[Federal Register Volume 61, Number 79 (Tuesday, April 23, 1996)]
[Notices]
[Pages 17960-18011]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 96-9711]
[[Page 17959]]
_______________________________________________________________________
Part II
Environmental Protection Agency
_______________________________________________________________________
Proposed Guidelines for Carcinogen Risk Assessment; Notice
Federal Register / Vol. 61, No. 79 / Tuesday, April 23, 1996 /
Notices
[[Page 17960]]
ENVIRONMENTAL PROTECTION AGENCY
[FRL-5460-3]
Proposed Guidelines for Carcinogen Risk Assessment
AGENCY: Environmental Protection Agency (EPA).
ACTION: Notice of Availability and Opportunity to Comment on Proposed
Guidelines for Carcinogen Risk Assessment.
-----------------------------------------------------------------------
SUMMARY: The U.S. Environmental Protection Agency (EPA) is today
publishing a document entitled Proposed Guidelines for Carcinogen Risk
Assessment (hereafter ``Proposed Guidelines''). These Proposed
Guidelines were developed as part of an interoffice guidelines
development program by a Technical Panel of the Risk Assessment Forum
within EPA's Office of Research and Development. These Proposed
Guidelines are a revision of EPA's 1986 Guidelines for Carcinogen Risk
Assessment (hereafter ``1986 cancer guidelines'') published on
September 24, 1986 (51 FR 33992). When final, these guidelines will
replace the 1986 guidelines.
In a future Federal Register notice, the Agency intends to publish
for comment how it will implement the Proposed Guidelines once they are
finalized. The plans will propose and seek comment on how the
Guidelines will be used for Agency carcinogen risk assessment and, in
particular, will address the impact of the Guidelines on the Agency's
existing assessments, and any mechanisms for handling reassessments
under finalized Guidelines.
DATES: The Proposed Guidelines are being made available for a 120-day
public review and comment period. Comments must be in writing and must
be postmarked by August 21, 1996. See Addresses section for guidance on
submitting comments.
ADDRESSES: The Proposed Guidelines will be made available in the
following ways:
(1) The electronic version will be accessible on EPA's Office of
Research and Development home page on the Internet at http://
www.epa.gov/ORD
(2) 3\1/2\'' high-density computer diskettes in Wordperfect 5.1
format will be available from ORD Publications, Technology Transfer and
Support Division, National Risk Management Research Laboratory,
Cincinnati, OH; telephone: 513-569-7562; fax: 513-569-7566. Please
provide the EPA No. (EPA/600/P-92/003Ca) when ordering.
(3) This notice contains the full draft document. In addition,
copies of the draft will be available for inspection at EPA
headquarters and regional libraries, through the U.S. Government
Depository Library program, and for purchase from the National
Technical Information Service (NTIS), Springfield, VA; telephone: 703-
487-4650, fax: 703-321-8547. Please provide the NTIS PB No. (PB96-
157599) ($35.00) when ordering.
SUBMITTING COMMENTS: Comments on the Proposed Guidelines may be mailed
or delivered to the Technical Information Staff (8623), NCEA-WA/OSG,
U.S. Environmental Protection Agency, 401 M Street, S.W., Washington,
DC 20460. Comments should be in writing and must be postmarked by the
date indicated. Please submit one unbound original with pages numbered
consecutively, and three copies. For attachments, provide an index,
number pages consecutively with the comment, and submit an unbound
original and three copies.
Please note that all technical comments received in response to
this notice will be placed in a public record. For that reason,
commenters should not submit personal information (such as medical data
or home address), Confidential Business Information, or information
protected by copyright. Due to limited resources, acknowledgments will
not be sent.
FOR FURTHER INFORMATION CONTACT: Technical Information Staff,
Operations and Support Group, National Center for Environmental
Assessment--Washington Office, telephone: 202-260-7345. Email inquiries
may be sent to [email protected].
SUPPLEMENTARY INFORMATION: In 1983, the National Academy of Sciences
(NAS)/National Research Council (NRC) published its report entitled
Risk Assessment in the Federal Government: Managing the Process (NRC,
1983). In that report, the NRC 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. The 1986 cancer guidelines were issued on September
24, 1986 (51 FR 33992). The Proposed Guidelines published today
continue the guidelines development process. These guidelines set forth
principles and procedures to guide EPA scientists in the conduct of
Agency cancer risk assessments and to inform Agency decisionmakers and
the public about these procedures.
Both the 1986 guidelines and the current proposal contain inference
guidance in the form of default inferences to bridge gaps in knowledge
and data. Research conducted in the past decade has elucidated much
about the nature of carcinogenic processes and continues to provide new
information. The intent of this proposal is to take account of
knowledge available now and to provide flexibility for the future in
assessing data and employing default inferences, recognizing that the
guidelines cannot always anticipate future research findings. Because
methods and knowledge are expected to change more rapidly than
guidelines can practicably be revised, the Agency will update specific
assessment procedures with peer-reviewed supplementary, technical
documents as needed. Further revision of the guidelines themselves will
take place when extensive changes are necessary.
Since 1986, the EPA has sponsored several workshops about revising
the cancer guidelines (U.S. EPA, 1989b, 1989c, 1994a). The Society for
Risk Analysis conducted a workshop on the subject in connection with
its 1992 annual meeting (Anderson et al., 1993). Participants in the
most recent workshop in 1994 reviewed an earlier version of the
guidelines proposed here and made numerous recommendations about
individual issues as well as broad recommendations about explanations
and perspectives that should be added. Most recently, the Committee on
the Environment and Natural Resources of the Office of Science and
Technology Policy reviewed the guidelines at a meeting held on August
15, 1995. The EPA appreciates the efforts of all participants in the
process and has tried to address their recommendations in this
proposal.
In addition, the recommendations of the NRC (1994) in Science and
Judgment in Risk Assessment have been addressed. Responses to these
recommendations are given generally in Appendix B as well as being
embodied in the Proposed Guidelines. Responses that explain the major
default assumptions adopted under these guidelines and the policy for
using and departing from these default assumptions appear in Section
1.3.
The Science Advisory Board also will review these Proposed
Guidelines at a meeting to be announced in a future Federal Register
notice. Following these reviews Agency staff will prepare summaries of
the public and SAB comments. Appropriate comments will be incorporated,
and the revised Guidelines will be submitted to the Risk Assessment
Forum for review. The
[[Page 17961]]
Agency will consider comments from the public, the SAB, and the Risk
Assessment Forum in its recommendations to the EPA Administrator.
Major Changes From the 1986 Guidelines
Characterizations
Increased emphasis on providing characterization discussions for
the hazard, dose response, and exposure sections is part of the
proposal. These discussions will summarize the assessments to explain
the extent and weight of evidence, major points of interpretation and
rationale, and strengths and weaknesses of the evidence and the
analysis, and to discuss alternative conclusions and uncertainties that
deserve serious consideration (U.S. EPA, 1995). They serve as starting
materials for the risk characterization process which completes the
risk assessment.
Weighing Evidence of Hazard
A major change is in the way hazard evidence is weighed in reaching
conclusions about the human carcinogenic potential of agents. In the
1986 cancer guidelines, tumor findings in animals or humans were the
dominant components of decisions. Other information about an agent's
properties, its structure-activity relationships to other carcinogenic
agents, and its activities in studies of carcinogenic processes was
often limited and played only a modulating role as compared with tumor
findings. In this proposal, decisions come from considering all of the
evidence. This change recognizes the growing sophistication of research
methods, particularly in their ability to reveal the modes of action of
carcinogenic agents at cellular and subcellular levels as well as
toxicokinetic and metabolic processes. The effect of the change on the
assessment of individual agents will depend greatly on the availability
of new kinds of data on them in keeping with the state of the art. If
these new kinds of data are not forthcoming from public and private
research on agents, assessments under these guidelines will not differ
significantly from assessments under former guidelines.
Weighing of the evidence includes addressing the likelihood of
human carcinogenic effects of the agent and the conditions under which
such effects may be expressed, as these are revealed in the
toxicological and other biologically important features of the agent.
(Consideration of actual human exposure and risk implications are done
separately; they are not parts of the hazard characterization). In this
respect, the guidelines incorporate recommendations of the NRC (1994).
In that report, the NRC recommends expansion of the former concept of
hazard identification, which rests on simply a finding of carcinogenic
potential, to a concept of characterization that includes dimensions of
the expression of this potential. For example, an agent might be
observed to be carcinogenic via inhalation exposure and not via oral
exposure, or its carcinogenic activity might be secondary to another
toxic effect. In addition, the consideration of evidence includes the
mode(s) of action of the agent apparent from the available data as a
basis for approaching dose response assessment.
Classification Descriptors
To express the weight of evidence for carcinogenic hazard
potential, the 1986 cancer guidelines provided summary rankings for
human and animal cancer studies. These summary rankings were integrated
to place the overall evidence in classification groups A through E,
Group A being associated with the greatest probability of human
carcinogenicity and Group E with evidence of noncarcinogenicity in
humans. Data other than tumor findings played a modifying role after
initial placement of an agent into a group.
These Proposed Guidelines take a different approach, consistent
with the change in the basic approach to weighing evidence. No interim
classification of tumor findings followed by modifications with other
data takes place. Instead, the conclusion reflects the weighing of
evidence in one step. Moreover, standard descriptors of conclusions are
employed rather than letter designations, and these are incorporated
into a brief narrative description of their informational basis. The
narrative with descriptors replaces the previous letter designation.
The descriptors are in three categories: ``known/likely,'' ``cannot be
determined,'' or ``not likely.'' For instance, using a descriptor in
context, a narrative could say that an agent is likely to be
carcinogenic by inhalation exposure and not likely to be carcinogenic
by oral exposure. The narrative explains the kinds of evidence
available and how they fit together in drawing conclusions, and points
out significant issues/strengths/limitations of the data and
conclusions. Subdescriptors are used to further refine the conclusion.
The narrative also summarizes the mode of action information underlying
a recommended approach to dose response assessment.
In considering revision of the former classification method, the
Agency has examined other possibilities that would retain the use of
letter and number designation of weights of evidence. The use of
standard descriptors within a narrative presentation is proposed for
three primary reasons. First, the proposed method permits inclusion of
explanations of data and of their strengths and limitations. This is
more consistent with current policy emphasis on risk characterization.
Second, it would take a large set of individual number or letter codes
to cover differences in the nature of contributing information (animal,
human, other), route of exposure, mode of action, and relative overall
weight. When such a set becomes large--10 to 30 codes--it is too large
to be a good communication device, because people cannot remember the
definitions of the codes so they have to be explained in narrative.
Third, it is impossible to predefine the course of cancer research and
the kinds of data that may become available. A flexible system is
needed to accommodate change in the underlying data and inferences, and
a system of codes might become out of date, as has the one in the 1986
cancer guidelines.
Dose Response Assessment
The approach to dose response assessment calls for analysis that
follows the conclusions reached in the hazard assessment as to
potential mode(s) of action. The assessment begins by analyzing the
empirical data in the range of observation. When animal studies are the
basis of the analysis, the estimation of a human equivalent dose
utilizes toxicokinetic data, if appropriate and adequate data are
available. Otherwise, default procedures are applied. For oral dose,
the default is to scale daily applied doses experienced for a lifetime
in proportion to body weight raised to the 0.75 power. For inhalation
dose, the default methodology estimates respiratory deposition of
particles and gases and estimates internal doses of gases with
different absorption characteristics. These two defaults are a change
from the 1986 cancer guidelines which provided a single scaling factor
of body weight raised to the 0.66 power. Another change from the 1986
guidelines is that response data on effects of the agent on
carcinogenic processes are analyzed (nontumor data) in addition to data
on tumor incidence. If appropriate, the analyses of data on tumor
incidence and on precursor effects may be combined, using
[[Page 17962]]
precursor data to extend the dose response curve below the tumor data.
Even if combining data is not appropriate, study of the dose response
for effects believed to be part of the carcinogenic influence of the
agent may assist in thinking about the relationship of exposure and
response in the range of observation and at exposure levels below the
range of observation.
