the implications of individual risk assessment conclusions, so that appropriate risk management
decisions can be made and explained.
While these guidelines are published Agency documents, they should not be interpreted as static, but as
the first step in the continuing process of identifying the best methods for assessing risk to environmental
pollutants. Consequently, the risk assessment guidelines are constantly undergoing Agency scrutiny and
will be revised in line with new methods and information, as appropriate.
This document presents the five guidelines as they originally appeared in the Federal Register but in a
format that is easier to read.
IV
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ABSTRACT
On September 24, 1986, the U.S. Environmental Protection Agency issued risk assessment guidelines
relating to five areas: carcinogenicity, mutagenicity, chemical mixtures, suspect developmental toxicants,
and estimating exposures (51 PR 33992-34054). The guidelines were developed to promote high technical
quality and Agencywide consistency in the risk assessment process. This document presents the five
guidelines as they originally appeared in the Federal Register but in a format that is easier to read.
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51 FR 33992
GUIDELINES FOR CARCINOGEN RISK
ASSESSMENT
SUMMARY:On September 24, 1986, the U.S.
Environmental Protection Agency issued the
following five guidelines for assessing the health
risks of environmental pollutants.
Guidelines for Carcinogen Risk Assessment
Guidelines for Estimating Exposures
Guidelines for Mutagenicity Risk Assessment
Guidelines for the Health Assessment of Suspect
Developmental Toxicants
Guidelines for the Health Risk Assessment of
Chemical Mixtures
This section contains the Guidelines for Carcinogen
Risk Assessment.
The Guidelines for Carcinogen Risk Assessment
(hereafter "Guidelines") are intended to guide
Agency evaluation of suspect carcinogens in line
with the policies and procedures established in the
statutes administered by the EPA. These Guidelines
were developed as part of an interoffice guidelines
development program under the auspices of the
Office of Health and Environmental Assessment
(OHEA) in the Agency's Office of Research and
Development. They reflect Agency consideration of
public and Science Advisory Board (SAB) comments
on the Proposed Guidelines for Carcinogen Risk
Assessment published November 23, 1984 (49 FR
46294).
This publication completes the first round of risk
assessment guidelines development. These
Guidelines will be revised, and new guidelines will
be developed, as appropriate.
FOR FURTHER INFORMATION CONTACT:
Dr. Robert E. McGaughy
Carcinogen Assessment Group
Office of Health and Environmental Assessment
(RD-689)
401M Street, S.W.
Washington, DC 20460
202-382-5898
SUPPLEMENTARY INFORMATION: In 1983,
the National Academy of Sciences (NAS) published
its book entitled Risk Assessment in the Federal
Government: Managing the Process. In that book,
the NAS recommended that Federal regulatory
agencies establish "inference guidelines" to ensure
consistency and technical quality in risk
assessments and to ensure that the risk assessment
process was maintained as a scientific effort
separate from risk management. A task force within
EPA accepted that recommendation and requested
that Agency scientists begin to develop such
guidelines.
General
The guidelines are products of a two-year
Agencywide effort, which has included many
scientists from the larger scientific community.
These guidelines set forth principles and procedures
to guide EPA scientists in the conduct of Agency risk
assessments, and to inform Agency decision makers
and the public about these procedures. In particular,
the guidelines emphasize that risk assessments will
be conducted on a case-by-case basis, giving full
consideration to all relevant scientific information.
This case-by-case approach means that Agency
experts review the scientific information on each
agent and use the most scientifically appropriate
interpretation to assess risk. The guidelines also
stress that this information will be fully presented
in Agency risk assessment documents, and that-
Agency scientists will identify the strengths and
weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well
as the scientific basis and rationale for each
assessment.
Finally, the guidelines are formulated in part to
bridge gaps in risk assessment methodology and
data. By identifying these gaps and the importance
of the missing information to the risk assessment
process, EPA wishes to encourage research and
analysis that will lead to new risk assessment
methods and data.
Guidelines for Carcinogen Risk Assessment
Work on the Guidelines for Carcinogen Risk
Assessment began in January 1984. Draft
guidelines were developed by Agency work groups
composed of expert scientists from throughout the
Agency. The drafts were peer-reviewed by expert
scientists, in the field of carcinogenesis from
universities, environmental groups, industry, labor,
and other governmental agencies. They were then
proposed for public comment in the FEDERAL
REGISTER (49 FR 46294). On November 9, 1984,
the Administrator directed that Agency offices use
the proposed guidelines in performing risk
assessments until final guidelines become available.
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After the close of the public comment period,
Agency staff prepared summaries of the comments,
analyses of the major issues presented by the
commentors, and proposed changes in the language
of the guidelines to deal with the issues raised.
These analyses were presented to review panels of
the SAB on March 4 and April 22-23, 1985, and to
the Executive Committee of the SAB on April 25-26,
1985. The SAB meetings were announced in the
FEDERAL REGISTER as follows: February 12
1985 (50 PR 5811) and April 4, 1985 (50 PR 13420
and 13421).
In a letter to the Administrator dated June 19,
1985, the Executive Committee generally concurred
on all five of the guidelines, but recommended
certain revisions, and requested that any revised
guidelines be submitted to the appropriate SAB
review panel chairman for review and concurrence
on behalf of the Executive Committee. As described
in the responses to comments (see Part B: Response
to the Public and Science Advisory Board
Comments), each guidelines document was revised,
where appropriate, consistent with the SAB
recommendations, and revised draft guidelines were
submitted to the panel chairmen. Revised draft
Guidelines for Carcinogen Risk Assessment were
concurred on in a letter dated February 7, 1986.
Copies of the letters are available at the Public
Information Reference Unit, EPA Headquarters
Library, as indicated elsewhere in this section.
Following this Preamble are two parts: Part A
contains the Guidelines and Part B, the Response to
the Public and Science Advisory Board Comments (a
summary of the major public comments, SAB
comments, and Agency responses to those
comments).
The Agency is continuing to study the risk
assessment issues raised in the guidelines and will
revise, these Guidelines in line with new information
as appropriate.
References, supporting documents, and
comments received on the proposed guidelines, as
well as copies of the final guidelines, are available
for inspection and copying at the Public Information
Reference Unit (202-382-5926), EPA Headquarters
Library, 401 M Street, S.W., Washington, DC,
between the hours of 8:00 a.m. and 4:30 p.m.
I certify that these Guidelines are not major
rules as defined by Executive Order 12291, because
they are nonbinding policy statements and have no
direct effect on the regulated community. Therefore,
they will have no effect on costs or prices, and they
will
[51FR33993J
have no other
significant adverse effects on the economy. These
Guidelines were reviewed by the Office of
Management and Budget under Executive Order
12291.
August 22, 1986
Lee M. Thomas,
Administrator
CONTENTS
Part A: Guidelines for Carcinogen Risk Assessment
/, Introduction
HJIazard Identification
A. Overview
B. Elements of Hazard Identification
1. Physical-Chemical Properties and Routes and
Patterns of Exposure
2, Structure-Activity Relationships
3, Metabolic and Pharmacokinetic Properties
4. Toxicologie Effects
5. Short-Term Tests
6.1.ong-Term Animal Studies
7. Human Studies
C. Weight of Evidence
D. Guidance for Dose-Response Assessment
E. Summary and Conclusion
///.Dose-Response Assessment, Exposure A ssessment, and Risk
Characterization
A.Uose-Response Assessment
1. Selection of Data
2. Choice of Mathematical Extrapolation
Model
3. Equivalent Exposure Units Among Species
B. Exposure Assessment
C. Risk Characterization
I. Options for Numerical Risk Estimates
2. Concurrent Exposure
3. Summary of Risk Characterization
IV. EPA Classification System for Categorizing Weight of
Evidence for Carcinogenicity from Human and Animal
Studies (Adapted from IARC)
A, AssessmentofWeightol'Evidence forCarcinogenicity from
Studies in Humans
B. Assessmentof Weighlof Evidence for Carcinogenicity from
Studies in Experimental Animals
C. Categorization of Overall Weight of Evidence for Human
Carcinogenicity
V.References
Part B: Response to Public and Science Advisory Board
Comments
/. Introduction
II. Office of Science and Technology Policy Report on
Chemical Carcinogens
III. Inference Guidelines
IV. Evaluation of Benign Tumors
V.TransplacentalandMultigenerationalAnimalBioassays
VI. Maximum Tolerated Dose
VII. Mouse Liver Tumors
VIII. Weight-of-Euidence Categories
XL Quantitative Estimates of Risk
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Part A: Guidelines for Carcinogen Risk
Assessment
/. Introduction
This is the first revision of the 1976 Interim
Procedures and Guidelines for Health Risk
Assessments of Suspected Carcinogens (U.S. EPA,
1976; Albert et al., 1977). The impetus for this
revision is the need to incorporate into these
Guidelines the concepts and approaches to
carcinogen risk assessment that have been
developed during the last ten years. The purpose of
these Guidelines is to promote quality and
consistency of carcinogen risk assessments within
the EPA and to inform those outside the EPA about
its approach to carcinogen risk assessment. These
Guidelines emphasize the broad but essential
aspects of risk assessment that are needed by
experts in the various disciplines required (e.g.,
toxicology, pathology, pharmacology, and statistics)
for carcinogen risk assessment. Guidance is given in
general terms since the science of carcinogenesis is
in a state of rapid advancement, and overly specific
approaches may rapidly become obsolete.
These Guidelines describe the general
framework to be followed in developing an analysis
of carcinogenic risk and some salient principles to be
used in evaluating the quality of data and in
formulating judgments concerning the nature and
magnitude of the cancer hazard from suspect
carcinogens. It is the intent of these Guidelines to
permit sufficient flexibility to accommodate new
knowledge and new assessment methods as they
emerge. It is also recognized that there is a need for
new methodology that has not been addressed in this
document in a number of areas, e.g., the
characterization of uncertainty. As this knowledge
and assessment methodology are developed, these
Guidelines will be revised whenever appropriate.
A summary of the current state of knowledge in
the field of carcinogenesis and a statement of broad
scientific principles of carcinogen risk assessment,
which was developed by the Office of Science and
Technology Policy (OSTP, 1985), forms an important
basis for these Guidelines; the format of these
Guidelines is similar to that proposed by the
National Research Council (NRC) of the National
Academy of Sciences in a book entitled Risk
Assessment in the Federal Government: Managing
the Process (NRC, 1983).
These Guidelines are to be used within the
policy framework already provided by applicable
EPA statutes and do not alter such policies. These
Guidelines provide general directions for analyzing
and organizing available data. They do not imply
that one kind of data or another is prerequisite for
regulatory action to control, prohibit, or allow the
use of a carcinogen.
Regulatory decision making involves two
components: risk assessment and risk management.
Risk assessment defines the adverse health
consequences of exposure to toxic agents. The risk
assessments will be carried out independently from
considerations of the consequences of regulatory
action. Risk management combines the risk
assessment with the directives of regulatory
legislation, together with socioeconomic, technical,
political, and other considerations, to reach a
decision as to whether or how much to control future
exposure to the suspected toxic agents.
Risk assessment includes one or more of the
following components: hazard identification, dose-
response assessment, exposure assessment, and risk
characterization (NRC, 1983).
Hazard identification is a qualitative risk
assessment, dealing with the process of determining
whether exposure to an agent has the potential to
increase the incidence of cancer. Kor purposes of
these Guidelines, both malignant and benign
tumors are used in the evaluation of the
carcinogenic hazard. The hazard identification
component qualitatively answers the question of
how likely an agent is to be a human carcinogen.
Traditionally, quantitative risk assessment has
been used as an inclusive term to describe all or
parts of dose-response assessment, exposure
assessment, and risk characterization. Quantitative
risk assessment can be a useful general term in
some circumstances, but the more explicit
terminology developed by the NRC (1983) is usually
preferred. The dose-response assessment defines the
relationship between the dose of an agent and the
probability of induction of a carcinogenic effect. This
component usually entails an extrapolation from the
generally high doses administered to experimental
animals or exposures noted in epidemiologic studies
to the exposure levels expected from human contact
with the agent in the environment; it also includes
considerations of the validity of these
extrapolations.
The exposure assessment identifies populations
exposed to the agent, describes their composition
and size, and presents the types, magnitudes,
frequencies, and durations of exposure to the agent.
[51 PR 339941
In risk characterization, the results of the
exposure assessment and the dose-response
assessment are combined to estimate quantitatively
the carcinogenic risk. As part of risk
characterization, a summary of the strengths and
weaknesses in the hazard identification, dose-
response, assessment, exposure assessment, and the
public health risk estimates are presented. Major
assumptions, scientific judgments, and, to the extent
possible, estimates of the uncertainties embodied in
the assessment are also presented, distinguishing
clearly between fact, assumption, and science policy.
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The National Research Council (NRC, 1983)
pointed out that there are many questions
encountered in the risk assessment process that are
unanswerable given current scientific knowledge.
To bridge the uncertainty that exists in these areas
where there is no scientific consensus, inferences
must be made to ensure that progress continues in
the assessment process. The OSTP (1985) reaffirmed
this position, and generally left to the regulatory
agencies the job of articulating these inferences.
Accordingly, the Guidelines incorporate judgmental
positions (science policies) based on evaluation of the
presently available information and on the
regulatory mission of the Agency. The Guidelines
are consistent with the principles developed by the
OSTP (1985), although in many instances are
necessarily more specific.
//. Hazard Identification
A. Overview
The qualitative assessment or hazard
identification part of risk assessment contains a
review of the relevant biological and chemical
information bearing on whether or not an agent may
pose a carcinogenic hazard. Since chemical agents
seldom occur in a pure state and are often
transformed in the body, the review should include
available information on contaminants, degradation
products, and metabolites.
Studies are evaluated according to sound
biological and statistical considerations and
procedures. These have been described in several
publications (Inleragency Regulatory Liaison
Group, 1979; OSTP, 1985; Peto et ah, 1980; Mantel,
1980; Mantel and Haenszel, 1959; Interdisciplinary
Panel on Carcinogenicity, 1984; National Center for
Toxicological Research, 1981; National Toxicology
Program, 1984; U.S. EPA, 1983a, 1983b, I983c;
Haseman, 1984). Results and conclusions
concerning the agent, derived from different types of
information, whether indicating positive or negative
responses, are melded together into a weight-of-
evidence determination. The strength of the
evidence supporting a potential human
carcinogenicity judgment is developed in a weight-
of-evidence stratification scheme.
B. Elements of Hazard Identification
Hazard identification should include a review of
.the following information to the extent that it is
available.
1. Physical-Chemical Properties and Routes and
Patterns of Exposure. Parameters relevant to
carcinogenesis, including physical state, physical-
chemical properties, and exposure pathways in the
environment should be described where possible.
2. Structure-Activity Relationships. This section
should summarize relevant structure-activity
correlations that support or argue against the
prediction of potential carcinogenicity.
3. Metabolic and Pharmacokinetic Properties.
This section should summarize relevant metabolic
information. Information such as whether the agent
is direct-acting or requires conversion to a reactive
carcinogenic (e.g., an electrophilic) species,
metabolic pathways for such conversions,
macromotecutar interactions, and fate (e.g.,
transport, storage, and excretion), as well as species
differences, should be discussed and critically
evaluated. Pharmacokinetic properties determine
the biologically effective dose and may be relevant to
hazard identification and other components of risk
assessment.
4. Toxicologic Effects. Toxicologic effects other
than carcinogenicity (e.g., suppression of the
immune system, endocrine disturbances, organ
damage) that are relevant to the evaluation of
carcinogenicity should be summarized. Interactions
with other chemicals or agents and with lifestyle
factors should be discussed. Prechronic and chronic
toxicity evaluations, as well as other test results,
may yield information on target organ effects,
pathophysiological reactions, and preneoplastic
lesions that bear on the evaluation of
carcinogenicity. Dose-response and time-to-response
analyses of these reactions may also be helpful.
5. Short-Term Tests. Tests for point mutations,
numerical and structural chromosome aberrations,
DNA damage/repair, and in vitro transformation
provide supportive evidence of carcinogenicity and
may give information on potential carcinogenic
mechanisms. A range of tests from each of the above
end points helps to characterize an agent's response
spectrum.
Short-term in vivo and in vitro tests that can
give indication of initiation and promotion activity
may also provide supportive evidence for
carcinogenicity. Lack of positive results in short-
term tests for genetic toxicity does not provide a
basis for discounting positive results in long-term
animal studies.
6. Long-Term Animal Studies. Criteria for the
technical adequacy of animal carcinogenicity
studies have been published (e.g., U.S. Food and
Drug Administration, 1982; Interagency Regulatory
Liaison Group, 1979; National Toxicology Program,
1984; OSTP, 1985; U.S. EPA, 1983a, 1983b, 1983c;
Feron et al., 1980; Mantel, 1980) and should be used
to judge the acceptability of individual studies.
Transplacental and multigenerational
carcinogenesis studies, in addition to more
conventional long-term animal studies, can yield
useful information about the carcinogenicity of
agents.
It is recognized that chemicals that induce
benign tumors frequently also induce malignant
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tumors, and that benign tumors often progress to
malignant tumors (Interdisciplinary Panel on
Carcinogenicity, 1984). The incidence of benign and
malignant tumors will be combined when
scientifically defensible (OSTP, 1985; Principle 8).
For example, the Agency will, in general, consider
the combination of benign and malignant tumors to
be scientifically defensible unless the benign tumors
are not considered to have the potential to progress
to the associated malignancies of the same
histogenic origin. If an increased incidence of benign
tumors is observed in the absence of malignant
tumors, in most cases the evidence will be
considered as limited evidence of carcinogenicity.
The weight of evidence that an agent is
potentially carcinogenic for humans increases (1)
with the increase in number of tissue sites affected
by the agent; (2) with the increase in number of
animal species, strains, sexes, and number of
experiments and doses showing a carcinogenic
response; (3) with the occurrence of clear-cut dose-
response relationships as well as a high level of
statistical significance of the increased tumor
incidence in treated compared to control groups; (4)
when there is a dose-related shortening of the time-
to-tumor occurrence or time to death with tumor;
and (5) when there is a dose-related increase in the
proportion of tumors that are malignant.
Long-term animal studies at or near the
maximum tolerated dose level (MTD) are used to
ensure an adequate power for the detection of
carcinogenic
[51 PR 33995]
activity (NTP,
1984; IARC, 1982). Negative long-term animal
studies at exposure levels above the MTD may not be
acceptable if animal survival is so impaired that the
sensitivity of the study is significantly reduced
below that of a conventional chronic animal study at
the MTD. The OSTP (1985; Principle 4) has stated
that,
The carcinogenic effects of agents may be influenced by non-
physiological responses (such as extensive organ damage, radical
disruption of hormonal function, saturation of metabolic-
pathways, formation of stones in the urinary tract, saturation of
DNA repair with a functional loss of the system) induced in the
model systems. Testing regimes inducing these responses should
be evaluated for their relevance to the human response to an
agent and evidence from such a study, whether positive or
negative, must be carefully reviewed.
Positive studies at levels above the MTD should be
carefully reviewed to ensure that the responses are
not due to factors which do not operate at exposure
levels below the MTD. Evidence indicating that high
exposures alter tumor responses by indirect
mechanisms that may be unrelated to effects at
lower exposures should be dealt with on an
individual basis. As noted by the OSTP (1985),
"Normal metabolic activation of carcinogens may
possibly also be altered and carcinogenic potential
reduced as a consequence [of high-dose testing]."
Carcinogenic responses under conditions of the
experiment should be reviewed carefully as they
relate to the relevance of the evidence to human
carcinogenic risks (e.g., the occurrence of bladder
tumors in the presence of bladder stones and
implantation site sarcomas). Interpretation of
animal studies is aided by the review of target organ
toxicity and other effects (e.g., changes in the
immune and endocrine systems) that may be noted
in prechronic or other toxicological studies. Time
and dose-related changes in the incidence of
preneoplastic lesions may also be helpful in
interpreting animal studies.
Agents that are positive in long-term animal
experiments and also show evidence of promoting or
cocarcinogenic activity in specialized tests should be
considered as complete carcinogens unless there is
evidence to the contrary because it is, at present,
difficult to determine whether an agent is only a
promoting or cocarcinogenic agent. Agents that
show positive results in special tests for initiation,
promotion, or cocarcinogenicity and no indication of
tumor response in well-conducted and well-designed
long-term animal studies should be dealt with on an
individual basis.
To evaluate carcinogenicity, the primary
comparison is tumor response in dosed animals as
compared with that in contemporary matched
control animals. Historical control data are often
valuable, however, and could be used along with
concurrent control data in the evaluation of
carcinogenic responses (Haseman et al., 1984). For
the evaluation of rare tumors, even small tumor
responses may be significant compared to historical
data. The review of tumor data at sites with high
spontaneous background requires special
consideration (OSTP, 1985; Principle 9). For
instance, a response that is significant with respect
to the experimental control group may become
questionable if the historical control data indicate
that the experimental control group had an
unusually low background incidence (NTP, 1984).
For a number of reasons, there are widely
diverging scientific views (OSTP, 1985; Ward et al.,
1979a, b; Tomatis, 1977; Nutrition Foundation,
1983) about the validity of mouse liver tumors as an
indication of potential carcinogenicity in humans
when such tumors occur in strains with high
spontaneous background incidence and when they
constitute the only tumor response to an agent.
These Guidelines take the position that when the
only tumor response is in the mouse liver and when
other conditions for a classification of "sufficient"
evidence in animal studies are met (e.g., replicate
studies, malignancy; see section IV), the data should
be considered as "sufficient" evidence of
carcinogenicity. It is understood that this
classification could be changed on a case-by-case
basis to "limited," if warranted, when factors such as
the following, are observed: an increased incidence
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J .
of tumors only in the highest dose group and/or only
at the end of the study; no substantial dose-related
increase in the proportion of tumors that are
malignant; the occurrence of tumors that are
predominantly benign; no dose-related shortening of
the time to the appearance of tumors; negative or
inconclusive results from a spectrum of short-term
tests for mutagenic activity; the occurrence of excess
tumors only in a single sex.
Data from all long-term animal studies are to be
considered in the evaluation of carcinogenicity. A
positive carcinogenic response in one
species/strain/sex is not generally negated by
negative results in other species/strain/sex.
Replicate negative studies that are essentially
identical in all other respects to a positive study may
indicate that the positive results are spurious.
Evidence for carcinogenic action should be based
on the observation of statistically significant tumor
responses in specific organs or tissues. Appropriate
statistical analysis should be performed on data
from long-term studies to help determine whether
the effects are treatment-related or possibly due to
chance. These should at least include a statistical
test for trend, including appropriate correction for
differences in survival. The weight to be given to the
level of statistical significance (the p-value) and to
other available pieces of information is a matter of
overall scientific judgment. A statistically
significant excess of tumors of all types in the
aggregate, in the absence of a statistically
significant increase of any individual tumor type,
should be regarded as minimal evidence of
carcinogenic action unless there are persuasive
reasons to the contrary.
1, Human Studies, Epidemiologic studies
provide unique information about the response of
humans who have been exposed to suspect
carcinogens. Descriptive epidemiologic studies are
useful in generating hypotheses and providing
supporting data, but can rarely be used to make a
causal inference. Analytical epidemiologic studies of
the case-control or cohort variety, on the other hand,
are especially useful in assessing risks to exposed
humans.
Criteria for the adequacy of epidemiologic
studies are well recognized. They include factors
such as the proper selection and characterization of
exposed and control groups, the adequacy of
duration and quality of follow-up, the proper
identification and characterization of confounding
factors and bias, the appropriate consideration of
latency effects, the valid ascertainment of the causes
of morbidity and death, and the ability to detect
specific effects. Where it can be calculated, the
statistical power to detect an appropriate outcome
should be included in the assessment.
The strength of the epidemiologic evidence for
carcinogenicity depends, among other things, on the
type of analysis and on the magnitude and
specificity of the response. The weight of evidence
increases rapidly with the number of adequate
studies that show comparable results on populations
exposed to the same agent under different
conditions.
It should be recognized that epidemiologic
studies are inherently capable of detecting only
comparatively large increases in the relative risk of
T51FR 33996]
cancer. Negative
results from such studies cannot prove the absence
of carcinogenic action; however, negative results
from a well-designed and well-conducted
epidemiologic study that contains usable exposure
data can serve to define upper limits of risk; these
are useful if animal evidence indicates that the
agent is potentially carcinogenic in humans.
C. Weight of Evidence
Evidence of possible carcinogenicity in humans
comes primarily from two sources: long-term animal
tests and epidemiologic investigations. Results from
these studies are supplemented with available
information from short-term tests, pharmacokinetic
studies, comparative metabolism studies, structure-
activity relationships, and other relevant toxicologic
studies. The question of how likely an agent is to be
a human carcinogen should be answered in the
framework of a weight-of-evidence judgment.
Judgments about the weight of evidence involve
considerations of the quality and adequacy of the
data and the kinds and consistency of responses
induced by a suspect carcinogen. There are three
major steps to charactering the weight of evidence
for carcinogenicity in humans: (1) characterization
of the evidence from human studies and from animal
studies individually, (2) combination of the
characterizations of these two types of data into an
indication of the overall weight of evidence for
human carcinogenicity, and (3) evaluation of all
supporting information to determine if the overall
weight of evidence should be modified.
EPA has developed a system for stratifying the
weight of evidence (see section IV). This
classification is not meant to be applied rigidly or
mechanically. At various points in the above
discussion, EPA has emphasized the need for an
overall, balanced judgment of the totality of the
available evidence. Particularly for well-studied
substances, the scientific data base will have a
complexity that cannot be captured by any
classification scheme. Therefore, the hazard
identification section should include a narrative
summary of the strengths and weaknesses of the
evidence as well as its categorization in the EPA
scheme.
The EPA classification system is, in general, an
adaptation of the International Agency for Research
on Cancer (IARC, 1982) approach for classifying the
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. 1
weight of evidence for human data and animal data.
The EPA classification system for the
characterization of the overall weight of evidence for
carcinogenicity (animal, human, and other
supportive data) includes: Group A ~ Carcinogenic
to Humans; Group B Probably Carcinogenic to
Humans; Group C Possibly Carcinogenic to
Humans; Group D -- Not Classifiable as to Human
Carcinogenicity; and Group E Evidence of Non-
Carcinogen icity for Humans.
The following modifications of the IARC
approach have been made for classifying human and
animal studies.
For human studies:
(1) The observation of a statistically significant
association between an agent and life-threatening
benign tumors in humans is included in the
evaluation of risks to humans.
(2) A "no data available" classification is added.
(3) A "no evidence of carcinogenicity"
classification is added. This classificaton indicates
that no association was found between exposure and
increased risk of cancer in well-conducted, well-
designed, independent analytical epidemiologic
studies.
For animal studies:
(1) An increased incidence of combined benign
and malignant tumors will be considered to provide
sufficient evidence of carcinogenicity if the other
criteria defining the "sufficient" classification of
evidence are met (e.g., replicate studies,
malignancy; see section IV). Benign and malignant
tumors will be combined when scientifically
defensible.
(2) An increased incidence of benign tumors
alone generally constitutes "limited" evidence of
carcinogenicity.
(3) An increased incidence of neoplasms that
occur with high spontaneous background incidence
(e.g., mouse liver tumors and rat pituitary tumors in
certain strains) generally constitutes "sufficient"
evidence of carcinogenicity, but may be changed to
"limited" when warranted by the specific
information available on the agent.
(4) A "no data available" classification has been
added.
(5) A "no evidence of carcinogenicity"
classification is also added. This operational
classification would include substances for which
there is no increased incidence of neoplasms in at
least two well-designed and well-conducted animal
studies of adequate power and dose in different
species.
D. Guidance for Dose-Response Assessment
The qualitative evidence for carcinogenesis
should be discussed for purposes of guiding the dose-
response assessment. The guidance should be given
in terms of the appropriateness and limitations of
specific studies as well as pharmacokinetic
considerations that should be factored into the dose-
response assessment. The appropriate method of
extrapolation should be factored in when the
experimental route of exposure differs from that
occurring in humans.
Agents that are judged to be in the EPA weight-
of-evidence stratification Groups A and B would be
regarded as suitable for quantitative risk
assessments. Agents that are judged to be in Group
C will generally be regarded as suitable for
quantitative risk assessment, but judgments in this
regard may be made on a case-by-case basis. Agents
that are judged to be in Groups D and E would not
have quantitative risk assessments.
E. Summary and Conclusion
The summary should present all of the key
findings in all of the sections of the qualitative
assessment and the interpretive rationale that
forms the basis for the conclusion. Assumptions,
uncertainties in the evidence, and other factors that
may affect the relevance of the evidence to humans
should be discussed. The conclusion should present
both the weight-of-evidence ranking and a
description that brings out the more subtle aspects of
the evidence that may not be evident from the
ranking alone.
///. Dose-Response Assessment, Exposure
Assessment, and Risk Characterization
After data concerning the carcinogenic
properties of a substance have been collected,
evaluated, and categorized, it is frequently desirable
to estimate the likely range of excess cancer risk
associated with given levels and conditions of
human exposure. The first step of the analysis
needed to make such estimations is the development
of the likely relationship between dose and response
(cancer incidence) in the region of human exposure.
This information on dose-response relationships is
coupled with information on the nature and
magnitude of human exposure to yield an estimate
of human risk. The risk-characterization step also
includes an interpretation of these estimates in light
of the biological, statistical, and exposure
assumptions and uncertainties that have arisen
throughout the process of assessing risk.
The elements of dose-response assessment are
described in section III.A. Guidance on human.
exposure assessment is provided in another EPA
[51 FR 339971
document (U.S.
EPA, 1986); however, section III.B. of these
Guidelines includes a brief description of the specific
type of exposure information that is useful for
carcinogen risk assessment. Finally, in section Ill.C.
on risk characterization, there is a description of the
manner in which risk estimates should be presented
so as to be most informative.
It should be emphasized that calculation of
quantitative estimates of cancer risk does not
1-7
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require that an agent be carcinogenic in humans.
The likelihood that an agent is a human carcinogen
is a function of the weight of evidence, as this has
been described in the hazard identification section of
these Guidelines. It is nevertheless important to
present quantitative estimates, appropriately
qualified and interpreted, in those circumstances in
which there is a reasonable possibility, based on
human and animal data, that the agent is
carcinogenic in humans.
It should be emphasized in every quantitative
risk estimation that the results are uncertain.
Uncertainties due to experimental and
epidemiologic variability as well as uncertainty in
the exposure assessment can be important. There
are major uncertainties in extrapolating both from
animals to humans and from high to low doses.
There are important species differences in uptake,
metabolism, and organ distribution of carcinogens,
as well as species and strain differences in target-
site susceptibility. Human populations are variable
with respect to genetic constitution, diet,
occupational and home environment, activity
patterns, and other cultural factors. Risk estimates
should be presented together with the associated
hazard assessment (section III.C.3.) to ensure that
there is an appreciation of the weight of evidence for
carcinogenicity that underlies the quantitative risk
estimates.
A. Dose-Response Assessment
1. Selection of Data. As indicated in section H.D.,
guidance needs to be given by the individuals doing
the qualitative assessment (lexicologists,
pathologists, pharmacologists, etc.) to those doing
the quantitative assessment as to the appropriate
data to be used in the dose-response assessment.
This is determined by the quality of the data, its
relevance to human modes of exposure, and other
technical details.
If available, estimates based on adequate human
epidemiologic data are preferred over estimates
based on animal data. If adequate exposure data
exist in a well-designed and well-conducted negative
epidemiologic study, it may be possible to obtain an
upper-bound estimate of risk from that study.
Animal-based estimates, if available, also should be
presented.
In the absence of appropriate human studies,
data from a species that responds most like humans
should be used, if information to this effect exists.
Where, for a given agent, several studies are
available, which may involve different animal
species, strains, and sexes at several doses and by
different routes of exposure, the following approach
to selecting the data sets is used: (1) The tumor
incidence data are separated according to organ site
and tumor type. (2) All biologically and statistically
acceptable data sets are presented. (3) The range of
the risk estimates is presented with due regard to
biological relevance (particularly in the case of
animal studies) and appropriateness of route of
exposure. (4) Because it is possible that human
sensitivity is as high as the most sensitive
responding animal species, in the absence of
evidence to the contrary, the biologically acceptable
data set from long-term animal studies showing the
greatest sensitivity should generally be given the
greatest emphasis, again with due regard to
biological and statistical considerations.
When the exposure route in the species from
which the dose-response information is obtained
differs from the route occurring in environmental
exposures, the considerations used in making the
route-to-route extrapolation must be carefully
described. All assumptions should be presented
along with a discussion of the uncertainties in the
extrapolation. Whatever procedure is adopted in a
given case, it must be consistent with the existing
metabolic and pharmacokinetic information on the
chemical (e.g., absorption efficiency via the gut and
lung, target organ doses, and changes in placental
transport throughout gestation for transplacental
carcinogens).
Where two or more significantly elevated tumor
sites or types are observed in the same study,
extrapolations may be conducted on selected sites or
types. These selections will be made on biological
grounds. To obtain a total estimate of carcinogenic
risk, animals with one or more tumor sites or types
showing significantly elevated tumor incidence
should be pooled and used for extrapolation. The
pooled estimates will generally be used in preference
to risk estimates based on single sites or types.
Quantitative risk extrapolations will generally not
be done on the basis of totals that include tumor sites
without statistically significant elevations.
Benign tumors should generally be combined
with malignant tumors for risk estimates unless the
benign tumors are not considered to have the
potential to progress to the associated malignancies
of the same histogenic origin. The contribution of
the benign tumors, however, to the total risk should
be indicated.
2. Choice of Mathematical Extrapolation Model.
Since risks at low exposure levels cannot be
measured directly either by animal experiments or
by epidemiologic studies, a number of mathematical
models have been developed to extrapolate from
high to low dose. Different extrapolation models,
however, may fit the observed data reasonably well
but may lead to large differences in the projected
risk at low doses.
As was pointed out by OSTP (1985; Principle
26),
No single mathematical procedure is recognized as the most
appropriate for low-dose extrapolation in carcinogenests. When
relevant biological evidence on mechanism of action exists
-------
employed should be consistent with the evidence. When data and
information are limited, however, and when much uncertainty
exists regarding the mechanism of carcinogenic action, models or
procedures which incorporate low-dose linearity are preferred
when compatible with the limited information.
At present, mechanisms of the carcinogenesis
process are largely unknown and data are generally
limited. If a carcinogenic agent acts by accelerating
the same carcinogenic process that leads to the
background occurrence of cancer, the added effect of
the carcinogen at low doses is expected to be
virtually linear (Crump et al., 1976).
The Agency will review each assessment as to
the evidence on carcinogenesis mechanisms and
other biological or statistical evidence that indicates
the suitability of a particular extrapolation model.
Goodness-of-fit to the experimental observations is
not an effective means of discriminating among
models (OSTP, 1985). A rationale will be included to
justify the use of the chosen model. In the absence of
adequate information to the contrary, the linearized
multistage procedure will be employed. Where
appropriate, the results of using various
extrapolation models may be useful for comparison
with the linearized multistage procedure. When
longitudinal data on tumor development are
available, time-to-tumor models may be used.
It should be emphasized that the linearized
multistage procedure leads to
[51 PR 33998]
a plausible upper
limit to the risk that is consistent with some
proposed mechanisms of carcinogenesis. Such an
estimate, however, does not necessarily give a
realistic prediction of the risk. The true value of the
risk is unknown, and may be as low as zero. The
range of risks, defined by the upper limit given by
the chosen model and the lower limit which may be
as low as zero, should be explicitly stated. An
established procedure does not yet exist for making
"most likely" or "best" estimates of risk within the
range of uncertainty defined by the upper and lower
limit estimates, if data and procedures become
available, the Agency will also provide "most likely"
or "best" estimates of risk. This will be most feasible
when human data are available and when exposures
are in the dose range of the data.
In certain cases, the linearized multistage
procedure cannot be used with the observed data as,
for example, when the data are nonmonotonic or
flatten out at high doses. In these cases, it may be
necessary to make adjustments to achieve low-dose
linearity.
When pharmacokinetic or metabolism data
are available, or when other substantial evidence on
the mechanistic aspects of the carcinogenesis
process exists, a low-dose extrapolation model other
than the linearized multistage procedure might be
considered more appropriate on biological grounds.
When a different model is chosen, the risk
assessment should clearly discuss the nature and
weight of evidence that led to the choice.
Considerable uncertainty will remain concerning
response at low doses; therefore, in most cases an
upper-limit risk estimate using the linearized
multistage procedure should also be presented.
3. Equivalent Exposure Units Among Species.
Low-dose risk estimates derived from laboratory
animal data extrapolated to humans are
complicated by a variety of factors that differ among
species and potentially affect the response to
carcinogens. Included among these factors are
differences between humans and experimental test
animals with respect to life span, body size, genetic
variability, population homogeneity, existence of
concurrent disease, pharmacokinetic effects such as
metabolism and excretion patterns, and the
exposure regimen.
The usual approach for making interspecies
comparisons has been to use standardized scaling
factors. Commonly employed standardized dosage
scales include mg per kg body weight per day, ppm
in the diet or water, mg per m2 body surface area per
day, and mg per kg body weight per lifetime. In the
absence of comparative toxicological, physiological,
metabolic, and pharmacokinetic data for a given
suspect carcinogen, the Agency takes the position
that the extrapolation on the basis of surface area is
considered to be appropriate because certain
pharmacological effects commonly scale according to
surface area (Dedrick, 1973; Freireich et al., 1966;
Pinkel, 1958).
B. Exposure Assessment
In order to obtain a quantitative estimate of the
risk, the results of the dose-response assessment
must be combined with an estimate of the exposures
to which the populations of interest are likely to be
subject. While the reader is referred to the
Guidelines for Estimating Exposures (U.S. EPA,
1986) for specific details, it is important to convey an
appreciation of the impact of the strengths and
weaknesses of exposure assessment on the overall
cancer risk assessment process.
At present there is no single approach to
exposure assessment that is appropriate for all
cases. On a case-by-case basis, appropriate methods
are selected to match the data on hand and the level
of sophistication required. The assumptions,
approximations, and uncertainties need to be clearly
stated because, in some instances, these will have a
major effect on the risk assessment.
In general, the magnitude, duration, and
frequency of exposure provide fundamental
information for estimating the concentration of the
carcinogen to which the organism is exposed. These
data are generated from monitoring information,
modeling results, and/or reasoned estimates. An
appropriate treatment of exposure should consider
1-9
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i .
the potential for exposure via ingestion, inhalation,
and dermal penetration from relevant sources of
exposures including multiple avenues of intake from
the same source.
Special problems arise when the human
exposure situation of concern suggests exposure
regimens, e.g., route and dosing schedule that are
substantially different from those used in the
relevant animal studies. Unless there is evidence to
the contrary in a particular case, the cumulative
dose received over a lifetime, expressed as average
daily exposure prorated over a lifetime, is
recommended as an appropriate measure of
exposure to a carcinogen. That is, the assumption is
made that a high dose of a carcinogen received over a
short period of time is equivalent to a corresponding
low dose spread over a lifetime. This approach
becomes more problematical as the exposures in
question become more intense but less frequent,
especially when there is evidence that the agent has
shown dose-rate effects.
An attempt should be made to assess the level of
uncertainty associated with the exposure
assessment which is to be used in a cancer risk
assessment. This measure of uncertainty should be
included in the risk characterization (section III.C.)
in order to provide the decision-maker with a clear
understanding of the impact of this uncertainty on
any final quantitative risk estimate. Subpopulations
with heightened susceptibility (either because of
exposure or predisposition) should, when possible, be
identified.
C. Risk Characterization
Risk characterization is composed of two parts.
One is a presentation of the numerical estimates of
risk; the other is a framework to help judge the
significance of the risk. Risk characterization
includes the exposure assessment and dose-response
assessment; these are used in the estimation of
carcinogenic risk. It may also consist of a unit-risk
estimate which can be combined elsewhere with the
exposure assessment for the purposes of estimating
cancer risk.
Hazard identification and dose-response
assessment are covered in sections II. and III.A., and
a detailed discussion of exposure assessment is
contained in EPA's Guidelines for Estimating
Exposures (U.S. EPA, 1986). This section deals with
the numerical risk estimates and the approach to
summarizing risk characterization.
1. Options for Numerical Risk Estimates.
Depending on the needs of the individual program
offices, numerical estimates can be presented in one
or more of the following three ways.
a. Unit Risk -- Under an assumption of low-dose
linearity, the unit cancer risk is the excess lifetime
risk due to a continuous constant lifetime exposure
of one unit of carcinogen concentration. Typical
exposure units include ppm or ppb in food or water,
mg/kg/day by ingestion, or ppm or ug/m3 in air.
b. Dose Corresponding to a Given Level of Risk
This approach can be useful, particularly when
using nonlinear extrapolation models where the
unit risk would differ at different dose levels.
c. Individual and Population Risks Risks may
be characterized either in terms of the excess
individual lifetime risks, the excess number of
cancers
[51 FR 33999]
produced per
year in the exposed population, or both.
Irrespective of the i.,,lions chosen, the degree
of precision and accuracy in the numerical risk
estimates currently do not permit more than one
significant figure to be presented.
2. Concurrent Exposure. In characterizing the
risk due to concurrent exposure to several
carcinogens, the risks are combined on the basis of
additivity unless there is specific information to the
contrary. Interactions of cocarcinogens, promoters,
and inititators with known carcinogens should be
considered on a case-by-case basis.
3. Summary of Risk Characterization,
Whichever method of presentation is chosen, it is
critical that the numerical estimates not be allowed
to stand alone, separated from the various
assumptions and uncertainties upon which they are
based. The risk characterization should contain a
discussion and interpretation of the numerical
estimates that affords the risk manager some
insight into the degree to which the quantitative
estimates are likely to reflect the true magnitude of
human risk, which generally cannot be known with
the degree of quantitative accuracy reflected in the
numerical estimates. The final risk estimate will be
generally rounded to one significant figure and will
be coupled with the EPA classification of the
qualitative weight of evidence. For example, a
lifetime individual risk of 2X10-* resulting from
exposure to a "probable human carcinogen" (Group
B2) should be designated as 2X10-4 [B2J . This
bracketed designation of the qualitative weight of
evidence should be included with all numerical risk
estimates (i.e., unit risks, which are risks at a
specified concentration or concentrations
corresponding to a given risk). Agency statements,
such as FEDERAL REGISTER notices, briefings,
and action memoranda, frequently include
numerical estimates of carcinogenic risk. It is
recommended that whenever these numerical
estimates are used, the qualitative weight-of-
evidence classification should also be included.
The section on risk characterization should
summarize the hazard identification, dose-response
assessment, exposure assessment, and the public
health risk estimates. Major assumptions, scientific
judgments, and, to the extent possible, estimates of
1-10
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the uncertainties embodied in the assessment are
presented.
IV. EPA Classification System for Categorizing
Weight of Evidence for Carcinogenicity from Human
and Animal Studies (Adapted from I ARC)
A. Assessment of Weight of Evidence for
Carcinogenicity from Studies in Humans
Evidence of Carcinogenicity from human studies
comes from three main sources:
1. Case reports of individual cancer patients who
were exposed to the agent(s).
2. Descriptive epidemiologic studies in which the
incidence of cancer in human populations was found
to vary in space or time with exposure to the
agent(s).
3. Analytical epidemiologic (case-control and
cohort) studies in which individual exposure to the
agent(s) was found to be associated with an
increased risk of cancer.
Three criteria must be met before a causal
association can be inferred between exposure and
cancer in humans:
1. There is no identified bias that could explain
the association.
2. The possibility of confounding has been
considered and ruled out as explaining the
association.
3. The association is unlikely to be due to
chance.
In general, although a single study may be
indicative of a cause-effect relationship, confidence
in inferring a causal association is increased when
several independent studies are concordant in
showing the association, when the association is
strong, when there is a dose-response relationship,
or when a reduction in exposure is followed by a
reduction in the incidence of cancer.
The weight of evidence for Carcinogenicity1 from
studies in humans is classified as:
1. Sufficient evidence of Carcinogenicity, which
indicates that there is a causal relationship between
the agent and human cancer.
2. Limited evidence of Carcinogenicity, which
indicates that a causal interpretation is credible, but
that alternative explanations, such as chance, bias,
or confounding, could not adequately be excluded.
1 For purposes of public health protection, agents
associated with life-threatening benign tumors in humans are
included in the evaluation.
2 An increased incidence of neoplasms that occur with high
spontaneous background incidence (e.g., mouse liver tumors
and rat pituitary tumors in certain strains) generally
constitutes "sufficient" evidence of Carcinogenicity, but may be
changed to "limited" when warranted by the specific
information available on the agent.
3 Benign and malignant tumors will be combined unless
the benign tumors are not considered to have the potential to
progress to the associated malignancies of the same histogenic
origin.
3. Inadequate evidence, which indicates that one
of two conditions prevailed: (a) there were few
pertinent data, or (b) the available studies, while
showing evidence of association, did not exclude
chance, bias, or confounding, and therefore a causal
interpretation is not credible.
4. No data, which indicates that data are not
available.
5. No evidence, which indicates that no
association was found between exposure and an
increased risk of cancer in well-designed and well-
conducted independent analytical epidemiologic
studies.
B. Assessment of Weight of Evidence for
Carcinogenicity from Studies in Experimental
Animals
These assessments are classified into five
groups:
1. Sufficient evidence2 of Carcinogenicity, which
indicates that there is an increased incidence of
malignant tumors or combined malignant and
benign tumors:3 (a) in multiple species or strains; or
(b) in multiple experiments (e.g., with different
routes of administration or using different dose
levels); or (c) to an unusual degree in a single
experiment with regard to high incidence, unusual
site or type of tumor, or early age at onset.
Additional evidence may be provided by data on
dose-response effects, as well as information from
short-term tests or on chemical structure.
2. Limited evidence of Carcinogenicity, which
means that the data suggest a carcinogenic effect
but are limited because: (a) the studies involve a
single species, strain, or experiment and do not meet
criteria for sufficient evidence (see section IV. B.l.c);
(b) the experiments are restricted by inadequate
dosage levels, inadequate duration of exposure to the
agent, inadequate period of follow-up, poor survival,
too few animals, or inadequate reporting; or (c) an
increase in the incidence of benign tumors only.
3. Inadequate evidence, which indicates that
because of major qualitative or quantitative
limitations, the studies cannot be interpreted as
showing either the presence or absence of a
carcinogenic effect.
4. No data, which indicates that data are not
available.
5. No evidence, which indicates that there is no
increased incidence of neoplasms in at least two
well-designed
[51 PR 34000]
and well-
conducted animal studies in different species.
The classifications "sufficient evidence" and
"limited evidence" refer only to the weight of the
experimental evidence that these agents are
carcinogenic and not to the potency of their
carcinogenic action.
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C, Categorization of Overall Weight of Evidence for
Human Carcinogenicity
The overall scheme for categorization of the
weight of evidence of carcinogenicity of a chemical
for humans uses a three-step process. (1) The weight
of evidence in human studies or animal studies is
summarized; (2) these lines of information are
combined to yield a tentative assignment to a
category (see Table 1); and (3) all relevant
supportive information is evaluated to see if the
designation of the overall weight of evidence needs
to be modified. Relevant factors to be included along
with the tumor information from human and animal
studies include structure-activity relationships;
short-term test findings; results of appropriate
physiological, biochemical, and lexicological
observations; and comparative metabolism and
pharmacokinetic studies. The nature of these
findings may cause one to adjust the overall
categorization of the weight of evidence.
The agents are categorized into five groups as
follows:
Group A Human Carcinogen
This group is used only when there is sufficient
evidence from epidemiologic studies to support a
causal association between exposure to the agents
and cancer.
Group B Probable Human Carcinogen
This group includes agents for which the weight
of evidence of human carcinogenicity based on
epidemiologic studies is "limited" and also includes
agents for which the weight of evidence of
carcinogenicity based on animal studies is
"sufficient." The group is divided into two
subgroups. Usually, Group Bl is reserved for agents
for which there is limited evidence of carcinogenicity
from epidemiologic studies. It is reasonable, for
practical purposes, to regard an agent for which
there is "sufficient" evidence of carcinogenicity in
animals as if it presented a carcinogenic risk to
humans. Therefore, agents for which there is
"sufficient" evidence from animal studies and for
which there is "inadequate evidence" or "no data"
from epidemiologic studies would usually be
categorized under Group B2.
Group C Possible Human Carcinogen
This group is used for agents with limited
evidence of carcinogenicity in animals in the
absence of human data. It includes a wide variety of
evidence, e.g., (a) a malignant tumor response in a
single well-conducted experiment that does not meet
conditions for sufficient evidence, (b) tumor
responses of marginal statistical significance in
studies having inadequate design or reporting, (c)
benign but not malignant tumors with an agent
showing no response in a variety of short-term tests
for mutagenicity, and (d) responses of marginal
statistical significance in a tissue known to have a
high or variable background rate.
Group D -- Not Classifiable as to Human
Carcinogenicity
This group is generally used for agents with
inadequate human and animal evidence of
carcinogenicity or for which no data are available.
Group E Evidence of Non-Carcinogenicity for
Humans
This group is used for agents that show no
evidence for carcinogenicity in at least two adequate
animal tests in different species or in both adequate
epidemiologic and animal studies.
The designation of an agent as being in Group E
is based on the available evidence and should not be
interpreted as a definitive conclusion that the agent
will not be a carcinogen under any circumstances.
V. References
Albert, R.E.,Train, R.E., and Anderson, E. 1977. Rationale
developed by the Environmental Protection Agency for the
assessment'of carcinogenic risks. J. Natl. Cancer Inst.
58:1537-1541.
Crump, K.S., Hoel, D.G., Langley, C.H., Peto R. 1976.
Fundamental carcinogenic processes and their implications
for low dose risk assessment. Cancer Res. 36:2973-2979.
Dedrick, R.L. 1973. Animal Scale Up. J. Pharmacokinet.
Biopharm. 1:435-461.
Feron, V.J., Grice, H.C., Griesemer, R., Peto R., Agthe, C., Althoff,
J., Arnold, D.L., Blumenthal, H., Cabral, J.R.P., Delia Porta,
G., Ito, N., Kimmerle, G., Kroes, R., Mohr, U., Napalkov,
N.P., Odashima, S., Page, N.P., Schramm, T., Steinhoff, D.,
Sugar, J.,Tomatis, L.Uehleke, H., and Vouk, V. 1980. Basic
requirements for long-term assays for carcinogenicity. In:
Long-term and short-term screening assays for carcinogens:
a critical appraisal. I ARC Monographs, Supplement 2. Lyon,
France: International Agency for Research on Cancer, pp 21-
83.
Freireich, E.J., Gehan, E.A., Rail, D.P., Schmidt, L.H., and
Skipper, H.E. 1966. Quantitative comparison of toxicity of
anticancer agents in mouse, rat, hamster, dog, monkey and
man. Cancer Chemother. Rep. 50:219-244.
Haseman, J.K. 1984. Statistical issues in the design, analysis and
interpretation of animal carcinogenicity studies. Environ.
Hea Ith Perspect. 58:385-392.
Haseman, J.K., Huff, J., andBoormnn,G.A. 1984. Use of
historical control data in carcinogenicity studies in rodents,
Toxicol. Pathol. 12:126-135.
Interagency Regulatory Liaison Group (IRLG), 1979. Scientific
basis for identification of potential carcinogens and
estimation of risks. J. Natl. Cancer Inst. 63:245-267.
Interdisciplinary Panel on Carcinogenicity. 1984. Criteria for
evidence of chemical carcinogenicity. Science 225:682-687.
International Agency for Research on Cancer (IAUC). 1982. IARC
Monographs on the
[51 PR 34001)
Evaluation of the
Carcinogenic Risk of Chemicals to Humans, Supplement 4. Lyon,
France: International Agency for Research on Cancer.
Mantel, N. 1980. Assessing laboratory evidence for neoplaslic
activity.Biometrics36:381-399.
Mantel, N., and Haenszel, W. 1959. Statistical aspects of the
analysis of data from retrospective studies of disease. J, Null.
Cancer Inst, 22:719-748.
National Center for Toxicological Research (NCTR). 1981.
Guidelines for statistical tests for carcinogenicity in chronic
bioassays. NCTR Biometry Technical Report 81-001.
Available from: National Center for Toxicological Research.
1-12
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TABLE 1.-ILLUSTRATIVE CATEGORIZATION OF EVIDENCE BASED ON ANIMAL AND HUMAN DATAI
Human evidence
Sufficient
Limited
Inadequate
No data
No evidence
Animal evidence
Sufficient
A
81
B2
B2
B2
Limited
A
B1
C
C
C
Inadequate
A
81
D
D
D
No data
A
B1
D
D
D
No evidence
A
B1
D
E
E
1 The above assignments are presented for illustrative purposes. There may be nuances in the classification of both
animal and human data indicating that different categorizations than those given in the table should be assigned.
Furthermore, these assignments are tentative and may be modified by ancillary evidence. In this regard all relevant
information should be evaluated to determine if the designation of the overall weight of evidence needs to be modified.
Relevant factors to be included along with the tumor data from human and animal studies include structure-activity
relationships, short-term test findings, results of appropriate physiological, biochemical, and toxicological observations, and
comparative metabolism and pharmacokinetic studies. The nature of these findings may cause an adjustment of the overall
categorization of the weight of evidence.
National Research Council (NRC). 1983. Risk assessment in the
Federal government: managing the process. Washington,
D.C.: National Academy Press.
National Toxicology Program. 1984. Report of the Ad Hoc Panel
on Chemical Carcinogenesis Testing and Evaluation of the
National Toxicology Program, Board of Scientific
Counselors. Available from: U.S. Government Printing
Office, Washington, D.C. 1984-421 -132:4726.
Nutrition Foundation. 1983. The relevance of mouse liver
hepatoma to human carcinogenic risk: a report of the
International Expert Advisory Committee to the Nutrition
Foundation. Available from: Nutrition Foundation. ISBN 0-
935368-37-x.
Office of Science and Technology Policy (OSTP). 1985. Chemical
carcinogens: review of the science and its associated
principles. Federal Register 50:10372-10442.
Peto, R., Pike, M., Day, N-, Gray, R., Lee, P., Parish, S., Peto, J.,
Richard, S., and Wahrendorf, J. 1980. Guidelines for simple,
sensitive, significant tests for carcinogenic effects in long-
term animal experiments. In: Monographs on the long-term
and short-term screening assays for carcinogens: a critical
appraisal. IARC Monographs, Supplement 2. Lyon, France:
International Agency for Research on Cancer, pp. 311 -426.
Pinkel, D. 1958. The use of body surface area as a criterion of drug
dosage in cancer chemotherapy. Cancer Res. 18:853-856.
Tomatis, L. 1977. The value of long-term testing for the
implementation of primary prevention. In: Origins of human
cancer. Hiatt, II.H., Watson, J.D., and Winstein, J.A., eds.
Cold Spring Harbor Laboratory, pp. 1339-1357.
U.S. Environmental Protection Agency (U.S. EPA). 1976. Interim
procedures and guidelines for health risk and economic
impact assessments of suspected carcinogens. Federal
Register 41:21402-21405.
U.S. Environmental Protection Agency (U.S. EPA). 1980. Water
quality criteria documents; availability. Federal Register
45:79318-79379.
U.S. Environmental Protection Agency (U.S. EPA). 1983a. Good
laboratory practices standards - toxicology testing. Federal
Register 48:53922.
U.S. Environmental Protection Agency (U.S. EPA). 19831).
Hazard evaluations: humans and domestic animals.
Subdivision F. Available from: NTIS, Springfield, VA. PB 83-
153916.
U.S. Environmental Protection Agency (U.S. EPA). 1983c. Health
effects test guidelines. Available from: NTIS, Springfield,
VA. PB 83-232984.
U.S. Environmental Protection Agency (U.S. EPA). 1986,Sept.
24.GuideIines for estimating exposures. Federal Register 51
(185): 34042-34054
U.S. Food and Drug Administration (U.S. FDA). 1982.
Toxicological principles for the safety assessment of direct
food additives and color additives used in food. Available
from: Bureau of Foods, U.S. Food and Drug Administration.
Ward, J.M., Griesemer, R.A., and Weisburger, E K. 1979a. The
mouse liver tumor as an eadpoint in carcinogenesis tests.
Toxicol. Appl. Pharmacol. 51:389-397.
Ward, J.M., Goodman, D.G., Squire, R.A. Chu, K.C., and Linhart,
M.S. 1979b. Neoplastic and nonneoplastic lesions in aging
(CsvBL/eNXCall/HeNlF! (BeCsF^ mice. J. Natl. Cancer
Inst. 63:849-854.
Part B: Response to Public and Science
Advisory Board Comments
/. Introduction
This section summarizes the major issues raised
during both the public comment period on the
Proposed Guidelines for Carcinogen Risk
Assessment published on November 23, 1984 (49 FR
46294), and also during the April 22-23, 1985,
meeting of the Carcinogen Risk Assessment
Guidelines Panel of the Science Advisory Board
(SAB).
In order to respond to these issues the Agency
modified the proposed guidelines in two stages.
First, changes resulting from consideration of the
public comments were made in a draft sent to the
SAB review panel prior to their April meeting.
Secondly, the guidelines were further modified in
response to the panel's recommendations.
The Agency received 62 sets of comments during
the public comment period, including 28 from
corporations, 9 from professional or trade
associations, and 4 from academic institutions. In
general, the comments were favorable. The
commentors welcomed the update of the 1976
guidelines and felt that the proposed guidelines of
,
1-13
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1985 reflected some of the progress that has occurred
in understanding the mechanisms of carcinogenesis.
Many commentors, however, felt that additional
changes were warranted.
The SAB concluded that the guidelines are
"reasonably complete in their conceptual framework
and are sound in their overall interpretation of the
scientific issues" (Report by the SAB
Carcinogenicity Guidelines Review Group, June 19,
1985). The SAB suggested various editorial changes
and raised some issues regarding the content of the
proposed guidelines, which are discussed below.
Based on these recommendations, the Agency has
modified the draft guidelines,
//. Office of Science and Technology Policy Report on
Chemical Carcinogens
Many commentors requested that the final
guidelines not be issued until after publication of the
report of the Office of Technology and Science Policy
(OSTP) on chemical carcinogens. They further
requested that this report be incorporated into the
final Guidelines for Carcinogen Risk Assessment.
The final OSTP report was published in 1985 (50
FR 10372). In its deliberations, the Agency reviewed
the final OSTP report and feels that the Agency's
guidelines are consistent with the principles
established by the OSTP. In its review, the SAB
agreed that the Agency guidelines are generally
consistent with the OSTP report. To emphasize this
consistency, the OSTP principles have been
incorporated into the guidelines when controversial
issues are discussed.
///. Inference Guidelines
Many commentors felt that the proposed
guidelines did not provide a sufficient distinction
between scientific fact and policy decisions. Others
felt that EPA should not attempt to propose firm
guidelines in the absence of scientific consensus. The
SAB report also indicated the need to "distinguish
recommendations based on scientific evidence from
those based on science policy decisions."
The Agency agrees with the recommendation
that policy, judgmental, or inferential decisions
should be clearly identified. In its revision of the
proposed guidelines, the Agency has included
phrases (e.g., "the Agency takes the position that")
to more clearly distinguish policy decisions.
The Agency also recognizes the need to establish
procedures for action on important issues in the
absence of complete scientific knowledge or
consensus. This need was acknowledged in'both the
National Academy of Sciences book entitled Risk
Management in the Federal Government: Managing
the Process and the OSTP report on chemical
carcinogens. As the NAS report states, "Risk
assessment is an analytic process that is firmly
based on scientific considerations, but it also
requires judgments to be made when the available
information is incomplete. These judgments
inevitably draw on both scientific and policy
considerations."
151FR 34002]
The judgments of the Agency have been based on
current available scientific information and on the
combined experience of Agency experts. These
judgments, and the resulting guidance, rely on
inference; however, the positions taken in these
inference guidelines are felt to be reasonable and
scientifically defensible. While all of the guidance is,
to some degree, based on inference, the guidelines
have attempted to distinguish those issues that
depended more oh judgment. In these cases, the
Agency has stated a position but has also retained
flexibility to accommodate new data or specific
circumstances that demonstrate that the proposed
position is inaccurate. The Agency recognizes that
scientific opinion will be divided on these issues.
Knowledge about carcinogens and
carcinogenesis is progressing at a rapid rate. While
these guidelines are considered a best effort at the
present time, the Agency has attempted to
incorporate flexibility into the current guidelines
and also recommends that the guidelines be revised
as often as warranted by advances in the field.
IV. Evaluation of Benign Tumors
Several commentors discussed the appropriate
interpretation of an increased incidence of benign
tumors alone or with an increased incidence of
malignant tumors as part of the evaluation of the
carcinogenicity of an agent. Some comments were
supportive of the position in the proposed guidelines,
i.e., under certain circumstances, the incidence of
benign and malignant tumors would be combined,
and an increased incidence of benign tumors alone
would be considered an indication, albeit limited, of
carcinogenic potential. Other commentors raised
concerns about the criteria that would be used to
decide which tumors should be combined. Only a few
commentors felt that benign tumors should never be
considered in evaluating carcinogenic potential.
The Agency believes that current information
supports the use of benign tumors. The guidelines
have been modified to incorporate the language of
the OSTP report, i.e., benign tumors will be
combined with malignant tumors when
scientifically defensible. This position allows
flexibility in evaluating the data base for each
agent. The guidelines have also been modified to
indicate that, whenever benign and malignant
tumors have been combined, and the agent is
considered a candidate for quantitative risk
extrapolation, the contribution of benign tumors to
the estimation of risk will be indicated.
V. Transplacental and Multigenerational Animal
Bioassays
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As one of its two proposals for additions to the
guidelines, the SAB recommended a discussion of
transplacental and multigenerational animal
bioassays for carcinogenicity.
The Agency agrees that such data, when
available, can provide useful information in the
evaluation of a chemical's potential carcinogenicity
and has stated this in the final guidelines. The
Agency has also revised the guidelines to indicate
that such studies may provide additional
information on the metabolic and pharmacokinetic
properties of the chemical. More guidance on the
specific use of these studies will be considered in
future revisions of these guidelines.
VI. Maximum Tolerated Dose
The proposed guidelines discussed the
implications of using a maximum tolerated dose
(MTD) in bioassays for carcinogenicity. Many
commentors requested that EPA define MTD. The
tone of the comments suggested that the
commentors were concerned about the uses and
interpretations of high-dose testing.
The Agency recognizes that controversy
currently surrounds these issues. The appropriate
text from the OSTP report has been incorporated
into the final guidelines which suggests that the
consequences of high-dose testing be evaluated on a
case-by-case basis.
VII. Mouse Liver Tumors
A large number of commentors expressed
opinions about the assessment of bioassays in which
the only increase in tumor incidence was liver
tumors in the mouse. Many felt that mouse liver
tumors were afforded too much credence, especially
given existing information that indicates that they
might arise by a different mechanism, e.g., tissue
damage followed by regeneration. Others felt that
mouse liver tumors were but one case of a high
background incidence of one particular type of
tumor and that all such tumors should be treated in
the same fashion.
The Agency has reviewed these comments and
the OSTP principle regarding this issue. The OSTP
report does not reach conclusions as to the treatment
of tumors with a high spontaneous background rate,
but states, as is now included in the text of the
guidelines, that these data require special
consideration. Although questions have been raised
regarding the validity of mouse liver tumors in
general, the Agency feels that mouse liver tumors
cannot be ignored as an indicator of carcinogenicity.
Thus, the position in the proposed guidelines has not
been changed: an increased incidence of only mouse
liver tumors will be regarded as "sufficient"
evidence of carcinogenicity if all other criteria, e.g.,
replication and malignancy, are met with the
understanding that this classification could be
changed to "limited" if warranted. The factors that
may cause this re-evaluation are indicated in the
guidelines.
VIII. Weight-of'Evidence Catagories
The Agency was praised by both the public and
the SAB for incorporating a weight-of-evidence
scheme into its evaluation of carcinogenic risk.
Certain specific aspects of the scheme, however,
were criticized.
1. Several commentors noted that while the text
of the proposed guidelines clearly states that EPA
will use all available data in its categorization of the
weight of the evidence that a chemical is a
carcinogen, the classification system in Part A,
section IV did not indicate the manner in which EPA
will use information other than data from humans
and long-term animal studies in assigning a weight-
of-evidence classification.
The Agency has added a discussion to Part A,
section IV.C. dealing with the characterization of
overall evidence for human carcinogenicity. This
discussion clarifies EPA's use of supportive
information to adjust, as warranted, the designation
that would have been made solely on the basis of
human and long-term animal studies.
2. The Agency agrees with the SAB and those
commentors who felt that a simple classification of
the weight of evidence, e.g., a single letter or even a
descriptive title, is inadequate to describe fully the
weight of evidence for each individual chemical. The
final guidelines propose that a paragraph
summarizing the data should accompany the
numerical estimate and weight-of-evidence
classification whenever possible.
3. Several commentors objected to the
descriptive title E (No Evidence of Carcinogenicity
for Humans) because they felt the title would be
confusing to people inexperienced with the
classification system. The title for Group E, No
Evidence of Carcinogenicity for Humans, was
thought by these commentors to suggest the absence
of data. This group, however, is intended to be
reserved for agents for which there exists credible
data demonstrating that the agent is not
carcinogenic.
Based on these comments and further
discussion, the Agency has changed the
[51 FR 34003]
title of Group E
to "Evidence of Non-Carcinogenicity for Humans."
4. Several commentors felt that the title for
Group C, Possible Human Carcinogen, was not
sufficiently distinctive from Group B, Probable
Human Carcinogen. Other commentors felt that
those agents that minimally qualified for Group C
would lack sufficient data for such a label.
The Agency recognizes that Group C covers a
range of chemicals and has considered whether to
subdivide Group C. The consensus of the Agency's
1-15
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Carcinogen Risk Assessment Committee, however,
is that the current groups, which are based on the
IARC categories, are a reasonable stratification and
should be retained at present. The structure of the
groups will be reconsidered when the guidelines are
reviewed in the future. The Agency also feels lhat
the descriptive title it originally selected best
conveys the meaning of the classification within the
context of EPA's past and current activities.
5. Some commentors indicated a concern about
the distinction between Bl and 132 on the basis of
epidemiologic evidence only. This issue has been
under discussion in the Agency and may be revised
in future versions of the guidelines.
6. Comments were also received about the
possibility of keeping the groups for animal and
human data separate without reaching a combined
classification. The Agency feels that a combined
classification is useful; thus, the combined
classification was retained in the final guidelines.
The SAB suggested that a table be added to Part
A, section IV to indicate the manner in which
human and animal data would be combined to
obtain an overall weight-of-evidence category. The
Agency realizes that a table that would present all
permutations of potentially available data would be
complex and possibly impossible to construct since
numerous combinations of ancillary data (e.g.,
genetic toxicity, pharmacokinetics) could be used to
raise or lower the weight-of-evidence classification.
Nevertheless, the Agency decided to include a table
to illustrate the most probable weight-of-evidence
classification that would be assigned on the basis of
standard animal and human data without
consideration of the ancillary data. While it is hoped
that this table will clarify the weight-of-evidence
classifications, it is also important to recognize that
an agent may be assigned to a final categorization
different from the category which would appear
appropriate from the table and still conform to the
guidelines.
IX. Quantitative Estimates of Risk
The method for quantitative estimates of
carcinogenic risk in the proposed guidelines received
substantial comments from the public. Five issues
were discussed by the Agency and have resulted in
modifications of the guidelines.
1. The major criticism was the perception that
EPA would use only one method for the
extrapolation of carcinogenic risk and would,
therefore, obtain one estimate of risk. Even
commentors who concur with the procedure usually
followed by EPA felt that some indication of the
uncertainty of the risk estimate should be included
with the risk estimate.
The Agency feels that the proposed guidelines
were not intended to suggest that EPA would
perform quantitative risk estimates in a rote or
mechanical fashion. As indicated by the OSTP
report and paraphrased in the proposed guidelines,
no single mathematical procedure has been
determined to be the most appropriate method for
risk extrapolation. The final guidelines quote rather
than paraphrase the OSTP principle. The guidelines
have been revised to stress the importance of
considering all available data in the risk assessment
and now state, "The Agency will review each
assessment as to the evidence on carcinogenic
mechanisms and other biological or statistical
evidence that indicates the suitability of a particular
extrapolation model." Two issues are emphasized:
First, the text now indicates the potential for
pharmacokinetic information to contribute to the
assessment of carcinogenic risk. Second, the final
guidelines state that time-to-tumor risk
extrapolation models may be used when
longitudinal data on tumor development are
available.
2. A number of commentors noted that the
proposed guidelines did not indicate how the
uncertainties of risk characterization would be
presented. The Agency has revised the proposed
guidelines to indicate that major assumptions,
scientific judgments, and, to the extent possible,
estimates of the uncertainties embodied in the risk
assessment will be presented along with the
estimation of risk.
3. The proposed guidelines stated that the
appropriateness of quantifying risks for chemicals in
Group C (Possible Human Carcinogen), specifically
those agents that were on the boundary of Groups C
and D (Not Classifiable as to Human
Carcinogenicity), would be judged on a case-by-case
basis. Some commentors felt that quantitative risk
assessment should not be performed on any agent in
Group C.
Group C includes a wide range of agents,
including some for which there are positive results
in one species in one good bioassay. Thus, the
Agency feels that many agents in Group C will be
suitable for quantitative risk assessment, but that
judgments in this regard will be made on a case-by-
case basis.