Whenever data are sufficient, a biologically based or case-specific
dose response model is developed to relate dose and response data in
the range of empirical observation. Otherwise, as a standard, default
procedure, a model is used to curve-fit the data. The lower 95%
confidence limit on a dose associated with an estimated 10% increased
tumor or relevant nontumor response (LED10) is identified. This
generally serves as the point of departure for extrapolating the
relationship to environmental exposure levels of interest when the
latter are outside the range of observed data. The environmental
exposures of interest may be measured ones or levels of risk management
interest in considering potential exposure control options. Other
points of departure may be more appropriate for certain data sets; as
described in the guidance, these may be used instead of the LED10.
Additionally, the LED10 is available for comparison with parallel
analyses of other carcinogenic agents or of noncancer effects of agents
and for gauging and explaining the magnitude of subsequent
extrapolation to low-dose levels. The LED10, rather than the
ED10 (the estimate of a 10% increased response), is the proposed
standard point of departure for two reasons. One is to permit easier
comparison with the benchmark dose procedure for noncancer health
assessment--also based on the lower limit on dose. Another is that the
lower limit, as opposed to the central estimate, accounts for
uncertainty in the experimental data. The issue of using a lower limit
or central estimate was discussed at a workshop held on the benchmark
procedure for noncancer assessment (Barnes et al., 1995) and at a
workshop on a previous version of this proposal (U.S. EPA, 1994b). The
latter workshop recommended a central estimate; the benchmark workshop
recommended a lower limit.
The second step of dose response assessment is extrapolation to
lower dose levels, if needed. This is based on a biologically based or
case-specific model if supportable by substantial data. Otherwise,
default approaches are applied that accord with the view of mode(s) of
action of the agent. These include approaches that assume linearity or
nonlinearity of the dose response relationship or both. The default
approach for linearity is to extend a straight line to zero dose, zero
response. The default approach for nonlinearity is to use a margin of
exposure analysis rather than estimating the probability of effects at
low doses. A margin of exposure analysis explains the biological
considerations for comparing the observed data with the environmental
exposure levels of interest and helps in deciding on an acceptable
level of exposure in accordance with applicable management factors.
The use of straight line extrapolation for a linear default is a
change from the 1986 guidelines which used the ``linearized
multistage'' (LMS) procedure. This change is made because the former
modeling procedure gave an appearance of specific knowledge and
sophistication unwarranted for a default. The proposed approach is also
more like that employed by the Food and Drug Administration (U.S. FDA,
1987). The numerical results of the straight line and LMS procedures
are not significantly different (Krewski et al., 1984). The use of a
margin of exposure approach is included as a new default procedure to
accommodate cases in which there is sufficient evidence of a nonlinear
dose response, but not enough evidence to construct a mathematical
model for the relationship. (The Agency will continue to seek a
modeling method to apply in these cases. If a modeling approach is
developed, it will be subject to peer review and public notice in the
context of a supplementary document for these guidelines.)
The public is invited to provide comments to be considered in EPA
decisions about the content of the final guidelines. After the public
comment period, the EPA Science Advisory Board will be asked to review
and provide advice on the guidelines and issues raised in comments. EPA
asks those who respond to this notice to include their views on the
following:
(1) The proposed guidance for characterization of hazard, including
the weight of evidence descriptors and weight of evidence narrative
which are major features of the proposal. There are three categories of
descriptors: ``known/likely,'' ``cannot be determined,'' and ``not
likely'' which are further refined by subdescriptors. It is felt that
these three descriptors will satisfactorily delineate the types of
evidence bearing on carcinogenicity as they are used with
subdescriptors in the context of a narrative of data and rationale.
However, an issue that has been discussed by external peer reviewers
and by EPA staff is whether the descriptor-subdescriptor called
``cannot be determined--suggestive evidence'' should become a separate,
fourth category called ``suggestive.'' The EPA may choose this course
in the final guidelines and requests comment. In considering this
issue, commenters may wish to refer not only to Sections 2.6.2. and
2.7.2. which cover the descriptors and narrative, but also to case
study example #6 in Section 2.6.3. and example narrative #2 in Appendix
A of the proposal. EPA asks commenters on this question to address the
rationale (science as well as policy) for leaving the categories of
descriptors as proposed or making the fourth category. How might the
coverage of a ``suggestive'' category be defined in order to be most
useful?
(2) The use of mode of action information in hazard
characterization and to guide dose response assessment is a central
part of the proposed approach to bringing new research on carcinogenic
processes to bear in assessments of environmental agents (Sections
1.3.2., 2.3.2., 2.5., 3.1.). The appropriate use of this information
now and in the future is important. EPA requests comment on the
treatment of such information in the proposal, including reliance on
peer review as a part of the judgmental process on its application.
(3) Uses of nontumor data in the dose response assessment and the
methodological and science policy issues posed are new to these
guidelines (Sections 1.3.2., 3.1.2.). EPA requests comment on both
issues.
(4) Dose response assessment is proposed to be considered in two
parts--range of observed data and range of extrapolation (Section
3.1.). The lower 95% confidence limit on a dose associated with a 10%
response (tumor or nontumor response) is proposed as a default point of
departure, marking the beginning of extrapolation. This is a parallel
to the benchmark procedure for evaluating dose-response of noncancer
health endpoints (Barnes et al., 1995). An alternative is to use the
central estimate of a 10% response. Another alternative is to use a 1%,
instead of a 10%, response when the observed data are tumor incidence
data. Does the generally larger sample size of tumor effect studies
support using a 1% response as compared with using 10% for smaller
studies? Are there other approaches for the point of departure that
might be considered?
(5) Discussions of default assumptions and other responses to the
1994 NRC report Science and Judgment in Risk
[[Page 17963]]
Assessment appear in Section 1.3.1. and Appendix B of the proposal,
respectively. Comments are requested on responses to the NRC
recommendations and how the guidelines as a whole address them.
Dated: April 10, 1996.
Carol M. Browner,
Administrator.
Contents
List of Figures
1. Introduction
1.1. Purpose and Scope of the Guidelines
1.2. Organization and Application of the Guidelines
1.2.1. Organization
1.2.2. Application
1.3. Use of Default Assumptions
1.3.1. Default Assumptions
1.3.2. Major Defaults
1.3.2.1. Is the Presence or Absence of Effects Observed in a
Human Population Predictive of Effects in Another Exposed Human
Population?
1.3.2.2. Is the Presence or Absence of Effects Observed in an
Animal Population Predictive of Effects in Exposed Humans?
1.3.2.3. How Do Metabolic Pathways Relate Across Species?
1.3.2.4. How Do Toxicokinetic Processes Relate Across Species?
1.3.2.5. What Is the Correlation of the Observed Dose Response
Relationship to the Relationship at Lower Doses?
1.4. Characterizations
2. Hazard Assessment
2.1. Overview of Hazard Assessment and Characterization
2.1.1. Analyses of Data
2.1.2. Cross-Cutting Topics for Data Integration
2.1.2.1. Conditions of Expression
2.1.2.2. Mode of Action
2.1.3. Presentation of Results
2.2. Analysis of Tumor Data
2.2.1. Human Data
2.2.1.1. Types of Studies
2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic
Studies
2.2.1.3. Criteria for Causality
2.2.1.4. Assessment of Evidence of Carcinogenicity from Human
Data
2.2.2. Animal Data
2.2.2.1. Long-Term Carcinogenicity Studies
2.2.2.2. Other Studies
2.2.3. Structural Analogue Data
2.3. Analysis of Other Key Data
2.3.1. Physicochemical Properties
2.3.2. Structure-Activity Relationships
2.3.3. Comparative Metabolism and Toxicokinetics
2.3.4. Toxicological and Clinical Findings
2.3.5. Mode of Action-Related Endpoints and Short-Term Tests
2.3.5.1. Direct DNA Effects
2.3.5.2. Secondary DNA Effects
2.3.5.3. Nonmutagenic and Other Effects
2.3.5.4. Criteria for Judging Mode of Action
2.4. Biomarker Information
2.5. Mode of Action--Implications for Hazard Characterization
and Dose Response
2.6. Weight of Evidence Evaluation for Potential Human
Carcinogenicity
2.6.1. Weight of Evidence Analysis
2.6.2. Descriptors for Classifying Weight of Evidence
2.6.3. Case Study Examples
2.7. Presentation of Results
2.7.1. Technical Hazard Characterization
2.7.2. Weight of Evidence Narrative
3. Dose Response Assessment
3.1. Dose Response Relationship
3.1.1. Analysis in the Range of Observation
3.1.2. Analysis in the Range of Extrapolation
3.1.3. Use of Toxicity Equivalence Factors and Relative Potency
Estimates
3.2. Response Data
3.3. Dose Data
3.3.1. Interspecies Adjustment of Dose
3.3.2. Toxicokinetic Analyses
3.3.3. Route-to-Route Extrapolation
3.3.4. Dose Averaging
3.4. Discussion of Uncertainties
3.5. Technical Dose Response Characterization
4. Technical Exposure Characterization
5. Risk Characterization
5.1. Purpose
5.2. Application
5.3. Presentation of Risk Characterization Summary
5.4. Content of Risk Characterization Summary
Appendix A
Appendix B
Appendix C
References
List of Figures
Figure 1-1. Decisions on Dose Response Assessment Approaches for the
Range of Extrapolation
Figure 1-2. Risk Characterization
Figure 2-1. Factors for Weighing Human Evidence
Figure 2-2. Factors for Weighing Animal Evidence
Figure 2-3. Factors for Weighing Other Key Evidence
Figure 2-4. Factors for Weighing Totality of Evidence
Figure 3-1. Graphical Presentation of Data and Extrapolations
1. Introduction
1.1. Purpose and Scope of the Guidelines
These guidelines revise and replace United States Environmental
Protection Agency (EPA) Guidelines for Carcinogen Risk Assessment
published in 51 FR 33992, September 24, 1986. The guidelines provide
EPA staff and decisionmakers with guidance and perspectives to develop
and use risk assessments. They also provide basic information to the
public about the Agency's risk assessment methods.
The guidelines encourage both regularity in procedures to support
consistency in scientific components of Agency decisionmaking and
innovation to remain up-to-date in scientific thinking. In balancing
these goals, the Agency relies on input from the general scientific
community through established scientific peer review processes. The
guidelines incorporate basic principles and science policies based on
evaluation of the currently available information. As more is
discovered about carcinogenesis, the need will arise to make
appropriate changes in risk assessment guidance. The Agency will revise
these guidelines when extensive changes are due. In the interim, the
Agency will issue special reports, after appropriate peer review, to
supplement and update guidance on single topics, (e.g., U.S. EPA,
1991b)
1.2. Organization and Application of the Guidelines
1.2.1. Organization
Publications of the Office of Science and Technology Policy (OSTP,
1985) and the National Research Council (NRC, 1983, 1994) provide
information and general principles about risk assessment. Risk
assessment uses available scientific information on the properties of
an agent \1\ and its effects in biological systems to provide an
evaluation of the potential for harm as a consequence of environmental
exposure to the agent. Risk assessment is one of the scientific
analyses available for consideration, with other analyses, in
decisionmaking on environmental protection. The 1983 and 1994 NRC
documents organize risk assessment information into four areas: hazard
identification, dose response assessment, exposure assessment, and risk
characterization. This structure appears in these guidelines, which
additionally emphasize characterization of evidence and conclusions in
each part of the assessment. In particular, the guidelines adopt the
approach of the NRC's 1994 report in adding a dimension of
characterization to the hazard identification step. Added to the
identification of hazard is an evaluation of the conditions under which
its expression is anticipated. The risk assessment questions addressed
in these guidelines are:
---------------------------------------------------------------------------
\1\ The term ``agent'' refers generally to any chemical
substance, mixture, or physical or biological entity being assessed,
unless otherwise noted.
---------------------------------------------------------------------------
For hazard--Can the agent present a carcinogenic hazard to
humans, and if so, under what circumstances?
For dose response--At what levels of exposure might
effects occur?
For exposure--What are the conditions of human exposure?
For risk--What is the character of the risk? How well do
data support conclusions about the nature and extent of the risk?
[[Page 17964]]
1.2.2. Application
The guidelines apply within the framework of policies provided by
applicable EPA statutes and do not alter such policies. The guidelines
cover assessment of available data. They do not imply that one kind of
data or another is prerequisite for regulatory action concerning any
agent. Risk management applies directives of regulatory legislation,
which may require consideration of potential risk, or solely hazard or
exposure potential, along with social, economic, technical, and other
factors in decisionmaking. Risk assessments support decisions, but to
maintain their integrity as decisionmaking tools, they are not
influenced by consideration of the social or economic consequences of
regulatory action.