4. A few commentors felt that EPA intended to
perform quantitative risk estimates on aggregate
tumor incidence. While EPA will consider an
increase in total aggregate tumors as suggestive of
potential carcinogenicity, EPA does not generally
intend to make quantitative estimates of
carcinogenic risk based on total aggregate tumor
incidence.
5. The proposed choice of body surface area as an
interspecies scaling factor was criticized by several
commentors who felt that body weight was also
appropriate and that both methods should be used.
The OSTP report recognizes that both scaling factors
are in common use. The Agency feels that the choice
of the body surface area scaling factor can be
1-16
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justified from the data on effects of drugs in various
species. Thus, EPA will continue to use this scaling
factor unless data on a specific agent suggest that a
different scaling factor is justified. The uncertainty
engendered by choice of scaling factor will be
included in the summary of uncertainties associated
with the assessment of risk mentioned in point 1,
above.
In the second of its two proposals for additions to
the proposed guidelines, the SAB suggested that a
sensitivity analysis be included in EPA's
quantitative estimate of a chemical's carcinogenic
potency. The Agency agrees that an analysis of the
assumptions and uncertainties inherent in an
assessment of carcinogenic risk must be accurately
portrayed. Sections of the final guidelines that deal
with this issue have been strengthened to reflect the
concerns of the SAB and the Agency. In particular,
the last paragraph of the guidelines states that
"major assumptions, scientific judgments, and, to
the extent possible, estimates of the uncertainties
embodied in the assessment" should be presented in
the summary characterizing the risk. Since the
assumptions and uncertainties will vary for each
assessment, the Agency feels that a formal
requirement for a particular type of sensitivity
analysis would be less useful than a case-by-case
evaluation of the particular assumptions and
uncertainties most significant for a particular risk
assessment.
1-17
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51FR34006
GUIDELINES FOR MUTAGENICITY RISK
ASSESSMENT
SUMMARY: On September 24, 1986, the U.S.
Environmental Protection Agency issued the
following five guidelines for assessing the health
risks of environmental pollutants.
Guidelines for Carcinogen Risk Assessment
Guidelines for Estimating Exposures
Guidelines for Mutagenieity Risk Assessment
Guidelines for the Health Assessment of Suspect
Developmental Toxicants
Guidelines for the Health Risk Assessment of
Chemical Mixtures
This section contains the Guidelines for
Mutagenieity Risk Assessment.
The Guidelines for Mutagenieity Risk
Assessment (hereafter "Guidelines") are intended to
guide Agency analysis of mutagenicity data in line
with the policies and procedures established in the
statutes administered by the EPA. These Guidelines
were developed as part of an interoffice guidelines
development program under the auspices of the
Office of Health and Environmental Assessment
(OHEA) in the Agency's Office of Research and
Development. They reflect Agency consideration of
public and Science Advisory Board (SAB) comments
on the Proposed Guidelines for Mutagenicity Risk
Assessment published November 23, 1984 (49 FR
46314).
This publication completes the first round of risk
assessment guidelines development. These
Guidelines will be revised, and new guidelines will
be developed, as appropriate.
FOR FURTHER INFORMATION CONTACT:
Dr. Lawrence R. Valcovic
Reproductive Effects Assessment Group
Office of Health and Environmental Assessment
(RD-689)
U. S. Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460
202-382-7303
SUPPLEMENTARY INFORMATION: In 1983,
the National Academy of Sciences (NAS) published
its book entitled Risk Assessment in the Federal
Government: Managing the Process. In that book,
the NAS recommended that federal regulatory
agencies establish "inference guidelines" to ensure
consistency and technical quality in risk
assessments and to ensure that the risk assessment
process was maintained as a scientific effort
separate from risk management. A task force within
EPA accepted that recommendation and requested
that Agency scientists begin to develop such
guidelines.
General
The guidelines are products of a two-year
Agencywide effort, which has included many
scientists from the larger scientific community.
These guidelines set forth principles and procedures
to guide EPA scientists in the conduct of Agency risk
assessments, and to inform Agency decision makers
and the public about these procedures. In particular,
the guidelines emphasize that risk assessments will
be conducted on a case-by-case basis, giving full
consideration to all relevant scientific information.
This case-by-case approach means that Agency
experts review the scientific information on each
agent and use the most scientifically appropriate
interpretation to assess risk. The guidelines also
stress that this information will be fully presented
in Agency risk assessment documents, and that
Agency scientists will identify the strengths and
weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well
as the scientific basis and rationale for each
assessment.
Finally, the guidelines are formulated in part to
bridge gaps in risk assessment methodology and
data. By identifying these gaps and the importance
of the missing information to the risk assessment
process, EPA wishes to encourage research and
analysis that will lead to. new risk assessment
methods and data.
Guidelines for Mutagenicity Risk Assessment
Work on the Guidelines for Mutagenicity Risk
Assessment began in January 1984. Draft
guidelines were developed by Agency work groups
composed of expert scientists from throughout the
Agency. The drafts were peer-reviewed by expert
scientists in the field of genetic toxicology from
universities, environmental groups, industry, labor,
and other governmental agencies. They were then
proposed for public comment in the FEDERAL
REGISTER (49 FR 46314). On November 9, 1984,
the Administrator directed that Agency offices use
the proposed guidelines in performing risk
assessments until final guidelines become available.
2-1
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After the close of the public comment period,
Agency staff prepared summaries of the comments,
analyses of the major issues presented by the
commentors, and preliminary Agency responses to
those comments. These analyses were presented to
review panels of the SAB on March 4 and April 22-
23, 1985, and to the Executive Committee of the
SAB on April 25-26, 1985. The SAB meetings were
announced in the FEDERAL REGISTER as follows:
February 12, 1985 (50 FR 5811) and April 4, 1985
(50 FR13420 and 13421).
In a letter to the Administrator dated June 19,
1985, the Executive Committee generally concurred
on all five of the guidelines, but recommended
certain revisions, and requested that any revised
guidelines be submitted to the appropriate SAB
review panel chairman for review and concurrence
on behalf of the Executive Committee. As described
in the responses to comments (see Part B: Response
to the Public and Science Advisory Board
Comments), each guidelines document was revised,
where appropriate, consistent with the SAB
recommendations, and revised draft guidelines were
submitted to the panel chairmen. Revised draft
Guidelines for Mutagenicity Risk Assessment were
concurred on in a letter dated September 24, 1985.
Copies of the letters are available at the Public
Information Reference Unit, EPA Headquarters
Library, as indicated elsewhere in this section.
Following this Preamble are two parts: Part A
contains the Guidelines and Part B, the Response to
the Public and Science Advisory Board Comments (a
summary of the major public comments, SAB
comments, and Agency responses to those
comments).
The Agency is continuing to study the risk
assessment issues raised in the guidelines and will
revise these Guidelines in line with new information
as appropriate.
References, supporting documents, and
comments received on the proposed guidelines, as
well as copies of the final guidelines, are available
for inspection and copying at the Public Information
Reference Unit (202-382-5926), EPA Headquarters
Library, 401 M Street, S.W., Washington, DC,
between the hours of 8:00 a.m. and 4:30 p.m.
I certify that these Guidelines are not major
rules as defined by Executive Order 12291, because
they are nonbinding policy statements and have no
direct effect on the regulated community. Therefore,
they will have no effect on costs or prices, and they
will have no other significant adverse effects on the
economy. These Guidelines were reviewed by the
Office of Management
[51FR34007J
and Budget
under Executive Order 12291.
August 22,1986
Lee M. Thomas,
Administrator
CONTENTS
Part A: Guidelines for Mutagenicily Itisk Assessment
/. Introduction
A.Concepts Relating to Heritable Mutagenie Risk
B.Test Systems
17. Qualitative Assessment (Hazard Identification)
A.Mutagenic Activity
B.Chemical Interactions in the Mammalian Gonad
C.Weight-of-Evidence Determination
///. Quantitative Assessment
A.Dose Response
B,Exposure Assessment
C.Risk Characterization
IV. References
Part B: Response to Public and Science Advisory Board
Comments
Part A: Guidelines for Mutagenicity Risk
Assessment
/. Introduction
This section describes the procedures that the
U.S. Environmental Protection Agency will follow
in evaluating the potential genetic risk associated
with human exposure to chemicals. The central
purpose of the health risk assessment is to provide a
judgment concerning the weight of evidence that an
agent is a potential human mutagen, capable of
inducing transmitted genetic changes, and, if so, to
provide a judgment on how great an impact this
agent is likely to have on public health. Regulatory
decision making involves two components: risk
assessment and risk management. Risk assessment
estimates the potential adverse health consequences
of exposure to toxic chemicals; risk management
combines the risk assessment with the directives of
the enabling regulatory legislationtogether with
socioeconomic, technical, political, and other
considerationsto reach a decision as to whether or
how much to control future exposure to the
chemicals. The issue of risk management will not be
dealt with in these Guidelines.
Risk assessment is comprised of the following
components: hazard identification, dose-response
assessment, exposure assessment, and risk
characterization (1). Hazard identification is the
qualitative risk assessment, dealing with the
inherent toxicity of a chemical substance. The
qualitative mutagenicity assessment answers the
question of how likely an agent is to be a human
mutagen. The three remaining components
2-2
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comprise quantitative risk assessment, which
provides a numerical estimate of the public health
consequences of exposure to an agent. The
quantitative mutagenicity risk assessment deals
with the question of how much mutational damage
is likely to be produced by exposure to a given agent
under particular exposure scenarios.
In a dose-response assessment, the relationship
between the dose of a chemical and the probability of
induction of an adverse effect is defined. The
component generally entails an extrapolation from
the high doses administered to experimental
animals or noted in some epidemiologic studies to
the low exposure levels expected from human
contact with the chemical in the environment.
The exposure assessment identifies populations
exposed to toxic chemicals, describes their
composition and size, and presents the types,
magnitudes, frequencies, and durations of exposure
to the chemicals. This component is developed
independently of the other components of the
mutagenicity assessment and is addressed in
separate Agency guidelines (2).
In risk characterization, the outputs of the
exposure assessment and the dose-response
assessment are combined to estimate quantitatively
the mutation risk, which is expressed as either
estimated increase of genetic disease per generation
or per lifetime, or the fractional increase in the
assumed background mutation rate of humans. In
each step of the assessment, the strengths and
weaknesses of the major assumptions need to be
presented, and the nature and magnitude of
uncertainties need to be characterized.
The procedures set forth in these Guidelines will
ensure consistency in the Agency's scientific risk
assessments for mutagenic effects. The necessity for
a consistent approach to the evaluation of mutagenic
risk from chemical substances arises from the
authority conferred upon the Agency by a number of
statutes to regulate potential mutagens. As
appropriate, these Guidelines will apply to statutes
administered by the Agency, including the Federal
Insecticide, Fungicide, and Rodenticide Act; the
Toxic Substances Control Act; the Clean Air Act; the
Federal Water Pollution Control Act; the Safe
Drinking Water Act; the Resource Conservation and
Recovery Act; and the Comprehensive
Environmental Response, Compensation, and
Liability Act. Because each statute is administered
by separate offices, a consistent Agency-wide
approach for performing risk assessments is
desirable.
The mutagenicity risk assessments prepared
pursuant to these Guidelines will be utilized with
the requirements and constraints of the applicable
statutes to arrive at regulatory decisions concerning
mutagenicity. The standards of the applicable
statutes and regulations may dictate that additional
considerations (e.g., the economic and social benefits
associated with use of the chemical substance) will
come into play in reaching appropriate regulatory
decisions.
The Agency has not attempted to provide in the
Guidelines a detailed discussion of the mechanisms
of mutagenicity or of the various test systems that
are currently in use to detect mutagenic potential.
Background information on mutagenesis and
mutagenicity test systems is available in
"Identifying and Estimating the Genetic Impact of
Chemical Mutagens", National Academy of Sciences
(NAS) Committee on Chemical Environmental
Mutagens (3), as well as in other recent publications
(4,5).
The Agency is concerned with the risk
associated with both germ-cell mutations and
somatic-cell mutations. Mutations carried in germ
cells may be inherited by future generations and
may contribute to genetic disease, whereas
mutations occurring in somatic cells may be
implicated in the etiology of several disease states,
including cancer. These Guidelines, however, are
only concerned with genetic damage as it relates to
germ-cell mutations. The use of mutagenicity test
results in the assessment of carcinogenic risk is
described in the Guidelines for Carcinogen Risk
Assessment (6).
As a result of the progress in the control of
infectious diseases, increases in average human life
span, and better procedures for identifying genetic
disorders, a considerable heritable genetic disease
burden has been recognized in the human
population. It is estimated that at least 10% of all
human disease is related to specific genetic
abnormalities, such as abnormal composition,
arrangement, or dosage of genes and chromosomes
(3, 7, 8). Such genetic abnormalities can lead to
structural or functional health impairments. These
conditions may be expressed in utero; at the time of
birth; or during infancy, childhood, adolescence, or
adult life; they may be chronic or acute in nature. As
a result, they often have a severe impact upon the
affected individuals and their families in terms of
physical and mental
[51 PR 34008]
suffering and
economic losses, and upon society in general, which
often becomes responsible for institutional care of
severely affected individuals. Some examples of
genetic disorders are Down and Klinefelter
syndromes, cystic fibrosis, hemophilia, sickle-cell
anemia, and achondroplastic dwarfism. Other
commonly recognized conditions that are likely to
have a genetic component include
hypercholesterolemia, hypertension, pyloric
stenosis, glaucoma, allergies, several types of
cancer, and mental retardation. These disorders are
only a few of the thousands that are at least partially
genetically determined (9).
I
2-3
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Estimation of the fraction of human genetic
disorders that result from new mutations is difficult,
although in certain specific cases insights are
available (10). It is clear that recurring mutation is
important in determining the incidence of certain
genetic disorders, such as some chromosomal
aberration syndromes (e.g., Down syndrome) and
rare dominant and X-Iinked recessive diseases (e.g.,
achondroplasia and hemophilia A). For other single-
factor disorders (e.g., sickle-cell anemia) and certain
multifactorial disorders (e.g., pyloric stenosis), the
contribution of new mutations to disease frequency
is probably small. However, it is generally
recognized that most newly-arising mutations that
are phenotypically expressed are in some ways
deleterious to the organism receiving them (3, 7, 8).
Adverse effects may be manifested at the
biochemical, cellular, or physiological levels of
organization. Although mutations are the building
blocks for further evolutionary change of species, it
is believed that increases in the mutation rate could
lead to an increased frequency of expressed genetic
disorders in the first and subsequent generations.
Life in our technological society results in
exposure to many natural and synthetic chemicals.
Some have been shown to have mutagenic activity
in mammalian and submammalian test systems,
and thus may have the potential to increase genetic
damage in the human population. Chemicals
exhibiting mutagenic activity in various test
systems have been found distributed among foods,
tobacco, drugs, food additives, cosmetics, industrial
compounds, pesticides, and consumer products. The
extent to which exposure to natural and synthetic
environmental agents may have increased the
frequency of genetic disorders in the present human
population and contributed to the mutational "load"
that will be transmitted to future generations is
unknown at this time. However, for the reasons
cited above, it seems prudent to limit exposures to
potential human mutagens.
A. Concepts Relating to Heritable Mutagenic Risk
These Guidelines are concerned with chemical
substances or mixtures of substances that can
induce alterations in the genome of either somatic or
germinal cells. The mutagenicity of physical agents
(e.g., radiation) is not addressed here. There are
several mutagenic end points of concern to the
Agency. These include point mutations (i.e.,
submicroscopic changes in the base sequence of
DNA) and structural or numerical chromosome
aberrations. Structural aberrations include
deficiencies, duplications, insertions, inversions,
and translocations, whereas numerical aberrations
are gains or losses of whole chromosomes (e.g.,
trisomy, monosomy) or sets of chromosomes
(haploidy, polyploidy).
Certain mutagens, such as alkylating agents,
can directly induce alterations in the DNA.
Mutagenic effects may also come about through
mechanisms other than chemical alterations of
DNA. Among these are interference with normal
DNA synthesis (as caused by some metal mutagens),
interference with DNA repair, abnormal DNA
methylation, abnormal nuclear division processes,
or lesions in non-DNA targets (e.g., protamine,
tubulin).
Evidence that an agent induces heritable
mutations in human beings could be derived from
epidemiologic data indicating a strong association
between chemical exposure and heritable effects. It
is difficult to obtain such data because any specific
mutation is a rare event, and only a small fraction of
the estimated thousands of human genes and
conditions are currently useful as markers in
estimating mutation rates. Human genetic
variability, small numbers o*f offspring per
individual, and long generation times further
complicate such studies. In addition, only disorders
caused by dominant mutations, some sex-linked
recessive mutations, and certain chromosome
aberrations can be detected in the first generation
after their occurrence. Conditions caused by
autosomal recessive disorders (which appear to
occur more frequently than dominant disorders) or
by polygenic traits may go unrecognized for many
generations. Therefore, in the absence of human
epidemiological data, it is appropriate to rely on data
from experimental animal systems as long as the
limitations of using surrogate and model systems
are clearly stated.
Despite species differences in metabolism, DNA
repair, and other physiological processes affecting
chemical mutagenesis, the virtual universality of
DNA as the genetic material and of the genetic code
provides a rationale for using various nonhuman
test systems to predict the intrinsic mutagenicity of
test chemicals. Additional support for the use of
nonhuman systems is provided by the observation
that chemicals causing genetic effects in one species
or test system frequently cause similar effects in
other species or systems. Evidence also exists that
chemicals can induce genetic damage in somatic
cells of exposed humans. For example, high doses of
mutagenic chemotherapeutic agents have been
shown to cause chromosomal abnormalities (11),
sister chromatic exchange (11), and, quite probably,
point mutations in human lymphocytes exposed in.
vivo (12). While these results are not in germ cells,
they do indicate that it is possible to induce
mutagenic events in human cells in vivo.
Furthermore, a wide variety of different types of
mutations have been observed in humans including
numerical chromosome aberrations, translocations,
base-pair substitutions, and frameshift mutations.
Although the cause of these mutations is uncertain,
it is clear from these observations that the human
germ-cell DNA is subject to the same types of
2-4
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mutational events that are observed in other species
and test systems.
Certain test systems offer notable advantages:
cost; anatomical, histological, and/or metabolic
similarities to humans; suitability for handling
large numbers of test organisms; a large data base;
or a basis for characterizing genetic events.
B. Test Systems
Many test systems are currently available that
can contribute information about the mutagenic
potential of a test compound with respect to various
genetic end points. These tests have recently been
evaluated through the EPA Gene-Tox Programs and
the results of Phase I have been published (5). The
Agency's Office of Pesticides and Toxic Substances
has published various testing guidelines for the
detection of mutagenic effects (13,14).
Test systems for detecting point mutations
include those in bacteria, eukaryotic
microorganisms, higher plants, insects, mammalian
somatic cells in culture, and germinal cells of intact
mammals. Data from heritable, mammalian germ-
cell tests provide the best experimental evidence
that a
[51FR 34009]
chemical is a
potential human germ-cell mutagen since these
tests require that mutations occur in germinal cells
and that they are transmitted to the next
generation. To date, the most extensively used test
for the induction of heritable mutation is the mouse
specific-locus test which measures the induction of
recessive mutations at seven loci concerned with
coat color and ear morphology. While this test has a
large data base compared to other germ-cell assays,
it is difficult to extrapolate results to humans since
recessive mutations may occur more frequently than
dominants, and the impact of recessive mutations is
not seen for many generations. Information on
frequencies of induced mutations resulting in health
disorders in the first generation may be obtained
from mouse systems designed to detect skeletal
abnormalities, cataracts, or general morphological
abnormalities. However, these assays have been
used to a relatively limited extent, and there is a
need for additional studies with known, chemical
germ-cell mutagens to further characterize the test
systems. Because large numbers of offspring must
usually be generated in the systems described above,
it is not expected that many chemicals will be tested
using these systems. To obtain data on a large
number of environmental chemicals, it will be
necessary to rely on other tests to identify and
characterize hazards from gene mutations.
Test systems for detecting structural
chromosome aberrations have been developed in a
variety of organisms including higher plants,
insects, fish, birds, and several mammalian species.
Many of these assays can be performed in vitro or in
vivo, and in either germ or somatic cells. Procedures
available for detecting structural chromosome
aberrations in mammalian germ cells include
measurement of heritable translocations or
dominant lethality, as well as direct cytogenetic
analyses of germ cells and early embryos in rodents.
Some chemicals may cause numerical
chromosome changes (i.e., aneuploidy) as their sole
mutagenic effect. These agents may not be detected
as mutagens if evaluated only in tests for DNA
damage, gene mutations, or chromosome breakage
and rearrangement. Therefore, it is important to
consider tests for changes in chromosome number in
the total assessment of mutagenic hazards.
Although tests for the detection of variation in the
chromosome number are still at an early stage of
development, systems exist in such diverse
organisms as fungi, Drosophila, mammalian cells in
culture, and intact mammals (e.g., mouse X-
chromosome loss assay). Aneuploidy can arise from
disturbances in a number of events affecting the
meiotic process (15, 16). Although the mechanisms
by which nondisjunction occurs are not well
understood, mitotic structures other than DNA may
be the target molecules for at least some
mechanisms of induced nondisjunction.
Other end points that provide information
bearing on the mutagenicity of a chemical can be
detected by a variety of test systems. Such tests
measure DNA damage in eukaryotic or prokaryotic
cells, unscheduled DNA synthesis in mammalian
somatic and germ cells, mitotic recombination and
gene conversion in yeast, and sister-chromatid
exchange in mammalian somatic and germ cells.
Results in these assays are useful because the
induction of these end points often correlates
positively with the potential of a chemical to induce
mutations.
In general, for all three end points (i.e., point
mutations and numerical and structural
aberrations), the Agency will place greater weight
on tests conducted in germ cells than in somatic
cells, on tests performed in vivo rather than in vitro,
in eukaryotes rather than prokaryotes, and in
mammalian species rather than in submammalian
species. Formal numerical weighting systems have
been developed (17); however, the Agency has
concluded that these do not readily accommodate
such variables as dose range, route of"exposure, and
magnitude of response.
The Agency anticipates that from time to time
somatic cell data from chemically exposed human
beings will be available (e.g., cytogenetic markers in
peripheral lymphocytes). When possible, the Agency
will use such data in conjunction with somatic and
germ cell comparisons from in vivo mammalian
experimental systems as a component in performing
risk assessments.
2-5
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The test systems mentioned previously are not
the only ones that will provide evidence of
mutagenicity or related DNA effects. These systems
are enumerated merely to demonstrate the breadth
of the available techniques for characterizing
mutagenic hazards, and to indicate the types of data
that the Agency will consider in its evaluation of
mutagenic potential of a chemical agent. Most
systems possess certain limitations that must be
taken into account. The selection and performance of
appropriate tests for evaluating the risks associated
with human exposure to any suspected mutagen will
depend on sound scientific judgment and experience,
and may necessitate consultation with geneticists
familiar with the sensitivity and experimental
design of the test system in question. In view of the
rapid advances in test methodology, the Agency
expects that both the number and quality of the tools
for assessing genetic risk to human beings will
increase with time. The Agency will closely monitor
developments in mutagenicity evaluation and will
refine its risk assessment scheme as better test
systems become available.
II. Qualitative Assessment (Hazard Identification)
The assessment of potential human germ-cell
mutagenic risk is a multistep process. The first step
is an analysis of the evidence bearing on a
chemical's ability to induce mutagenic events, while
the second step involves an analysis of its ability to
produce these events in the mammalian gonad. All
relevant information is then integrated into a
weight-of-evidence scheme which presents the
strength of the information bearing on the
chemical's potential ability to produce mutations in
human germ cells. For chemicals demonstrating this
potential, one may decide to proceed with an
evaluation of the quantitative consequences of
mutation following expected human exposure.
For hazard identification, it is clearly desirable
to have data from mammalian germ-cell tests, such
as the mouse specific-locus test for point mutations
and the heritable translocation or germ-cell
cytogenetic tests for structural chromosome
aberrations. It is recognized, however, that in most
instances such data will not be available, and
alternative means of evaluation will be required. In
such cases the Agency will evaluate the evidence
bearing on the agent's mutagenic activity and the
agent's ability to interact with or affect the
mammalian gonadal target. When evidence exists
that an agent possesses both these attributes, it is
reasonable to deduce that the agent is a potential
human germ-cell mutagen.
While mammalian germ-cell assays are
presently primarily performed on male animals, a
chemical cannot be considered to be a non-mutagen
for mammalian germ cells unless it is shown to be
negative in both sexes. Furthermore, because most
mammalian germ-cell assays are performed in mice,
it is noteworthy that the data from ionizing
radiation suggest that the female mouse immature
oocyte may not be an appropriate surrogate for the
same stage in the human female in mutagenicity
testing. However,
[51 FR 34010]
mutagenicity
data on the maturing and mature oocyte of the
mouse may provide a useful model for human risk
assessment.
A. Mutagenic Activity
In evaluating chemicals for mutagenic activity,
a number of factors will be considered: 1) genetic end
points (e.g., gene mutations, structural or numerical
chromosomal aberrations) detected by the test
systems, 2) sensitivity and predictive value of the
test systems for various classes of chemical
compounds, 3) number of different test systems used
for detecting each genetic end point, 4) consistency of
the results obtained in different test systems and
different species, 5) aspects of the dose-response
relationship, and 6) whether the tests are conducted
in accordance with appropriate test protocols agreed
upon by experts in the field.
B. Chemical Interactions in the Mammalian Gonad
Evidence for chemical interaction in the
mammalian gonad spans a range of different types
of findings. Each chemical under consideration
needs to be extensively reviewed since this type of
evidence may be part of testing exclusive of
mutagenicity per se (e.g., reproduction, metabolism,
and mechanistic investigations). Although it is not
possible to classify clearly each type of information
that may be available on a chemical, two possible
groups are illustrated.
1. Sufficient evidence of chemical interaction is
given by the demonstration that an agent interacts
with germ-cell DNA or other chromatin
constituents, or that it induces such end points as
unscheduled DNA synthesis, sister-chromatid
exchange, or chromosomal aberrations in germinal
cells.
2. Suggestive evidence will include the finding of
adverse gonadal effects such as sperm abnormalities
following acute, subchronic, or chronic toxicity
testing, or findings of adverse reproductive effects
such as decreased fertility, which are consistent
with the chemical's interaction with germ cells.
C. Weight-of-Evidence Determination
The evidence for a chemical's ability to produce
mutations and to interact with the germinal target
are integrated into a weight-of-evidence judgment
that the agent may pose a hazard as a potential
human germ-cell mutagen. All information bearing
on the subject, whether indicative of potential
concern or not, must be evaluated. Whatever
2-6
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evidence may exist from humans must also be
factored into the assessment.
All germ-cell stages are important in evaluating
chemicals because some chemicals have been shown
to be positive in postgonial stages but not in gonia
(18). When human exposures occur, effects on
postgonial stages should be weighted by the relative
sensitivity and the duration of the stages. Chemicals
may show positive effects for some end points and in
some test systems, but negative responses in others.
Each review must take into account the limitations
in the testing and in the types of responses that may
exist.
To provide guidance as to the categorization of
the weight of evidence, a classification scheme is
presented to illustrate, in a simplified sense, the
strength of the information bearing on the potential
for human germ-cell mutagenicity. It is not possible
to illustrate all potential combinations of evidence,
and considerable judgment must be exercised in
reaching conclusions. In addition, certain responses
in tests that do not measure direct mutagenic end
points (e.g., SCE induction in mammalian germ
cells) may provide a basis for raising the weight of
evidence from one category to another. The
categories are presented in decreasing order of
strength of evidence.
1. Positive data derived from human germ-cell
mutagenicity studies, when available, will
constitute the highest level of evidence for human
mutagenicity.
2. Valid positive results from studies on
heritable mutational events (of any kind) in
mammalian germ cells.
3. Valid positive results from mammalian germ-
cell chromosome aberration studies that do not
include an intergeneration test.
4. Sufficient evidence for a chemical's
interaction with mammalian germ cells, together
with valid positive mutagenicity test results from
two assay systems, at least one of which is
mammalian (in. vitro or in uiuo). The positive results
may both be for gene mutations or both for
chromosome aberrations; if one is for gene
mutations and the other for chromosome
aberrations, both must be from mammalian
systems.
5. Suggestive evidence for a chemical's
interaction with mammalian germ cells, together
with valid positive mutagenicity evidence from two
assay systems as described under 4, above.
Alternatively, positive mutagenicity evidence of less
strength than defined under 4, above, when
combined with sufficient evidence for a chemical's
interaction with mammalian germ cells.
6. Positive mutagenicity test results of less
strength than defined under 4, combined with
suggestive evidence for a chemical's interaction with
mammalian germ cells.
7. Although definitive proof of non-mutagenicity
is not possible, a chemical could be classified
operationally as a non-mutagen for human germ
cells, if it gives valid negative test results for all end
points of concern.
8.Inadequate evidence bearing on either
mutagenicity or chemical interaction with
mammalian germ cells.
///. Quantitative Assessment
The preceding section addressed primarily the
processes of hazard identification, i.e., the
determination of whether a substance is a potential
germ-cell mutagen. Often, no further data will be
available, and judgments will need to be based
mainly on qualitative criteria. Quantitative risk
assessment is a two-step process: determination of
the heritable effect per unit of exposure (dose-
response) and the relationship between mutation
rate and disease incidence. The procedures that are
presently accepted for the estimation of an increase
in disease resulting from increased mutation have
been described (3,7, 8). Dose-response information is
combined with anticipated levels and patterns of
human exposure in order to derive a quantitative
assessment (risk characterization).
A. Dose Response
Dose-response assessments can presently only
be performed using data from in uiuo, heritable
mammalian germ-cell tests, until such time as other
approaches can be demonstrated to have equivalent
predictability. The morphological specific-locus and
biochemical specific-locus assays can provide data
on the frequencies of recessive mutations induced by
different chemical exposure levels, and similar data
can be obtained for heritable chromosomal damage
using the heritable translocation test. Data on the
frequencies of induced mutations resulting in health
disorders in the first generation may be obtained
from mouse systems designed to detect skeletal
abnormalities, cataracts, or general morphological
abnormalities. Assays that directly detect heritable
health effects in the first generation may provide the
best basis for predicting human health risks that
result from mutagen exposure. The experimental
data on induced mutation frequency are usually
obtained at exposure levels much higher than those
that will be experienced by human beings. An
assessment of human risk is obtained by
extrapolating the induced mutation frequency or the
observed phenotypic
[51FR34011]
effect downward
to- the approximate level of anticipated human
exposure. In performing these extrapolations, the
Agency will place greater weight on data derived
from exposures and exposure rates that most closely
simulate those experienced by the human
population under study.
The Agency will strive to use the most
appropriate extrapolation models for risk analysis
2-7
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and will be guided by the available data and
mechanistic considerations in this selection.
However, it is anticipated that for tests involving
germ cells of whole mammals, few dose points will
be available to define dose-response functions. The
Agency is aware that for at least one chemical that
has been tested for mutations in mammalian germ
cells, there exist departures from linearity at low
exposure and exposure rates in a fashion similar to
that seen for ionizing radiation that has a low linear
energy transfer (19). The Agency will consider all
relevant models for gene and chromosomal
mutations in performing low-dose extrapolations
and will choose the most appropriate model. This
choice will be consistent both with the experimental
data available and with current knowledge of
relevant mutational mechanisms.
An experimental approach for quantitative
assessment of genetic risk, which may have utility
in the future, uses molecular dosimetry data from
intact mammals in conjunction with mutagenicity
and dosimetry data from other validated test
systems (20). The intact mammal is used primarily
for relating the exposure level for a given route of
administration of a chemical to germ-cell dose, i.e.,
the level of mutagen-DNA interactions. This
information is then used in conjunction with results
obtained from mutagenicity test systems in which
the relationship between the induction of mutations
and chemical interactions with DNA can be derived.
With mutagen-DNA interactions as the common
denominator, a relationship can be constructed
between mammalian exposure and the induced
mutation frequency. The amount of DNA binding
induced by a particular chemical agent may often be
determined at levels of anticipated human exposure.
For some mutagenic events, DNA may not
necessarily be the critical target. Interaction of
chemicals with other macromoleeules, such as
tubulin, which is involved in the separation of
chromosomes during nuclear division, can lead to
chromosomal nondisjunction. At present, general
approaches are not available for dose-response
assessments for these types of mutations. Ongoing
research should provide the means to make future
assessments on chemicals causing aneuploidy.
B. Exposure Asssessmen.t
The exposure assessment identifies populations
exposed to toxic chemicals; describes their
composition and size; and presents the types,
magnitudes, frequencies, and durations of exposure
to the chemicals. This component is developed
independently of the other components of the
mutagenicity assessment (2).
C. Risk Characterization
In performing mutagenicity risk assessments, it
is important to consider each genetic end point
individually. For example, although certain
chemical substances that interact with DNA may
cause both point and chromosomal mutations, it is
expected that the ratio of these events may differ
among chemicals and between doses for a given
chemical. Furthermore, transmissible chromosomal
aberrations are recoverable with higher frequencies
from meiotic and postmeiotic germ-cell stages,
which have a brief life span, than in spermatogonial
stem cells, which can accumulate genetic damage
throughout the reproductive life of an individual.
For these reasons, when data are available, the
Agency, to the best extent possible, will assess risks
associated with all genetic end points.
Any risk assessment should clearly delineate
the strengths and weaknesses of the data, the
assumptions made, the uncertainties in the
methodology, and the rationale used in reaching the
conclusions, e.g., similar or different routes of
exposure and metabolic differences between humans
and test animals. When possible, quantitative risk
assessments should be expressed in terms of the
estimated increase of genetic disease per generation,
or the fractional increase in the assumed
background spontaneous mutation rate of humans
(7). Examples of quantitative risk estimates have
been published (7, 8, 21); these examples may be of
use in performing quantitative risk assessments for
mutagens.
IV. References
1. Committee on the Institutional Means for Assessment of
the Risks to Public Health. 1983. Risk assessment in the Federal
government: managing the process. Washington, DC: National
Academy Press.
2. U.S. Environmental Protection Agency. 1986, Sept. 24.
Guidelines for estimating exposures. Federal Register 51(185):
34042-34054.
3. Committee on Chemical Environmental Mutagens,
National Academy of Sciences. 1982. Identifying and estimating
the genetic impact of chemical mutagens. Washington, DC:
National Academy Press.
4. Committee 1 Final Report. 1983. Screening strategy for
chemicals that are potential germ-cell mutagens in mammals.
Mutat. Res. 114:117-177.
5. A complete reference of all Gene-Tox publications is
available from the TSCA Industry Assistance Office (TS-794),
Office of Toxic Substances, U.S. Environmental Protection
Agency, Washington, DC 20460.
6. U.S. Environmental Protection Agency. 1986, Sept. 24.
Guidelines for carcinogen risk assessment. Federal Register
51(185):33992-34003.