Not every EPA assessment has the same scope or depth. Agency staff
often conduct screening-level assessments for priority-setting or
separate assessments of hazard or exposure for ranking purposes or to
decide whether to invest resources in collecting data for a full
assessment. Moreover, a given assessment of hazard and dose response
may be used with more than one exposure assessment that may be
conducted separately and at different times as the need arises in
studying environmental problems in various media. The guidelines apply
to these various situations in appropriate detail given the scope and
depth of the particular assessment. For example, a screening assessment
may be based almost entirely on structure-activity relationships and
default assumptions. As more data become available, assessments can
replace or modify default assumptions accordingly. These guidelines do
not require that all of the kinds of data covered here be available for
either assessment or decisionmaking. The level of detail of an
assessment is a matter of Agency management policy regarding the
applicable decisionmaking framework.
1.3. Use of Default Assumptions
The National Research Council, in its 1983 report on the science of
risk assessment (NRC, 1983), recognized that default assumptions are
necessarily made in risk assessments where gaps exist in general
knowledge or in available data for a particular agent. These default
assumptions are inferences based on general scientific knowledge of the
phenomena in question and are also matters of policy concerning the
appropriate way to bridge uncertainties that concern potential risk to
human health (or, more generally, to environmental systems) from the
agent under assessment.
EPA's 1986 guidelines for cancer risk assessment (EPA, 1986) were
developed in response to the 1983 NRC report. The guidelines contained
a number of default assumptions. They also encouraged research and
analysis that would lead to new risk assessment methods and data and
anticipated that these would replace defaults. The 1986 guidelines did
not explicitly discuss how to depart from defaults. In practice, the
agency's assessments routinely have employed defaults and, until
recently, only occasionally departed from them.
In its 1994 report on risk assessment, the NRC supported continued
use of default assumptions (NRC, 1994). The NRC report thus validated a
central premise of the approach to risk assessment that EPA had evolved
in preceding years--the making of science policy inferences to bridge
gaps in knowledge--while at the same time recommending that EPA develop
more systematic and transparent guidelines to inform the public of the
default inferences EPA uses in practice. It recommended that the EPA
review and update the 1986 guidelines in light of evolving scientific
information and experience in practice in applying those guidelines,
and that the EPA explain the science and policy considerations
underlying current views as to the appropriate defaults and provide
general criteria to guide preparers and reviewers of risks assessments
in deciding when to depart from a default. Pursuant to this
recommendation, the following discussion presents descriptions of the
major defaults and their rationales. In addition, it presents general
policy guidance on using and departing from defaults in specific risk
assessments.
1.3.1. Default Assumptions
The 1994 NRC report contains several recommendations regarding
flexibility and the use of default options:
EPA should continue to regard the use of default options
as a reasonable way to deal with uncertainty about underlying
mechanisms in selecting methods and models for use in risk assessment.
EPA should explicitly identify each use of a default
option in risk assessments.
EPA should clearly state the scientific and policy basis
for each default option.
The Agency should consider attempting to give greater
formality to its criteria for a departure from default options in order
to give greater guidance to the public and to lessen the possibility of
ad hoc, undocumented departures from default options that would
undercut the scientific credibility of the Agency's risk assessments.
At the same time, the Agency should be aware of the undesirability of
having its guidelines evolve into inflexible rules.
EPA should continue to use the Science Advisory Board and
other expert bodies. In particular, the Agency should continue to make
the greatest possible use of peer review, workshops, and other devices
to ensure broad peer and scientific participation to guarantee that its
risk assessment decisions will be based on the best science available
through a process that allows full public discussion and peer
participation by the scientific community.
In the 1983 report (p. 28), NAS defined the use of ``inference
options'' (default options) as a means to bridge inherent uncertainties
in risk assessment. These options exist when the assessment encounters
either ``missing or ambiguous information on a particular substance''
or ``gaps in current scientific theory.'' Since there is no instance in
which a set of data on an agent or exposure is complete, all risk
assessments must use general knowledge and policy guidance to bridge
data gaps. Animal toxicity data are used, for example, to substitute
for human data because we do not test human beings. The report
described the components of risk assessment in terms of questions
encountered during analysis for which inferences must be made. The
report noted (p. 36) that many components ``* * * lack definitive
scientific answers, that the degree of scientific consensus concerning
the best answer varies (some are more controversial than others), and
that the inference options available for each component differ in their
degree of conservatism. The choices encountered in risk assessment
rest, to various degrees, on a mixture of scientific fact and
consensus, on informed scientific judgment, and on policy
determinations (the appropriate degree of conservatism)* * *.'' The
report did not note that the mix varies significantly from case to
case. For instance, a question that arises in hazard identification is
how to use experimental animal data when the routes of exposure differ
between animals and humans. A spectrum of inferences could be made,
ranging from the most conservative, or risk adverse one that effects in
animals from one route may be seen in humans by another route, to an
intermediate, conditional inference that such translation of effects
will be assumed if the agent is absorbed by humans through the second
route, to
[[Page 17965]]
a nonconservative view that no inference is possible and the agent's
effects in animals must be tested by the second route. The choice of an
inference, as the report observed, comes from more than scientific
thinking alone. While the report focused mainly on the idea of
conservatism of public health as a science policy rationale for making
the choice, it did not evaluate other considerations. These include
such things as the matters of time and resources and whether the
analysis is for an important decision required to be made soon or is
simply a screening or ranking effort. For a screening analysis, one
might make several ``worst case'' inferences to determine if, even
under those conditions, risk is low enough that a problem can be
eliminated from further consideration. In the above discussion
concerning inferences about route-to-route extrapolation, one might use
the most conservative one for screening.
These revised guidelines retain the use of default assumptions as
recommended in the 1994 report. Generally, these defaults remain public
health conservative, but in some instances, they have been modified to
reflect the evolution of scientific knowledge since 1986.
In addition, the guidelines reflect evaluation of experience in
practice in applying defaults and departing from them in individual
risk assessments conducted under the 1986 guidelines. The application
and departure from defaults and the principles to be used in these
judgments have been matters of debate among practitioners and reviewers
of risk assessments. Some observers believe that in practice EPA risk
assessors have been too resistant to considering departures; others
question whether proposed departures have been adequately supported.
Some cases in which departures have been considered have been generally
accepted, while others have been controversial. The guidelines here are
intended to be both explicit and more flexible than in the past
concerning the basis for making departures from defaults, recognizing
that expert judgment and peer review are essential elements of the
process.
In response to the recommendations of the 1994 report, these
guidelines call for identification of the default assumptions used
within assessments and for highlighting significant issues about
defaults within characterization summaries of component analyses in
assessment documents. As to the use of peer review to aid in making
judgments about applying or departing from defaults, we agree with the
NRC recommendation. The Agency has long made use of workshops, peer
review of documents and guidelines, and consultations as well as formal
peer review by the Science Advisory Board (SAB). In 1994, the
Administrator of EPA published formal guidance for peer review of EPA
scientific work products that increases the amount of peer review for
risk assessments as well as other work, as a response to the NRC report
and to SAB recommendations (U.S. EPA, 1994b).
The 1994 NRC report recommended that EPA should consider adopting
principles or criteria that would give greater formality and
transparency to decisions to depart from defaults. The report named
several possible criteria for such principles (p. 7): ``* * *
[P]rotecting the public health, ensuring scientific validity,
minimizing serious errors in estimating risks, maximizing incentives
for research, creating an orderly and predictable process, and
fostering openness and trustworthiness. There might be additional
relevant criteria* * *.'' The report indicated, however, that the
committee members had not reached consensus on a single criterion to
address the key issue of how much certainty or proof a risk assessor
must have in order to justify departing from a default. Appendix N of
the report contains two presentations of alternative views held by some
committee members on this issue. One view, known as ``plausible
conservatism,'' suggested that departures from defaults should not be
made unless new information improves the understanding of a biological
process to the point that relevant experts reach consensus that the
conservative default assumption concerning that process is no longer
plausible. The same criterion was recommended where the underlying
scientific mechanism is well understood, but where a default is used to
address missing data. In this case, the default should not be replaced
with case-specific data unless it is the consensus of relevant experts
that the proffered data make the default assumption no longer
plausible. Another view, known as the ``maximum use of scientific
information'' approach, acknowledged that the initial choice of
defaults should be conservative but argued that conservatism should not
be a factor in determining whether to depart from the default in favor
of an alternate biological theory or alternate data. According to this
view, it should not be necessary to reach expert consensus that the
default assumption had been rendered implausible; it should be
sufficient that risk assessors find the alternate approach more
plausible than the default.
The EPA is not adopting a list of formal decision criteria in the
sense of a checklist based on either view. It would not be helpful to
generate a checklist of uniform criteria for several reasons. First,
risk assessments are highly variable in content and purpose. Screening
assessments may be purposely ``worst case'' in their default
assumptions to eliminate problems from further investigation.
Subsequent risk assessments based on a fuller data set can discard
worst-case default assumptions in favor of plausibly conservative
assumptions and progressively replace or modify the latter with data.
No uniform checklist will fit all cases. Second, a checklist would
likely become more a source of rote discussion than of enlightenment
about the process.
Instead, these guidelines use a combination of principles and
process in the application of and departure from default assumptions.
The guidelines provide a framework of default assumptions to allow risk
assessment to proceed when current scientific theory or available case-
specific data do not provide firm answers in a particular case, as the
1983 report outlined. Some of the default assumptions bridge large gaps
in fundamental knowledge which will be filled by basic research on the
causes of cancer and on other biological processes, rather than by
agent-specific testing. Other default assumptions bridge smaller data
gaps that can feasibly be filled for a single agent, such as whether a
metabolic pathway in test animals is like (default) or unlike that in
humans.
The decision to use a default, or not, is a choice considering
available information on an underlying scientific process and agent-
specific data, depending on which kind of default it is. Generally, if
a gap in basic understanding exists, or if agent-specific data are
missing, the default is used without pause. If data are present, their
evaluation may reveal inadequacies that also lead to use of the
default. If data support a plausible alternative to the default, but no
more strongly than they support the default, both the default and its
alternative are carried through the assessment and characterized for
the risk manager. If data support an alternative to the default as the
more reasonable judgment, the data are used. (This framework of choices
is not wholly applicable to screening assessments. As mentioned above,
screening assessments may appropriately use ``worst case'' inferences
to determine if, even under
[[Page 17966]]
those conditions, risk is low enough that a problem can be eliminated
from further consideration.)
Scientific peer review, peer consultative workshops and similar
processes are the principal ways determining the strength of thinking
and generally accepted views within the scientific community about the
application of and departure from defaults and about judgments
concerning the plausibility and persuasiveness of data in a particular
case. The choices made are explicitly discussed in the assessment, and
if a particular choice raises a significant issue, it is highlighted in
the risk characterization.
The discussion of major defaults in these guidelines together with
the explicit discussion of the choice of inferences within the
assessment and the processes of peer review and peer consultation will
serve the several goals stated in the 1994 report. One is to encourage
research, since results of research efforts will be considered. Another
is to allow timely decisionmaking, when time is a constraint, by
supporting completion of the risk assessment using defaults as needed.
Another is to be flexible, using new science as it develops. Finally,
the use of public processes of peer consultation and peer review will
ensure that discipline of thought is maintained to support trust in
assessment results.
Experience has shown that the most difficult part of the framework
of choices is the judgment of whether a data analysis is both
biologically plausible and persuasive as applied to the case at hand.
There is no set of rules for making this judgment in all cases. Two
criteria that apply in these guidelines are that the underlying
scientific principle has been generally accepted within the scientific
community and that supportive experiments are available that test the
application of the principle to the agent under review. For example,
mutagenicity through reactivity with DNA has been generally accepted as
a carcinogenic influence for many years. This acceptance, together with
evidence of such mutagenicity in experiments on an agent, provides
plausible and persuasive support for the inference that mutagenicity is
a mode of action for the agent.
Judgments about plausibility and persuasiveness of analyses vary
according to the scientific nature of the default. An analysis of data
may replace a default or modify it. An illustration of the former is
development of EPA science policy on the issue of the relevance for
humans of male rat kidney neoplasia involving alpha 2u globulin (U.S.
EPA, 1991b). The 1991 EPA policy gives guidance on the kind of
experimental findings that demonstrate whether the alpha 2u globulin
mechanism is present and responsible for carcinogenicity in a
particular case. Before this policy guidance was issued, the default
assumption was that neoplasia in question was relevant to humans and
indicated the potential for hazard to humans. A substantial body of
data was developed by public and private research groups as a
foundation for the view that the alpha 2u globulin-induced response was
not relevant to humans. These studies first addressed the alpha 2u
globulin mechanism in the rat and whether this mechanism has a
counterpart in the human being, both were large research efforts. The
resulting data presented difficulties; some reviewers were concerned
that the mechanism in the rat appeared to be understood only in
outline, not in detail, and felt that the data were insufficient to
show the lack of a counterpart mechanism in humans. It was particularly
difficult to support a negative such as the nonexistence of a mechanism
in humans because so little is known about what the mechanisms are in
humans. Despite these concerns, in its 1991 policy guidance, EPA
concluded that the alpha 2u globulin-induced response in rats should be
regarded as not relevant to humans (i.e., as not indicating human
hazard).
One lesson in the development and peer review of this policy is
that if the default concerns an inherently complex biological question,
large amounts of work will be required to replace the default. A second
is that addressing a negative is difficult. A third is that ``proof''
in the strict sense of having laid all reasonable doubt to rest is not
required. Instead, an alternative may displace a default when it is
generally accepted in peer review as the most reasonable judgment. The
issue of relevance may not always be so difficult. It would be an
experimentally easier task, for example, to determine whether
carcinogenesis in an animal species is due to a metabolite of the agent
in question that is not produced in humans.
When scientific processes are understood but case-specific data are
missing, defaults can be constructed to be modified by experimental
data, even if data do not suffice to replace them entirely. For
example, the approaches adopted in these guidelines for scaling dose
from experimental animals to humans are constructed to be either
modified or replaced as data become available on toxicokinetic
parameters for the particular agent being assessed. Similarly, the
selection of an approach or approaches for dose response assessment is
based on a series of decisions that consider the nature and adequacy of
available data in choosing among alternative modeling and default
approaches.
The 1994 NRC report notes (p. 6) that ``[a]s scientific knowledge
increases, the science policy choices made by the Agency and Congress
should have less impact on regulatory decisionmaking. Better data and
increased understanding of biological mechanisms should enable risk
assessments that are less dependent on conservative default assumptions
and more accurate as predictions of human risk.'' Undoubtedly, this is
the trend as scientific understanding increases. However, some gaps in
knowledge and data will doubtless continue to be encountered in
assessment of even data-rich cases, and it will remain necessary for
risk assessments to continue using defaults within the framework set
forth here.
1.3.2. Major Defaults
This discussion covers the major default assumptions commonly
employed in a cancer risk assessment and adopted in these guidelines.
They are predominantly inferences necessary to use data observed under
empirical conditions to estimate events and outcomes under
environmental conditions. Several inferential issues arise when effects
seen in a subpopulation of humans or animals are used to qualitatively
infer potential effects in the population of environmentally exposed
humans. Several more inferential issues arise in extrapolating the
exposure-effect relationship observed empirically to lower-exposure
environmental conditions. The following issues cover the major default
areas. Typically, an issue has some subissues; they are introduced
here, but are discussed in greater detail in subsequent sections.
Is the presence or absence of effects observed in a human
population predictive of effects in another exposed human population?
Is the presence or absence of effects observed in an
animal population predictive of effects in exposed humans?
How do metabolic pathways relate across species?
How do toxicokinetic processes relate across species?
What is the correlation of the observed dose response
relationship to the relationship at lower doses?
[[Page 17967]]
1.3.2.1. Is the Presence or Absence of Effects Observed in a Human
Population Predictive of Effects in Another Exposed Human Population?
When cancer effects in exposed humans are attributed to exposure to an
exogenous agent, the default assumption is that such data are
predictive of cancer in any other exposed human population. Studies
either attributing cancer effects in humans to exogenous agents or
reporting no effects are often studies of occupationally exposed
humans. By sex, age, and general health, workers are not representative
of the general population exposed environmentally to the same agents.
In such studies there is no opportunity to observe whether infants and
children, males, or females who are under represented in the study, or
people whose health is not good, would respond differently. Therefore,
it is understood that this assumption could still underestimate the
response of certain sensitive human subpopulations, i.e. biologically
vulnerable parts of the population may be left out of risk assessments
(NRC, 1993a, 1994). Consequently, this is a default that does not err
on the side of public health conservatism, as the 1994 NRC report also
recognizes.
On the one hand, if effects are seen in a worker population, this
may be in fact indicative of heightened effects in sensitive
subpopulations. There is not enough knowledge yet to form a basis for
any generally applicable, qualitative inference to compensate for this
knowledge gap. In these guidelines, this problem is left to case-by-
case analysis, to be attended to as future research and information on
particular agents allow. When information on a sensitive subpopulation
exists, it will be used. The topic of variability is addressed further
in the discussion of quantitative default assumptions about dose
response relationships below. On the other hand, when cancer effects
are not found in an exposed human population, this information by
itself is not generally sufficient to conclude that the agent poses no
carcinogenic hazard to this or other populations of potentially exposed
humans. This is because epidemiologic studies usually have low power to
detect and attribute responses (section 2.2.1.). This may be
particularly true when extrapolating null results from a healthy,
worker population to other potentially sensitive exposed humans. Again,
the problem is left to case-by-case analysis.
1.3.2.2. Is the Presence or Absence of Effects Observed in an
Animal Population Predictive of Effects in Exposed Humans? The default
assumption is that positive effects in animal cancer studies indicate
that the agent under study can have carcinogenic potential in humans.
Thus, if no adequate human data are present, positive effects in animal
cancer studies are a basis for assessing the carcinogenic hazard to
humans. This assumption is a public health conservative policy, and it
is both appropriate and necessary given that we do not test for
carcinogenicity in humans. The assumption is supported by the fact that
nearly all of the agents known to cause cancer in humans are
carcinogenic in animals in tests with adequate protocols (IARC, 1994;
Tomatis et al., 1989; Huff, 1994). Moreover, almost one-third of human
carcinogens were identified subsequent to animal testing (Huff, 1993).
Further support is provided by research on the molecular biology of
cancer processes, which has shown that the mechanisms of control of
cell growth and differentiation are remarkably homologous among species
and highly conserved in evolution. Nevertheless, the same research
tools that have enabled recognition of the nature and commonality of
cancer processes at the molecular level also have the power to reveal
differences and instances in which animal responses are not relevant to
humans (Linjinsky, 1993; U.S. EPA, 1991b). Under these guidelines,
available mode of action information is studied for its implications in
both hazard and dose response assessment and its effect on default
assumptions.
There may be instances in which the use of an animal model would
identify a hazard in animals that is not truly a hazard in humans
(e.g., the alpha-2u-globulin association with renal neoplasia in male
rats (U.S. EPA, 1991b)). The extent to which animal studies may yield
false positive indications for humans is a matter of scientific debate.
To demonstrate that a response in animals is not relevant to any human
situation, adequate data to assess the relevancy issue must be
available.
Animal studies are conducted at high doses in order to provide
statistical power, the highest dose being one that is minimally toxic
(maximum tolerated dose). Consequently, the question often arises
whether a carcinogenic effect at the highest dose may be a consequence
of cell killing with compensatory cell replication or of general
physiological disruption, rather than inherent carcinogenicity of the
tested agent. There is little doubt that this may happen in some cases,
but skepticism exists among some scientists that it is a pervasive
problem (Ames and Gold, 1990; Melnick et al., 1993a; Melnick et al.,
1993b; Barrett, 1993). In light of this question, the default
assumption is that effects seen at the highest dose tested are
appropriate for assessment, but it is necessary that the experimental
conditions be scrutinized. If adequate data demonstrate that the
effects are solely the result of excessive toxicity rather than
carcinogenicity of the tested agent per se, then the effects may be
regarded as not appropriate to include in assessment of the potential
for human carcinogenicity of the agent. This is a matter of expert
judgment, considering all of the data available about the agent
including effects in other toxicity studies, structure-activity
relationships, and effects on growth control and differentiation.
When cancer effects are not found in well-conducted animal cancer
studies in two or more appropriate species and other information does
not support the carcinogenic potential of the agent, these data provide
a basis for concluding that the agent is not likely to possess human
carcinogenic potential, in the absence of human data to the contrary.
This default assumption about lack of cancer effects is not public
health conservative. For instance, the tested animal species may not be
predictive of effects in humans; arsenic shows only minimal or no
effect in animals, while it is clearly positive in humans. (Other
information, such as absence of mutagenic activity or absence of
carcinogenic activity among structural analogues, can increase the
confidence that negative results in animal studies indicate a lack of
human hazard.) Also, it is recognized that animal studies (and
epidemiologic studies as well) have very low power to detect cancer
effects. Detection of a 10% tumor incidence is generally the limit of
power with currently conducted animal studies (with the exception of
rare tumors that are virtually markers for a particular agent, e.g.,
angiosarcoma caused by vinyl chloride).
Target organs of carcinogenesis for agents that cause cancer in
both animals and humans are most often concordant at one or more sites
(Tomatis et al., 1989; Huff, 1994). However, concordance by site is not
uniform. The default assumption is that target organ concordance is not
a prerequisite for evaluating the implications of animal study results
for humans. This is a public health conservative science policy. The
mechanisms of control of cell growth and differentiation are concordant
among species, but there are marked differences among species in the
way control is managed in various tissues. For example, in humans,
mutation of the tumor suppressor gene
[[Page 17968]]
p53 is one of the most frequently observed genetic changes in tumors.
This tumor suppressor is also observed to be operating in some rodent
tissues, but other growth control mechanisms predominate in rodents.
Thus, an animal response may be due to changes in a control that are
relevant to humans, but appear in animals in a different way. However,
it is appropriate under these guidelines to consider the influences of
route of exposure, metabolism, and, particularly, hormonal modes of
action that may either support or not support target organ concordance
between animals and humans. When data allow, these influences are
considered in deciding whether the default remains appropriate in
individual instances (NRC, 1994, p. 121). An exception to the basic
default of not assuming site concordance exists in the context of
toxicokinetic modeling. Site concordance is inherently assumed when
these models are used to estimate delivered dose in humans based on
animal data.
As in the approach of the National Toxicology Program and the
International Agency for Research on Cancer, the default is to include
benign tumors observed in animal studies in the assessment of animal
tumor incidence if they have the capacity to progress to the
malignancies with which they are associated. This treats the benign and
malignant tumors as representative of related responses to the test
agent, which is scientifically appropriate. This is a science policy
decision that is somewhat more conservative of public health than not
including benign tumors in the assessment. Nonetheless, in assessing
findings from animal studies, a greater proportion of malignancy is
weighed more heavily than a response with a greater proportion of
benign tumors. Greater frequency of malignancy of a particular tumor
type in comparison with other tumor responses observed in an animal
study is also a factor to be considered in selecting the response to be
used in dose response assessment.
Benign tumors that are not observed to progress to malignancy are
assessed on a case-by-case basis. There is a range of possibilities for
their overall significance. They may deserve attention because they are
serious health problems even though they are not malignant; for
instance, benign tumors may be a health risk because of their effect on
the function of a target tissue such as the brain. They may be
significant indicators of the need for further testing of an agent if
they are observed in a short term test protocol, or such an observation
may add to the overall weight of evidence if the same agent causes
malignancies in a long term study. Knowledge of the mode of action
associated with a benign tumor response may aid in the interpretation
of other tumor responses associated with the same agent. In other
cases, observation of a benign tumor response alone may have no
significant health hazard implications when other sources of evidence
show no suggestion of carcinogenicity.
1.3.2.3. How Do Metabolic Pathways Relate Across Species? The
default assumption is that there is a similarity of the basic pathways
of metabolism and the occurrence of metabolites in tissues in regard to
the species-to-species extrapolation of cancer hazard and risk. If
comparative metabolism studies were to show no similarity between the
tested species and humans and a metabolite(s) were the active form,
there would be less support for an inference that the animal
response(s) relates to humans. In other cases, parameters of metabolism
may vary quantitatively between species; this becomes part of deciding
on an appropriate human equivalent dose based on animal studies,
optimally in the context of a toxicokinetic model.
1.3.2.4. How Do Toxicokinetic Processes Relate Across Species? A
major issue is how to estimate human equivalent doses in extrapolating
from animal studies. As a default for oral exposure, a human equivalent
dose is estimated from data on another species by an adjustment of
animal oral dose by a scaling factor of body weight to the 0.75 power.