7. National Research Council, Advisory Committee on the
Biological Effects of Ionizing Radiations. 1972. The effects on
populations of exposure to low levels of ionizing radiation. tBEIR
I) Washington, DC: National Academy of Sciences.
National Research Council, Advisory Committee on the
Biological Effects of Ionizing Radiations. 1977. Considerations of
health benefit-cost analysis for activities involving ionizing
radiation exposure and alternatives. {BEIR II) Washington, DC:
National Academy of Sciences.
National Research Council, Advisory Committee on the
Biological Effects of Ionizing Radiations. 1980. The effects on
populations of exposure to low levels of ionizing radiation. (BEIR
III) Washington, DC: National Academy of Sciences.
8. United Nations General Assembly. 1958. Report of the
United Nations Scientific Committee on the Effects of Atomic
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Radiation. Official records of the General Assembly. Thirteenth
Session. Supplement No. 17 (A/3838)'. New York: United Nations
United Nations General Assembly. 1962. Report of the
United Nations Scientific Committee on the Effects of Atomic
Radiation. Official records of the General Assembly. Seventeenth
Session. Supplement No. 1 6 (A/52 1 6). New York: United Nations.
United Nations General Assembly. 1966. Report of the
United Nations Scientific Committee on the Effects of Atomic
Radiation. Official records of the General Assembly. Twenty-first
Session. Supplement No. 14 (A/63 14). New York: United Nations.
United Nations General Assembly. 1969. Report of the
United Nations Scientific Committee on the effects of Atomic
Radiation. Official records of the General Assembly Twenty-
fourth Session. Supplement No. 13 (A/7613). New York: United
Nations.
United Nations General Assembly. 1972. Report of the
United Nations Scientific Committee on the Effects of Atomic
Radiation. Ionizing radiation: levels and effects. Vol. II Official
records of the General Assembly. Twenty-seventh Session
Supplement No. 25 (A/8725). New York: United Nations
United Nations General Assembly. 1982. Report of the
United Nations Scientific Committee on the Effects of Atomic
Radiation. Sources and biological effects. New York- United
Nations.
9. McKusick. V.A. 1983. Mendelian inheritance in man-
catalogs of autosomal dominant, autosomal recessive and x-linked
[51 FR 34012]
phenotypes
Baltimore, MD: Johns Hopkins University Press.
10. Crow, J.F., and C. Denniston. 1981. The mutation
component of genetic damage. Science 2 1 2:888-893
11. Musilova, J., K. Michalova, and J. Urban. 1979 Sister
chromatid exchanges and chromosomal breakage in patients
treated with cytostatics. Mutat. Res. 67:289-294.
12. Strauss, G.H., and R J. Albertini. 1979. Enumeration of
6- thioguanme-resistant peripheral blood lymphocytes in man as
a potential test for somatic cell mutations arising in. viuo. Mutat.
Kes. 61:353-379.
13. U.S. Environmental Protection Agency. 1983 OTS
health effects test guidelines. EPA 560/6/82-001. Natl Tech
Inform. Serv. PB82-232984.
ioQo14nU'S" Environmental Protection Agency. November 24
1982. Pesticides registration; proposed data requirements'
Federal Register 47:53 192- 53 203.
1 5. Parker, D.R., and J.H. Williamson. 1974. Some radiation
effects on segregation in Drosophila. Genetics 78-163-171
16 Grell, R F. 1979. Origin of meiotic nondisjunction in
Drosophila females. Environ. Health Perspect. 31 -33-39
17. Russell^ L B C.S. Aaron, F. de Serres, W.M. Generoso,
K.L Kannan, M. Shelby, J. Springer, and P. Voytek. 1984
Evaluation of existing mutagenicity bioassays for purposes of
genetic risk assessment. Mutat. Res. 134:143-157.
18. Russell, L.B., R.B. Gumming, and P.R. Hunsicker 1984
bpecilic locus mutation rates in the mouse following inhalation of
ethylene oxide, and application of results to estimation of genetic
risk. Mutat. Res. 129:381-388.
19. Russell, W.L., P.R. Hunsicker, D.A. Carpenter, C V
Cornell, and G.M. Gum. 1982. Effecl of dose fractionation on the
ethylmtrosourea induction of specific-locus mutations in mouse
spermatogonia. Proc. Natl. Acad.Sci. 79:3592-3593.
20. Lee, W.R. 1979. Dosimetry of chemical mutagens in
eukaryote germ- cells. In. A. Hollaender and F.J. de Serres, eds
Chemical mutagens: principles and methods for their detection
Vol. 5, New York: Plenum Press, pp. 177-202.
j 21J Ehlini' U-H- and A- Neuhauser. 1979. Procarbazine-
L« o«trslec lc"locus mutations »n male mice. Mutat. Res.
5-256.
Part B: Response to Public and Science
Advisory Board Comments
This section summarizes some of the issues
raised in public and Science Advisory Board (SAB)
comments on the Proposed Guidelines for
Mutagenicity Risk Assessment published on
November 23, 1984 (49 FR 46314). Unlike the other
guidelines published on the same date, the Proposed
Guidelines for Mutagenicity Risk Assessment
contained a detailed section dealing with public
comments received in response to the original
proposal of 1980 (45 FR 74984). Several of the
comments received in response to the proposed
guidelines of 1984 were similar to those received in
response to the proposed guidelines of 1980. Those
comments are not addressed here because the
position of the Agency on those issues has been
presented in the responses included with the 1984
proposed guidelines (49 FR 46315- 46316).
A total of 44 comments were received in
response to the proposed guidelines of 1984: 21 from
manufacturers of regulated products, 10 from
associations, 9 from government agencies, 2 from
educational institutions, 1 from an individual, and 1
from a private consulting firm. The proposed
guidelines and the public comments received were
transmitted to the Agency's SAB prior to its public
review of the proposed guidelines held April 22-23,
1985. The majority of the comments were favorable
and expressed the opinion that the proposed
guidelines accurately represent the existing state of
knowledge in the field of mutagenesis. Several
commentors offered suggestions for further
clarification of particular issues, and many of the
suggestions have been incorporated.
The two areas thai received the most
substantive comments were the sections concerning
Weight-of-Evidence Determination and Dose
Response. The comments on the proposed weight-of-
evidence scheme ranged from suggestions for the
elimination of a formal scheme to the expansion of
the scheme to cover more potential data
configurations. The SAB recommended an eight-
level rank ordering scheme to define levels of
evidence relating to human germ-cell mutagenicity
The Agency has incorporated this scheme into the
Guidelines. Some commentors and the SAB
suggested that the molecular dosimetry approach to
dose-response data be presented as a concept that
may be useful in the future rather than being
available for use now. The Agency agrees that the
data base at the present time is too sparse to
recommend a general application of this approach to
a wide range of chemical classes, and the Guidelines
have been changed to reflect this. It should be noted,
however, that the Agency strongly supports the'
development of molecular dosimetry methodologies
as they relate to both an understanding of dose-
response relationships and to methods for studying
human exposure. A number of comments suggesting
clarifications and editorial changes have been
incorporated and the references have been
expanded.
2-9
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I
51 FR 34014
GUIDELINES FOR THE HEALTH RISK
ASSESSMENT OF CHEMICAL MIXTURES
SUMMARY: On September 24, 1986, the U.S.
Environmental Protection Agency issued the
following five guidelines for assessing the health
risks of environmental pollutants.
Guidelines for Carcinogen Risk Assessment
Guidelines for Estimating Exposures
Guidelines for Mutagenicity Risk Assessment
Guidelines for the Health Assessment of Suspect
Developmental Toxicants
Guidelines for the Health Risk Assessment of
Chemical Mixtures
This section contains the Guidelines for the Health
Risk Assessment of Chemical Mixtures.
The Guidelines for the Health Risk Assessment
of Chemical Mixtures (hereafter "Guidelines") are
intended to guide Agency analysis of information
relating to health effects data on chemical mixtures
in line with the policies and procedures established
in the statutes administered by the EPA. These
Guidelines were developed as part of an interoffice
guidelines development program under the auspices
of the Office of Health and Environmental
Assessment (OHEA) in the Agency's Office of
Research and Development. They reflect Agency
consideration of public and Science Advisory Board
(SAB) comments on the Proposed Guidelines for the
Health Risk Assessment of Chemical Mixtures
published January 9,1985 (50 FR 1170).
This publication completes the first round of risk
assessment guidelines development. These
Guidelines will be revised, and new guidelines will
be developed, as appropriate.
FOR FURTHER INFORMATION CONTACT:
Dr. Richard Hertzberg
Methods Evaluation and Development Staff
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
26 W. St. Clair Street
Cincinnati, OH 45268
513-569-7582
SUPPLEMENTARY INFORMATION: In 1983,
the National Academy of Sciences (NAS) published
its book entitled Risk Assessment in the Federal
Government: Managing the Process. In that book,
the NAS recommended that Federal regulatory
agencies establish "inference guidelines" to ensure
consistency and technical quality in risk
assessments and to ensure that the risk assessment
process was maintained as a scientific effort
separate from risk management. A task force within
EPA accepted that recommendation and requested
that Agency scientists begin to develop such
guidelines.
General
The guidelines are products of a two-year
Agencywide effort, which has included many
scientists from the larger scientific community.
These guidelines set forth principles and procedures
to guide EPA scientists in the conduct of Agency risk
assessments, and to inform Agency decision makers
and the public about these procedures. In particular,
the guidelines emphasize that risk assessments will
be conducted on a case-by-case basis, giving full
consideration to all relevant scientific information.
This case-by-case approach means that Agency
experts review the scientific information on each
agent and use the most scientifically appropriate
interpretation to assess risk. The guidelines also
stress that this information will be fully presented
in Agency risk assessment documents, and that
Agency scientists will identify the strengths and
weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well
as the scientific basis and rationale for each
assessment.
Finally, the guidelines are formulated in part to
bridge gaps in risk assessment methodology and
data. By identifying these gaps and the importance
of the missing information to the risk assessment
process, EPA wishes to encourage research and
analysis that will lead to new risk assessment
methods and data.
Guidelines for the Health Risk Assessment of
Chemical Mixtures
Work on the Guidelines for the Health Risk
Assessment of Chemical Mixtures began in January
1984. Draft guidelines were developed by Agency
work groups composed of expert scientists from
throughout the Agency. The drafts were peer-
reviewed by expert scientists in the fields of
toxicology, pharmacokinetics, and statistics from
universities, environmental groups, industry, labor,
and other governmental agencies. They were then
proposed for public comment in the FEDERAL
REGISTER (50 FR 1170). On November 9,1984, the
Administrator directed that Agency offices use the
3-1
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proposed guidelines in performing risk assessments
until final guidelines become available. After the
close of the public comment period, Agency staff
prepared summaries of the comments, analyses of
the major issues presented by the commentors, and
preliminary Agency responses to those comments.
These analyses were presented to review panels of
the SAB on March 4 and April 22-23, 1985, and to
the Executive Committee of the SAB on April 25-26,
1985. The SAB meetings were announced in the
FEDERAL REGISTER as follows: February 12,
1985 (50 FR 5811) and April 4, 1985 (50 FR 13420
and 13421).
In a letter to the Administrator dated June 19,
1985, the Executive Committee generally concurred
on all five of the guidelines, but recommended
certain revisions, and requested that any revised
guidelines be submitted to the appropriate SAB
review panel chairman for review and concurrence
on behalf of the Executive Committee. As described
in the responses to comments (see Part B: Response
to the Public and Science Advisory Board
Comments), each guidelines document was revised,
where appropriate, consistent with the SAB
recommendations, and revised draft guidelines were
submitted to the panel chairmen. Revised draft
Guidelines for the Health Risk Assessment of
Chemical Mixtures were concurred on in a letter
dated August 16, 1985. Copies of the letters are
available at the Public Information Reference Unit,
EPA Headquarters Library, as indicated elsewhere
in this section.
Following this Preamble are two parts: Part A
contains the Guidelines and Part B, the Response to
the Public and Science Advisory Board Comments (a
summary of the major public comments, SAB
comments, and Agency responses to those
comments).
The SAB requested that the Agency develop a
technical support document for these Guidelines.
The SAB identified the need for this type of
document due to the limited knowledge on
interactions of chemicals in biological systems.
Because of this, the SAB commented that progress
in improving risk assessment will be particularly
dependent upon progress in the science of
interactions.
Agency staff have begun preliminary work on
the technical support document and expect it to be
completed by early 1987. The Agency is continuing
to study the risk assessment issues raised in the
guidelines and will revise these Guidelines in line
with new information as appropriate.
[51 FR 340151
References, supporting documents, and
comments received on the proposed guidelines, as
well as copies of the final guidelines, are available
for inspection and copying at the Public Information
Reference Unit (202-382-5926), EPA Headquarters
Library, 401 M Street, S.W., Washington, DC,
between the hours of 8:00 a.m. and 4:30 p.m.
I certify that these Guidelines are not major
rules as defined by Executive Order 12291, because
they are nonbinding policy statements and have no
direct effect on the regulated community. Therefore,
they will have no effect on costs or prices, and they
will have no other significant adverse effects on the
economy. These Guidelines were reviewed by the
Office of Management and Budget under Executive
Order 12291.
August 22,1986
Lee M. Thomas,
Administrator
CONTENTS
Part A: Guidelines for the Health Risk Assessment of
Chemical Mixtures
I. Introduction
II. Proposed Approach
A.Data Available on the Mixture of Concern
B .Data Available on Similar Mixtures
C.Data Available Only on Mixture Components
1. Systemic Toxicants
2. Carcinogens
3. Interactions
4. Uncertainties
a. Health Effects
b. Exposure Uncertainties
c. Uncertainties Regarding Composition of
the Mixture
///.Assumptions and Limitations
A,Information on Interactions
B.Additivity Models
IV. Mathematical Models and the Measurement of Joint Action
A.Dose Addition
B.Response Addition
C.Interactions
V, References
Part B: Response to Public and Science Advisory Board
Comments
/. Introduction
//.Recommended Procedures
A.Definitions
B.Mixtures of Carcinogens and Systemic Toxicants
///. Additivity Assumption
A.Complex Mixtures
B.Dose Additivity
C.lnterpretationof the Hazard Index
D.Use of Interaction Data
IV. Uncertainties and the Sufficiency of the Data Base
V. Need for a Technical Support Document
3-2
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^^m^^m
T
Part A: Guidelines for the Health Risk
Assessment of Chemical Mixtures
I. Introduction
The primary purpose of this document is to
generate a consistent Agency approach for
evaluating data on the chronic and subchronic
effects of chemical mixtures. It is a procedural guide
that emphasizes broad underlying principles of the
various science disciplines (toxicology,
pharmacology, statistics) necessary for assessing
health risk from chemical mixture exposure.
Approaches to be used with respect to the analysis
and evaluation of the various data are also
discussed.
It is not the intent of these Guidelines to
regulate any social or economic aspects concerning
risk of injury to human health or the environment
caused by exposure to a chemical agent(s). All such
action is addressed in specific statutes and federal.
legislation and is independent of these Guidelines.
While some potential environmental hazards
involve significant exposure to only a single
compound, most instances of environmental
contamination involve concurrent or sequential
exposures to a mixture of compounds that may
induce similar or dissimilar effects over exposure
periods ranging from short-term to lifetime. For the
purposes of these Guidelines, mixtures will be
defined as any combination of two or more chemical
substances regardless of source or of spatial or
temporal proximity. In some instances, the mixtures
are highly complex consisting of scores of compounds
that are generated simultaneously as by-products
from a single source or process (e.g., coke oven
emissions and diesel exhaust). In other cases,
complex mixtures of related compounds are
produced as commercial products (e.g., PCBs,
gasoline and pesticide formulations) and eventually
released to the environment. Another class of
mixtures consists of compounds, often unrelated
chemically or commercially, which are placed in the
same area for disposal or storage, eventually come
into contact with each other, and are released as a
mixture to the environment. The quality and
quantity of pertinent information available for risk
assessment varies considerably for different
mixtures. Occasionally, the chemical composition of
a mixture is well characterized, levels of exposure to
the population are known, and detailed toxicologic
data on the mixture are available. Most frequently,
not all components of the mixture are known,
exposure data are uncertain, and toxicologic data on
the known components of the mixture are limited.
Nonetheless, the Agency may be required to take
action because of the number of individuals at
potential risk or because of the known toxicologic
effects of these compounds that have been identified
in the mixture.
The prediction of how specific mixtures of
toxicants will interact must be based on an
understanding of the mechanisms of such
interactions. Most reviews and texts that discuss
toxicant interactions attempt to discuss the
biological or chemical bases of the interactions (e.g.,
Klaassen and Doull, 1980; Levine, 1973; Goldstein
et al., 1974; NRC, 1980a; Veldstra, 1956; Withey,
1981). Although different authors use somewhat
different classification schemes when discussing the
ways in which toxicants interact, it generally is
recognized that toxicant interactions may occur
during any of the toxicologic processes that take
place with a single compound: absorption,
distribution, metabolism, excretion, and activity at
the receptor site(s). Compounds may interact
chemically, yielding a new toxic component or
causing a change in the biological availability of the
existing component. They may also interact by
causing different effects at different receptor sites.
Because of the uncertainties inherent in
predicting the magnitude and nature of toxicant
interactions, the assessment of health risk from
chemical mixtures must include a thorough
discussion of all assumptions. No single approach is
recommended in these Guidelines. Instead,
guidance is given for the use of several approaches
depending on the nature and quality of the data.
Additional mathematical details are presented in
section IV.
In addition to these Guidelines, a supplemental
technical support document is being developed
which will contain a thorough review of all available
information on the toxicity of chemical mixtures and
a discussion of research needs.
//. Proposed Approach
No single approach can be recommended to risk
assessments for multiple chemical exposures.
Nonetheless, general guidelines can be
recommended depending on the type of mixture, the
known toxic effects of its components, the
availability of toxicity data on the mixture or
similar mixtures,
[51 FR 34016]
the known or
anticipated interactions among components of the
mixture, and the quality of the exposure data. Given
the complexity of this issue and the relative paucity
of empirical data from which sound generalizations
can be constructed, emphasis must be placed on
flexibility, judgment, and a clear articulation of the
assumptions and limitations in any risk assessment
that is developed. The proposed approach is
summarized in Table 1 and Figure 1 and is detailed
below. An alphanumeric scheme for ranking the
quality of the data used in the risk assessment is
given in Table 2.
3-3
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A. Data Available on the Mixture of Concern
For predicting the effects of subchronic or
chronic exposure to mixtures, the preferred
approach usually will be to use subchronic or chronic
health effects data on the mixture of concern and
adopt procedures similar to those used for single
compounds, either systemic toxicants or carcinogens
(see U.S. EPA, 1986a-1986c). The risk assessor must
recognize, however, that dose-response models used
for single compounds are often based on biological
mechanisms of the toxicity of single compounds, and
may not be as well justified when applied to the
mixture as a whole. Such data are most likely to be
available on highly complex mixtures, such as coke
oven emissions or diesel exhaust, which are
generated in large quantities and associated with or
suspected of causing adverse health effects.
Attention should also be given to the persistence of
the mixture in the environment as well as to the
variability of the mixture composition over time or
from different sources of emissions. If the
components of the mixture are known to partition
into different environmental compartments or to
degrade or transform at different rates in the
environment, then those factors must also be taken
into account, or the confidence in and applicability of
the risk assessment is diminished.
TABLE 1.-- RISK ASSESSMENT APPROACH FOR
CHEMICAL MIXTURES
1. Assess the quality of the data on interactions,
health effects, and exposure (see Table 2).
a. If adequate, proceed to Step 2.
b. If inadequate, proceed to Step 14.
2. Health effects information is available on the
chemical mixture of concern .
a. If yes, proceed to Step 3.
b. If no, proceed to Step 4.
3. Conduct risk assessment on the mixture of
concern based on health effects data on the mixture.
Use the same procedures as those for single
compounds. Proceed to Step 7 (optional) and Step 12.
4. Health effects information is available on a
mixture that is similar to the mixture of concern.
a. If yes, proceed to Step 5.
b. If no, proceed to Step 7.
5. Assess the similarity of the mixture on which
health effects data are available to the mixture of
concern, with emphasis on any differences in
components or proportions of components, as well as
the effects that such differences would have on
biological activity.
a. If sufficiently similar, proceed to Step 6.
b. If not sufficiently similar, proceed to Step 7.
6. Conduct risk assessment on the mixture of
concern based on health effects data on the similar
mixture. Use the same procedures as those for single
compounds. Proceed to Step 7 (optional) and Step 12.
7. Compile health effects and exposure
information on the components of the mixture.
8. Derive appropriate indices of acceptable
exposure and/or risk on the individual components
in the mixture. Proceed to Step 9.
9. Assess data on interactions of components in
the mixtures.
a. If sufficient quantitative data are available on
the interactions of two or more components in the
mixture, proceed to Step 10.
b. If sufficient quantitative data are not
available, use whatever information is available to
qualitatively indicate the nature of potential
interactions. Proceed to Step 11.
10. Use an appropriate interaction model to
combine risk assessments on compounds for which
data are adequate, and use an additivity assumption
for the remaining compounds. Proceed to Step 11
(optional) and Step 12.
11. Develop a risk assessment based on an
additivity approach for all compounds in the
mixture. Proceed to Step 12.
12. Compare risk assessments conducted in
Steps 5, 8, and 9. Identify and justify the preferred
assessment, and quantify uncertainty, if possible.
Proceed to Step 13.
13. Develop an integrated summary of the
qualitative and quantitative assessments with
special emphasis on uncertainties and assumptions.
Classify the overall quality of the risk assessment,
as indicated in Table 2. Stop.
14. No risk assessment can be conducted because
of inadequate data on interactions, health effects, or
exposure. Qualitatively assess the nature of any
potential hazard and detail the types of additional
data necessary to support a risk assessment. Stop.
Note. -- Several decisions used here, especially
those concerning adequacy of data and similarity
between two mixtures, are not precisely
characterized and will require considerable
judgment. See text.
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51 FR 34017
1. Assess quality of data using Table 2.
I. Interactions
II.Health Effects
III. Exposure
Adequate
2. Data on mixture of concern?
3. Risk assessment using data
on mixture of concern.
Inadequate
14. Qualitatively assess hazard.
No quantitative risk assessment.
1
ilar mixture?
Y
S.Mixtures sufficiently similar?
1 Y
N
N
1
lixture components
8. Indices of acceptability and
risk based on component data.
1
6. Risk assessment using data
on similar mixtures.
9.Sufficient information to
quantify interactions?
10.Risk assessment with interactions
quantified where appropriate.
Use additivity for all components.
Optional
11. Risk assessment using additivity
for all components.
12. Compare risk assessment from
steps 3,6,10,11 as appropriate.
Identify preferred assessment.
13. Develop integrated summary including
discussion of uncertainties.
Figure 1. Flow chart of the risk assessment approach in Table 1. Note that it may be desirable to conduct all three
assessments when possible (i.e., using data on the mixture, a similar mixture ,orthe components) in order to make
the fullest use of the available data. See text for further discussion.
3-5
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51 FR 34018
TABLE 2. -- CLASSIFICATION SCHEME FOR THE
QUALITY OF THE RISK ASSESSMENT OF THE
MIXTURE*
Information on Interactions
I. Assessment is based on data on the mixture of
concern.
II. Assessment is based on data on a sufficiently
similar mixture.
III. Quantitative interactions of components are
well characterized.
IV. The assumption of additivity is justified
based on the nature of the health effects and on the
number of component compounds.
V. An assumption of additivity cannot be
justified, and no quantitative risk assessment can be
conducted.
Health Effects Information
A. Full health effects data are available and
relatively minor extrapolation is required.
B. Full health effects data are available but
extensive extrapolation is required for route or
duration of exposure or for species differences. These
extrapolations are supported by pharmacokinetic
considerations, empirical observations, or other
relevant information.
C. Full health effects data are available, but
extensive extrapolation is required for route or
duration of exposure or for species differences. These
extrapolations are not directly supported by the
information available.
D. Certain important health effects data are
lacking and extensive extrapolations are required
for route or duration of exposure or for species
differences.
E. A lack of health effects information on the
mixture and its components in the mixture
precludes a quantitative risk assessment.
Exposure Information1'
1. Monitoring information either alone or in
combination with modeling information is sufficient
to accurately characterize human exposure to the
mixture or its components.
2. Modeling information is sufficient to
reasonably characterize human exposure to the
mixture or its components.
3. Exposure estimates for some components are
lacking, uncertain, or variable. Information on
health effects or environmental chemistry suggest
that this limitation is not likely to substantially
affect the risk assessment.
4. Not all components in the mixture have been
identified or levels of exposure are highly uncertain
or variable. Information on health effects or
environmental chemistry is not sufficient to assess
the effect of this limitation on the risk assessment.
5. The available exposure information is
insufficient for conducting a risk assessment.
B. Data Available on Similar Mixtures
If the risk assessment is based on data from a
single mixture that is known to be generated with
varying compositions depending on time or different
emission sources, then the confidence in the
applicability of the data to a risk assessment also is
diminished. This can be offset to some degree if data
are available on several mixtures of the same
components that have different component ratios
which encompass the temporal or spatial differences
in composition of the mixture of concern. If such data
are available, an attempt should be made to
determine if significant and systematic differences
exist among the chemical mixtures. If significant
differences are noted, ranges of risk can be estimated
based on the toxicologic data of the various
mixtures. If no significant differences are noted,
then a single risk assessment may be adequate,
although the range of ratios of the components in the
mixtures to which the risk assessment applies
should also be given.
If no data are available on the mixtures of
concern, but health effects data are available on a
similar mixture (i.e., a mixture having the same
components but in slightly different ratios, or
having several common components but lacking one
or more components, or having one or more
additional components), a decision must be made
whether the mixture on which health effects data
are available is or is not "sufficiently similar" to the
mixture of concern to permit a risk assessment. The
determination of "sufficient similarity" must be
made on a case-by-case basis, considering not only
the uncertainties associated with using data on a
dissimilar mixture but also the uncertainties of
using other approaches such as additivity. In
determining reasonable similarity, consideration
should be given to any information on the
components that differ or are contained in markedly
different proportions between the mixture on which
health effects data are available and the mixture of
concern. Particular emphasis should be placed on
any toxicologic or pharmacokinetic data on the
components or the mixtures which would be useful
in assessing the significance of any chemical
difference between the similar mixture and the
mixtures of concern.
Even if a risk assessment can be made using
data on the mixtures of concern or a reasonably
similar mixture, it may be desirable to conduct a
risk assessment based on toxicity data on the
a See text for discussion of sufficient similarity,
adequacy of data, and justification for additivity
assumptions.
b See the Agency's Guidelines for Estimating
Exposures (U.S. EPA, 1986d) for more complete
information on performing exposure assessments
and evaluating the quality of exposure data.
3-6
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i
ft
components in the mixture using the procedure
outlined in section II.B. In the case of a mixture
containing carcinogens and toxicants, an approach
based on the mixture data alone may not be
sufficiently protective in all cases. For example, this
approach for a two-component mixture of one
carcinogen and one toxicant would use toxicity data
on the mixture of the two compounds. However, in a
chronic study of such a mixture, the presence of the
toxicant could mask the activity of the carcinogen.
That is to say, at doses of the mixture sufficient to
induce a carcinogenic effect, the toxicant could
induce mortality so that at the maximum tolerated
dose of the mixture, no carcinogenic effect could be
observed. Since carcinogenicity is considered by the
Agency to be a nonthreshold effect, it may not be
prudent to construe the negative results of such a
bioassay as indicating the absence of risk at lower
doses. Consequently, the mixture approach should
be modified to allow the risk assessor to evaluate the
potential for masking, of one effect by another, on a
case-by-case basis.
C. Data Available Only on Mixture Components
If data are not available on an identical or
reasonably similar mixture, the risk assessment
may be based on the toxic or carcinogenic properties
of the components in the mixture. When little or no
quantitative information is available on the
potential interaction among the components,
additive models (defined in the next section) are
recommended for systemic toxicants. Several studies
have demonstrated that dose additive models often
predict reasonably well the toxicities of mixtures
composed of a substantial variety of both similar and
dissimilar compounds (Pozzani el al., 1959; Smyth et
al., 1969, 1970; Murphy, 1980). The problem of
multiple toxicant exposure has been addressed by
the American Conference of Governmental
Industrial Hygienists (ACGIH, 1983), the
Occupational Safety and Health Administration
(OSHA, 1983), the World Health Organization
(WHO, 1981), and the National Research Council
(NRC, 1980a, b). Although the focus and purpose of
each group was somewhat different, all groups that
recommended an approach elected to adopt some
type of dose additive model. Nonetheless, as
discussed in section IV, dose additive models are not
the most biologically plausible approach if the
compounds do not have the same mode of toxicologic
action. Consequently, depending on the nature of the
risk assessment and the available information on
modes of action and patterns of joint action, the
[51 PR 34019]
most reasonable
additive model should be used.
1. Systemic Toxicants. For systemic toxicants,
the current risk assessment methodology used by
the Agency for single compounds most often results
in the derivation of an exposure level which is not
anticipated to cause significant adverse effects.
Depending on the route of exposure, media of
concern, and the legislative mandate guiding the
risk assessments, these exposure levels may be
expressed in a variety of ways such as acceptable
daily intakes (ADIs) or reference doses (RfDs), levels
associated with various margins of safety (MOS), or
acceptable concentrations in various media. For the
purpose of this discussion, the term "acceptable
level" (AL) will be used to indicate any such criteria
or advisories derived by the Agency. Levels of
exposure (E) will be estimates obtained following
the most current Agency Guidelines for Estimating
Exposures (U.S. EPA, 1986d). For such estimates,
the "hazard index" (HI) of a mixture based on the
assumption of dose addition may be defined as:
HI =
where:
+ E2/AL2 +...+
(H-l)
Ej = exposure level to the ith toxicant* and
ALj = maximum acceptable level for the ith
toxicant.
Since the assumption of dose addition is most
properly applied to compounds that induce the same
effect by similar modes of action, a separate hazard
index should be generated for each end point of
concern. Dose addition for dissimilar effects does not
have strong scientific support, and, if done, should
be justified on a case-by-case basis in terms of
biological plausibility.
The assumption of dose addition is most clearly
justified when the mechanisms of action of the
compounds under consideration are known to be the
same. Since the mechanisms of action for most
compounds are not well understood, the justification
of the assumption of dose addition will often be
limited to similarities in pharmacokinetic and
toxicologic characteristics. In any event, if a hazard
index is generated, the quality of the experimental
evidence supporting the assumption of dose addition
must be clearly articulated.
The hazard index provides a rough measure of
likely toxicity and requires cautious interpretation.
The hazard index is only a numerical indication of
the nearness to acceptable limits of exposure or the
degree to which acceptable exposure levels are
exceeded. As this index approaches unity, concern
for the potential hazard of the mixture increases. If
the index exceeds unity, the concern is the same as if
an individual chemical exposure exceeded its
acceptable level by the same proportion. The hazard
index does not define dose-response relationships,
and its numerical value should not be construed to
be a direct estimate of risk. Nonetheless, if sufficient
data are available to derive individual acceptable
levels for a spectrum of effects (e.g., MFO induction,
*See the Agency's guidelines (U.S. EPA, 1986d) for
information on how to estimate this value.
3-7
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variabilities of the acceptable levels are known, or if
the acceptable levels are given as ranges (e.g.,
associated with different margins of safety), then the
hazard index should be presented with
corresponding estimates of variation or range. Most
studies on systemic toxicity report only descriptions
of the effects in each dose group. If dose-response
curves are estimated for systemic toxicants,
however, dose-additive or response-additive
assumptions can be used, with preference given to
the most biologically plausible assumption (see
section IV for the mathematical details).
2. Carcinogens. For carcinogens, whenever
linearity of the individual dose-response curves has
been assumed (usually restricted to low doses), the
increase in risk P (also called excess or incremental
risk), caused by exposure d, is related to
carcinogenic potency B, as:
P = dB
(H-2)
For multiple compounds, this equation may be
generalized to:
P =
(H-3)
This equation assumes independence of action by
the several carcinogens and is equivalent to the
assumption of dose addition as well as to response
addition with completely negative correlation of
tolerance, as long as P < 1 (see section IV).
Analogous to the procedure used in equation II-1 for
systemic toxicants, an index for n carcinogens can be
developed by dividing exposure levels (E) by doses
(DR) associated with a set level of risk:
HI = Ei/DR1 + E2/DR2+...-l-En/DRn (II-4)
Note that the less linear the dose-response curve is,
the less appropriate equations H-3 and II-4 will be,
perhaps even at low doses. It should be emphasized
that because of the uncertainties in estimating dose-
response relationships for single compounds, and the
additional uncertainties in combining the individual
estimate to assess response from exposure to
mixtures, response rates and hazard indices may.
have merit in comparing risks but should not be
regarded as measures of absolute risk.
3.Interactions. None of the above equations
incorporates any form of synergistic or antagonistic
interaction. Some types of information, however,
may be available that suggest that two or more
components in the mixture may interact. Such
information must be assessed in terms of both its
relevance to subchronic or chronic hazard and its
suitability for quantitatively altering the risk
assessment.
For example, if chronic or subehronic toxieity or
carcinogenicity studies have been conducted that
permit a quantitative estimation of interaction for
two chemicals, then it may be desirable to consider
using equations detailed in section IV, or
modifications of these equations, to treat the two
compounds as a single toxicant with greater or
lesser potency than would be predicted from
additivity. Other components of the mixture, on
which no such interaction data are available, could
then be separately treated in an additive manner.
Before such a procedure is adopted, however, a
discussion should be presented of the likelihood that
other compounds in the mixture may interfere with
the interaction of the two toxicants on which
quantitative interaction data are available. If the
weight of evidence suggests that interference is
likely, then a quantitative alteration of the risk
assessment may not be justified. In such cases, the
risk assessment may only indicate the likely nature
of interactions, either synergistic or antagonistic,
and not quantify their magnitudes.
Other types of information, such as those
relating to mechanisms of toxicant interaction, or
quantitative estimates of interaction between two
chemicals derived from acute studies, are even less
likely to be of use in the quantitative assessment of
long-term health risks. Usually it will be
appropriate only to discuss these types of
information, indicate the relevance of the
information to subchronic or chronic exposure, and
indicate, if possible, the nature of potential
interactions, without attempting to quantify their
magnitudes.
When the interactions are expected to have a
minor influence on the mixture's toxicity, the
assessment should indicate, when possible, the
compounds most responsible for the predicted
toxicity. This judgment should be based on predicted
toxicity of each component,
[51 PR 34020]
based on
exposure and toxic or carcinogenic potential. This
potential alone should not be used as an indicator of
the chemicals posing the most hazard.