This adjustment factor is used because it represents scaling of
metabolic rate across animals of different size. Because the factor
adjusts for a parameter that can be improved on and brought into more
sophisticated toxicokinetic modeling, when such data become available,
the default assumption of 0.75 power can be refined or replaced.
For inhalation exposure, a human equivalent dose is estimated by
default methodologies that provide estimates of lung deposition and of
internal dose. The methodologies can be refined to more sophisticated
forms with data on toxicokinetic and metabolic parameters of the
specific agent. This default assumption, like the one with oral
exposure, is selected in part because it lays a foundation for
incorporating better data. The use of information to improve dose
estimation from applied, to internal, to delivered dose is encouraged,
including use of toxicokinetic modeling instead of any default, where
data are available. Health conservatism is not an element in choosing
the default.
For a route-to-route of exposure extrapolation, the default
assumption is that an agent that causes internal tumors by one route of
exposure will be carcinogenic by another route if it is absorbed by the
second route to give an internal dose. This is a qualitative assumption
and is considered to be public health conservative. The rationale is
that for internal tumors an internal dose is significant no matter what
the route of exposure. Additionally, the metabolism of the agent will
be qualitatively the same for an internal dose. The issue of
quantitative extrapolation of the dose-response relationship from one
route to another is addressed case by case. Quantitative extrapolation
is complicated by considerations such as first-pass metabolism, but is
approachable with empirical data. Adequate data are necessary to
demonstrate that an agent will act differently by one route versus
another route of exposure.
1.3.2.5. What Is the Correlation of the Observed Dose Response
Relationship to the Relationship at Lower Doses? The overriding
preferred approach is to use a biologically based or case-specific
model for both the observed range and extrapolation below that range
when there are sufficient data. While biologically based models are
still under development, it is likely that they will be used more
frequently in the future. The default procedure for the observed range
of data, when the preferred approach cannot be used, is to use a curve-
fitting model.
In the absence of data supporting a biologically based or case-
specific model for extrapolation outside of the observed range, the
choice of approach is based on the view of mode of action of the agent
arrived at in the hazard assessment. A linear default approach is used
when the mode of action information is supportive of linearity or,
alternatively, is insufficient to support a nonlinear mode of action.
The linear approach is used when a view of the mode of action indicates
a linear response, for example, when a conclusion is made that an agent
directly causes alterations in DNA, a kind of interaction that not only
theoretically requires one reaction, but also is likely to be additive
to ongoing, spontaneous gene mutation. Other kinds of activity may have
linear implications, e.g., linear rate-limiting steps, that support a
linear procedure also. The linear approach is to draw a straight line
between a point of departure from observed data, generally, as a
default, the LED10, and the origin (zero dose, zero response).
Other points of
[[Page 17969]]
departure may be more appropriate for certain data sets; these may be
used instead of the LED10. This approach is generally considered
to be public health conservative. The LED10 is the lower 95% limit
on a dose that is estimated to cause a 10% response. This level is
chosen to account (conservatively) for experimental variability.
Additionally, it is chosen because it rewards experiments with better
designs in regard to number of doses and dose spacing, since these
generally will have narrower confidence limits. It is also an
appropriate representative of the lower end of the observed range
because the limit of detection of studies of tumor effect is about 10%.
The linear default is thought to generally produce an upper bound
on potential risk at low doses, e.g., a 1/100,000 to 1/1,000,000 risk;
the straight line approach gives numerical results about the same as a
linearized multistage procedure (Krewski et al., 1984). This upper
bound is thought to cover the range of human variability although, in
some cases, it may not completely do so (Bois et al., 1995). The EPA
considers the linear default to be inherently conservative of public
health, without addition of another factor for human variability. In
any case, the size of such a factor would be hard to determine since a
good empirical basis on which to construct an estimate does not
currently exist. The question of what may be the actual variability in
human sensitivity is one that the 1994 NRC report discussed as did the
1993 NRC report on pesticides in children and infants. The NRC has
recommended research on the question, and the EPA and other agencies
have begun such research.
When adequate data on mode of action show that linearity is not the
most reasonable working judgment and provide sufficient evidence to
support a nonlinear mode of action, the default changes to a different
approach--a margin of exposure analysis--which assumes that
nonlinearity is more reasonable. The departure point is again generally
the LED10. A margin of exposure analysis compares the LED10
with the dose associated with the environmental exposure(s) of interest
by computing the ratio between the two.
The purpose of a margin of exposure analysis is to provide the risk
manager with all available information on how much reduction in risk
may be associated with reduction in exposure from the point of
departure. This is to support the risk manager's decision as to what
constitutes an acceptable margin of exposure, given requirements of the
statute under which the decision is being made. There are several
factors to be considered. (For perspective, keep in mind that a
sufficient basis to support this nonlinear procedure often will include
data on responses that are precursors to tumor effects. This means that
the point of departure may well be from these biological response data
rather than tumor incidence data, e.g., hormone levels, mitogenic
effects.) One factor to consider is the slope of the dose response
curve at the point of departure. A steeper slope implies an apparent
greater reduction in risk as exposure decreases. This may support a
smaller margin of exposure. Conversely, a shallow slope may support use
of a greater margin of exposure. A second factor is the nature of the
response used in the assessment--A precursor effect or frank toxicity
or tumor response. The latter two may support a greater margin of
exposure. A third factor is the nature and extent of human variability
in sensitivity to the phenomenon. A fourth factor is the agent's
persistence in the body. Greater variability or persistence argue for
greater margins of exposure. A fifth factor is human sensitivity to the
phenomenon as compared with experimental animals. The size of the
margin of exposure that is acceptable would increase or decrease as
this factor increases or decreases. If human variability cannot be
estimated based on data, it should be considered to be at least 10-
fold. Similarly, if comparison of species sensitivities cannot be
estimated from available data, humans can be considered to be 10-fold
more sensitive. If it is found that humans are less sensitive than
animals a factor that is a fraction no smaller than \1/10\ may be
assumed. The 10-fold factors are moderately conservative, traditional
ones used for decades in the assessment of toxicological effects. It
should not be assumed that the numerical factors are the sole
components for determination of an acceptable margin of exposure. Each
case calls for individual judgment. It should be noted that for cancer
assessment the margin of exposure analysis begins from a point of
departure that is adjusted for toxicokinetic differences between
species to give a human equivalent dose. Since the traditional factor
for interspecies difference is thought to contain a measure for
toxicokinetics as well as sensitivity to effect, the result of
beginning with a human equivalent dose is to add some conservatism. The
ultimate judgment whether a particular margin of exposure is acceptable
is a risk management decision under applicable law, rather than being
inherent in the risk assessment. Nonetheless, the risk assessor is
responsible for providing scientific rationale to support the the
decision.
When the mode of action information indicates that the dose
response may be adequately described by both a linear and a nonlinear
approach, then the default is to present both the linear and margin of
exposure analyses. An assessment may use both linear and nonlinear
approaches either for responses that are thought to result from
different modes of action or for presenting considerations for a
response that appears to be very different at high and low doses due to
influence of separate modes of action. Also, separate approaches may be
used for different induced responses (i.e. tumor types) from the same
agent. These would also be carried forward and presented in the
assessment. Figure 1-1 presents the decision points in deciding on a
dose response approach or approaches.
BILLING CODE 6560-50-P
[[Page 17970]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.000
BILLING CODE 6560-50-C
A default assumption is made that cumulative dose received over a
lifetime, expressed as a lifetime average daily dose, is an appropriate
measure of dose. This assumes that a high dose of such an agent
received over a shorter period of time is equivalent to a low dose
spread over a lifetime. This is thought to be a relatively public
health conservative assumption and has empirical support (Monro, 1992).
An example of effects of short-term, high exposure that results in
subsequent cancer development is treatment of cancer patients with
certain chemotherapeutic agents. An example of cancer from long-term
exposure to an agent of relatively low potency is smoking. Whether the
cumulative dose measure is exactly the correct measure in both such
instances is not certain and should be assessed case by case and
altered when data are available to support another approach. Other
measures of dose that consider dose rate and duration are appropriate,
e.g., when an agent acts by causing cell toxicity or hormone
disruption. In these cases both agent concentration and duration are
likely to be important, because such effects are generally observed to
be reversible at cessation of short-term exposure.
1.4. Characterizations
The risk characterization process first summarizes findings on
hazard, dose response, and exposure characterizations, then develops an
integrative analysis of the whole risk case. It ends in a nontechnical
Risk Characterization Summary. The Risk Characterization Summary is a
presentation for risk managers who may or may not be familiar with the
scientific details of cancer assessment. It also provides information
for other interested readers. The initial steps in the risk
characterization process are to make building blocks in the form of
characterizations of the assessments of hazard, dose response, and
exposure. The individual assessments and characterizations are then
integrated to arrive at risk estimates for exposure scenarios of
interest. There are two reasons for individually characterizing the
hazard, dose response, and exposure assessments. One is that they are
often done by different people than those who do the integrative
analyses. The second is that there is very often a lapse of time
between the conduct of hazard and dose response analyses and the
conduct of exposure assessment and integrative analysis. Thus, it is
necessary to capture characterizations of assessments as the
assessments are done to avoid the need to go back and reconstruct them.
Figure 1-2 shows the relationships of analyses. The figure does not
necessarily correspond to the number of documents involved; there may
be one or several. ``Integrative analysis'' is a generic term. At EPA,
the documents of various programs that contain integrative analyses
have other names such as the ``Staff Paper'' that discusses air quality
criteria issues. In the following sections, the elements of this figure
are discussed.
BILLING CODE 6560-50-P
[[Page 17971]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.001
BILLING CODE 6560-50-C
[[Page 17972]]
2. Hazard Assessment
2.1. Overview of Hazard Assessment and Characterization
2.1.1. Analyses of Data
The purpose of hazard assessment is to review and evaluate data
pertinent to two questions: (1) whether an agent may pose a
carcinogenic hazard to human beings and (2) under what circumstances an
identified hazard may be expressed (NRC, 1994, p. 142). Hazard
assessment is composed of analyses of a variety of data that may range
from observations of tumor responses to analysis of structure-activity
relationships. The purpose of the assessment is not simply to assemble
these separate evaluations; its purpose is to construct a total case
analysis examining the biological story the data reveal as a whole
about carcinogenic effects, mode of action, and implications of these
for human hazard and dose response evaluation. Weight of evidence
conclusions come from the combined strength and coherence of inferences
appropriately drawn from all of the available evidence. To the extent
that data permit, hazard assessment addresses the mode of action
question as both an initial step in considering appropriate approaches
to dose response assessment and as a part of identifying human hazard
potential.
The topics in this section include analysis of tumor data, both
animal and human, and analysis of other key information about
properties and effects that relate to carcinogenic potential. The
section addresses how information can be used to evaluate potential
modes of action. It also provides guidance on performing a weight of
evidence evaluation.
2.1.2. Cross-Cutting Topics for Data Integration
Two topics are included in the analysis of each kind of available
data: first, gathering information from available data about the
conditions of expression of hazard and second, gathering perspectives
on the agent's potential mode of action.
2.1.2.1. Conditions of Expression. Information on the significance
of the route of exposure may be available from human or animal studies
on the agent itself or on structural analogues. This information may be
found in studies of the agent or analogue for toxicological endpoints
other than cancer under acute or subchronic or chronic exposure
regimens. Studies of metabolism or toxicokinetics of the agent
similarly may provide pertinent data.
Each kind of data is also examined for information on conditions
that affect expression of carcinogenic effect such as presence or
absence of metabolic pathways. If carcinogenicity is secondary to
another toxic effect, the physiological or tissue changes that mark the
other toxicity are examined. Comparison of metabolic processes and
toxicity processes in humans and animals also bears on the relevance of
animal responses to human hazard. Included in the examination are the
questions of the potential range of human variability and whether any
special sensitivity may occur because of age, sex, preexisting disease,
or other condition.
2.1.2.2. Mode of Action. Information on an agent's potential
mode(s) of action is important in considering the relevance of animal
effects to assessment of human hazard. It also plays an important role
in selecting dose response approach(es), which are generally either
biologically based models or case-specific models incorporating mode of
action data or default procedures based on more limited data that
support inferences about the likely shape of the dose response curve.
Each kind of data may provide some insight about mode of action and
insights are gathered from each to be considered together as discussed
in section 2.4. In Appendix C, is a background discussion of some of
the development of views about carcinogenic processes.
2.1.3. Presentation of Results. Presentation of the results of
hazard assessment follows Agency guidance as discussed in section 2.7.
The results are presented in a technical hazard characterization that
serves as a support to later risk characterization. It includes:
a summary of the evaluations of hazard data,
the rationales for its conclusions, and
an explanation of the significant strengths or limitations
of the conclusions.
Another presentation feature is the use of a weight of evidence
narrative that includes both a conclusion about the weight of evidence
of carcinogenic potential and a summary of the data on which the
conclusion rests. This narrative is a brief summary that replaces the
alphanumerical classification system used in EPA's previous guidelines.
2.2. Analysis of Tumor Data
Evidence of carcinogenicity comes from finding tumor increases in
humans or laboratory animals exposed to a given agent, or from finding
tumors following exposure to structural analogues to the compound under
review. The significance of observed or anticipated tumor effects is
evaluated in reference to all of the other key data on the agent. This
section contains guidance for analyzing human and animal studies to
decide whether there is an association between exposure to an agent or
a structural analogue and occurrence of tumors. Note that the use of
the term ``tumor'' here is generic, meaning malignant neoplasms or a
combination of malignant and corresponding benign neoplasms.
Observation of only benign neoplasias may or may not have
significance. Benign tumors that are not observed to progress to
malignancy are assessed on a case-by-case basis. There is a range of
possibilities for their overall significance. They may deserve
attention because they are serious health problems even though they are
not malignant; for instance, benign tumors may be a health risk because
of their effect on the function of a target tissue such as the brain.
They may be significant indicators of the need for further testing of
an agent if they are observed in a short term test protocol, or such an
observation may add to the overall weight of evidence if the same agent
causes malignancies in a long term study. Knowledge of the mode of
action associated with a benign tumor response may aid in the
interpretation of other tumor responses associated with the same agent.
In other cases, observation of a benign tumor response alone may have
no significant health hazard implications when other sources of
evidence show no suggestion of carcinogenicity.
2.2.1. Human Data
Human data may come from epidemiologic studies or case reports.
Epidemiology is the study of the distributions and causes of disease
within human populations. The goals of cancer epidemiology are to
identify differences in cancer risk between different groups in a
population or between different populations, and then to determine the
extent to which these differences in risk can be attributed causally to
specific exposures to exogenous or endogenous factors. Epidemiologic
data are extremely useful in risk assessment because they provide
direct evidence that a substance produces cancer in humans, thereby
avoiding the problem of species to species inference. Thus, when
available human data are extensive and of good quality, they are
generally preferable over animal data and should be given
[[Page 17973]]
greater weight in hazard characterization and dose response assessment,
although both are utilized.
Null results from a single epidemiologic study cannot prove the
absence of carcinogenic effects because they can arise either from
being truly negative or from inadequate statistical power, inadequate
design, imprecise estimates, or confounding factors. However, null
results from a well-designed and well-conducted epidemiologic study
that contains usable exposure data can help to define upper limits for
the estimated dose of concern for human exposure if the overall weight
of the evidence indicates that the agent is potentially carcinogenic in
humans.
Epidemiology can also complement experimental evidence in
corroborating or clarifying the carcinogenic potential of the agent in
question. For example, observations from epidemiologic studies that
elevated cancer incidence occurs at sites corresponding to those at
which laboratory animals experience increased tumor incidence can
strengthen the weight of evidence of human carcinogenicity. On the
other hand, strong nonpositive epidemiologic data alone or in
conjunction with compelling mechanistic information can lend support to
a conclusion that animal responses may not be predictive of a human
response. Furthermore, the advent of biochemical or molecular
epidemiology may help improve understanding of the mechanisms of human
carcinogenesis.
2.2.1.1. Types of Studies. The major types of cancer epidemiologic
studies are analytical epidemiologic studies and descriptive or
correlation epidemiologic studies. Each study type has well-known
strengths and weaknesses that affect interpretation of study results as
summarized below (Kelsey et al., 1986; Lilienfeld and Lilienfeld, 1979;
Mausner and Kramer, 1985; Rothman, 1986).
Analytical epidemiologic studies are most useful for identifying an
association between human exposure and adverse health effects.
Analytical study designs include case-control studies and cohort
studies. In case-control studies, groups of individuals with (cases)
and without (controls) a particular disease are identified and compared
to determine differences in exposure. In cohort studies, a group of
``exposed'' and ``nonexposed'' individuals are identified and studied
over time to determine differences in disease occurrence. Cohort
studies can either be performed prospectively or retrospectively from
historical records.
Descriptive or correlation epidemiologic studies (sometimes called
ecological studies) examine differences in disease rates among
populations in relation to age, gender, race, and differences in
temporal or environmental conditions. In general, these studies can
only identify patterns or trends in disease occurrence over time or in
different geographical locations but cannot ascertain the causal agent
or degree of exposure. These studies, however, are often very useful
for generating hypotheses for further research.
Biochemical or molecular epidemiologic studies are studies in which
laboratory methods are incorporated in analytical investigations. The
application of techniques for measuring cellular and molecular
alterations due to exposure to specific environmental agents may allow
conclusions to be drawn about the mechanisms of carcinogenesis. The use
of biological biomarkers in epidemiology may improve assessment of
exposure and internal dose.
Case reports describe a particular effect in an individual or group
of individuals who were exposed to a substance. These reports are often
anecdotal or highly selected in nature and are of limited use for
hazard assessment. However, reports of cancer cases can identify
associations particularly when there are unique features such as an
association with an uncommon tumor (e.g., vinyl chloride and
angiosarcoma or diethylstilbestrol and clear-cell carcinoma of the
vagina).
2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic Studies.
Criteria for assessing the adequacy of epidemiologic studies are well
recognized. Characteristics that are desirable in these studies include
(1) clear articulation of study objectives or hypothesis, (2) proper
selection and characterization of the exposed and control groups, (3)
adequate characterization of exposure, (4) sufficient length of follow-
up for disease occurrence, (5) valid ascertainment of the causes of
cancer morbidity and mortality, (6) proper consideration of bias and
confounding factors, (7) adequate sample size to detect an effect, (8)
clear, well-documented, and appropriate methodology for data collection
and analysis, (9) adequate response rate and methodology for handling
missing data, and (10) complete and clear documentation of results.
Ideally, these conditions should be satisfied, where appropriate, but
rarely can a study meet all of them. No single criterion determines the
overall adequacy of a study. The following discussions highlight the
major factors included in an analysis of epidemiologic studies.
Population Issues. The ideal comparison would be between two
populations that differ only in exposure to the agent in question.
Because this is seldom the case, it is important to identify sources of
bias inherent in a study's design or data collection methods. Bias can
arise from several sources, including noncomparability between
populations of factors such as general health (McMichael, 1976), diet,
lifestyle, or geographic location; differences in the way case and
control individuals recall past events; differences in data collection
that result in unequal ascertainment of health effects in the
populations; and unequal follow-up of individuals. Both acceptance of
studies for assessment and judgment of their strengths or weaknesses
depend on identifying their sources of bias and the effects on study
results.
Exposure Issues. For epidemiologic data to be useful in determining
whether there is an association between health effects and exposure to
an agent, there must be adequate characterization of exposure
information. In general, greater weight should be given to studies with
more precise and specific exposure estimates.
Questions to address about exposure are: What can one reliably
conclude about the level, duration, route, and frequency of exposure of
individuals in one population as compared with another? How sensitive
are study results to uncertainties in these parameters?
Actual exposure measurements are not available for many
retrospective studies. Therefore, surrogates are often used to
reconstruct exposure parameters when historical measurements are not
available. These may involve attributing exposures to job
classifications in a workplace or to broader occupational or geographic
groupings. Use of surrogates carries a potential for misclassification
in that individuals may be placed in the incorrect exposure group.
Misclassification generally leads to reduced ability of a study to
detect differences between study and referent populations.
When either current or historical monitoring data are available,
the exposure evaluation includes consideration of the error bounds of
the monitoring and analytic methods and whether the data are from
routine or accidental exposures. The potentials for misclassification
and measurement errors are amenable to both qualitative and
quantitative analysis. These are essential analyses for judging a
study's results because exposure estimation is
[[Page 17974]]
the most critical part of a retrospective study.
Biological markers potentially offer excellent measures of exposure
(Hulka and Margolin, 1992; Peto and Darby, 1994). Validated markers of
exposure such as alkylated hemoglobin from exposure to ethylene oxide
(van Sittert et al., 1985) or urinary arsenic (Enterline et al., 1987)
can greatly improve estimates of dose. Markers closely identified with
effects promise to greatly increase the ability of studies to
distinguish real effects from bias at low levels of relative risk
between populations (Taylor et al., 1994; Biggs et al., 1993) and to
resolve problems of confounding risk factors.
Confounding Factors. Because epidemiologic studies are mostly
observational, it is not possible to guarantee the control of
confounding variables, which may affect the study outcome. A
confounding variable is a risk factor, independent of the putative
agent, that is distributed unequally among the exposed and unexposed
populations (e.g., smoking habits, lifestyle). Adjustment for possible
confounding factors can occur either in the design of the study (e.g.,
matching on critical factors) or in the statistical analysis of the
results. The influence of a potential confounding factor is limited by
the effect of the exposure of interest. For example, a twofold effect
of an exposure requires that the confounder effect be at least as big.
The latter may not be possible due to the presentation of the data or
because needed information was not collected during the study. In this
case, indirect comparisons may be possible. For example, in the absence
of data on smoking status among individuals in the study population, an
examination of the possible contribution of cigarette smoking to
increased lung cancer risk may be based on information from other
sources such as the American Cancer Society's longitudinal studies
(Hammand, 1966; Garfinkel and Silverberg, 1991). The effectiveness of
adjustments contributes to the ability to draw inferences from a study.
Different studies involving exposure to an agent may have different
confounding factors. If consistent increases in cancer risk are
observed across a collection of studies with different confounding
factors, the inference that the agent under investigation was the
etiologic factor is strengthened, even though complete adjustment for
confounding factors cannot be made and no single study supports a
strong inference.
It also may be the case that the agent of interest is a risk factor
in conjunction with another agent. This relationship may be revealed in
a collection of studies such as in the case of asbestos exposure and
smoking.
Sensitivity. Sensitivity, or the ability of a study to detect real
effects, is a function of several factors. Greater size of the study
population(s) (sample size) increases sensitivity, as does greater
exposure (levels and duration) of the population members. Because of
the often long latency period in cancer development, sensitivity also
depends on whether adequate time has elapsed since exposure began for
effects to occur. A unique feature that can be ascribed to the effects
of a particular agent (such as a tumor type that is seen only rarely in
the absence of the agent) can increase sensitivity by permitting
separation of bias and confounding factors from real effects.
Similarly, a biomarker particular to the agent can permit these
distinctions. Statistical reanalyses of data, particularly an
examination of different exposure indices, can give insight on
potential exposure-response relationships. These are all factors to
explore in statistical analysis of the data.
Statistical Considerations. The analysis applies appropriate
statistical methods to ascertain whether or not there is any
significant association between exposure and effects. A description of
the method or methods should include the reasons for their selection.
Statistical analyses of the potential effects of bias or confounding
factors are part of addressing the significance of an association, or
lack of one, and whether a study is able to detect any effect.
The analysis augments examination of the results for the whole
population with exploration of the results for groups with
comparatively greater exposure or time since first exposure. This may
support identifying an association or establishing a dose response
trend. When studies show no association, such exploration may apply to
determining an upper limit on potential human risk for consideration
alongside results of animal tumor effects studies.
Combining Statistical Evidence Across Studies. Meta-analysis is a
means of comparing and synthesizing studies dealing with similar health
effects and risk factors. It is intended to introduce consistency and
comprehensiveness into what otherwise might be a more subjective review
of the literature. When utilized appropriately, meta-analysis can
enhance understanding of associations between sources and their effects
that may not be apparent from examination of epidemiologic studies
individually. Whether to conduct a meta-analysis depends on several
issues. These include the importance of formally examining sources of
heterogeneity, the refinement of the estimate of the magnitude of an
effect, and the need for information beyond that provided by individual
studies or a narrative review. Meta-analysis may not be useful in some
circumstances. These include when the relationship between exposure and
disease is obvious without a more formal analysis, when there are only
a few studies of the key health outcomes, when there is insufficient
information from available studies related to disease, risk estimate,
or exposure classification, or when there are substantial confounding
or other biases that cannot be adjusted for in the analysis (Blair et
al., 1995; Greenland, 1987; Peto, 1992).