4.Uncertainties. For each risk assessment, the
uncertainties should be clearly discussed and the
overall quality of the risk assessment should be
characterized. The scheme outlined in Table 2
should be used to express the degree of confidence in
the quality of the data on interaction, health effects,
and exposure.
a. Health EffectsIn some cases, when health
effects data are incomplete, it may be possible to
argue by analogy or quantitative structure-activity
relationships that the compounds on which no
health effects data are available are not likely to
significantly affect the toxicity of the mixture. If a
risk assessment includes such an argument, the
limitations of the approach must be clearly
articulated. Since a methodology has not been
adopted for estimating an acceptable level (e.g.,
ADI) or carcinogenic potential for single compounds
based either on quantitative structure-activity
relationships or on the results of short-term
screening tests, such methods are not at present
3-8
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recommended as the sole basis of a risk assessment
on chemical mixtures.
b. Exposure UncertaintiesThe general
uncertainties in exposure assessment have been
addressed in the Agency's Guidelines for Estimating
Exposures (U.S. EPA, 1986d). The risk assessor
should discuss these exposure uncertainties in terms
of the strength of the evidence used to quantify the
exposure. When appropriate, the assessor should
also compare monitoring and modeling data and
discuss any inconsistencies as a source of
uncertainty. For mixtures, these uncertainties may
be increased as the number of compounds of concern
increases.
If levels of exposure to certain compounds known
to be in the mixture are not available, but
information on health effects and environmental
persistence and transport suggest that these
compounds are not likely to be significant in
affecting the toxicity of the mixture, then a risk
assessment can be conducted based on the
remaining compounds in the mixture, with
appropriate caveats. If such an argument cannot be
supported, no final risk assessment can be
performed until adequate monitoring data are
available. As an interim procedure, a risk
assessment may be conducted for those components
in the mixture for which adequate exposure and
health effects data are available. If the interim risk
assessment does not suggest a hazard, there is still
concern about the risk from such a mixture because
not all components in the mixture have been
considered.
c. Uncertainties Regarding Composition of the
MixtureIn perhaps a worst case scenario,
information may be lacking not only on health
effects and levels of exposure, but also on the
identity of some components of the mixture.
Analogous to the procedure described in the previous
paragraph, an interim risk assessment can be
conducted on those components of the mixture for
which adequate health effects and exposure
information are available. If the risk is considered
unacceptable, a conservative approach is to present
the quantitative estimates of risk, along with
appropriate qualifications regarding the
incompleteness of the data. If no hazard is indicated
by this partial assessment, the risk assessment
should not be quantified until better health effects
and monitoring data are available to adequately
characterize the mixture exposure and potential
hazards.
777. Assumptions and Limitations
A. Information on Interactions
Most of the data available on toxicant
interactions are derived from acute toxicity studies
using experimental animals in which mixtures of
two compounds were tested, often in only a single
combination. Major areas of uncertainty with the
use of such data involve the appropriateness of
interaction data from an acute toxicity study for
quantitatively altering a risk assessment for
subchronic or chronic exposure, the appropriateness
of interaction data on two component mixtures for
quantitatively altering a risk assessment on a
mixture of several compounds, and the accuracy of
interaction data on experimental animals for
quantitatively predicting interactions in humans.
The use of interaction data from acute toxicity
studies to assess the potential interactions on
chronic exposure is highly questionable unless the
mechanism(s) of the interaction on acute exposure
were known to apply to low-dose chronic exposure.
Most known biological mechanisms for toxicant
interactions, however, involve some form of
competition between the chemicals or phenomena
involving saturation of a receptor site or metabolic
pathway. As the doses of the toxicants are decreased,
it is likely that these mechanisms either no longer
will exert a significant effect or will be decreased to
an extent that cannot be measured or approximated.
The use of information from two-component
mixtures to assess the interactions in a mixture
containing more than two compounds also is
questionable from a mechanistic perspective. For
example, if two compounds are known to interact,
either synergistically or antagonistically, because of
the effects of one compound on the metabolism or
excretion of the other, the addition of a third
compound which either chemically alters or affects
the absorption of one of the first two compounds
could substantially alter the degree of the toxicologic
interaction. Usually, detailed studies quantifying
toxicant interactions are not available on
multicomponent mixtures, and the few studies that
are available on such mixtures (e.g., Gullino et al.,
1956) do not provide sufficient information to assess
the effects of interactive interference.
Concerns with the use of interaction data on
experimental mammals to assess interactions in
humans is based on the increasing appreciation for
systematic differences among species in their
response to individual chemicals. If systematic
differences in toxic sensitivity to single chemicals
exist among species, then it seems reasonable to
suggest that the magnitude of toxicant interactions
among species also may vary in a systematic
manner. Consequently, even if excellent chronic
data are available on the magnitude of toxicant
interactions in a species of experimental mammal,
there is uncertainty that the magnitude of the
interaction will be the same in humans. Again, data
are not available to properly assess the significance
of this uncertainty.
Last, it should be emphasized that none of the
models for toxicant interaction can predict the
magnitude of toxicant interactions in the absence of
extensive data. If sufficient data are available to
3-9
-------
estimate interaction coefficients as described in
section IV, then the magnitude of the toxicant
interactions for various proportions of the same
components can be predicted. The availability of an
interaction ratio (observed response divided by
predicted response) is useful only in assessing the
magnitude of the toxicant interaction for the specific
proportions of the mixture which was used to
generate the interaction ratio.
The basic assumption in the recommended
approach is that risk assessments on chemical
mixtures are best conducted using toxicologic data
on the mixture of concern or a reasonably similar
mixture. While such risk
[51 FR 34021}
assessments do
not formally consider toxicologic interactions as part
of a mathematical model, it is assumed that
responses in experimental mammals or human
populations noted after exposure to the chemical
mixture can be used to conduct risk assessments on
human populations. In bioassays of chemical
mixtures using experimental mammals, the same
limitations inherent in species-to-species
extrapolation for single compounds apply to
mixtures. When using health effects data on
chemical mixtures from studies on exposed human
populations, the limitations of epidemiologic studies
in the risk assessment of single compounds also
apply to mixtures. Additional limitations may be
involved when using health effects data on chemical
mixtures if the components in the mixture are not
constant or if the components partition in the
environment.
B. Additivity Models
If sufficient data are not available on the effects
of the chemical mixture of concern or a reasonably
similar mixture, the proposed approach is £o assume
additivity. Dose additivity is based on the
assumption that the components in the mixture
have the same mode of action and elicit the same
effects. This assumption will not hold true in most
cases, at least for mixtures of systemic toxicants. For
systemic toxicants, however, most single compound
risk assessments will result in the derivation of
acceptable levels, which, as currently defined,
cannot be adapted to the different forms of response
additivity as described in section IV.
Additivity models can be modified to incorporate
quantitative data on toxicant interactions from
subchronic or chronic studies using the models given
in section IV or modifications of these models. If this
approach is taken, however, it will be under the
assumption that other components in the mixture do
not interfere with the measured interaction. In
practice, such subchronic or chronic interactions
data seldom will be available. Consequently, most
risk assessments (on mixtures) will be based on an
assumption of additivity, as long as the components
elicit similar effects.
Dose-additive and response-additive
assumptions can lead to substantial errors in risk
estimates if synergistic or antagonistic interactions
occur. Although dose additivity has been shown to
predict the acute toxicities of many mixtures of
similar and dissimilar compounds (e.g., Pozzani et
al., 1959; Smyth et al., 1969, 1970; Murphy, 1980),
some marked exceptions have been noted. For
example, Smyth et al. (1970) tested the interaction
of 53 pairs of industrial chemicals based on acute
lethality in rats. For most pairs of compounds, the
ratio of the predicted LDso to observed LDso did not
vary by more than a factor of 2. The greatest
variation was seen with an equivolume mixture of
morpholine and toluene, in which the observed LDso
was about five times less than the LDso predicted by
dose addition. In a study by Hammond et al. (1979),
the relative risk of lung cancer attributable to
smoking was 11, while the relative risk associated
with asbestos exposure was 5. The relative risk of
lung cancer from both smoking and asbestos
exposure was 53, indicating a substantial
synergistic effect. Consequently, in some cases,
additivity assumptions may substantially
underestimate risk. In other cases, risk may be
overestimated. While this is certainly an
unsatisfactory situation, the available data on
mixtures are insufficient for estimating the
magnitude of these errors. Based on current
information, additivity assumptions are expected to
yield generally neutral risk estimates (i.e., neither
conservative nor lenient) and are plausible for
component compounds that induce similar types of
effects at the same sites of action.
TV. Mathematical Models and the Measurement of
Joint Action
The simplest mathematical models for joint
action assume no interaction in any mathematical
sense. They describe either dose addition or response
addition and are motivated by data on acute lethal
effects of mixtures of two compounds.
A. Dose Addition
Dose addition assumes that the toxicants in a
mixture behave as if they were dilutions or
concentrations of each other, thus the true slopes of
the dose-response curves for the individual
compounds are identical, and the response elicited
by the mixture can be predicted by summing the
individual doses after adjusting for differences in
potency; this is defined as the ratio of equitoxic
doses, Probit transformation typically makes this
ratio constant at all doses when parallel straight
lines are obtained. Although this assumption can be
applied to any model (e.g., the one-hit model in NRC,
1980b), it has been most often used in toxicology
with the log-dose probit response model, which will
be used to illustrate the assumption of dose addition.
3-10
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Suppose that two toxicants show the following log-
dose probit response equations:
Y2= l."2 + 31ogZ2 (IV-2)
where Yj is the probit response associated with a
dose of Z, (i = 1,2). The potency, p, of toxicant #2 with
respect to toxicant #1 is defined by the quantity
7,1/7,2 when Yi = Y2 (that is what is meant by
equitoxic doses). In this example, the potency, p, is
approximately 2. Dose addition assumes that the
response, Y, to any mixture of these two toxicants
can be predicted by:
= 0.3 + 31og(Zl + pZ2)
(IV-3)
Thus, since p is defined as Zi/Z2, equation IV-3
essentially converts Z2 into an equivalent dose of Z
by adjusting for the difference in potency. A more
generalized form of this equation for any number of
toxicants is:
Y = ai + b log(fi 4- S f;
where:
+ blogZ (IV-4)
ai = the y-intercept of the dose-response equation
for toxicant #1
b = the slope of the dose-response lines for the
toxicants
fi = the proportion of the ith toxicant in the mixture
Pi = the potency of the ith toxicant with respect to
toxicant #1 (i.e., Zi/Zi), and
Z = the sum of the individual doses in the mixture.
A more detailed discussion of the derivation of the
equations for dose addition is presented by Finney
(1971).
B. Response Addition
The other form of additivity is referred to as
response addition. As detailed by Bliss (1939), this
type of joint action assumes that the two toxicants
act on different receptor systems and that the
correlation of individual tolerances may range from
completely negative (r = -l) to completely positive
(r=+l). Response addition assumes that the
response to a given concentration of a mixture of
toxicants is completely determined by the responses
to the components and the pairwise correlation
coefficient. Taking P as the proportion of organisms
responding to a mixture of two toxicants which
evoke individual responses of PI and P2, then
P = PI if r = 1 and PI S: P2
P = P2 if r = 1 and P! < P2
P = Pl + P2(l-P1)ifr = 0
= -landP<;
(IV-5)
(IV-6)
(IV-7)
(IV-8)
More generalized mathematical models for this form
of joint action have been given by Plackett and
Hewlett (1948).
C. Interactions
All of the above models assume no interactions
and therefore do not incorporate measurements of
synergistic or antagonistic effects. For measuring
toxicant interactions for mixtures of two compounds,
Finney (1942) proposed the
[51 FR 34022]
following
modification of equation IV-4 for dose addition:
Y = ai + b logto + pf2 + K[pfif2]0-5) + b logZ
(IV-9)
where ai, b, fi, fa, p, and Z are defined as before, and
K is the coefficient of interaction. A positive value of
K indicates synergism, a negative value indicates
antagonism, and a value of zero corresponds to dose
addition as in equation IV-4. Like other proposed
modifications of dose addition (Hewlett, 1969), the
equation assumes a consistent interaction
throughout the entire range of proportions of
individual components. To account for such
asymmetric patterns of interaction as those observed
by Alstott et al. (1973), Durkin (1981) proposed the
following modification to equation IV-9:
Y = ai + b log(fi + Pf2 + Kif1[pfif2]0-5
+ K2f2[pfif2]0-5) + b log Z (IV-10)
in which K(pfif2)°-5 is divided into two components,
Kifi(pfif2)0-5 and K2f2(pf!f2)0.5. Since KI and K2
need not have the same sign, apparent instances of
antagonism at one receptor site and synergism at
another receptor site can be estimated. When KI and
K2 are equal, equation IV-10 reduces to equation IV-
9. It should be noted that to obtain a reasonable
number of degrees of freedom in the estimation of K
in equation IV-9 or KI and K2 in equation IV-10, the
toxicity of several different combinations of the two
components must be assayed along with assays of
the toxicity of the individual components. Since this
requires experiments with large numbers of
animals, such analyses have been restricted for the
most part to data from acute bioassays using insects
(e.g., Finney, 1971) or aquatic organisms (Durkin,
1979). Also, because of the complexity of
experimental design and the need for large numbers
of animals, neither equation IV-9 nor equation IV-
10 has been generalized or applied to mixtures of
more than two toxicants. Modifications of response-
additive models to include interactive terms have
also been proposed, along with appropriate
statistical tests for the assumption of additivity
(Korn and Liu, 1983; Wahrendorf et al., 1981).
In the epidemiologic literature, measurements
of the extent of toxicant interactions, S, can be
expressed as the ratio of observed relative risk to
relative risk predicted by some form of additivity
assumption. Analogous to the ratio of interaction in
classical toxicology studies, S = 1 indicates no
interaction, S > 1 indicates synergism, and S < 1
indicates antagonism. Several models for both
3-11
-------
additive and multiplicative risks have been
proposed (e.g., Hogan et al., 1978; NRC, 1980b;
Walter, 1976). For instance, Rothman (1976) has
discussed the use of the following measurement of
toxicant interaction based on the assumption of risk
additivity:
where RJQ is the relative risk from compound #1 in
the absence of compound #2, RQJ is the relative risk
from compound #2 in the absence of compound #1,
and RH is the relative risk from exposure to both
compounds. A multiplicative risk model adapted
from Walter and Holford (1978, equation 4) can be
stated as:
S 13 /{T} O \ f t\T 1 O\
JtviiAK}Qiv0i/ ilv-lAJ
As discussed by both Walter and Holford (1978) and
Rothman (1976), the risk-additive model is
generally applied to agents causing diseases while
the multiplicative model is more appropriate to
agents that prevent disease. The relative merits of
these and other indices have been the subject of
considerable discussion in the epidemiologic
literature (Hogan et al., 1978; Kupper and Hogan,
1978; Rothman, 1978; Rothman et al., 1980; Walter
and Holford, 1978). There seems to be a consensus
that for public health concerns regarding causative
(toxic) agents, the additive model is more
appropriate.
Both the additive and multiplicative models
assume statistical independence in that the risk
associated with exposure to both compounds in
combination can be predicted by the risks associated
with separate exposure to the individual compounds.
As illustrated by Siemiatycki and Thomas (1981) for
multistage carcinogenesis, the better fitting
statistical model will depend not only upon actual
biological interactions, but also upon the stages of
the disease process which the compounds affect.
Consequently, there is no a priori basis for selecting
either type of model in a risk assessment. As
discussed by Stara et al. (1983), the concepts of
multistage carcinogenesis and the effects of
promoters and cocarcinogens on risk are extremely
complex issues. Although risk models for promoters
have been proposed (e.g., Burns et al., 1983), no
single approach can be recommended at this time.
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tosicities of 1-methylxanthine, ethanol, and 1-
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Bliss, C.I. 1939. The toxicity of poisons applied jointly. Ann. Appl.
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Burns, F., R. Albert, E. Altschuler, and E. Morris. 1983. Approach
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Durkin, P.B. 1979. Spent chtoruiation liquor and chlorophenotics:
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Finney, D.J. 1971. Probit analysis, 3rd ed. Cambridge, Great
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Goldstein, A., L, Aronow, and S.M. Kalman. 1974. Principles of
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Gullino, P., M. Winitz, S.M. Birnbaum, J. Cornfield, M.C. Otey,
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Hammond, E.G., I.V. Selikoff.and H.Seidman. 1979. Asbestos
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mixtures. Biometrics. 25:477-487.
Hogan, M.D., L. Kupper, B. Most, and J. Haseman. 1978.
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Klaassen, C.D., and J. Doull. 1980. Evaluation of safety:
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Levine, R.E. 1973. Pharmacology: drug actions and reactions.
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Murphy, S.D. 1980. Assessment of the potential for toxic
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NRC(National Research Council). 1980b. Principles of
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OSHA (Occupational Safety and Health Administration). 1983.
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Rothman, K., S. Greenland, and A. Walker. 1980. Concepts of
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Siemiatycki, J., and D.C. Thomas. 1981. Biological models and
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Smyth, H.F., C.S. Weil, J.S. West, and C.P. Carpenter. 1969. An
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j equivolume mixtures. Toxicol. Appl. Pharmacol. 17:498-503.
Stara, J.P., D. Mukerjee, R. McGaughy, P. Durkin, and M.L.
' Dourson. 1983. The current use of studies on promoters and
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reference to the combination of structural analogues.
Pharmacol. Rev. 8:339-387.
Wahrendorf, J., R. Zentgrof, and C.C. Brown. 1981.
Optimal designs for the analysis of interactive effects of two
| carcinogens or other toxicants. Biometrics. 37:45-54.
Walter, S.D. 1976. The estimation and interpretation of
! attributable risk in health research. Biometrics. 32:829-849.
Walter, S.D., and T.R. Holford. 1978. Additive,multiplicative,
I and other models for disease risks. Am. J. Epidemiol.
j 108:341-346.
4 Withey, J.R. 1981. Toxicodynamics and biotransformation. In:
i| International Workshop on the Assessmentof Multichemical
Contamination.Milan, Italy. (Draft copy courtesy of J.R.
Withey)
WHO (World Health Organization). 1981. Health effects of
combined exposures in the work environment. WHO Tech.
Report Series No. 662.
Part B: Response to Public and Science
Advisory Board Comments
/. Introduction
This section summarizes some of the major
issues raised in public comments on the Proposed
Guidelines for the Health Risk Assessment of
Chemical Mixtures published on January 9, 1985
(50 FR 1170). Comments were received from 14
individuals or organizations. An issue paper
reflecting public and external review comments was
presented to the Chemical Mixtures Guidelines
Panel of the Science Advisory Board (SAB) on March
4, 1985. At its April 22-23, 1985, meeting, the SAB
Panel provided the Agency with additional
suggestions and recommendations concerning the
Guidelines. This section also summarizes the issues
raised by the SAB.
The SAB and public commentors expressed
diverse opinions and addressed issues from a variety
of perspectives. In response to comments, the Agency
has modified or clarified many sections of the
Guidelines, and is planning to develop a technical
support document in line with the SAB
recommendations. The discussion that follows
highlights significant issues raised in the comments,
and the Agency's response to them. Also, many
minor recommendations, which do not warrant
discussion here, were adopted by the Agency.
//. Recommended Procedures
A. Definitions
Several comments were received concerning the
lack of definitions for certain key items and the
general understandability of certain sections.
Definitions have been rewritten for several terms
and the text has been significantly rewritten to
clarify the Agency's intent and meaning.
Several commentors noted the lack of a precise
definition of "mixture," even though several classes
of mixtures are discussed. In the field of chemistry,
the term "mixture" is usually differentiated from
true solutions, with the former defined as
nonhomogeneous multicomponent systems. For
these Guidelines, the term "mixture" is defined as
"...any combination of two or more chemicals
regardless of spatial or temporal homogeneity of
source" (section 1). These Guidelines are intended to
cover risk assessments for any situation where the
population is exposed or potentially exposed to two
or more compounds of concern. Consequently, the
introduction has been revised to clarify the intended
breadth of application.
Several commentors expressed concern that
"sufficient similarity" was difficult to define and
that the Guidelines should give more details
concerning similar mixtures. The Agency agrees
and is planning research projects to improve on the
definition. Characteristics such as composition and
toxic end-effects are certainly important, but the
best indicators of similarity in terms of risk
assessment have yet to be determined. The
discussion in the Guidelines emphasizes case-by-
case judgment until the necessary research can be
performed. The Agency considered but rejected
adding an example, because it is not likely that any
single example would be adequate to illustrate the
variety in the data and types of judgments that will
be required in applying this concept. Inclusion of
examples is being considered for the technical
support document.
B. Mixtures of Carcinogens and Systemic Toxicants
The applicability of the preferred approach for a
mixture of carcinogens and systemic
(noncarcinogenic) toxicants was a concern of several
public commentors as well as the SAB. The Agency
realizes that the preferred approach of using test
data on the mixture itself may not be sufficiently
protective in all cases. For example, take a simple
two-component mixture of one carcinogen and one
toxicant. The preferred approach would lead to using
toxicity data on the mixture of the two compounds.
However, it is possible to set the proportions of each
component so that in a chronic bioassay of such a
mixture, the presence of the toxicant could mask the
activity of the carcinogen. That is to say, at doses of
3-13
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the mixture sufficient for the carcinogen to induce
tumors in the small
[51FR34024]
exper imenta 1
group, the toxicant could induce mortality. At a
lower dose in the same study, no adverse effects
would be observed, including no carcinogenic effects.
The data would then suggest use of a threshold
approach. Since carcinogenicity is considered by the
Agency to be a nonthreshold effect, it may not be
prudent to construe the negative results of such a
bioassay as indicating the absence of risk at lower
doses. Consequently, the Agency has revised the
discussion of the preferred approach to allow the risk
assessor to evaluate the potential for masking of
carcinogenicity or other effects on a case-by-case
basis. Another difficulty occurs with such a mixture
when the risk assessment needs to be based on data
for the mixture components. Carcinogens and
systemic toxicants are evaluated by the Agency
using different approaches and generally are
described by different types of data: response rates
for carcinogens vs. effect descriptions for toxicants.
The Agency recognizes this difficulty and
recommends research to develop a new assessment
model for combining these dissimilar data sets into
one risk estimate. One suggestion in the interim is
to present separate risk estimates for the dissimilar
end points, including carcinogenic, teratogenic,
mutagenic, and systemic toxicant components,
///. Additivity Assumption
Numerous comments were received concerning
the assumption of additivity, including:
a. the applicability of additivity to "complex"
mixtures;
b. the use of dose additivity for compounds that
induce different effects;
c. the interpretation of the Hazard Index; and
d. the use of interaction data.
Parts of the discussion in the proposed guidelines
concerning the use of additivity assumptions were
vague and have been revised in the final Guidelines
to clarify the Agency's intent and position.
A. Complex Mixtures
The issue of the applicability of an assumption of
additivity to complex mixtures containing tens or
hundreds of components was raised in several of the
public comments. The Agency and its reviewers
agree that as the number of compounds in the
mixture increases, an assumption of additivity will
become less reliable in estimating risk. This is based
on the fact that each component estimate of risk or
an acceptable level is associated with some error and
uncertainty. With current knowledge, the
uncertainly will increase as the number of
components increases. In any event, little
experimental data are available to determine the
general change in the error as the mixture contains
more components. The Agency has decided that a
limit to the number of components should not be set
in these Guidelines. However, the Guidelines do
explicitly state that as the number of compounds in
the mixture increases, the uncertainty associated
with the risk assessment is also likely to increase.
B. Dose Additivity
Commentors were concerned about what
appeared to be a recommendation of the use of dose
additivity for compounds that induce different
effects. The discussion following the dose additivity
equation was clarified to indicate that the act of
combining all compounds, even if they induce
dissimilar effects, is a screening procedure and not
the preferred procedure in developing a hazard
index. The Guidelines were further clarified to state
that dose (or response) additivity is theoretically
sound, and therefore best applied for assessing
mixtures of similar acting components that do not
interact.
C. Interpretation of the Hazard Index
Several comments addressed the potential for
misinterpretation of the hazard index, and some
questioned its validity, suggesting that it mixes
science and value judgments by using "acceptable"
levels in the calculation. The Agency agrees with
the possible confusion regarding its use and has
revised the Guidelines for clarification. The hazard
index is an easily derived restatement of dose
additivity, and is, therefore, most accurate when
used with mixture components that have similar
toxic action. When used with components of
unknown or dissimilar action, the hazard index is
less accurate and should be interpreted only as a
rough indication of concern. As with dose addition,
the uncertainty associated with the hazard index
increases as the number of components increases, so
that it is less appropriate for evaluating the toxicity
of complex mixtures.
D. Use of Interaction Data
A few commentors suggested that any
interaction data should be used to quantitatively
alter the risk assessment. The Agency disagrees.
The current information on interactions is meager,
with only a few studies comparing response to the
mixture with that predicted by studies on
components. Additional uncertainties include
exposure variations due to changes in composition,
mixture dose, and species differences in the extent of
the interaction. The Agency is constructing an
interaction data base in an attempt to answer some
of these issues. Other comments concerned the use of
different types of interaction data. The Guidelines
restrict the use of interaction data to that obtained
from whole animal bioassays of a duration
appropriate to the risk assessment. Since such data
are frequently lacking, at least for chronic or
subchronic effects, the issue is whether to allow for
the use of other information such as acute data, in
3-14
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vitro data, or structure-activity relationships to
quantitatively alter the risk assessment, perhaps by
use of a safety factor. The Agency believes that
sufficient scientific support does not exist for the use
of such data in any but a qualitative discussion of
possible synergistic or antagonistic effects.
TV. Uncertainties and the Sufficiency of the Data
Base
In the last two paragraphs of section II of the
Guidelines, situations are discussed in which the
risk assessor is presented with incomplete toxicity,
monitoring, or exposure data. The SAB, as well as
several public commentors, recommended that the
"risk management" tone of this section be modified
and that the option of the risk assessor to decline to
conduct a risk assessment be made more explicit.
This is a difficult issue that must consider not
only the quality of the available data for risk
assessment, but also the needs of the Agency in risk
management. Given the types of poor data often
available, the risk assessor may indicate that the
risk assessment is based on limited information and
thus contains no quantification of risk. Nonetheless,
in any risk assessment, substantial uncertainties
exist. It is the obligation of the risk assessor to
provide an assessment, but also to ensure that all
the assumptions and uncertainties are articulated
clearly and quantified whenever possible.
The SAB articulated several other
recommendations related to uncertainties, all of
which have been followed in the revision of the
Guidelines. One recommendation was that the
summary procedure table also be presented as a flow
chart so that all options are clearly displayed. The
SAB further recommended the development of a
system to express the level of confidence in the
various steps of the risk assessment.
The Agency has revised the summary table to
present four major options: risk assessment using
data on the mixture
[51FR34025]
itself, data on a'
similar mixture, data on the mixture's components,
or declining to quantify the risk when the data are
inadequate. A flow chart of this table has also been
added to more clearly depict the various options and
to suggest the combining of the several options to
indicate the variability and uncertainties in the risk
assessment.
To determine the adequacy of the data, the SAB
also recommended the development of a system to
express the level of confidence associated with
various steps in the risk assessment process. The
Agency has developed a rating scheme to describe
data quality in three areas: interaction, health
effects, and exposure. This classification provides a
range of five levels of data quality for each of the
three areas. Choosing the last level in any area
results in declining to perform a quantitative risk
assessment due to inadequate data. These last
levels are described as follows:
Interactions:
An assumption of additivity cannot be justified,
and no quantitative risk assessment can be
conducted.
Health effects:
A lack of health effects information on the
mixture and its components precludes a
quantitative risk assessment.
Exposure:
The available exposure information is
insufficient for conducting a risk assessment.
Several commentors, including the SAB,
emphasized the importance of not losing these
classifications and uncertainties farther along in the
risk management process. The discussion of
uncertainties has been expanded in the final
Guidelines and includes the recommendation that a
discussion of uncertainties and assumptions be
included at every step of the regulatory process that
uses risk assessment.
Another SAB comment was that the Guidelines
should include additional procedures for mixtures
with more than one end point or effect. The Agency
agrees that these are concerns and revised the
Guidelines to emphasize these as additional
uncertainties worthy of further research.
V. Need fora Technical Support Document
The third major SAB comment concerned the
necessity for a separate technical support document
for these Guidelines. The SAB pointed out that the
scientific and technical background from which
these Guidelines must draw their validity is so
broad and varied that it cannot reasonably be
synthesized within the framework of a brief set of
guidelines. The Agency is developing a technical
support document that will summarize the available
information on health effects from chemical
mixtures, and on interaction mechanisms, as well as
identify and develop mathematical models and
statistical techniques to support these Guidelines.
This document will also identify critical gaps and
research needs.
Several comments addressed the need for
examples on the Use of the Guidelines. The Agency
has decided to include examples in the technical
support document.
Another issue raised by the SAB concerned the
identification of research needs. Because little
emphasis has been placed on the toxicology of
mixtures until recently, the information on
mixtures is limited. The SAB pointed out that
identifying research needs is critical to the risk
assessment process, and the EPA should ensure that
these needs are considered in the research planning
process. The Agency will include a section in the
ifl
iff
if
ill
3-15
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technical support document that identifies research
needs regarding both methodology and data.
3-16
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HI
51 FR 34028
GUIDELINES FOR THE HEALTH
ASSESSMENT OF SUSPECT
DEVELOPMENTAL TOXICANTS
SUMMARY:On September 24, 1986, the U.S.
Environmental Protection Agency issued the
following five guidelines for assessing the health
risks of environmental pollutants.
Guidelines for Carcinogen Risk Assessment
Guidelines for Estimating Exposures
Guidelines for Mutagenicity Risk Assessment
Guidelines for the Health Assessment of Suspect
Developmental Toxicants
Guidelines for the Health Risk Assessment of
Chemical Mixtures
This section contains the Guidelines for the Health
Assessment of Suspect Developmental Toxicants.
The Guidelines for the Health Assessment of
Suspect Developmental Toxicants (hereafter
"Guidelines") are intended to guide Agency analysis
of developmental toxicity data in line with the
policies and procedures established in the statutes
administered by the EPA. These Guidelines were
developed as part of an interoffice guidelines
development program under the auspices of the
Office of Health and Environmental Assessment
(OHEA) in the Agency's Office of Research and
Development. They reflect Agency consideration of
public and Science Advisory Board (SAB) comments
on the Proposed Guidelines for the Health
Assessment of Suspect Developmental Toxicants
published November 23,1984 (49 FR 46324).
This publication completes the first round of risk
assessment guidelines development. These
Guidelines will be revised, and new guidelines will
be developed, as appropriate.
FOR FURTHER INFORMATION CONTACT:
Dr. Carole A. Kimmel
Reproductive Effects Assessment Group
Office of Health and Environmental Assessment
(RD-689)
U. S. Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460
202-382-7331
SUPPLEMENTARY INFORMATION: In 1983,
the National Academy of Sciences (NAS) published
its book entitled Risk Assessment in the Federal
Government: Managing the Process. In that book,
the NAS recommended that Federal regulatory
agencies establish "inference guidelines" to ensure
consistency and technical quality in risk
assessments and to ensure that the risk assessment
process was maintained as a scientific effort
separate from risk management. A task force within
EPA accepted that recommendation and requested
that Agency scientists begin to develop such
guidelines.
General
The guidelines are products of a two-year
Agency wide effort, which has included many
scientists from the larger scientific community.
These guidelines set forth principles and procedures
to guide EPA scientists in the conduct of Agency risk
assessments, and to inform Agency decision makers
and the public about these procedures. In particular,
the guidelines emphasize that risk assessments will
be conducted on a case-by-case basis, giving full
consideration to all relevant scientific information.
This case-by-case approach means that Agency
experts review the scientific information on each
agent and use the most scientifically appropriate
interpretation to assess risk. The guidelines also
stress that this information will be fully presented
in Agency risk assessment documents, and that
Agency scientists will identify the strengths and
weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well
as the scientific basis and rationale for each
assessment.
Finally, the guidelines are formulated in part to
bridge gaps in risk assessment methodology and
data. By identifying these gaps and the importance
of the missing information to the risk assessment
process, EPA wishes to encourage research and
analysis that will lead to new risk assessment
methods and data.
Guidelines for the Health Assessment of
Suspect Developmental Toxicants
Work on the Guidelines for the Health
Assessment of Suspect Developmental Toxicants
began in January 1984. Draft guidelines were
developed by Agency work groups composed of
expert scientists from throughout the Agency. The
drafts were peer-reviewed by expert scientists in the
field of developmental toxicology from universities,
environmental groups, industry, labor, and other
governmental agencies. They were then proposed for
public comment in the FEDERAL REGISTER (49
FR 46324). On November 9,1984, the Administrator
directed that Agency offices use the proposed
guidelines in performing risk assessments until
final guidelines become available.
4-1
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After the close of the public comment period,
Agency staff prepared summaries of the comments,
analyses of the major issues presented by the
commentorsj and preliminary Agency responses to
those comments. These analyses were presented to
review panels of the SAB on March 4 and April 22-
23, 1985, and to the Executive Committee of the
SAB on April 25-26, 1985. The SAB meetings were
announced in the FEDERAL REGISTER as follows:
February 12, 1985 (50 FR 5811) and April 4, 1985
(50 FR 13420 and 13421).
In a letter to the Administrator dated June 19,
1985, the Executive Committee generally concurred
on all five of the guidelines, but recommended
certain revisions, and requested that any revised
guidelines be submitted to the appropriate SAB
review panel chairman for review and concurrence
on behalf of the Executive Committee. As described
in the responses to comments (see Part B: Response
to the Public and Science Advisory Board
Comments), each guidelines document was revised,
where appropriate, consistent with the SAB
recommendations, and revised draft guidelines were
submitted to the panel chairmen. Revised draft
Guidelines for the Health Assessment of Suspect
Developmental Toxicants were concurred on in a
letter dated July 26, 1985. Copies of the letters are
available at the Public Information Reference Unit,
EPA Headquarters Library, as indicated elsewhere
in this section.
Following this Preamble are two parts: Part A
contains the Guidelines and Part B, the Response to
the Public and Science Advisory Board Comments (a
summary of the major public comments, SAB
comments, and Agency responses to those
comments).
The SAB suggested that the Agency pursue
additional follow-up work on quantitative risk
assessment. Several efforts are currently underway
within the Agency on quantitative risk assessment
models and procedures, the relationship of maternal
and developmental toxicity, and the evaluation and
interpretation of postnatal studies. In addition, a
document addressing research needs is being
prepared to highlight those areas that are in need of
further study.
The Agency is continuing to study the risk
assessment issues raised in the guidelines and will
revise these Guidelines in line with new information
as appropriate.
References, supporting documents, and
comments received on the proposed
[51 FR 34029}
guidelines, as
well as copies of the final guidelines, are available
for inspection and copying at the Public Information
Reference Unit (202-382-5926), EPA Headquarters
Library, 401 M Street, S.W., Washington, DC,
between the hours of 8:00 a.m. and 4:30 p.m.
I certify that these Guidelines are not major
rules as defined by Executive Order 12291, because
they are nonbinding policy statements and have no
direct effect on the regulated community. Therefore,
they will have no effect on costs or prices, and they
will have no other significant adverse effects on the
economy. These Guidelines were reviewed by the
Office of Management and Budget under Executive
Order 12291.
August 22,1986
Lee M. Thomas,
Administrator
CONTENTS
Part A: Guidelines for the Health Assessment of Suspect
Developmental Toxicants
I.Introduction
II. Definitions and Terminology
III.Qualitative Assessment (Hazard Identification of
Developmental Toxicants
A. Laboratory Animal Studies of Developmental Toxicity:
End Points and Their Interpretation
1. End Points of Maternal Toxicity
2. End Points of Developmental Toxicity
3. Functional Developmental Toxicity
4. Overall Evaluation of Maternal and Developmental
Toxicity
5. Short-term Testing in Developmental Toxicity
&Jn Vivo Mammalian Developmental Toxicity Screen
b. In Vitro Developmental Toxicity Screens
6. Statistical Considerations
B. Human Studies
C. Other Considerations
1. Pharmacokinetics
2. Comparisons of Molecular Structure
D. Weight-of- Evidence Determination
TV. Quantitative Assessment
A. Dose-Response Assessment
B. Exposure Assessment
C. Risk Characterization
V, References
Part B: Response to Public and Science Advisory Board
Comments
I. Introduction
II. Coordination With Other Guidelines
A. Other Risk Assessment Guidelines
B. Coordination With Testing Guidelines
///. Definitions
TV. Qualitative Assessment
A. Maternal and Developmental Toxicity
B. Functional Developmental Toxicity
C. Short-Term Testing
D. Comparisons of Molecular Structure
V. Quantitative Assessment
4-2
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it
-J
Part A: Guidelines for the Health Assessment of
Suspect Developmental Toxicants
I. Introduction
These Guidelines describe the procedures that
the U.S. Environmental Protection Agency will
follow in evaluating potential developmental
toxicity associated with human exposure to
environmental toxicants. In 1980, the Agency
sponsored a conference that addressed issues related
to such evaluations (1) and provided some of the
scientific basis for these risk assessment Guidelines.
The Agency's authority to regulate substances that
have the potential to interfere adversely with
human development is derived from a number of
statutes which are implemented through multiple
offices within the Agency. Because many different
offices evaluate developmental toxicity, there is a
need for intra-Agency consistency in the approach to
assess these types of effects. The procedures
described here will promote consistency in the
Agency's assessment of developmental toxic effects.
The developmental toxicity assessments
prepared pursuant to these Guidelines will be
utilized within the requirements and constraints of
the applicable statutes to arrive at regulatory
decisions concerning developmental toxicity. These
Guidelines provide a general format for analyzing
and organizing the available data for conducting
risk assessments. The Agency previously has issued
testing guidelines (2, 3) that provide protocols
designed to determine the potential of a test
substance to induce structural and/or other
abnormalities in the developing conceptus. These
risk assessment Guidelines do not change any
statutory or regulatory prescribed standards for the
type of data necessary for regulatory action, but
rather provide guidance for the interpretation of
studies that follow the testing guidelines, and in
addition, provide limited information for
interpretation of other studies (e.g., epidemiologic
data, functional developmental toxicity studies, and
short-term tests) which are not routinely required,
but which may be encountered when reviewing data
on particular agents. Moreover, risk assessment is
just one component of the regulatory process and
defines the adverse health consequences of exposure
to a toxic agent. The other component, risk
management, combines risk assessment with the
directives of the enabling regulatory legislation,
together with socioeconomic, technical, political,
and other considerations, to reach a decision as to
whether or how much to control future exposure to
the suspected toxic agent. The issue of risk
management will not be addressed in these
Guidelines.
The background incidence of developmental
defects in the human population is quite large. For
example, approximately 50% of human conceptuses
fail to reach term (4); approximately 3% of newborn
children are found to have one or more significant
congenital malformations at birth, and by the end of
the first postnatal year, about 3% more are found to
have serious developmental defects (5,6). Of these, it
is estimated that 20% of human developmental
defects are of known genetic transmission, 10% are
attributable to known environmental factors, and
the remainder result from unknown causes (7).
Approximately 7.4% of children are reduced in
weight at birth (i.e., below 2500 g) (8). Exposure to
agents affecting development can result in multiple
manifestations (malformation, functional
impairment, altered growth, and/or lethality).
Therefore, assessment efforts should encompass a
wide array of adverse developmental end points,
such as spontaneous abortions, stillbirths,
malformations, early postnatal mortality, and other
adverse functional or physical changes that are
manifested postnatally.
Numerous agents have been shown to be
developmental toxicants in animal test systems (9).
Several of them have also been shown to be the cause
of adverse developmental effects in humans,
including alcohol, aminopterin, busulfan,
chlorobiphenyls, diethylstilbestrol, isotretinoin,
organic mercury, thalidomide, and valproic acid (10,
11, 12, 13). Although a number of agents found to be
positive in animal studies have not shown clear
evidence of hazard in humans, usually the human
data available are inadequate to determine a cause
and effect relationship. Comparisons of human and
animal data have been made for a limited number of
agents that are positive in humans (13, 14). In these
comparisons, there was almost always concordance
of effects between humans and at least one species
tested; also, the minimally effective dose (MED) for
the most sensitive animal species was
approximately 0.5 to 50 times the human
[51 FR 34030]
MED, not
accounting for differences in the incidence of effect
at the MED. Thus, there is some limited basis for
estimating the risk of exposure to human
development based on data from animal studies.
The National Research Council (15) has defined
risk assessment as being comprised of some or all of
the following components: hazard identification,
dose-response assessment, exposure assessment, and
risk characterization. In general, the process of
assessing the risk of human developmental toxicity
may be adapted to this format. However, due to
special considerations in assessing developmental
toxicity, which will be discussed later in these
Guidelines, it is not always possible to follow the
exact standards as defined for each component.
Hazard identification is the qualitative risk
assessment in which all available experimental
animal and human data are used to determine if an
agent is likely to cause developmental toxicity. In
considering developmental toxicity, these
Guidelines will address not only malformations, but
4-3
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also fetal wastage, growth alteration, and functional
abnormalities that may result from developmental
exposure to environmental agents.
The dose-response assessment defines the
relationship of the dose of an agent and the
occurrence of developmental toxic effects. According
to the National Research Council (15), this
component would usually include the results of an
extrapolation from high doses administered to
experimental animals or noted in epidemiologic
studies to the low exposure levels expected for
human contact with the agent in the environment.
Since at present there are no mathematical
extrapolation models that are generally accepted for
developmental toxicity, the Agency, for the most
part, uses uncertainty (safety) factors and margins
of safety, which will be discussed in these
Guidelines. Appropriate models are being sought by
the Agency for application to data in this area.
The exposure assessment identifies populations
exposed to the agent, describes their composition
and size, and presents the types, magnitudes,
frequencies, and durations of exposure to the agent.
In risk characterization, the exposure
assessment and the dose-response assessment are
combined to estimate some measure of the risk of
developmental toxicity. As part of risk
characterization, a summary of the strengths and
weaknesses in each component of the assessment are
presented along with major assumptions, scientific
judgments, and, to the extent possible, estimates of
the uncertainties.
//. 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 ToxicologyThe study of adverse
effects on the developing organism that may result
from exposure prior to conception (either parent),
during prenatal development, or postnatally to the
time of sexual maturation. Adverse developmental
effects may be detected at any point in the 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.
Embryotoxicity and FetotoxicityA.ny toxic effect
on the conceptus as a result of prenatal exposure; the
distinguishing feature between the two terms is the
stage of development during which the injury
occurred. The terms, as used here, include
malformations and variations, altered growth, and
in utero death.
Altered GrowthAn alteration in offspring
organ or body weight or size. Changes in body
weight may or may not be accompanied by a change
in crown-rump length and/or in skeletal ossification.
Altered growth can be induced at any stage of
development, may be reversible, or may result in a
permanent change.
Functional Developmental ToxicologyThe study
of the causes, mechanisms, and manifestations 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 VariationsA 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 since 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, deformations, and
aberrations.
///. Qualitative Assessment (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
the conceptus that may be produced by
developmental exposure to toxicants include death,
structural abnormality, altered growth, and
functional deficits. Of these, the first three types of
effects are traditionally measured in laboratory
animals using the conventional developmental
toxicity (also called teratogenicity or Segment II)
testing protocol as well as in other study protocols,
such as the multigeneration study. Functional
deficits are seldom evaluated in routine studies of
environmental agents. This section will discuss the
end points examined in routinely used protocols as
well as the evaluation of data from other types of
studies, including functional studies and short-term
tests. Transplacental carcinogenesis, another type of
developmental effect, will not be discussed in detail
here since, at present, it is considered more
appropriate to use the Guidelines for Carcinogen
Risk Assessment (16) for assessing the human risk
for these types of effects. Also, mutational events
may occur as part of developmental toxicity, and in
practice, are difficult to discriminate from other
possible mechanisms of developmental toxicity. The
Guidelines for Mutagenicity Risk Assessment (17)
should be consulted in cases where genetic damage
is suspected.
4-4
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A. Laboratory Animal Studies of Developmental
Toxicity: End Points and Their Interpretation
The most commonly used protocol for assessing
developmental toxicity in laboratory animals
involves the administration of a test substance to
pregnant animals (usually mice, rats, or rabbits)
during the period of major organogenesis,
evaluation of maternal responses throughout
pregnancy, and examination of the dam and the
uterine contents just prior to term (2, 3, 18, 19, 20).
Other protocols may use exposure periods of one to a
few days to investigate periods of particular
sensitivity for induction of anomalies in specific
organs or organ systems (21).In
[51 FR 34031]
addition,
developmental toxicity may be evaluated in studies
involving exposure of one or both parents prior to
conception, of the conceptus during pregnancy and
over several generations, or of offspring during the
late prenatal and early postnatal periods. 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 will be used
in conjunction with those to be published in the
future by EPA on male and female reproductive
toxicity.
Study designs should include a high dose, which
produces some maternal or adult toxicity (i.e., a level
which at the least produces marginal but
significantly reduced body weight, weight gain, or
specific organ toxicity, and at the most produces no
more than 10% mortality); a low dose, which
demonstrates a no observed effect level (NOEL) for
adult and offspring effects; and at least one
intermediate dose level. A concurrent control group
treated with the vehicle used for agent
administration should be included. The route of
exposure should be based on expected human
exposure considerations, 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, the multigeneration study, and, on
occasion, in postnatal studies. Other end points
specifically related to reproductive toxicity will be
covered in the relevant reproductive toxicity
guidelines. The fourth section deals with the
integrated evaluation of all data, including the
relative effects of exposure on maternal animals and
their offspring, which is important in assessing the
level of concern about a particular agent.
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 of other end points can be
observed which may give an indication of the subtle
effects of an agent. For example, in well-conducted
studies, the fertility and gestation indices provide
information on the general fertility rate of the
animal stock used and are important indicators of
toxic effects if treatment begins prior to mating or
implantation. Changes in gestation length may
indicate effects on the process of parturition.
Table l.-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 pups)
Body Weight
Treatment days (at least first, middle, and last
treatment days)
Sacrifice day
Body Weight Change
Throughout gestation
During treatment (including increments of time
within treatment period)
Post-treatment to sacrifice
Corrected maternal (body weight change
throughout gestation minus gravid uterine
weight or litter weight at sacrifice)
Organ Weights (in cases of suspected specific organ
toxicity)
Absolute
Relative to body weight
Food and Water Consumption (where relevant)
Clinical Evaluations (on days of treatment and
at sacrifice)
Types and incidence of clinical signs
Enzyme markers
Clinical chemistries
Gross Necropsy and Histopathology
Body weight and the change in body weight are
viewed collectively as indicators of maternal toxicity
for most species, although these end points may not
be as useful in rabbits, because body weight changes
in 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 during treatment could occur that would not
be reflected in the total weight change throughout
gestation, because of compensatory weight gain that
may occur following treatment but before sacrifice.
For this reason, changes in weight during treatment
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can be examined as another indicator of maternal
toxicity.
Changes in maternal body weight corrected for
gravid uterine weight at sacrifice may indicate
whether the effect is primarily maternal or 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 gain which would indicate primarily an
intrauterine effect. Conversely, a change in
corrected weight gain and no change in gravid
uterine weight suggests primarily maternal toxieity
and little or no 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 should also be
determined. For example, changes in relative and
absolute organ weights may be signs of a maternal
effect when an agent is suspected of causing specific
organ toxicity. 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 an agent is suspected of affecting
appetite, water intake, or excretory function.
Clinical evaluations of toxicity may also be used as
indicators of maternal toxicity. Daily clinical
observations may be useful in describing the profile
of 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.
2. End Points of Developmental Toxicity. Because the
maternal animal, and not the conceptus, is the
individual treated during gestation, data generally
should be calculated as incidence per litter 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 Toxicity.
Litters with implants
No. implantation sites/dam
No. corpora lutea (CL)/dama
Percent preimplantation loss
(CL - implantations) x IQQa
CL
No. and percent live offspring/litter
No. and percent resorptions/litter
No. and percent litters with resorptions
[51 PR 34032]
No, and percent late fetal deaths/litter
No. and percent nonlive (late fetal deaths +
resorptions) implants/litter
No. and percent litters with nonlive implants
No. and percent affected (nonlive + malformed)
implants/litter
No. and percent litters with affected implants
No. and percent litters with total resorptions
No. and percent stillbirths/litter
Litters with live offspring^
No. and percent litters with live offspring
No. and percent live offspring/litter
Viability of offspring*:
Sex ratio/litter
Mean offspring body weight/litter0
Mean male body weight/litter^
Mean female body weight/littere
No. and percent externally malformed
offspring/litter
No. and percent visceral ly malformed
offspring/litter
No. and percent skeletally malformed
offspring/litter
No. and percent malformed offspring/litter
No. and percent litters with malformed offspring
No. and percent malformed males/litter
No. and percent malformed females/litter
No. and percent offspring with
variations/litter
No. and percent litters having offspring with
variations
Types and incidence of individual malformations
Types and incidence of individual variations
Individual offspring and their malformations
and variations (grouped according to litter
and dose)
Clinical signs0
Gross necropsy and histopathology
a Important when treatment begins prior to
implantation. May be difficult in mice.
b Offspring refers both to fetuses observed prior
to term 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 prior to implantation,
an increase in preimplantation loss could indicate
an adverse effect either on the developing blastocyst
or on the process of implantation itself. If treatment
begins around the time of implantation (i.e., day 6 of
gestation in the mouse, rat, or rabbit), an increase in
preimplantation loss probably reflects normal
variability in the animals being used, but the data
should be examined carefully to determine whether
or not the effect is dose related. If preimplantation
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loss is related to dose in 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. The number and percent
resorptions or late fetal deaths per litter gives some
indication of when the conceptus died, and the
number and percent nonlive implants per litter
(postimplantation 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 loss
is found after exposure to an agent, the data may be
compared not only with concurrent controls, but also
with recent historical control data, since there is
considerable interlitter variability in the incidence
of postimplantation loss (22). 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 studies 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 sometimes reflects a better dose-
response relationship than does the incidence of
nonlive or malformed offspring taken individually.
This is especially true at the high end of the dose-
response curve in cases when the incidence of
nonlive implants per litter is greatly increased. In
such cases, the malformation rate may appear to
decrease because only unaffected offspring have
survived. If the incidence of prenatal 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 in 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 unusual
occurrence.
A change in offspring body weight is a sensitive
indicator of developmental toxicity, in part because
it is a continuous variable. In some cases, offspring
weight reduction may be the only indicator of
developmental toxicity; if so, there is always a
question remaining as to whether weight reduction
is a permanent or transitory effect. A permanent
weight change may be considered more severe than
a transitory change, although little is known about
the long-term consequences of short-term fetal or
neonatal weight changes. When fetal or neonatal
weight reduction is the only indicator of
developmental toxicity, data from the two-
generation reproduction study (2), if available, may
be useful for evaluating these parameters. Ideally,
follow-up studies to evaluate postnatal viability,
growth, and survival through weaning should be
conducted. 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 is
greater than that of females in the more commonly
used laboratory animals.
Live offspring should be examined for 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
offspring may be indicated by a change in one or
more of the following end points: the incidence of
malformed offspring per litter, the number and
percent of litters with malformed offspring, or the
number of offspring or litters with a particular
malformation that appears to increase with dose as
indicated by the incidence of individual types of
malformations.
[51 PR 34033]
Other ways of
examining the data include the incidence of
external, visceral, and skeletal malformations
which may indicate which general systems are
affected. A listing of individual offspring with their
malformations and variations may give an
indication of the pattern of developmental
deviations. All of these methods of expressing and
examining the data are valid for determining the
effects of an agent on structural development.
However, care must be taken to avoid counting
offspring more than once in evaluating any single
end point based on number or percent of offspring or
litters. The incidence of individual types of
malformations and variations should be examined
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for significant changes which may be masked if the
data on all malformations and variations are pooled.
Appropriate historical control data are helpful in the
interpretation of malformations and variations,
especially those that normally occur at a low
incidence apparently unrelated to dose in an
individual study. Although a dose-related increase
in malformations is interpreted as an adverse
developmental effect of exposure to an agent, the
significance of anatomical variations is more
difficult to determine, and must take into account
what is known about developmental stage (e.g., with
skeletal ossification), background incidence of
certain variations (e.g., 12 or 13 pairs of ribs in
rabbits), or other strain - or species-specific factors.
However, if variations are significantly increased in
a dose-related manner, these should also be
evaluated as a possible indication of developmental
toxicity. The Interagency Regulatory Liaison Group
noted that dose-related increases in defects, which
may occur spontaneously, are as relevant as dose-
related increases in any other developmental
toxicity end points (23),
3. Functional Developmental Toxicology.
Developmental effects, which are inducible 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 result 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 (24).
At present, such testing is not routinely required in
the United States. However, 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 at birth are unknown, and
further data on postnatal development and function
may contribute valuable information. When
regulatory statutes permit, studies designed to
evaluate adverse fetal or neonatal outcomes have
been requested (e.g., the Office of Pesticide
Programs has sometimes requested postnatal
studies where the reversibility of study findings
were at issue). 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. Less work has
been done on other functional systems, but sufficient
data have accumulated to indicate that the
cardiopulmonary, immune, endocrine, digestive,
urinary, nervous, and reproductive systems are
subject to alterations in functional competence (25,
26). Currently, there are no standard, testing
procedures, although some attempts are being made
to standardize and evaluate tests and protocols (27).
The functional evaluation of specific systems often
involves highly specialized training and equipment.
The routine use of such test procedures may not
always be practical, but may be extremely
important in determining the nature of a suspected
alteration in terms of its biological significance and
dose-response relationship.
The interpretation of data from functional
developmental toxicology studies is limited due to
the lack of knowledge about the underlying
toxicological mechanisms and their significance.
However, since such data are sometimes
encountered in the risk assessment of particular
agents, some guidance is provided here concerning
general concepts of study design and evaluation.
a. Several aspects of study design are similar to
those important in standard developmental toxicity
studies (e.g., a dose-response approach with the
highest dose producing minimal overt maternal or
perinatal toxicity, number of litters large enough for
adequate statistical power, randomization of
animals to dose groups, litter generally considered
the statistical unit, etc.).
b. A replicate study design provides added
confidence in the interpretation of data.
c. Use of a pharmacological challenge may be
valuable in evaluating function and "unmasking"
effects not otherwise detectable, particularly in the
case of organ systems that are endowed with a
reasonable degree of functional reserve capacity.
d. 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. (28)
have discussed this with relation to behavioral end
points.
e. A battery of functional tests usually provides
a more thorough evaluation of the functional
competence of an animal; tests conducted at several
ages may provide more information about
maturational changes.
f. 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 difficult at present, there are at least three ways
in which the data from these studies may be useful
for risk assessment purposes: 1) to help elucidate the
long-term consequences of fetal and neonatal
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;
4-8
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f
f
and 3) for existing environmental agents, to focus on
organ systems to be evaluated in exposed human
populations.
4. Overall Evaluation of Maternal and
Developmental Toxiclty. As discussed previously,
individual end points are evaluated in
developmental toxicity studies, but an integrated
evaluation must be done considering all maternal
and developmental end points in order to interpret
the data fully. Developmental toxicity is considered
to be an increase in the incidence of malformed
offspring, decreased viability (prenatal or
postnatal), altered growth, and/or functional
deficits.
The level of concern for a developmental toxic
effect is related to several issues, including the
relative toxicity of an agent to the offspring versus
the adult animal, and the long-term consequences of
findings in the fetus or neonate. Those agents which
produce developmental toxicity at a dose that is not
toxic to the maternal animal are of greatest concern
because the developing organism appears to be
selectively affected or more sensitive than the adult.
However, when developmental effects are produced
only
[51 FR 34034]
at maternally
toxic doses, the types of developmental effects should
be examined carefully, and not discounted as being
secondary to maternal toxicity. Current information
is inadequate to assume that developmental effects
at maternally toxic doses result only from the
maternal toxicity; rather, when the lowest observed
effect level 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 minimally 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).
Approaches for ranking agents for their
selective developmental toxicity are being
developed; Schardein (10) has reviewed several of
these. Of current interest are approaches that
develop ratios relating an adult toxic dose to a
developmental toxic dose (29, 30,31, 32). Ratios near
unity indicate that developmental toxicity occurs
only at doses producing maternal toxicity; as the
ratio increases, there is a greater likelihood of
developmental effects occurring without maternal
manifestations. Although further exploration and
validation are necessary, such approaches may
ultimately help in identifying those agents that pose
the greatest threat and should be given higher
priority for further testing (33).
5. Short-term Testing in Developmental Toxicity. The
need for short-term tests for developmental toxicity
has arisen from 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 contribution to the
overall testing process: 1) an in vivo mammalian
screen, and 2) a variety of in vitro systems.
Currently, neither approach is considered as a
replacement for routine in. vivo developmental
toxicity testing in experimental animals, and should
not be used to make the final decision as to whether
an agent is a positive or negative developmental
toxicant; rather, such tests may be useful as tools for
assigning priorities for further, more extensive
testing. Although such short-term tests are not
routinely required, data are sometimes encountered
in the review of chemicals; the comments are
provided here for guidance in the evaluation of such
data.
a. In Vivo Mammalian Developmental Toxicity
Screen. The most widely studied in vivo approach is
that developed by Chernoff and Kavlock (34) which
uses the pregnant mouse. This approach is based on
the hypothesis that a prenatal injury, which results
in altered development, will be manifested
postnatally as reduced viability and/or impaired
growth. In general, the test substance is
administered over the period of major organogenesis
at a single dose level that will elicit some degree of
maternal toxicity. A second lower dose level may be
used which potentially will reduce the chances of
false positive results. The pups are counted and
weighed shortly after birth, and again after 3-4
days. End points that are considered in the
evaluation include: general maternal toxicity
(including survival and weight gain), litter size, and
viability, weight,' and gross malformations in the
offspring. Basic priority-setting categories for more
extensive testing have been suggested: 1) agents
that induce perinatal death should receive highest
priority, 2) agents inducing perinatal weight
changes should be ranked lower in priority, and 3)
agents inducing no effect should receive the lowest
priority (34). Another scheme that has been
proposed applies a numerical ranking to the results
as a means of prioritizing agents for further testing
(35,36).
The mouse was chosen originally for this test
because of its low cost, but the procedure should be
easily applicable to other species. However, the test
will only predict the potential for developmental
toxicity of an agent in the species utilized and does
not improve the ability to extrapolate risk to other
species, including humans. The Office of Toxic
Substances has developed testing guidelines for this
procedure (37). Although the testing guidelines are
available, such procedures are not routinely
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required, and further validation is currently being
carried out (38).
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. These systems have long
been used to assess events associated with normal
and abnormal development, but only recently have
they been considered for their potential as screens in
testing (39, 40, 41). Many of these systems are now
being evaluated for their ability to predict the
developmental toxicity of various agents in intact
mammalian systems. This validation process
requires certain considerations in study design,
including defined end points for toxicity and an
understanding of the system's ability to handle
various test agents (40,42). A list of agents for use in
such validation studies has been developed (43).
6. Statistical Considerations. In the assessment
of developmental toxicity data, statistical
considerations require special attention. Since the
litter is generally considered the experimental unit
in most developmental toxicity studies, the
statistical analyses should be designed to analyze
the relevant data based on incidence per litter 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 but allow use of
individual offspring data and an evaluation of both
within and between litter variance as well as dose
effects. Nonparametric and categorical procedures
have also been widely used for binomial or incidence
data. In addition, tests for dose-response trends can
be applied. Although a single statistical approach
has not been agreed upon, a number of factors
important in the analysis of developmental toxicity
data have been discussed (23,44).
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 20-30 Utters 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 between
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 that
a study will demonstrate a true effect), which is
limited by the sample size used in 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
(45) have shown that the number of litters needed to
detect a 5 or 10% change was dramatically lower for
fetal weight (a continuous variable with low
variability) than for resorptions (a binomial
response with high variability). With the current
recommendation in
[51 FR 34035J
testing protocols
being 20 rodents per dose group (2, 3), 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 6 times the in utero death rate, and a
decrease of 0.15 to 0.25 times the fetal weight. Thus,
even within the same study, the ability to detect a
change in fetal weight is much greater than for the
other 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 point(s)
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 ire
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
Because of the ethical considerations involved,
studies with deliberate dosing of humans are not
done. Therefore, dose-effect developmental toxicity
data from humans are limited to those available
from occupational, environmental, or therapeutic
exposures. While animal studies provide dose-
response data that can be used in the extrapolation
of risk to humans, good epidemiologic data provide
the best information for assessing human risk.
The category of "human studies" includes both
epidemiologic studies and other reports of cases or
clusters of events. While case reports have been
important in identifying several human teratogens,
they are potentially of greater value in identifying
topics for further investigation (46). The data from
case reports are often of an anecdotal or highly
selected nature, and thus are of limited usefulness
for risk assessment except when a unique defect is
produced, as with thalidomide, or when the agent is
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so potent as to greatly increase the incidence of a
particular defect(s).
As there are many different designs for
epidemiologic studies, simple rules for their
evaluation do not exist. The assessment of
epidemiologic studies requires a sophisticated level
of understanding of the appropriate epidemiologic
and statistical methods and interpretation of the
findings. Factors that increase a study's usefulness
for risk assessment include such things as the
examination of multiple end points and exposure
levels, the validity of the data, and proper control of
other risk factors, effect modifiers, and confounders
in the study design and/or analysis. A more in-depth
discussion can be found elsewhere (47).
As described earlier, a single developmental
toxicant can result in multiple end points
(malformations, functional impairment, altered
growth, and/or lethality). These end points can be
thought of as sequential competing risks. For
example, a malformed fetus spontaneously aborted
would not be observed in a study of births with
malformations (48). Very early conceptus losses may
not be identified in human populations, whereas in
most laboratory animal studies, all resorption sites
can be identified. Many epidemiologic studies,
especially of the case-control design, have focused on
one end point, possibly missing a true effect of
exposure. Furthermore, some studies have selected
one type or class of malformations to study. Since an
agent can result in different spectra of
malformations following exposure at different times
in the pregnancy (49), limiting a study to one class of
malformation may give misleading results.
Malformations can be meaningfully grouped only if
there is a logical underlying teratogenic mechanism
or pathogenetic pathway. As a minimum,
malformations, deformations, and disruptions
should be separated.
The power, or probability of a study to detect a
true effect, is dependent upon the size of the study
group, the frequency of the outcome in the general
population, and the level of excess risk to be
identified. Rarer outcomes, such as malformations,
require thousands of pregnancies to have a high
probability of detecting an increase in risk. More
common outcomes, such as fetal loss, require
hundreds of pregnancies to have the same
probability (8, 23, 50, 51, 52, 53). The confidence one
has in the results of a study with negative findings is
directly related to the power of the study to detect
clinically meaningful differences in incidence for the
end points studied.
As in animal studies, pregnancies within the
same family (or litter) are not independent events.
In animal studies, the litter is generally used as the
unit of measure. This approach is difficult in
humans since the pregnancies are sequential, with
the risk factors changing for the different
pregnancies (23, 51, 54). If more than one pregnancy
per family is included, and this is often necessary
due to small study groups, the use of non-
independent observations overestimates the true
size of the population at risk and artificially
increases the significance level (54).
Other criteria for evaluating epidemiologic
studies include the following (23, 50, 52, 55, 56, 57,
58):
1. The potential for complete or relatively
complete ascertainment of events for study. This can
vary by outcome and by data source; for example, if
hospital records are used, early fetal losses will be
underascertained, but a more complete list of
pregnancies could be obtained by interviewing the
women. Congenital malformations can be more
completely ascertained using hospital records than
birth certificates. Studies with relatively complete
ascertainment of events, or at least low
probability of unbiased ascertainment, should carry
more weight.
2. Validity (accuracy) of the data. Recall of past
events in interviews may be faulty, while hospital
files contain data recorded at the time of the event
(but may be incomplete). Validation of interview
data with an independent source, where possible,
increases confidence in the results of the study.
3. Collection of data on other risk factors, effect
modifiers, and confounders. Data on smoking,
alcohol consumption, drug use, and environmental
and occupational exposure, etc., during pregnancy
should be examined and controlled for in the study
design and/or analysis where appropriate. The
analytic techniques used to control for these factors
require careful consideration in their application
and interpretation.
C. Other Considerations
1. Pharmacokinetics. Extrapolation of data between
species can be aided considerably by the availability
of data on the pharmacokinetics of a particular
agent in the species tested and, if possible, in
humans. Information on half-lives, placental
metabolism and transfer, and concentrations of the
parent compound and metabolites in the
[51 FR 34036]
maternal animal
and conceptus 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 (59, 60),
determining dqsimetry 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
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useful in determining the contribution of specific
pharmacokinetic parameters to the effects observed
(61).
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 available from studies discussed
previously, whether indicative of potential concern
or not, must be evaluated and factored into the risk
assessment. The types of data may vary from
chemical to chemical, and certain types of data may
be more relevant than other types in performing
developmental toxicity assessments. The primary
considerations are the human data (which are
seldom available) and the experimental animal
data. The qualitative assessment for developmental
toxicity should include statements concerning the
quality of the data, the resolving power of the
studies, the number and types of end points
examined, the relevance of route and timing of
exposure, the appropriateness of the dose selection,
the replication of the effects, the number of species
examined, and the availability of human case
reports, case series, and/or epidemiologic study data.
In addition, pharmacokinetic data and structure-
activity considerations, as well as other factors that
may affect the quality, 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.
IV. Quantitative Assessment
Risk assessment involves the description of the
nature and often the magnitude of potential human
risk, including a description of any attendant
uncertainty. In the final phase of the risk
assessment (risk characterization), the results of the
qualitative evaluation (hazard identification), the
dose-response, and the exposure assessments are
combined to give qualitative and/or quantitative
estimates of the developmental toxicity risk. A
summary of the strengths and weaknesses of the
hazard identification, dose-response assessment,
and exposure assessment should be discussed. Major
assumptions, scientific judgments, and, to the extent
possible, estimates of the uncertainties in the
assessment also should be presented.
A. Dose-Response Assessment
When quantitative human dose-effect data are
available and with sufficient range of exposure,
dose-response relationships may be examined!
However, such data have rarely been available;
thus, other methods have been used in
developmental toxicology for estimating exposure
levels that are unlikely to produce adverse effects in
humans. 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 three-
dose studies. As mentioned earlier (section III. A. 2.),
however, traditional dose-response relationships
may not always be observed for some end points. For
example, as the exposure level rises,
embryo/fetolethal levels may be reached, resulting
in an observed decrease in malformations with
increasing dose (49, 51). The potential for this
relationship indicates that dose-response
relationships for individual end points as well as
combinations of end points (e.g., dead and
malformed combined) must be carefully examined
and interpreted.
Although dose-response data are important in
this area, the approaches frequently employed in
attempts to extrapolate to humans has involved
simply the use of uncertainty (safety) factors and
margins of safety, which in some respects are
conceptually similar. However, uncertainty factors
and margins of safety are computed differently and
are often used in different regulatory situations. The
choice of approach is dependent upon many factors,
including the statute involved, the situation being
addressed, the data base used, and the needs of the
decision-maker. The final uncertainty factor used
and the acceptability of the margin of safety are risk
management decisions, but the scientific issues that
must be taken into account are addressed here.
The uncertainty factor approach results in a
calculated exposure level believed to be unlikely to
cause any toxic developmental response in humans.
The size of the uncertainty factor will vary from
agent to agent and will require the exercise of
scientific judgment (10, 62), taking into account
interspecies differences, the nature and extent of
human exposure, the slope of the dose-response
curve, the types of developmental effects observed,
and the relative dose levels for maternal and
developmental toxicity in the test species. The
uncertainty factor selected is then divided into the
NOEL for the most sensitive end point obtained from
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" I
12 i
K
E
the most appropriate and/or sensitive mammalian
species examined to obtain an acceptable exposure
level. Currently, there is no one laboratory animal
species that can be considered most appropriate for
predicting risk to humans (10). Each agent should be
considered on a case-by-case basis.
The margin of safety approach derives a ratio of
the NOEL from the most sensitive species to the
estimated human exposure level from all potential
sources (63). The adequacy of the margin of safety is
then considered, based on the weight of evidence,
including the nature and quality of the hazard and
exposure data, the number of species affected, dose-
response relationships, and other factors such as
benefits of the agent.
Although the standard study design for a
developmental toxicity study calls for a low dose that
demonstrates a NOEL, there may be circumstances
where a risk assessment is based on the results of a
study in which a NOEL for developmental toxicity
could not be identified. Rather, the lowest dose
administered caused significant effect(s) and was
identified as the lowest observed effect level (LOEL).
In circumstances where only a LOEL is available, it
may be appropriate to apply
[51FR34037]
an additional
uncertainty factor. The magnitude of this additional
factor is dependent upon scientific judgment. In
some instances, additional studies may be needed to
strengthen the confidence in this additional
uncertainty factor.
B. Exposure Assessment
The results of the dose-response assessment are
combined with an estimate of human exposure in
order to obtain a quantitative estimate of risk. The
Guidelines for Estimating Exposures are published
separately (64) and will not be discussed in detail
here. In general, the exposure assessment describes
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 relevant to developmental toxicity
are duration and period of exposure as 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.,
chronic exposure is not a necessary prerequisite for
developmental toxicity to be manifested). Also, it
should be recognized that exposure of almost any
segment of the human population (i.e., fertile men
and women, the conceptus, and the child up to the
age of sexual maturation) may lead to risk to the
developing organism.
Data on exposure to humans may be qualitative
or quantitative. The qualitative data could be
surrogate data, such as employment or residence
histories; quantitative or dose data are frequently
not available. Exposures at different stages of the
reproductive process can result in different outcomes
(49). In laboratory studies, these time periods can be
carefully controlled. In human studies, especially
retrospective ones, linking of specific time periods
and specific exposures, even on a qualitative level,
may be difficult due to errors of recall or record
keeping (where records are available). The increased
probability of misclassification of exposure status
may affect the ability of a study to recognize a true
effect (8, 23,52, 65,66).