2.2.1.3. Criteria for Causality. A causal interpretation is
enhanced for studies to the extent that they meet the criteria
described below. None of the criteria is conclusive by itself, and the
only criterion that is essential is the temporal relationship. These
criteria are modeled after those developed by Bradford Hill in the
examination of cigarette smoking and lung cancer (Rothman, 1986) and
they need to be interpreted in the light of all other information on
the agent being assessed.
Temporal relationship: The development of cancers require
certain latency periods, and while latency periods vary, existence of
such periods is generally acknowledged. Thus, the disease has to occur
within a biologically reasonable time after initial exposure. This
feature must be present if causality is to be considered.
Consistency: Associations occur in several independent
studies of a similar exposure in different populations, or associations
occur consistently for different subgroups in the same study. This
feature usually constitutes strong evidence for a causal interpretation
when the same bias or confounding is not also duplicated across
studies.
Magnitude of the association: A causal relationship is
more credible when the risk estimate is large and precise (narrow
confidence intervals).
Biological gradient: The risk ratio (i.e., the ratio of
the risk of disease or death among the exposed to the risk of the
unexposed) increases with increasing exposure or dose. A strong dose
response relationship across several categories of exposure, latency,
and duration is supportive for causality given that confounding is
unlikely to be correlated with exposure. The absence of a dose response
relationship,
[[Page 17975]]
however, is not by itself evidence against a causal relationship.
Specificity of the association: The likelihood of a causal
interpretation is increased if an exposure produces a specific effect
(one or more tumor types also found in other studies) or if a given
effect has a unique exposure.
Biological plausibility: The association makes sense in
terms of biological knowledge. Information is considered from animal
toxicology, toxicokinetics, structure-activity relationship analysis,
and short-term studies of the agent's influence on events in the
carcinogenic process considered.
Coherence: The cause-and-effect interpretation is in
logical agreement with what is known about the natural history and
biology of the disease, i.e., the entire body of knowledge about the
agent.
2.2.1.4. Assessment of Evidence of Carcinogenicity from Human Data.
In the evaluation of carcinogenicity based on epidemiologic studies, it
is necessary to critically evaluate each study for the confidence in
findings and conclusions as discussed under section 2.2.1.2. All
studies that are properly conducted, whether yielding positive or null
results, or even suggesting protective carcinogenic effects, should be
considered in assessing the totality of the human evidence. Although a
single study may be indicative of a cause-effect relationship,
confidence in inferring a causal relationship is increased when several
independent studies are concordant in showing the association, when the
association is strong, and when other criteria for causality are also
met. Conclusions about the overall evidence for carcinogenicity from
available studies in humans should be summarized along with a
discussion of strengths or limitations of the conclusions.
2.2.2. Animal Data
Various kinds of whole animal test systems are currently used or
are under development for evaluating potential carcinogenicity. Cancer
studies involving chronic exposure for most of the life span of an
animal are generally accepted for evaluation of tumor effects (Tomatis
et al., 1989; Rall, 1991; Allen et al., 1988; but see Ames and Gold,
1990). Other studies of special design are useful for observing
formation of preneoplastic lesions or tumors or investigating specific
modes of action.
2.2.2.1. Long-Term Carcinogenicity Studies. The objective of long-
term carcinogenesis bioassays is to determine the carcinogenic
potential and dose response relationships of the test agent. Long-term
rodent studies are designed to examine the production of tumors as well
as preneoplastic lesions and other indications of chronic toxicity that
may provide evidence of treatment-related effects and insights into the
way the test agent produces tumors. Current standardized long-term
studies in rodents test at least 50 animals per sex per dose group in
each of three treatment groups and in a concurrent control group,
usually for 18 to 24 months, depending on the rodent species tested
(OECD, 1981; U.S. EPA, 1983a; U.S. EPA, 1983b; U.S. EPA, 1983c). The
high dose in long-term studies is generally selected to provide the
maximum ability to detect treatment-related carcinogenic effects while
not compromising the outcome of the study due to excessive toxicity or
inducing inappropriate toxicokinetics (e.g., overwhelming
detoxification or absorption mechanisms). The purpose of two or more
lower doses is to provide some information on the shape of the dose
response curve. Similar protocols have been and continue to be used by
many laboratories worldwide.
All available studies of tumor effects in whole animals are
considered, at least preliminarily. The analysis discards studies
judged to be wholly inadequate in protocol, conduct, or results.
Criteria for the technical adequacy of animal carcinogenicity studies
have been published and should be used as guidance to judge the
acceptability of individual studies (NTP, 1984; OSTP, 1985). Care is
taken to include studies that provide some evidence bearing on
carcinogenicity or help interpret effects noted in other studies even
if they have some limitations of protocol or conduct. Such limited, but
not wholly inadequate, studies can contribute as their deficiencies
permit. The findings of long-term rodent bioassays are always
interpreted in conjunction with results of prechronic studies along
with toxicokinetic and metabolism studies and other pertinent
information, if available. Evaluation of tumor effects requires
consideration of both biological and statistical significance of the
findings (Haseman, 1984, 1985, 1990, 1995). The following sections
highlight the major issues in the evaluation of long-term
carcinogenicity studies.
Dosing issues. In order to obtain the most relevant information
from a long-term carcinogenicity study, it is important to require
maximization of exposure to the test material. At the same time, there
is a need for caution in using excessive high dose levels that would
confound the interpretation of study results to humans. The high dose
is conventionally defined as a dose that produces some toxic effects
without either unduly affecting mortality from effects other than
cancer or producing significant adverse effects on the nutrition and
health of the test animals (OECD, 1981; NRC, 1993b). It should be noted
that practical upper limits have been established to avoid the use of
excessive high doses in long-term carcinogenicity studies (e.g., 5% of
the test substance in the feed for dietary studies [OECD, 1981]).
Evaluating the appropriateness of the high dose in carcinogenicity
studies is based on scientific judgment using all available relevant
information. In general, if the test agent does not appear to cause any
specific target organ toxicity or perturbation of physiological
function, an adequate high dose would be a dose that causes no more
than 10% reduction of body weight gain over the life span of the
animals. On the other hand, significant increases in mortality from
effects other than cancer is accepted as clear evidence of frank
toxicity, which indicates that an adequate high dose may have been
exceeded. Other signs of treatment-related toxicity that may indicate
that an adequate high dose has been exceeded include the following: (a)
Reduction of body weight gain of 10% or greater, (b) significant
increases in abnormal behavioral and clinical signs, (c) significant
changes in hematology or clinical chemistry, (d) saturation of
absorption and detoxification mechanisms, or (e) marked changes in
organ weight, morphology, and histopathology.
For dietary studies, weight gain reductions should be evaluated as
to whether there is a palatability problem or an issue with food
efficiency; certainly, the latter is a toxic manifestation. In the case
of inhalation studies with respirable particles, evidence of impairment
of normal clearance of particles from the lung should be considered
along with other signs of toxicity to the respiratory airways to
determine whether the high exposure concentration has been
appropriately selected. For dermal studies, evidence of skin irritation
may indicate that an adequate high dose has been reached.
Interpretation of carcinogenicity study results is profoundly
affected by exposure conditions, especially by inappropriate dose
selection. This is particularly important in studies that are
nonpositive for carcinogenicity, since failure to reach a sufficient
dose reduces the sensitivity of a study. A lack of tumorigenic
responses at exposure levels that cause significant impairment
[[Page 17976]]
of animal survival may also not be acceptable as negative findings
because of the reduced sensitivity of the study. On the other hand,
overt toxicity or inappropriate toxicokinetics due to excessive high
doses may result in tumor effects that are secondary to the toxicity
rather than directly attributable to the agent.
There are several possible outcomes regarding the study
interpretation of the significance and relevance of tumorigenic effects
associated with exposure or dose levels below, at, or above an adequate
high dose. General guidance is given here that should not be taken as
prescriptive; for each case, the information at hand is evaluated and a
rationale should be given for the position taken.
Adequate high dose: If an adequate high dose has been
utilized, tumor effects are judged positive or negative depending on
the presence or absence of significant tumor incidence increases,
respectively.
Excessive high dose: If toxicity or mortality is excessive
at the high dose, interpretation depends on the finding of tumors or
not.
(a) Studies that show tumor effects only at excessive doses may be
compromised and may or may not carry weight, depending on the
interpretation in the context of other study results and other lines of
evidence. Results of such studies, however, are generally not
considered suitable for risk extrapolation.
(b) Studies that show tumors at lower doses, even though the high
dose is excessive and may be discounted, should be evaluated on their
own merits.
(c) If a study does not show an increase in tumor incidence at a
toxic high dose and appropriately spaced lower doses are used without
such toxicity or tumors, the study is generally judged as negative for
carcinogenicity.
Inadequate high dose: Studies of inadequate sensitivity
where an adequate high dose has not been reached may be used to bound
the dose range where carcinogenic effects might be expected.
Statistical Considerations. The main aim of statistical evaluation
is to determine whether exposure to the test agent is associated with
an increase of tumor development. Statistical analysis of a long-term
study should be performed for each tumor type separately. The incidence
of benign and malignant lesions of the same cell type, usually within a
single tissue or organ, are considered separately and are combined when
scientifically defensible (McConnell et al., 1986).
Trend tests and pairwise comparison tests are the recommended tests
for determining whether chance, rather than a treatment-related effect,
is a plausible explanation for an apparent increase in tumor incidence.
A trend test such as the Cochran-Armitage test (Snedecor and Cochran,
1967) asks whether the results in all dose groups together increase as
dose increases. A pairwise comparison test such as the Fisher exact
test (Fisher, 1932) asks whether an incidence in one dose group is
increased over the control group. By convention, for both tests a
statistically significant comparison is one for which p <0.05 that the
increased incidence is due to chance. Significance in either kind of
test is sufficient to reject the hypothesis that chance accounts for
the result. A statistically significant response may or may not be
biologically significant and vice versa. The selection of a
significance level is a policy choice based on a trade-off between the
risks of false positives and false negatives. A significance level of
greater or less than 5% is examined to see if it confirms other
scientific information. When the assessment departs from a simple 5%
level, this should be highlighted in the risk characterization. A two-
tailed test or a one-tailed test can be used. In either case a
rationale is provided.
Considerations of multiple comparisons should also be taken into
account. Haseman (1983) analyzes typical animal bioassays testing both
sexes of two species and concludes that, because of multiple
comparisons, a single tumor increase for a species-sex-site combination
that is statistically significant at the 1% level for common tumors or
5% for rare tumors corresponds to a 7-8% significance level for the
study as a whole. Therefore, animal bioassays presenting only one
significant result that falls short of the 1% level for a common tumor
may be treated with caution.
Concurrent and Historical Controls. The standard for determining
statistical significance of tumor incidence comes from a comparison of
tumors in dosed animals as compared with concurrent control animals.
Additional insights about both statistical and biological significance
can come from an examination of historical control data (Tarone, 1982;
Haseman, 1995). Historical control data can add to the analysis
particularly by enabling identification of uncommon tumor types or high
spontaneous incidence of a tumor in a given animal strain.
Identification of common or uncommon situations prompts further thought
about the meaning of the response in the current study in context with
other observations in animal studies and with other evidence about the
carcinogenic potential of the agent. These other sources of information
may reinforce or weaken the significance given to the response in the
hazard assessment. Caution should be exercised in simply looking at the
ranges of historical responses because the range ignores differences in
survival of animals among studies and is related to the number of
studies in the database.
In analyzing results for uncommon tumors in a treated group that
are not statistically significant in comparison to concurrent controls,
the analyst can use the experience of historical controls to conclude
that the result is in fact unlikely to be due to chance. In analyzing
results for common tumors, a different set of considerations comes into
play. Generally speaking, statistically significant increases in tumors
should not be discounted simply because incidence rates in the treated
groups are within the range of historical controls or because incidence
rates in the concurrent controls are somewhat lower than average.