Exposure may be defined at a specific point in
time, or the cumulative lifetime exposure up to a
specific point in time. Each of these definitions
carries an implicit assumption about the underlying
relationship between exposure and outcome. For
example, a cumulative exposure measure assumes
that total lifetime exposure is important, with a
greater probability of effect with greater total
exposure; a dichotomous exposure measure (ever
exposed versus never exposed) assumes an
irreversible effect of exposure; and exposure at a
specific time in the reproductive process assumes
that only concurrent exposure is important. The
appropriate exposure depends on the outcome(s)
studied, the biologic mechanism affected by
exposure, and the half-life of the exposure. Unbiased
misclassification of exposure, due either to poor data
or to an inappropriate exposure variable, may result
in missing an effect of the agent under study.
C. Risk Characterization
Many uncertainties have been pointed out in
these Guidelines which 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 risk assessment for
developmental toxicity should be accompanied by
statements concerning the strength of the hazard
evaluation (see section III. D. for more detail) as well
as dose-response relationships, estimates of human
exposure, and any other factors that affect the
quality and precision of the assessment. The dose-
response and exposure data are combined to
estimate risk based on a NOEL for any adverse
developmental effect. The uncertainty factor
selected or margin of safety calculated should be
sufficiently qualified as to the assumptions used and
the accuracy of the estimates.
At present, there are no mathematical models
that are generally accepted for estimating
developmental toxicity responses below the applied
dose range. This is due primarily to a lack of
understanding of the biological mechanisms
underlying developmental toxicity,
intra/interspecies differences in the types of
developmental events, the influence of maternal
effects on the dose-response curve, and whether or
not a threshold exists below which no effect will be
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produced by an agent. Many developmental
lexicologists assume a threshold for most
developmental effects; this assumption is based
largely on the biological rationale that the embryo is
known to have some capacity for repair of the
damage or insult (49), and that most developmental
deviations are probably multifactorial in nature
(67). The existence of a NOEL in an animal 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 test that are not
associated with a significant, increase in effect. The
use of NOELs and uncertainty factors or margins of
safety are attempts to ensure that the allowable
levels are below those that will produce a significant
increase in developmental effects.
Discussions of risk extrapolation procedures
have noted that further work is needed to improve
mathematical tools for developing estimates of
potential human developmental risk (62, 68). Gaylor
(69) has suggested an approach for controlling risk
that combines the use of mathematical models for
low-dose estimation of risk with the application of
an uncertainty factor based on a preselected level of
allowable 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 controlling
risk. Several such approaches are being examined.
For the most part, the Agency will continue to use
uncertainty factors and margins of safety as
described above. Other appropriate methods for
expressing risk are being sought and will be applied
if considered acceptable.
These Guidelines summarize the procedures
that the U.S. Environmental Protection Agency will
follow in evaluating the potential for agents to cause
developmental toxicity. These Guidelines will be
reviewed and updated as advances are made in the
field, since it is evident that our ability to evaluate
and predict human developmental toxicity is
imprecise. Further studies that 1) 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 fetal toxicity and the concept of a
threshold in developmental toxicity, will provide for
the development of improved mathematical models
to more precisely assess risk.
V. References
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Alan R. Liss, Inc., pp. 81-97.
67. Fraser, F.C. 1977. Relation of animal studies to the
problem in man. In: J.G. Wilson and F.C. Fraser, eds. Handbook of
teratology, Vol. I. New York, NY: Plenum Press, pp.75-96.
68. Environmental Health Criteria 30. 1984. Principles for
evaluating health risks to progeny associated with exposure to
chemicals during pregnancy. World Health Organization,
Geneva, Switzerland, pp. 111-114.
4-15
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69. Gaylor, D.W. 1983. The use of safety factors for
controlling risk. J. Toxicbl. Environ. Health 11:329-336.
Part B: Response to Public and Science
Advisory Board Comments
1. Introduction
This section summarizes some of the issues
raised in public comments on the Proposed
Guidelines for the Health Assessment of Suspect
Developmental Toxicants published November 23,
1984 (49 FR 46324). Comments were received from
44 individuals or organizations. The Agency's initial
summary of comments was presented to the
Developmental Toxicity Guidelines Panel of the
Science Advisory Board (SAB) at its organizational
meeting on March 4, 1985. At its April 22-23, 1985,
meeting, the Panel provided the Agency with its
suggestions and recommendations concerning the
Guidelines.
The SAB and public comments were diverse and
addressed issues from a variety of perspectives. In
general, the comments were favorable and in
support of the Guidelines. The SAB Panel noted that
the field of developmental toxicology is particularly
weak with respect to quantitative assessment and
recommended that further efforts be given to
developing alternative methods for quantitative
estimates of risk for developmental toxicity. They
also indicated that further discussion of the
relationship of maternal toxicity to fetal toxicity
could be added. Concern was expressed that these
Guidelines be coordinated with the reproductive
toxicity guidelines which are currently being
developed.
In response to the comments, the Agency has
modified or clarified many sections of the
Guidelines. For purposes of this discussion, only the
most significant issues reflected by the public and
SAB comments are discussed. Several minor
recommendations, which do not warrant discussion
here, were considered by the Agency in the revision
of these Guidelines.
77. Coordination With Other Guidelines
A. Other Risk Assessment Guidelines
Several commentors raised concerns about
aspects of developmental toxicity (e.g., paternally-
mediated effects, effects of subchronic exposures,
transplaeental carcinogenesis, etc.) that were not
covered in these Guidelines, and how these
Guidelines will integrate with those on male and
female reproductive toxicity which are still under
development.
The Guidelines have been revised to indicate
that developmental toxicity may result from several
different types of exposure, including parental
exposure prior to conception, acute or subacute
exposure during organogenesis, perinatal and
postnatal development to the time of sexual
maturation, or subchronic exposure as would be the
case in multigeneration studies. These Guidelines
provide information for interpreting developmental
effects related to any of the types of exposure
mentioned above. End points of developmental
toxicity, which are measured in multigeneration
studies, have been added to Table 2 and discussed in
the text. Transplaeental carcinogenesis, although
considered a developmental effect, will be evaluated
and assessed in terms of human risk according to the
Guidelines for Carcinogen Risk Assessment. Careful
attention will be paid to integrating these
developmental toxicity risk assessment Guidelines
and the male and female reproductive toxicity risk
assessment guidelines, which are currently being
written, so that overlapping material is not in
conflict, and no pertinent information is overlooked.
Since the developmental and reproductive toxicity
guidelines are being developed by Agency
committees that have overlapping membership
within the Agency, such integration will be ensured.
B. Coordination With Testing Guidelines
Several commentors indicated that these
Guidelines did not make clear enough the fact that
testing guidelines are already in place and that
these guidelines were intended only for the purposes
of risk assessment.
The Guidelines have been revised to indicate
that they do not constitute any changes in current
testing guidelines, but rather they are intended to
provide guidance for the interpretation of studies
that follow the testing guidelines. In addition,
limited information is provided for interpretation of
other studies (e.g., functional developmental toxicity
studies and short-term tests) which are not routinely
required or for which there are no current testing
guidelines, but which may be encountered when
reviewing data on particular agents.
7/7. Definitions
Several questions were raised about definitions
of terminology, due to lack of clarity or inconsistency
with other parts of these Guidelines or the testing
guidelines.
As indicated in the Guidelines, there are
differences in the use of terms in the field of
developmental toxicology, and the terms have been
defined so that the reader may understand how the
terms are being used. Several minor changes in the
definitions have been made
[51 FR 34040]
to make them
more consistent. For example, the definition for
developmental toxicology has been expanded to
include the wide range of exposure situations that
may result in developmental effects. The term
functional teratology has been changed to functional
developmental toxicology, and the term
4-16
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teratogenicity has been discussed in the section on
malformations and variations.
IV. Qualitative Assessment
A. Maternal and Developmental Toxicity
Several commentors noted the need for a better
discussion of how maternal toxicity affects the
evaluation of developmental toxic effects.
The Agency has taken the approach in these
Guidelines of discussing in detail the individual end
points of maternal and offspring toxicity, then
giving guidance relating to an overall evaluation of
the data in Part A, section III.A.4. This approach is
consistent with the philosophy reflected in the
Guidelines as follows: Those agents that cause
developmental effects at doses lower than those
causing maternal toxicity are of greatest 'concern,
but developmental effects at doses that also produce
maternal toxicity shoud not be discounted as
secondary to maternal effects. Rather, when the
lowest observed effect level (LOEL) is the same for
maternal and developmental toxicity, it may
indicate similar sensitivities to the agent, and
maternal effects may be reversible while
developmental effects may be permanent.
B. Functional Developmental Toxicity
Several commentors raised concern about the
premature use of functional data in the risk
assessment process. On the other hand, the SAB
Panel felt that these tests were very valuable in
assessing developmental toxicity.
The Agency does not routinely require such
testing, and these Guidelines do not suggest
requirements. However, in the review of data on
existing chemicals, such data are sometimes
encountered and must be evaluated by the Agency.
The discussion in the Guidelines is intended to
delineate the current state of the art, and to indicate
to what extent the data currently may be used for
risk assessment purposes.
C. Short-Term Testing
Several commentors stressed the need for
further refinement, validation, and comparative
testing to determine the credibility of short-term
tests for developmental toxicity. The
appropriateness of single dose level screens for the
purpose of prioritization was endorsed by the SAB
Panel with the reservation that too many false
positives might occur, and that positive agents in
these screens would be permanently labelled as
positive developmental toxicants.
Since data from these types of test procedures
may be encountered in the assessment of chemicals,
the Agency felt it appropriate to give guidance as to
how these should be evaluated. The Guidelines have
been revised to clearly indicate that these tests are
not routinely required, shoud not be considered as a
replacement for routine in viuo developmental
toxicity testing in mammals, and should not be used
to make the final decision as to whether an agent is a
positive or negative developmental toxicant.
D. Comparisons of Molecular Structure
Comments suggested that not much is known
about structure-activity relationships for
developmental toxicants, and that this procedure
should not be used except in the case of hormone
analogs.
A statement has been added to indicate that
structure-activity relationships have not been well-
studied in developmental toxicology, but under
certain circumstances, e.g., in the case of the
premanufacturing notice process (TSCA, section 5),
the evaluation of molecular structure is one of
several procedures used by the Agency to evaluate
potential toxicity and to support requests for testing
of new chemicals.
V. Quantitative Assessment
Most comments related to the appropriateness of
using uncertainty (safety) factors, margins of safety,
and no observed effect levels (NOELs). Some
commentors- felt that the concept of threshold was
not adequately discussed in the Guidelines.
These Guidelines are intended to reflect current
Agency policy and practice. Although more
quantitative assessment of developmental toxicity
data are desirable, and efforts are currently ongoing
within the Agency to evaluate other approaches, the
current practice is to use the NOEL (or the LOEL if a
NOEL is not available), and to apply an uncertainty
factor or to calculate the margin of safety. This
practice is based in large part on the lack of
understanding of the biological mechanisms
involved. The uncertainty factor used or
acceptability of the margin of safety are considered
risk management decisions, but the scientific issues
that must be taken into account are discussed in
these Guidelines. An experimentally determined
NOEL does not prove or disprove the existence of a
threshold, although many developmental
toxicologists assume a threshold for most
developmental effects because of known repair
capabilities in developing systems and the fact that
many developmental alterations are multifactorial
in nature.
4-17
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51 FR 34042
GUIDELINES FOR ESTIMATING
EXPOSURES
SUMMARY: On September 24, 1986, the U.S.
Environmental Protection Agency issued the
following five guidelines for assessing the health
risks of environmental pollutants.
Guidelines for Carcinogen Risk Assessment
Guidelines for Estimating Exposures
Guidelines for Mutagenicity Risk Assessment
Guidelines for the Health Assessment of Suspect
Developmental Toxicants
Guidelines for the Health Risk Assessment of
Chemical Mixtures
This section contains the Guidelines for Estimating
Exposures.
The Guidelines for Estimating Exposures
(hereafter "Guidelines") are intended to guide
Agency analysis of exposure assessment data in line
with the policies and procedures established in the
statutes administered by the EPA. These Guidelines
were developed as part of an interoffice guidelines
development program under the auspices of the
Office of Health and Environmental Assessment
(OHEA) in the Agency's Office of Research and
Development. They reflect Agency consideration of
public and Science Advisory Board (SAB) comments
on the Proposed Guidelines for Exposure
Assessment published November 23, 1984 (49 FR
46304).
This publication completes the first round of risk
assessment guidelines development. These
Guidelines will be revised, and new guidelines will
be developed, as appropriate.
FOR FURTHER INFORMATION CONTACT:
Dr. Richard V. Moraski
Exposure Assessment Group
Office of Health and Environmental Assessment
(RD-689)
U. S. Environmental Protection Agency
401M Street, S.W.
Washington, DC 20460
202-475-8923
SUPPLEMENTARY INFORMATION: In 1983,
the National Academy of Sciences (NAS) published
its book entitled Risk Assessment in the Federal
Government: Managing the Process. In that book,
the NAS recommended that Federal regulatory
agencies establish "inference guidelines" to ensure
consistency and technical quality in risk
assessments and to ensure that the risk assessment
process was maintained as a scientific effort
separate from risk management. A task force within
EPA accepted that recommendation and requested
that Agency scientists begin to develop such
guidelines.
General
The guidelines are products of a two-year
Agencywide effort, which has included many
scientists from the larger scientific community.
These guidelines set forth principles and procedures
to guide EPA scientists in the conduct of Agency risk
assessments, and to inform Agency decision makers
and the public about these procedures. In particular,
the guidelines emphasize that risk assessments will
be conducted on a case-by-case basis, giving full
consideration to all relevant scientific information.
This case-by-case approach means that Agency
experts review the scientific information on each
agent and use the most scientifically appropriate
interpretation to assess risk. The guidelines also
stress that this information will be fully presented
in Agency risk assessment documents, and that
Agency scientists will identify the strengths and
weaknesses of each assessment by describing
uncertainties, assumptions, and limitations, as well
as the scientific basis and rationale for each
assessment.
Finally, the guidelines are formulated in part to
bridge gaps in risk assessment methodology and
data. By identifying these gaps and the importance
of the missing information to the risk assessment
process, EPA wishes to encourage research and
analysis that will lead to new risk assessment
methods and data.
Guidelines for Estimating Exposures
Work on the Guidelines for Estimating
Exposures began in January 1984. Draft guidelines
were developed by Agency work groups composed of
expert scientists from throughout the Agency. The
drafts were peer-reviewed by expert scientists in the
field of exposure assessment from universities,
environmental groups, industry, labor, and other
governmental agencies. They were then proposed for
public comment in the FEDERAL REGISTER (49
FR 46304). On November 9,1984, the Administrator
directed that Agency offices use the proposed
guidelines in performing risk assessments until
final guidelines become available.
5-1
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After the close of the public comment period,
Agency staff prepared summaries of the comments,
analyses of the major issues presented by the
commentors, and preliminary Agency responses to
those comments. These analyses were presented to
review panels of the SAB on March 4 and April 22-
23, 1985, and to the Executive Committee of the
SAB on April 25-26, 1985. The SAB meetings were
announced in the FEDERAL REGISTER as follows:
February 12, 1985 (50 FR 5811) and April 4, 1985
(50 FR 13420 and 13421).
In a letter to the Administrator dated June 19,
1985, the Executive Committee generally concurred
on all five of the guidelines, but recommended
certain revisions, and requested that any revised
guidelines be submitted to the appropriate SAB
review panel chairman for review and concurrence
on behalf of the Executive Committee. As described
in the responses to comments (see Part B: Response
to the Public and Science Advisory Board
Comments), each guidelines document was revised,
where appropriate, consistent with the SAB
recommendations, and revised draft guidelines were
submitted to the panel chairmen. Revised draft
Guidelines for Estimating Exposures were
concurred on in a letter dated January 13, 1986.
Copies of the letters are available at the Public
Information Reference Unit, EPA Headquarters
Library, as indicated elsewhere in this section.
Following this Preamble are two parts: Part A
contains the Guidelines and Part B, the Response to
the Public and Science Advisory Board Comments (a
summary of the major public comments, SAB
comments, and Agency responses to those
comments).
The SAB requested that the Agency develop
guidelines on the principles for the measurement of
pollutant concentrations in the various
environmental media and for the uses of
environmental measurements for exposure
assessment. This effort is currently underway.
The Agency also will provide technical support
documents that contain detailed technical
information needed to implement the Guidelines.
Two of these technical reports entitled
"Development of Statistical Distributions or Ranges
of Standard Factors Used in Exposure Assessments"
(available from the National Technical Information
Service, PB85-242667) and "Methodology for
Characterization of Uncertainty in Exposure
Assessments" (available from the National
Technical Information Service, PB85-240455) are
currently available. Technical support documents
will be revised periodically to reflect improvements
in exposure assessment methods and new
information or experience.
[51 FR 34043]
The Agency is continuing to study the risk
assessment issues raised in the Guidelines and will
revise these Guidelines in line with new
information, as appropriate.
References, supporting documents, and
comments received on the proposed guidelines, as
well as copies of the final guidelines, are available
for inspection and copying at the Public Information
Reference Unit (202-382-5926), EPA Headquarters
Library, 401 M Street, S.W., Washington, DC,
between the hours of 8:00 a.m. and 4:30 p.m.
I certify that these Guidelines are not major
rules as defined by Executive Order 12291, because
they are nonbinding policy statements and have no
direct effect on the regulated community. Therefore,
they will have no effect on costs or prices, and they
will have no other significant adverse effects on the
economy. These Guidelines were reviewed by the
Office of Management and Budget under Executive
Order 12291.
August 22,1986
Lee M. Thomas,
Administrator
CONTENTS
Part A: Guidelines for Estimating Exposure
/. Introduction
II. General Guidelines and Principles
A. Exposure and Dose
B. Decision Path to Determine Scope of the Assessment
C. Uncertainty
III.Organization and Contents of an Exposure Assessment
A. Overview
B. Detailed Explanation of Outline
1. Executive Summary
2. Introduction (Purpose and Scope)
3. General Information for Each Chemical or Mixture
4. Sources
5. Exposure Pathways and Environmental Fate
6. Measured or Estimated Concentrations
7. Exposed Populations
8. Integrated Exposure Analysis
9, References
10. Appendices
Part B: Response to Public and Science Advisory Board
Comments
/. Introduction
II. General Information
A. Acceptable Latitude of Approach
B. Technical Nature of Guidelines
C. Measurements vs. Modeling
///. Data Aoailability and Uncertainty Analysis
A. Information Uses
B. Worst-Case Estimates
TV. Evaluation of Uncertainties
A. Uncertainty Analysis
B. Population Characterization
V. Clarification ofTerminology
A. Exposure vs. Dose
B. Mixtures and Synergism
C. Removal and Creation Steps
VI. Purpose, Philosophy, and Results
5-2
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Part A: Guidelines for Estimating Exposures
7, Introduction
These Guidelines provide the Agency with a
general approach and framework for carrying out
human or nonhuman exposure assessments for
specified pollutants. The Guidelines have been
developed to assist future assessment activities and
encourage improvement in those EPA programs
that require, or could benefit from, the use of
exposure assessments. The Guidelines are
procedural. They should be followed to the extent
possible in instances where exposure assessment is a
required element in the regulatory process or where
exposure assessments are carried out on a
discretionary basis by EPA management to support
regulatory or programmatic decisions.
This document, by laying out a set of questions
to be considered in carrying out an exposure
assessment, should help avoid inadvertent mistakes
of omission. Ideally, exposure assessments are based
on measured data. EPA recognizes that gaps in data
will be common, but the Guidelines will
nevertheless serve to assist in organizing the data
that are available, including new data developed as
part of the exposure assessment. In the absence of
sufficient reliable data and the time to obtain
appropriate measurements, exposure assessments
may be based on validated mathematical models.
Whenever possible, exposure assessments based on
modeling should be complemented by reliable
measurements. Furthermore, it is understood that
the level of detail found in the exposure assessments
depends on the scope of the assessment.
These Guidelines should also promote
consistency among various exposure assessment
activities that are carried out by the Agency.
Consistency with respect to common physical,
chemical, and biological parameters, with respect to
assumptions about typical exposure situations, and
with respect to the characterization of uncertainty of
estimates, will enhance the comparability of results
and enable the Agency to improve the state-of-the-
art of exposure assessment over time through the
sharing of common data and experiences.
It is recognized that the main objective of an
exposure assessment is to provide reliable data
and/or estimates for a risk assessment. Since a risk
assessment requires the coupling of exposure
information and toxicity or effects information, the
exposure assessment process should be coordinated
with the toxicity/effects assessment. This document
provides a common approach to format, which
should simplify the process of reading and
evaluating exposure assessments and thereby
increase their utility in assessing risk.
As the Agency performs more exposure
assessments, the Guidelines will be revised to reflect
the benefit of experience.
71. General Guidelines and Principles
A. Exposure and Dose
Exposure has been defined by Committee E-47,
Biological Effects and Environmental Fate, of the
American Society for Testing and Materials, as the
contact with a chemical or physical agent. The
magnitude of the exposure is determined by
measuring or estimating the amount of an agent
available at the exchange boundaries, i.e., lungs,
gut, skin, during some specified time. Exposure
assessment is the determination or estimation
(qualitative or quantitative) of the magnitude,
frequency, duration, and route of exposure.
Exposure assessments may consider past, present,
and future exposures with varying techniques for
each phase, e.g., modeling of future exposures,
measurements of existing exposure, and biological
accumulation for past exposures. Exposure
assessments are generally combined with
environmental and health effects data in performing
risk assessments.
In considering the exposure of a subject to a
chemical agent, there are several related processes.
The contact between the subject of concern and the
agent may lead to the intake of some of the agent. If
absorption occurs, this constitutes an uptake (or an
absorbed dose). When biological tissue or fluid
measurements indicate the presence of a chemical,
exposures may be estimated from these data.
Presence of a chemical in such biological samples is
the most direct indication that an exposure has
occurred. The route of exposure generally impacts
the extent of absorption and should be considered in
performing risk assessments.
[51 FR 34044]
B. Decision Path to Determine Scope of the
Assessment
The first step in preparing an exposure
assessment should be the circumscription of the
problem at hand to minimize effort by use of a
narrowing process. A decision path that describes
this process is shown in Figure 1. As illustrated in
Figure 1, the preliminary assessment and the in-
depth assessment are two major phases in this logic
path.
[51FR34046]
The preliminary assessment phase should
commence by considering what risk is under study.
Within this framework, a data base should be
compiled from readily available scientific data and
exposure information based on manufacturer,
processor, and user practices. Next, the most likely
areas of exposure (manufacturing, processing,
consumer, distribution, disposal, water and food,
etc.) should be identified. The preliminary exposure
assessments should be based on data derived from
environmental measurements. When a limited
amount of measurement data is available, estimates
may be based on modeling. Since a complete data
search may not be possible, well identified
5-3
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51FR34045
REGULATORY CONCERN
SCIENTIFIC DATA:
' POPULATION
EXPOSURE
PRODUCT LIFE CYCLE
GENERAL INFORMATION
GATHERING
PRELIMINARY EXPOSURE
ASSESSMENT
MOST PROBABLE AREAS OF EXPOSURE
PRELIMINARY EXPOSURE ASSESSMENT
HAZARD IDENTIFICATION:
TOXICITY
ENV. CONC, ETC,
PRELIMINARY RISK ANALYSIS
DECISION
BEGIN IN-DEPTH
EXPOSURE ASSESSMENT
X
NO NEED FOR FURTHER
EXPOSURE ASSESSMENT
MULTI-DISCIPLINARY
PEER REVIEW
DESIGN ASSESSMENT STUDY PLAN
COMPREHENSIVE DATA GATHERING
IN-DEPTH EXPOSURE
ASSESSMENT
CONDUCT REFINED EXPOSURE MODELING
IN-DEPTH EXPOSURE ASSESSMENT
DECISION
SCIENCE PANEL
REVIEW
HAZARD INPUT
Figure 1. Decision path for exposure assessment.
5-4
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assumptions and order of magnitude estimates may
be used to further narrow the exposure areas of
concern.
Data from this preliminary exposure assessment
can then be coupled with toxicity information to
perform a preliminary risk analysis. As a result of
this analysis, a decision will be made that either an
in-depth exposure assessment is necessary or that
there is no need for further exposure information.
The organization and contents of an in-depth
exposure assessment are given in the following
section.
In assembling the information base for either a
preliminary assessment or a more detailed
assessment, its adequacy should be ascertained by
addressing the following considerations:
Availability of information in every area
needed for an adequate assessment;
Quantitative and qualitative nature of the
data;
Reliability of information;
Limitations on the ability to assess exposure.
C. Uncertainty
Exposure assessments are based on
measurements, simulation model estimates, and
assumptions about parameters used in
approximating actual exposure conditions. Actual
measurements should be used whenever possible.
Both data and assumptions contain varying degrees
of uncertainty which influence the accuracy of
exposure assessments. Consequently, evaluation of
uncertainty is an important part of all exposure
assessments.
The uncertainty analyses performed will vary
depending on the scope of the assessment, the
quantity and quality of measurements, and the type
and complexity of mathematical models used. A
discussion of the types of analyses used for
quantifying uncertainties in exposures is presented
in the next section.
///. Organization and Contents of an Exposure
Assessment
A. Overview
A suggested outline for an exposure assessment
document is given in Exhibit 1. The five major topics
to be addressed within most exposure assessments
are as follows: Source(s), Exposure Pathways,
Measured or Estimated Concentrations and
Duration, Exposed Population(s), and Integrated
Exposure Analysis. These five topics are appropriate
for exposure assessments in general, whether the
assessments are of global, national, regional, local,
site specific, workplace related, or other scope. The
topics are appropriate for exposure assessments on
new or existing chemicals and radionuclides. They
are also applicable to both single media and
multimedia assessments. Since exposure
assessments are performed at different levels of
detail, the extent to which any assessment contains
items listed in Exhibit 1 depends upon its scope. The
outline is a guide to organize the data whenever they
are available.
Exhibit l.-Suggested Outline for an Exposure Assessment
1. Executive Summary
2. Introduction
a. Purpose
b. Scope
3. General Information for Each Chemical or Mixture
a. Identity
(l)Molecular formula and structure, synonyms, and
Chemical Abstracts Service (CAS) number
(2) Description of grades, contaminants, and additives
(3) Other identify ing characteristics
b. Chemical and Physical Properties
4. Sources
a. Characterization of Production and Distribution
b. Uses
c. Disposal
d. Summary of Environmental Releases
5. Exposure Pathways and Environmental Fate
a. Transport and Transformation
b. Identification of Principal Pathways of Exposure
c. Predicting Environmental Distribution
6. Measured or Estimated Concentrations
a. Uses of Measurements
b. Estimation of Environmental Concentrations
7. Exposed Populations
a. Human Populations
(1) Population size and characteristics
(2) Population location
(3) Population habits
b. Nonhuman Populations (where appropriate)
(1) Population size and characteristics
(2) Population location
(3) Population habits
8. Integrated Exposure Analysis
a. Calculation of Exposure
(1) Identification of-the exposed population and critical
elements of the ecosystem
(2) Identification of pathways of exposure
b. Human Dosimetry and Biological Measurements
c. Developmentof Exposure Scenarios and Profiles
d. Evaluation of Uncertainty
(1) Introduction
(2) Assessments based on limited initial data
(3)Assessments based on subjective estimates of input
variable distributions
(4) Assessments based on data for model input variables
(5) Assessments based on data for exposure
(6) Summary
9. References
10. Appendices
B. Detailed Explanation of Outline
1.Executive Summary. The "Executive
Summary" should be written so that it can stand on
its own as a miniature report. Its main focus should
be on a succinct description of the procedures used,
assumptions employed, and summary tables or
charts of the results. A brief discussion of the
uncertainties associated with the results should be
included.
2. Introduction (Purpose and Scope). This section
should state the intended purpose of the exposure
assessment and identify the agent being
5-5
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investigated, the types of sources and exposure
routes included, and the populations of concern.
3. General Information for Each Chemical or
Mixture.
a. Identity.
(1) Molecular formula and structure,
synonyms, and Chemical Abstracts Service number.
(2) Description of grades, contaminants, and
additives.
(3) Other identifying characteristics.
b. Chemical and Physical Properties. This
subsection should provide a summary description of
the chemical and physical properties of the agent.
Particular attention should be paid to the features
that would affect its behavior in the environment.
4, Sources. The points at which a substance is
believed to enter the environment should be
described, along with any known rates of entry.
(Points of entry may be indoors as well as outdoors;
environments include indoor settings such as offices
as well as outdoor environments.) A detailed
exposure assessment should include a study of
sources, production, uses, destruction/disposal, and
environmental release of a substance. The studies
[51FR34047]
should include a
description of human activities with respect to the
substance and the environmental releases resulting
from those activities. It should account for the
controlled mass flow of the substance from creation
to destruction and provide estimates of
environmental releases at each step in this flow.
Seasonal variations in environmental releases
should also be examined. All sources of the
substance should be accounted for with the sum of
the uses, destruction, and the environmental
releases. The environmental releases can be
described in terms of geographic and temporal
distribution and the receiving environmental media,
with the form identified at the various release
points.
a.Characterization of Production and
Distribution. All sources of the substance's release
to the environment, consistent with the scope of the
assessment, should be included, such as production,
extraction, processing, imports, stockpiles,
transportation, accidental/ incidental production as
a side reaction, and natural sources. The sources
should be located, and activities involving exposure
to the substance should be identified.
b. Uses. The substance should be traced from its
sources through various uses (with further follow-up
on the products made to determine the presence of
the original material as an impurity), e.g., exports,
stockpile increases, etc.
c. Disposal. This subsection should contain an
evaluation of disposal sites and destruction
processes, such as incineration of industrial
chemical waste, incineration of the substance as
part of an end-use item in municipal waste,
landfilling of wastes, biological destruction, or
destruction in the process of using the end product.
Hazardous contaminants of the substance may be
included, and products containing the substance as a
contaminant may be followed from production
through destruction/disposal.
d. Summary of Environmental Releases.
Estimates should be made of the quantities of the
substance released to the various environmental
media. Sources of release to the environment include
production, use, distribution/transport, natural
sources, disposal, and contamination of other
products. Environmental releases should be
presented at a reasonable level of detail. Extremely
detailed exposure estimates would attempt to specify
the following information for each significant
emission source: location, amount of the substance
being released as a function of time to each
environmental medium, physical characteristics of
the emission source, and the physical and chemical
form of the substance being released. Evaluation of
the uncertainties associated with the emission
estimates should be given. A detailed discussion of
the procedures for estimating uncertainty is
presented in section 8.d.
5. Exposure Pathways and Environmental Fate.
The exposure pathways section should address how
an agent moves from the source to the exposed
population or subject. For a less detailed assessment,
broad generalizations on environmental pathways
and fate may be made. In the absence of data, e.g.,
for new substances, fate estimates may have to be
predicted by analogy with data from other
substances. Fate estimates may also be made by
using measurements and/or models and laboratory-
derived process rate coefficients. At any level of
detail, certain pathways may be judged insignificant
and not pursued further.
For more detailed assessments involving
environmental fate, the analysis of sources
described previously should provide the amount and
rate of emissions to the environment, and possibly
the locations and form of the emissions. The
environmental pathways and fate analysis follows
the substance from its point of initial environmental
release, through the environment, to its ultimate
fate. It may result in an estimation of the geographic
and temporal distribution of concentrations of the
substance in the various contaminated
environmental media.
a. Transport and Transformation. The
substance, once released to the environment, may be
transported (e.g., convected downstream in water or
on suspended sediment, through the atmosphere,
etc.) or physically transformed (e.g., volatilized,
melted, absorbed/desorbed, etc.); may undergo
chemical transformation, such as photolysis,
hydrolysis, oxidation, and reduction; may undergo
biotransformation, such as biodegradation; or may
accumulate in one or more media. Thus, the
environmental behavior of a substance should be
5-6
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evaluated before exposures are assessed. Factors
that should be addressed include:
How does the agent behave in air, water, soil,
. and biological media? Does it bioaccumulate or
biodegrade? Is it absorbed or taken up by plants?
What are the principal mechanisms for change
or removal in each of the environmental media?
Does the agent react with other compounds in
the environment?
Is there intermedia transfer? What are the
mechanisms for intermedia transfer? What are the
rates of the intermedia transfer or reaction
mechanisms?
How long might the agent remain in each
environmental medium? How does its concentration
change with time in each medium?
What are the products into which the agent
might degrade or change in the environment? Are
any of these degradation products ecologically or
biologically harmful? What is the environmental
behavior of the harmful products?
Is a steady-state concentration distribution in
the environment, or in specific segments of the
environment, achieved? If not, can the nonsteady-
state distribution be described?
What is the resultant distribution in the
environment-for different media, different types or
forms of the agent, for different geographical areas,
at different times or seasons?
b. Identification of Principal Pathways of
Exposure. The principal pathway analysis should
evaluate the sources, locations, and types of
environmental releases, together with
environmental behavioral factors, to determine the
significant routes of human and environmental
exposure to the substance. Thus, by listing the
important characteristics of the environmental
release (entering media, emission rates, etc.) and the
agent's behavior (intermedia transfer, persistence,
etc.) after release to each of the entering media, it
should be possible to follow the movement of the
agent from its initial release to its subsequent fate
in the environment. At any point in the
environment, human or environmental exposure
may occur. Pathways that result in major
concentrations of the agent and high potential for
human or environmental contact are the principal
exposure pathways.
c. Predicting Environmental Distribution.
Models may be used to predict environmental
distributions of chemicals. Model estimates of
environmental distribution of chemicals are based
on measurements whenever feasible. In predicting
environmental distributions of chemicals, available
measurements must be considered.
In this section an estimation is made, using
appropriate models, of representative concentrations
of the agent in different environmental media, and
its time-dependence in specific geographical
locations (e.g., river basins, streams, etc.).
6. Measured or Estimated Concentrations.
a. Uses of Measurements. Measurements are
used to identify releases (source terms) and, in the
[51 PR 34048]
exposure
pathways and fate assessments, to quantitatively
estimate both release rates and environmental
concentrations. Some examples of uses of
measurements are: sampling of stacks or discharge
pipes for emissions to the environment, testing of
products for chemical or radionuclide content,
testing of products for chemical or radioactive
releases, sampling of appropriate points within a
manufacturing plant to determine releases from
industrial processes or practices, sampling of
potentially exposed populations using personal
dosimeters, and sampling of solid waste for chemical
or radionuclide content. These data should be
characterized as to accuracy, precision, and
representativeness. If actual environmental
measurements are unavailable, concentrations can
be estimated by various means, including the use of
fate models (see previous section) or, in the case of
new chemicals, by analogy with existing chemicals.
Measurements are a direct source of information
for exposure analysis. Furthermore, reliable
measurements can be used to calibrate or
extrapolate models or calculations to assess
environmental distributions. However,
environmental pathway and fate analysis may be
needed in addition to the measured data for the
following reasons: for most pollutants, particularly
organic and new chemicals, measurements are
limited; analysis of measured data does not often
yield relationships between environmental releases
and environmental concentration distribution in
media or geographic locations that have not been
measured; analysis of measurements does not
provide information on how and where biota
influence the environmental distribution of a
pollutant; and measured concentrations may not be
traceable to individual sources.
b. Estimation of Environmental Concentrations.
Concentrations of agents should be estimated for all
environmental media that might contribute to
significant exposures. Generally, the environmental
concentrations are estimated from measurements,
mathematical models, or a combination of the two. If
environmental measurements are not limited by
sample size or inaccuracies, then exposure
assessments based on measurements have
precedence over estimates based on models.
The concentrations must be estimated and
presented in a format consistent with available dose-
response information. In some cases an estimate of
annual average concentration will be sufficient,
while in other cases the temporal distribution of
concentrations may be required. Future
environmental concentrations resulting from
current or past releases may also be projected. In
some cases, both the temporal and geographic
distributions of the concentration may be assessed.
5-7
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Moreover, if the agent has natural sources, the
contribution of these to environmental
concentrations may be relevant. These
"background" concentrations may be particularly
important when the results of tests of toxic effects
show a threshold or distinctly nonlinear dose-
response.
The uncertainties associated with the estimated
concentrations should be evaluated by an analysis of
the uncertainties of the model parameters and input
variables. When the estimates of the environmental
concentrations are based on mathematical models,
the model results must be compared to available
measurements, and any significant discrepancies
should be discussed. Reliable, analytically-
determined values must be given precedence over
estimated values whenever significant discrepancies
are found.
7. Exposed Populations. Populations selected for
study may be done a priori, but frequently the
populations will be identified as a result of the
sources and fate studies. Prom an analysis of the
distribution of the agent, populations and
subpopulations (i.e., collections of subjects) at
potentially high exposure can be identified, which
will then form the basis for the populations studied,
Subpopulations of high sensitivity, such as pregnant
women, infants, chronically ill, etc., may be studied
separately.
Census and other survey data may be used to
identify and describe the population exposed to
various contaminated environmental media.
Depending on the characteristics of available
toxieological data, it may be appropriate to describe
the exposed population by other characteristics such
as species, subspecies-age-sex distribution, and
health status.
In many cases, exposed populations can be
described only generally. In some cases, however,
more specific information may be available on
matters such as the following:
a. Human Populations
(1) Population size and characteristics (e.g.,
trends, sex/age distribution)
(2) Population location
(3) Population habits transportation habits,
eating habits, recreational habits, workplace habits,
product use habits, etc.
b. Nonhuman Populations (where appropriate)
(1) Population size and characteristics (e.g.,
species, trends)
(2) Population location
(3) Population habits
8. Integrated Exposure Analysis. The integrated
exposure analysis combines the estimation of
environmental concentrations (sources and fate
information) with the description of the exposed
population to yield exposure profiles. Data should be
provided on the size of the exposed populations;
duration, frequency, and intensity of exposure; and
routes of exposure. Exposures should be related to
sources.
For more detailed assessments, the estimated
environmental concentrations should be considered
in conjunction with the geographic distribution of
the human and environmental populations. The
behavioral and biological characteristics of the
exposed populations should be considered, and the
exposures of populations to various concentration
profiles should be estimated. The results can be
presented in tabular or graphic form, and an
estimate of the uncertainty associated with them
should be provided.
a. Calculation of Exposure. The calculation of
exposure involves two major aspects:
(1) Identification of the exposed population and
critical elements of the ecosystem.
The estimate of environmental concentrations
also should give the geographical areas and
environmental media contaminated. The stated
purpose of the assessment should have described the
human and environmental subjects for which
exposures are to be calculated. If the subjects are not
listed, the contaminated geographical areas and
environmental media can be evaluated to determine
subject populations. The degree of detail to be used
in defining the exposed population distribution
depends on the concentration gradient over
geographic areas.
(2) Identification of pathways of exposure.
(a) Identification and description of the routes by
which the substances travel from production site,
through uses, through environmental
releases/sources, through transport and fate
processes, to the target population.
(b) Quantitative estimates of the amounts of the
chemical following each exposure pathway. Such
estimates allow the various pathways ,to be put in
the perspective of relative importance.
From the geographic and temporal distribution
of environmental
[51 FR 34049]
concentrations,
the exposed population, the behavioral
characteristics, and the critical elements of the
ecosystem, exposure distributions can be estimated.
The results of exposure calculation should be
presented in a format that is consistent with the
requirements of the dose-response functions which
may later be used in a risk assessment. For example,
when health risks caused by exposure over extended
durations are considered, average daily exposure
over the duration of exposure usually is calculated.
When lifetime risks are considered, average daily
exposure over a lifetime usually is calculated. In
contrast, when health risks caused by exposures
over short durations are considered, exposure rates
are calculated over short time intervals to ensure
that peak risks are defined. Many exposure
assessments are based on the average exposure
occurring over the exposure period. The range of
possible exposures Is usually divided into intervals,
5-8
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and the exposures within each interval are counted.
The results can be presented in tabular form or as a
histogram.
The population residing in a specific geographic
area may be exposed to a substance from several
exposure routes. For each exposure route, exposure
of individuals in these populations may be
determined by summing the contribution of all
sources to the exposure route. When exposures
involve more than one exposure route, the relative
amounts of a substance absorbed is usually route
dependent. Consequently, total absorbed dose
estimates must account for these differences.
Because EPA regulates sources of releases, the
contribution to exposures from each type of source
being considered should be displayed. Exposure
estimates should be presented for each significant
exposure route, and the results should be tabulated
in such a way that total externally applied and
absorbed dose can be determined.
b. Human Dosimetry and Biological
Measurements. Biological measurements of human
body fluids and tissues for substances or their
metabolites can be used to estimate current or past
exposure to chemicals. When analytical methods are
available, chemicals that have been absorbed into
the body can be measured in body tissue and fluid.
TABLE 1.-EXPOSURE ASSESSMENT INFORMATION
Such measurements may be used to estimate human
exposure if the chemical substances leave in the
body reliable indicators of exposure. Furthermore,
although a compound may be relatively easy to
detect in body tissue, for some compounds,
attributing body burdens to specific environmental
releases may be difficult because of limited ability to
obtain environmental measurements or appropriate
metabolic data.
c. Development of Exposure Scenarios and
Profiles. Depending on the scope of the exposure
assessment, the total exposure may be fractionated
into one or more "exposure scenarios" to facilitate
quantification. As an example, Table 1 lists seven
very broad scenarios: Occupational, Consumer,
Transportation, Disposal, Food, Drinking Water,
and Ambient. For each of the scenarios, the major
topics necessary to quantify exposure include
sources, pathways, measurements, and population
characteristics. Investigation of only one scenario
may be necessary for the scope of some assessments.
For example, a pesticide application exposure
assessment may consider the occupational scenario
which would address the exposure to applicators and
populations in the vicinity of the site. An exposure
assessment around a hazardous waste site may focus
on the disposal scenario. The exposure assessment
NEEDS FOR VARIOUS EXPOSURE SCENARIOS
Exposure scenario
Occupational
(chemical
production).
Consumer (direct use
of chemical or
inadvertent use).
Transportation/
storage/spills.
Disposal (include
incineration,
landfill).
Food
Drinking water
Ambient
Sources
Site/plant locations,
in-plant/on-site
materials balance.
Consumption rates,
distribution pattern
amounts in
products.
Patterns of
distribution and
transportation;
models for spills.
Materials balance
around disposal
method,
efficiency, releases to
environment.
Food chain,
packaging,
additives.
Groundwater,
surface water,
distribution system.
Releases to
environment; air,
land, water.
Fate
Physical and
chemical properties
models.
Physical and
chemical properties,
shelf life release
rates, models.
Physical and
chemical properties,
environmental fate
models.
Fate within disposal
process;
environmental fate
of releases; models.
Food chain models,
fate during
preparation or
processing of food.
Leach rates from
pipes, chlorination
processes, fate in
water; models.
Environmental fate
models.
Population Characteristics
Workers, families, population
around sites/plants.
Consumers
Storage, transportation
workers, general population
in area.
Workers at site of disposal,
general population around
site.
General population,
nonhuman population.
General population.
General population,
nonhuman population.
Measurement
In-plant/on-site
releases, ambient
levels surrounding
site/plants; human
dosimetry.
Levels in products
releases.
Releases, ambient
levels.
Releases, levels at
various points within
process, ambient
levels.
Levels in food,
feedstuff; food
chain sampling.
Levels in drinking
water, groundwater,
surface water,
treatment plants.
Ambient air, water,
soil, etc.; human
dosimetry.
5-9
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also may consider other scenarios. The more
extensive and comprehensive the scope, the more
scenarios are usually involved.
It will usually be advantageous in performing an
exposure assessment to identify exposure scenarios,
quantify the exposure in each scenario, and then
integrate the scenarios to estimate total exposure. In
this "integrated exposure analysis," the summation
of independent exposures from different scenarios
(keeping exposure routes separate) often will result
in a breakout of exposure by subpopulations, since
the individual scenarios usually treat exposure by
subpopulation. Therefore, the integration of the
scenarios, or integrated exposure analysis, will often
result in an exposure profile.
For each exposed subpopulation, exposure
profiles should include the size of the group, the
make-up of the group (age, sex, etc.), the source of
the agent, the exposure pathways, the frequency and
the intensity of exposure by each route (dermal,
inhalation, etc.), the duration of exposure, and the
form of the agent when exposure occurs.
Assumptions and uncertainties associated with each
scenario and profile should be clearly discussed.
d. Evaluation of Uncertainty.
(1) Introduction. Often an exposure assessment
progresses through several stages of refinement. The
purpose of these Guidelines is to present methods
appropriate for characterization of uncertainty for
assessments at various stages of refinement, from
assessments based on limited initial data to those
based on extensive data.
The appropriate method for characterizing
uncertainty for an exposure assessment depends
upon the underlying parameters being estimated,
the type and extent of data available, and the
estimation procedures utilized.
[51 FR 34050]
The uncertainty
of interest is always with regard to the population
characteristic being estimated. For example, when
the population distribution of exposures is being
estimated, characterization of uncertainty addresses
the possible differences between the estimated
distribution of exposure and the true population
distribution of exposure.
An exposure assessment quantifies contact of
a substance with affected population members
(human or nonhuman subjects). The measure of
contact (e.g., environmental level or absorbed dose)
depends upon what is needed to predict risk. An
integrated exposure assessment quantifies this
contact via all routes of exposure (inhalation,
ingestion, and dermal) and all exposure pathways
(e.g., occupational exposure, exposure from
consumption of manufactured goods, etc.). The
exposed population generally is partitioned into
subpopulations such that the likely exposure of all
members of a subpopulation is attributable to the
same sources. The exposure for each member of a
subpopulation is then the sum of exposures over a
fixed set of sources and pathways. The measured or
estimated exposures for members of a subpopulation
are ideally used to estimate the subpopulation
distribution of exposure or characteristics thereof.
However, a lack of sufficient information sometimes
precludes estimation of the subpopulation
distributions of exposure and only summary
measures of this distribution, such as the mean,
minimum, maximum, etc., are estimated. In each
case, characterization of uncertainty for the
exposure assessment primarily addresses
limitations of the data and the estimation
procedures. The proportions of the population
members in the individual subpopulations are
usually estimated and can be used (by combining
estimated distributions for the subpopulations) to
estimate the distribution of exposure for the total
population. Uncertainty concerning the sizes of the
subpopulations should be addressed by discussing
limitations of the data and estimation methods as
well as by tabulating confidence interval estimates
for the population sizes whenever possible.
(2) Assessments based on limited initial data.
The initial exposure assessment for a substance may
be based on limited data for exposure and/or input
variables for an exposure prediction model (i.e., an
equation that expresses exposure as a function of one
or more input variables). These data might be either
extant data or data produced by an initial small-
scale study. The limited initial data frequently are
insufficient to permit estimation of the entire
distribution of exposure. Instead, summary
measures of this distribution, such as the mean,
minimum, and maximum, are usually estimated.
If the assessment is based on measured
exposures, the methods used to characterize
uncertainty depend mainly upon whether or not the
data result from a probability sample for which the
probability of inclusion is known for each sample
member. Characterization of uncertainty for an
assessment based on a probability sample of
exposures is discussed later in section 8.d.(5). If the
measured exposures are not based on a probability
sample, acknowledgement that no strictly valid
statistical inferences can be made beyond the units
actually in the sample is one aspect of the
characterization of uncertainty. If inference
procedures are implemented, the assumptions upon
which these inferences are based (e.g., treatment of
the sample as if it were a simple random sample, or
assumption of an underlying model) should be
explicitly stated and justified. The data collection
methods and inherent limitations of the data should
also be discussed.
An initial exposure assessment also may be
based on limited data, such as estimated ranges, for
input variables for an exposure prediction model.
The exposure prediction model would be derived
from a postulated exposure scenario that describes
the pathways from sources to contact with
population members. If the data were only sufficient
to support estimates of the ranges of the input
variables, the exposure assessment might be limited
5-10
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to a sensitivity analysis. The purpose of the
sensitivity analysis would be to identify influential
model input variables and develop bounds on the
distribution of exposure. A sensitivity analysis
would estimate the range of exposures that would
result as individual model input variables were
varied from their minumum to their maximum
possible values with the other input variables held
at fixed values, e.g., their midranges. The overall
minimum and maximum possible exposures usually
would be estimated also. For an exposure
assessment of this type, the uncertainty would be
characterized by describing the limitations of the
data used to estimate possible ranges of model input
variables and by discussing justification for the
model. Justification of the model should include a
description of the exposure scenario, choice of model
input variables, and the functional form of the
model. Sensitivity to the model formulation also can
be investigated by replicating the sensitivity
analysis for plausible alternative models.
The sensitivity analysis can be enhanced by
computing the predicted exposures that result from
all possible input variable combinations. If each
input variable has only a finite set of possible
values, the set of all possible combinations of the
input variables can be formed, and the predicted
exposure can be computed for each combination.
These exposure predictions can be used to form a
distribution of exposures by counting the number of
occurrences at each exposure level or interval of
exposures. This is equivalent to estimating the
distribution of exposures that results from treating
all input variable combinations as equally likely.
This procedure can also be applied by transforming
continuous input variables into discrete ones and
representing them by equally spaced points. In the
limit, as the equal spaces become small and the
number of points becomes large, the distribution of
exposure that results from counting occurrences of
exposure levels is equivalent to estimating the
distribution of exposures that results from
statistically independent, continuous input
variables with uniform distributions on the
estimated ranges. This estimated distribution of
exposure values can be produced by Monte Carlo
simulation, one of the methods of mathematical
statistics. The Monte Carlo method consists of
randomly generating input variate values and using
these to compute corresponding exposure levels,
generating an exposure distribution via many
iterations. Interpretation of statistics based on this
exposure distribution would be in terms of the
equally likely input variable combinations. For
example, the 95th percentile of this distribution
would be the exposure level exceeded by only 5% of
the exposures resulting from treating all
combinations of input variable values as equally
likely. Although this distribution of exposures
cannot be interpreted as an estimate of the
population distribution (unless the input variables
actually are statistically independent and uniformly
distributed), it provides additional information for
making regulatory decisions. Characterization of
uncertainty would include a discussion of
limitations of the data and justification for the
model as discussed above. Sensitivity to model
formulation could also be investigated by estimating
the distribution of exposure that results from using
the
[51FR34051]
same uniform
input variable distributions with plausible
alternative models and comparing the estimated
percentiles.
(3) Assessments based on subjective estimates
of input variable distributions. If a model has been
formulated that expresses exposure as a function of
one or more input variables, the methods of
mathematical statistics, such as Monte Carlo
simulation, can be used to estimate the population
distribution of exposure from an estimate of the joint
distribution of the model input variables. Ideally,
model input variables should be represented by
empirically-validated probability distributions. In
some cases, it may be possible to formulate an
estimate of the joint distribution of model input
variables from discussions with subject matter
experts (e.g., via histograms for statistically-
independent input variables). The estimated
population distribution of exposure will be
equivalent to the distribution discussed in section
8.d.(2) for equally likely combinations of input
variable values only when the input variable
distributions supported are independent uniform
distributions. When qualitative knowledge of input
variable distributions is used to estimate the
population distribution of exposure, uncertainty is
characterized by discussing justification for the
presumed model and input variable distributions.
Alternative models and/or alternative input
variable distributions also should be discussed.
Sensitivity to these alternatives can be investigated
by estimating the distributions of exposure that
result from plausible alternatives and comparing
the percentiles of the estimated exposure
distributions. All available data, even if data are
limited, should be used to validate the presumed
input variable distributions and the predicted
distribution of exposure.
(4) Assessments based on data for model input
variables. The exposure assessment based on an
estimate of the joint probability distribution for
model input variables can be refined by collecting
sample survey data for model input variables for a
sample of population members. The population
distribution of exposure can then be estimated by
computing the expected exposure for each sample
member based on the model. These expected
exposures can be used to directly compute confidence
interval estimates for percentiles of the exposure
distribution. Alternatively, the sample survey data
can be used to compute joint confidence interval
estimates for percentiles of the input variable
5-11
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distribution, which can then be used to generate
confidence interval estimates for percentiles of the
exposure distribution. In either case, the interval
estimates for percentiles of the exposure distribution
are a useful quantitative characterization of
uncertainty.
Characterization of uncertainty for the
exposure assessment would contain a thorough
discussion of limitations of the data and justification
for the model used to compute expected exposures.
The design of the sample survey used to produce the
data base should also be discussed. If a probability
sample were not used, the lack of a probability
sample would be an additional source of uncertainty.
Any assumptions used in computing the confidence
interval estimates, such as independence of model
input variables, should be explicitly stated and
justified. Sensitivity to model formulation can be
investigated by estimating the distribution of
exposure for plausible alternative models and
comparing the estimated percentiles, if sample
survey data have been collected for the input
variables of the alternative models. Appropriate
available data for exposure should be used to
validate the predicted distribution of exposure. If
specific probability distributions have been
presumed for any model input variables, the data for
these variables should be used to test for goodness of
fit for these distributions.
(5) Assessments based on data for exposure. A
major reduction in the uncertainty associated with
an exposure assessment can be achieved by directly
measuring the exposure for a sufficiently large
sample of members of the affected population. This
reduction in uncertainty is achieved by eliminating
the use of a model to predict exposure. The measured
exposure levels can be used to directly estimate the
population distribution of exposure and confidence
interval estimates for percentiles of the exposure
distribution. Direct confidence interval estimates
also can be computed for other characteristics of the
exposure distribution, such as the mean exposure.
These confidence interval estimates are then
the primary characterization of uncertainty for the
exposure assessment. Limitations of the data and
design of the sample survey used to collect the data
also should be discussed. If the sample was not a
probability sample, this would again be an
additional source of uncertainty.
(6) Summary. A summary of the primary
methods recommended for characterizing
uncertainty in exposure assessments is presented in
Table 2. Virtually all exposure assessments, except
those based on measured exposure levels for a
probability sample of population members, rely
upon a model to predict exposure. The model may be
any mathematical function, simple or complex, that
expresses an individual's exposure as a function of
one or more input variables. Whenever a model that
has not been validated is used as the basis for an
exposure assessment, the uncertainty associated
with the exposure assessment may be substantial.
The primary characterization of uncertainty is at
least partly qualitative in this case, i.e., it includes a
description of the assumptions inherent in the model
and their justification. Plausible alternative models
should be discussed. Sensitivity of the exposure
assessment to model formulation can be
investigated by replicating the assessment for
plausible alternative models,
[51 FR 34052]
When an exposure assessment is based on
directly measured exposure levels for a probability
sample of population members, uncertainty can be
greatly reduced and described quantitatively. In this
case, the primary sources of uncertainty are
measurement errors and sampling errors. The
effects of these sources of error are measured
quantitatively by confidence interval estimates of
percentiles of the exposure distribution. Moreover,
the sampling errors can be limited by taking a large
sample.
Whenever it is not feasible to take a large
sample, it is sometimes possible to obtain at least
some data for exposure and model input variables.
These data should be used to assess goodness of fit of
the model and/or presumed distributions of input
variables. This substantially reduces the amount of
quantitative uncertainty for estimation of the
distribution of exposure and is strongly
recommended. It is recognized, however, that it may
not be feasible to collect such data.
9.References. The references should contain a
listing of all reports, documents, articles,
memoranda, contacts, etc. that have been cited in
the report.
10.Appendices. The appendices may contain
such items as memoranda and letters that are not
readily accessible, other tables of measurements,
detailed lists of emission sources, detailed tables of
exposures, process flow diagrams, mathematical
model formulations, or any other item that may be
needed to describe or document the exposure
assessment.
Part B: Response to Public and Science
Advisory Board Comments
/. Introduction
This section summarizes some of the issues
raised in public comments on the Proposed
Guidelines for Exposure Assessment published
November 23, 1984 (49 FR 46304). Comments were
received from 29 individuals or organizations. The
Agency's initial summary of comments was
presented to the Exposure Assessment Guidelines
Review Group of the Science Advisory Board (SAB)
on March 4, 1985. At its April 22-23,1985, meeting,
the panel provided the Agency with suggestions and
recommendations concerning the Guidelines.
The SAB and public commentors expressed
diverse opinions and addressed issues from a variety
of perspectives. While most commentors supported
the Guidelines, two urged withdrawal of the
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TABLE 2.-SUMMARY OF PRIMARY METHODS FOR CHARACTERIZING UNCERTAINTY FOR ESTIMATING
EXPOSURES
Type and extent of data
Measured exposures
fora large sample
of population
members.
Measured exposures for a
small sample of
population members.
Measured model input
variables for a large
sample of population
members.
Estimated distributions
of model input
variables.
Limited data for model
input variables.
Population characteristic
being estimated
Distribution of exposure.
Summary parameter(s) of
the exposure
distribution, e.g..
meanorapercentile.
Distribution of exposure.
Distribution of exposure.
Minimum, maximum, and
range of the exposure
distribution.
Primary methods for characterizing uncertainty
Qualitative methods
1. Limitations of the
survey design and
measurement
techniques.
1. Limitations of the
survey design and.
measurement
techniques.
1. Limitations of the
survey.designand
measurement
techniques.
2. Validity of the
exposure model.
1. Validity of the
exposure model.
2. Limitations of the data.
or other basis for the
input variable
distributions.
1 . Limitations of the data.
2. Validity of the
exposure model.
Quantitative methods
1 . Confidence interval estimates for
percentiles of the exposure
distribution.
2. Goodness of fit for exposure models.
if any have been postulated.
1 . Confidence interval estimate for the
summary parameter(s).
2. Goodness of fit for exposure models,.
if any have been postulated.
1. Confidence interval estimates for
percentiles of the exposure
distribution.
2. Goodness of fit for input variable
distribution functions, if any have
been postulated.
3. Estimated distribution of exposure
based on alternative models.
1 . Confidence interval estimates for
percentiles of the exposure
distribution.
2. Goodness of fit for input variable
distributions, if input variable data
are available.
3. Estimated distribution of exposure
based on alternative models.
If input variable data are very limited.
e.g., some extant data collected for
other purposes, quantitative
characterization of uncertainty may
not be possible.
document. The SAB Panel recommended that
supplementary guidelines be written on the use of
measurements in preparing exposure assessments.
In addition, the Panel wished to see a greater
emphasis in the current Guidelines on the use of
measured data rather than models in generating
exposure assessments. The Panel recommended that
the technical support document entitled
"Methodology for Characterization of Uncertainty in
Exposure Assessments" be expanded with additional
examples.
In response to the comments, the Agency has
modified or clarified many sections of the
Guidelines, and is planning to develop
supplementary guidance in line with the SAB
recommendations. The discussion that follows
highlights significant issues raised in the comments,
and the Agency's response to them. Also, many
minor recommendations, which do not warrant
discussion here, were adopted by the Agency.
//. General Information
A. Acceptable Latitude of Approach
Some commentors believe the Guidelines are too
general and allow too much latitude in choice of
approach and do not assure that "all" data, sources,
limitations, etc. are considered before an exposure
assessment is conducted. Others suggested that the
Agency specify models to be used while others
thought that only measured data should be allowed.
The Guidelines were developed to provide
assistance in carrying out exposure assessments.
The approach suggested is deliberately general in
order to accommodate the development of exposure
assessments with different levels of detail depending
on the scope of the assessment. The Agency does not
agree with the inclusion of such restrictive
terminology as "in all cases." We cannot foresee all
possible cases. We believe reasonable flexibility is a
necessary ingredient for the proper implementation
of the Guidelines while relying on uncertainty and
sensitivity analyses to put the quality of the
approach in perspective.
B. Technical Nature of Guidelines
Some commentors believe the language of the
document is too technical for the lay person to
understand; one commentor expressed misgivings
concerning the "state-of-the-art" methods available
for conducting exposure assessments.
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While the Agency recognizes that the public has
an interest in the Guidelines and invites comments
from the public, the Guidelines are intended for use
by technical/professional people. Providing
guidelines written in lay terms would result in
insufficient technical specifications to the
professionals in the development of scientifically
acceptable exposure assessments.
The Agency believes that the suggested
procedures and methods in the Guidelines are
commonly accepted. The Guidelines do not suggest
the use of ad hoc, untested, and unvalidated
procedures, but stress the use of the best scientific
methods available with maximum analysis of
existing data. This is both a scientific and practical
approach that reflects the level of consensus within
the Agency.
[51 PR 34053]
C. Measurements vs. Modeling
Some commentors support the use of
measurements alone to develop an exposure
assessment. Some believed there should be no data
restraints; others thought all data should be
validated. Other commentors argued for the use of
simulation model estimates without measurements.
One commentor objected to the use of unvalidated
models to perform exposure assessments. In its
review, the SAB strongly encouraged the Agency to
develop a supplement to the current Guidelines on
the development and use of measurements for
exposure assessments.
The Agency encourages the use of validated
measurements when available. The Guidelines
specifically state that "Reliable, analytically
determined values should be given precedence over
estimated values. . ." and analytically determined
values ", . .can be used to calibrate. . . models. . .to
assess environmental distribution." Furthermore, in
practice, exposure assessments performed by the
Agency use published models with varying degrees
of testing and validation. It is our belief that
transport process models have been adequately
validated over many years in most cases.
Furthermore, the Agency has revised the
Guidelines to reflect the SAB suggestions that
exposure assessments based on reliable measured
data are preferred over model estimates whenever
feasible.
III. Data Availability and Uncertainty Analysis
A. Information Uses
Some commentors asked for guidance in the use
of information that may be false and how to deal
with the potential situation when different models
give different results. Others asked for model
selection criteria.
The Guidelines clearly state the considerations
that need to be addressed when assembling
information bases for exposure assessments. Two
considerations are: qualitative and quantitative
nature of the data and the reliability of the
information. Whether the exposure assessment is
based on measurements or simulation model
estimates, an evaluation of uncertainties associated
with the data including source data and
.assumptions is necessary and important.
When there is uncertainty in the scientific facts,
it is Agency policy to err on the side of public safety.
The Agency intends to be realistic, but will not
arbitrarily select midranges of environmental
distributions that may compromise human health.
In addition, quality assurance is an important
matter that requires detailed attention. The
collection of measured data and the development of
methods to collect measurements are done by
another office within the EPA. These issues will be
handled by the Office of Acid Deposition,
Environmental Monitoring, and Quality Assurance
as they develop the supplemental guidelines for
measurement of exposure.
Substantial work is currently being done on the
development of mathematical model selection
criteria. Results of these efforts will be published as
a technical support document containing detailed
information to further implement the Guidelines.
B. Worst-Case Estimates
A few commentors were concerned that worst-
case estimates would be used when data are
nonexistent or limited. The Guidelines do not
encourage the use of worst-case assessments, but
rather the development of realistic assessments
based on the best data available.
A technical support document and a substantial
section of the Guidelines currently discuss
evaluation of uncertainty in order to produce
objective assessments using the best (not worst-case)
estimates available either for preliminary or in-
depth exposure assessments. However, the Agency
will err on the side of public health when evaluating
uncertainties when data are limited or nonexistent.
IV. Evaluation of Uncertainties
A. Uncertainty Analysis
Many commentors felt that the sections of the
Guidelines that dealt with uncertainty needed
amplification while some sections as written were
confusing. Some urged that uncertainty evaluation
be presented and documented for each section within
a specific exposure scenario in order to judge the
overall plausibility of the assessment in reaching
regulatory decisions.
Since the accuracy of an exposure assessment is
influenced by the degrees of uncertainty contained
in both data and assumptions, the Guidelines call for
the evaluation of these uncertainties. The technical
support document, Methodology for
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Characterization of Uncertainty in Exposure
Assessments (available from the National Technical
Information Service, PB85-240455), describes in
detail how such analyses can be performed. The
Guidelines suggest that the uncertainty
characterization include a discussion of the
limitations of the data and estimation procedures as
the justification for the model chosen. A sensitivity
analysis of the exposure assessment is appropriate if
the data were only able to support the estimates of
ranges of the input variables. By identifying model
input variables that determine the bounds on the
distribution of exposure, the range of exposure,
which results as individual model input variables
are varied from minimum to maximum possible
values as other variables remain constant,
constitutes the sensitivity analysis. Further
sensitivity of model formulation can be examined by
repeating the sensitivity analysis for plausible
alternative models.
Nothing in the Guidelines precludes estimation
of uncertainty for each specific exposure scenario.
The Agency has encouraged the evaluation of
uncertainty in each aspect of the exposure
assessment, which could impact the total risk
estimate. It is important to estimate the level of
uncertainty in risk assessments so that decisions
based on risk assessment will reflect total
uncertainty. The information presented in the
Guidelines or the technical support documents
properly and adequately describes the extent and
quality of appropriate uncertainty analysis.
Recognizing that the basis for the decision to refine a
preliminary exposure assessment involves risk
management, the Agency, at the suggestion of many
commentors, decided to strike from the Guidelines
the paragraph beginning "If the maximum possible
exposure...." in section III.B.8.d.(2).
B. Population Characterization
The Guidelines state that identification of
populations and subpopulations at potentially high
exposure forms the basis of the populations to be
studied. Separate studies of sensitive subpopulation
can also be included. Population characteristics,
such as age and/or sex distributions, can be derived
from the use of geographic and activity-specific data.
Uncertainty related to estimation of a population
characteristic include a discussion of the data
limitations and the estimation procedures. In
addition, uncertainty in estimating sizes of sensitive
subpopulations should include estimates of
confidence intervals.
Some commentors suggested the inclusion of
additional characteristics, such as occupational and
life style factors, and the inclusion of additional
guidance concerning potential pitfalls when
conducting population exposure
[51 FR 34054]
assessments.
Others expressed concern that the exposure of a
particular subpopulation would be combined with
other exposures to produce an average exposure
level for the general population.
The section describing population
characterization encompasses, in general terms, the
many characteristics that may be available,
including life style factors, to describe exposed
populations. The Agency agrees that there are
difficulties associated with epidemiologic studies.
The relationship between exposure assessments and
epidemiologic studies is currently being
investigated and will be the subject of a future
technical support document and the further
refinement of the Guidelines.
V. Clarification of Terminology
A. Exposure vs. Dose
Commentors expressed concern with the
American Society for Testing and Materials (ASTM)
definition of exposure. Concern was also raised
about the assertion that exposures can be estimated
when biological tissues for fluid measurements
indicate the presence of a chemical. Some
commentors found difficulty in the wording of the
last sentence in section H.A., specifically "The route
of exposure.. .impacts.. .the overall exposure "
It is the Agency's opinion that the members who
served on the ASTM Committee E-47 had expertise
in exposure assessment. The scientists and
engineers cumulatively possessed many years of
experience in exposure assessment. In addition, no
technical society has presented an alternate
definition of exposure. The Agency will consider
changing the definition if a reasonable alternate
definition is written and agreed upon by the
scientific community.
The Agency agrees with the commentors who
were concerned that the wording provided in the
Guidelines that the presence of a chemical in
biological tissue can be used to estimate exposure is
not correct in all cases. Consequently, the word
"can" was changed to "may" to reflect the current
level of understanding between tissue residue and
exposure (II.A., 2nd paragraph, 4th sentence). The
Agency agrees with several commentors' concerns
that the route of exposure impacts the overall
absorbed dose, not the overall exposure, and the
Guidelines reflect this change (H.A., last sentence).
B. Mixtures and Synergism
Some commentors thought more discussion was
necessary on the effect of chemical mixtures and
potential synergistic effect on exposure. The
Guidelines for the Health Risk Assessment of
Chemical Mixtures includes a discussion of chemical
synergism. The Agency recognizes the need to do
further work in the area of exposure to mixtures. It
is recommended that this be identified as an area
requiring further research.
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These Guidelines stress the need to determine
the products into which the chemical might degrade
or react in the environment and to determine if any
of these products are ecologically or biologically
harmful.
C. Removal and Creation Steps
Some commentors urged that more emphasis be
placed on changes that occur once the materials
have entered the ambient environment. Other
commentors argued that our current understanding
will not allow a comprehensive treatment,
particularly for metabolic processes.
These Guidelines state the need to address how a
chemical agent moves from the source to the exposed
population, which may result in the estimation of
geographic and temporal distributions in various
environmental media. The Guidelines also state the
need to know such factors as, for example, whether
the chemical agent bioaccumulates or by what
mechanism the agent is removed from each medium
and the role of any degradation products on
ecological safety. We have already stated that
guidance for analysis of metabolism data is an area
of ongoing research which includes consideration of
metabolism data in the calculation of whole
organism dose from one species to another.
VI, Purpose, Philosophy, and Results
Several commentors raised questions related to
the basic style of the Guidelines. Among the issues
raised were:
the role of exposure assessment in risk
assessment/risk management (many comments
directed to appropriateness of Figure 1);
statutory/regulatory authority and uses of
results; and
the need for peer review of assessments and
periodic updating of Guidelines.
A deliberate effort to separate risk assessment
from risk management has been made. The
management of complex issues such as procedural
issues, which include coordination or linkage among
divisions in the Agency, are best dealt with by
management and not in Guidelines.
The decision pathway (Figure 1) was included in
the Guidelines at the recommendation of the SAB. It
has drawn many comments. The changes suggested
would include additional detail and steps that would
diminish the value of the graphic. However, the
figure has been truncated to remove risk
management steps.
In order to remain consistent with the
separation of risk assessment and risk management,
any directions to consider applicable laws or
regulatory decisions have been stricken from the
Guidelines.
The Agency agrees that peer review is an
important aspect of the assessment process.
However, emergency cases may not allow peer
review in preliminary assessments. All
nonemergency exposure assessments have been peer
reviewed and will continue to be peer reviewed.
Finally, it is clearly stated in the Guidelines that
periodic revision of the document will be done to
reflect the benefit of experience and knowledge.
7 - 748-121/67049
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