Random assignment of animals to groups and proper statistical
procedures provide assurance that statistically significant results are
unlikely to be due to chance alone. However, caution should be used in
interpreting results that are barely statistically significant or in
which incidence rates in concurrent controls are unusually low in
comparison with historical controls.
In cases where there may be reason to discount the biological
relevance to humans of increases in common animal tumors, such
considerations should be weighed on their own merits and clearly
distinguished from statistical concerns.
When historical control data are used, the discussion needs to
address several issues that affect comparability of historical and
concurrent control data. Among these issues are the following: genetic
drift in the laboratory strains; differences in pathology examination
at different times and in different laboratories (e.g., in criteria for
evaluating lesions; variations in the techniques for preparation or
reading of tissue samples among laboratories); comparability of animals
from different suppliers. The most relevant historical data come from
the same laboratory and same supplier, gathered within 2 or 3 years one
way or the other of the study under review; other data should be used
only with extreme caution.
Assessment of Evidence of Carcinogenicity from Long-Term Animal
Studies. In general, observation of tumor effects under different
circumstances lends support to the
[[Page 17977]]
significance of the findings for animal carcinogenicity. Significance
is a function of the number of factors present, and for a factor such
as malignancy, the severity of the observed pathology. The following
observations add significance to the tumor findings:
uncommon tumor types
tumors at multiple sites
tumors by more than one route of administration
tumors in multiple species, strains, or both sexes
progression of lesions from preneoplastic to benign to
malignant
reduced latency of neoplastic lesions
metastases
unusual magnitude of tumor response
proportion of malignant tumors
dose-related increases
These guidelines adopt the science policy position that tumor
findings in animals indicate that an agent may produce such effects in
humans. Moreover, the absence of tumor findings in well-conducted,
long-term animal studies in at least two species provides reasonable
assurance that an agent may not be a carcinogenic concern for humans.
Each of these is a default assumption that may be adopted, when
appropriate, after evaluation of tumor data and other key evidence.
Site concordance of tumor effects between animals and humans is an
issue to be considered in each case. Thus far, there is evidence that
growth control mechanisms at the level of the cell are homologous among
mammals, but there is no evidence that these mechanisms are site
concordant. Moreover, agents observed to produce tumors in both humans
and animals have produced tumors either at the same (e.g., vinyl
chloride) or different sites (e.g., benzene) (NRC, 1994). Hence, site
concordance is not assumed a priori. On the other hand, certain
processes with consequences for particular tissue sites (e.g.,
disruption of thyroid function) may lead to an anticipation of site
concordance.
2.2.2.2. Other Studies. Various intermediate-term studies often use
protocols that screen for carcinogenic or preneoplastic effects,
sometimes in a single tissue. Some involve the development of various
proliferative lesions, like foci of alteration in the liver
(Goldsworthy et al., 1986). Others use tumor endpoints, like the
induction of lung adenomas in the sensitive strain A mouse (Maronpot et
al., 1986) or tumor induction in initiation-promotion studies using
various organs such as the bladder, intestine, liver, lung, mammary
gland, and thyroid (Ito et al., 1992). In these tests, the selected
tissue is, in a sense, the test system rather than the whole animal.
Important information concerning the steps in the carcinogenic process
and mode of action can be obtained from ``start/stop'' experiments. In
these protocols, an agent is given for a period of time to induce
particular lesions or effects, then stopped to evaluate the progression
or reversibility of processes (Todd, 1986; Marsman and Popp, 1994).
Assays in genetically engineered rodents may provide insight into
the chemical and gene interactions involved in carcinogenesis (Tennant
et al., 1995a). These mechanistically based approaches involve
activated oncogenes that are introduced (transgenic) or tumor
suppressor genes that are deleted (knocked-out). If appropriate genes
are selected, not only may these systems provide information on
mechanisms, but the rodents typically show tumor development earlier
than the standard bioassay. Transgenic mutagenesis assays also
represent a mechanistic approach for assessing the mutagenic properties
of agents as well as developing quantitative linkages between exposure,
internal dose, and mutation related to tumor induction (Morrison and
Ashby, 1994; Sisk et al., 1994; Hayward et al., 1995). These systems
use a stable genomic integration of a lambda shuttle vector that
carries a lacI target gene and a lacZ reporter gene.
The support that these studies give to a determination of
carcinogenicity rests on their contribution to the consistency of other
evidence about an agent. For instance, benzoyl peroxide has promoter
activity on the skin, but the overall evidence may be less supportive
(Kraus et al., 1995). These studies also may contribute information
about mode of action. One needs to recognize the limitations of these
experimental protocols such as short duration, limited histology, lack
of complete development of tumors, or experimental manipulation of the
carcinogenic process that may limit their contribution to the overall
assessment. Generally, their results are appropriate as aids in the
assessment for interpreting other toxicological evidence (e.g., rodent
chronic bioassays), especially regarding potential modes of action.
With sufficient validation, these studies may partially or wholly
replace chronic bioassays in the future (Tennant et al., 1995).
2.2.3. Structural Analogue Data
For some chemical classes, there is significant information
available on the carcinogenicity of analogues, largely in rodent
bioassays. Analogue effects are instructive in investigating
carcinogenic potential of an agent as well as identifying potential
target organs, exposures associated with effects, and potential
functional class effects or modes of action. All appropriate studies
are included and analyzed, whether indicative of a positive effect or
not. Evaluation includes tests in various animal species, strains, and
sexes; with different routes of administration; and at various doses,
as data are available. Confidence in conclusions is a function of how
similar the analogues are to the agent under review in structure,
metabolism, and biological activity. This confidence needs to be
considered to ensure a balanced position.
2.3. Analysis of Other Key Data
The physical, chemical, and structural properties of an agent, as
well as data on endpoints that are thought to be critical elements of
the carcinogenic process, provide valuable insights into the likelihood
of human cancer risk. The following sections provide guidance for
analyses of these data.
2.3.1. Physicochemical Properties
Physicochemical properties affect an agent's absorption, tissue
distribution (bioavailability), biotransformation, and degradation in
the body and are important determinants of hazard potential (and dose
response analysis). Properties to analyze include, but are not limited
to, the following: molecular weight, size, and shape; valence state;
physical state (gas, liquid, solid); water or lipid solubility, which
can influence retention and tissue distribution; and potential for
chemical degradation or stabilization in the body.
An agent's potential for chemical reaction with cellular
components, particularly with DNA and proteins, is also important. The
agent's molecular size and shape, electrophilicity, and charge
distribution are considered in order to decide whether they would
facilitate such reactions.
2.3.2. Structure-Activity Relationships
Structure-activity relationship (SAR) analyses and models can be
used to predict molecular properties, surrogate biological endpoints,
and carcinogenicity. Overall, these analyses provide valuable initial
information on agents, which may strengthen or weaken the concern for
an agent's carcinogenic potential.
Currently, SAR analysis is useful for chemicals and metabolites
that are believed to initiate carcinogenesis through covalent
interaction with DNA (i.e., DNA-reactive, mutagenic, electrophilic, or
proelectrophilic
[[Page 17978]]
chemicals) (Ashby and Tennant, 1991). For organic chemicals, the
predictive capability of SAR analysis combined with other toxicity
information has been demonstrated (Ashby and Tennant, 1994). The
following parameters are useful in comparing an agent to its structural
analogues and congeners that produce tumors and affect related
biological processes such as receptor binding and activation,
mutagenicity, and general toxicity (Woo and Arcos, 1989):
nature and reactivity of the electrophilic moiety or
moieties present,
potential to form electrophilic reactive intermediate(s)
through chemical, photochemical, or metabolic activation,
contribution of the carrier molecule to which the
electrophilic moiety(ies) is attached,
physicochemical properties (e.g., physical state,
solubility, octanol-water partition coefficient, half-life in aqueous
solution),
structural and substructural features (e.g., electronic,
stearic, molecular geometric),
metabolic pattern (e.g., metabolic pathways and activation
and detoxification ratio), and
possible exposure route(s) of the agent.
Suitable SAR analysis of non-DNA-reactive chemicals and of DNA-
reactive chemicals that do not appear to bind covalently to DNA
requires knowledge or postulation of the probable mode(s) of action of
closely related carcinogenic structural analogues (e.g., receptor-
mediated, cytotoxicity-related). Examination of the physicochemical and
biochemical properties of the agent may then provide the rest of the
information needed in order to make an assessment of the likelihood of
the agent's activity by that mode of action.
2.3.3. Comparative Metabolism and Toxicokinetics
Studies of the absorption, distribution, biotransformation, and
excretion of agents permit comparisons among species to assist in
determining the implications of animal responses for human hazard
assessment, supporting identification of active metabolites,
identifying changes in distribution and metabolic pathway or pathways
over a dose range, and making comparisons among different routes of
exposure.
If extensive data are available (e.g., blood/tissue partition
coefficients and pertinent physiological parameters of the species of
interest), physiologically based pharmacokinetic models can be
constructed to assist in a determination of tissue dosimetry, species-
to-species extrapolation of dose, and route-to-route extrapolation
(Connolly and Andersen, 1991; see section 3.2.2). If it is not contrary
to available data, it is assumed as a default that toxicokinetic and
metabolic processes are qualitatively comparable between species.
Discussion of the defaults regarding quantitative comparison and their
modifications appears in section 3.
The qualitative question of whether an agent is absorbed by a
particular route of exposure is important for weight of evidence
classification discussed in section 2.7.1. Decisions whether route of
exposure is a limiting factor on expression of any hazard, in that
absorption does not occur by a route, are based on studies in which
effects of the agent, or its structural analogues, have been observed
by different routes, on physical-chemical properties, or on
toxicokinetics studies.
Adequate metabolism and pharmacokinetic data can be applied toward
the following as data permit. Confidence in conclusions is enhanced
when in vivo data are available.
Identifying metabolites and reactive intermediates of
metabolism and determining whether one or more of these intermediates
are likely to be responsible for the observed effects. This information
on the reactive intermediates will appropriately focus SAR analysis,
analysis of potential modes of action, and estimation of internal dose
in dose response assessment (D'Souza et al., 1987; Krewski et al.,
1987).
Identifying and comparing the relative activities of
metabolic pathways in animals with those in humans. This analysis can
provide insights for extrapolating results of animal studies to humans.
Describing anticipated distribution within the body and
possibly identifying target organs. Use of water solubility, molecular
weight, and structure analysis can support qualitative inferences about
anticipated distribution and excretion. In addition, describing whether
the agent or metabolite of concern will be excreted rapidly or slowly
or will be stored in a particular tissue or tissues to be mobilized
later can identify issues in comparing species and formulating dose
response assessment approaches.
Identifying changes in toxicokinetics and metabolic
pathways with increases in dose. These changes may result in important
differences in disposition of the agent or its generation of active
forms of the agent between high and low dose levels. These studies play
an important role in providing a rationale for dose selection in
carcinogenicity studies.
Determining bioavailability via different routes of
exposure by analyzing uptake processes under various exposure
conditions. This analysis supports identification of hazards for
untested routes. In addition, use of physicochemical data (e.g.,
octanol-water partition coefficient information) can support an
inference about the likelihood of dermal absorption (Flynn, 1990).
In all of these areas, attempts are made to clarify and describe as
much as possible the variability to be expected because of differences
in species, sex, age, and route of exposure. The analysis takes into
account the presence of subpopulations of individuals who are
particularly vulnerable to the effects of an agent because of
toxicokinetic or metabolic differences (genetically or environmentally
determined) (Bois et al., 1995).
2.3.4. Toxicological and Clinical Findings
Toxicological findings in experimental animals and clinical
observations in humans are an important resource to the cancer hazard
assessment. Such findings provide information on physiological effects,
effects on enzymes, hormones, and other important macromolecules as
well as on target organs for toxicity. Given that the cancer process
represents defects in terminal differentiation, growth control, and
cell death, developmental studies of agents may provide an
understanding of the activity of an agent that carries over to cancer
assessment. Toxicity studies in animals by different routes of
administration support comparison of absorption and metabolism by those
routes. Data on human variability in standard clinical tests may
provide insight into the range of human sensitivity and common
mechanisms to agents that affect the tested parameters.
2.3.5. Mode of Action-Related Endpoints and Short-Term Tests
A myriad of biochemical and biological endpoints relevant to the
carcinogenic process provide important information in determining
whether a cancer hazard exists and include, but are not limited to,
mutagenesis, inhibition of gap junctional intercellular communication,
increased cell proliferation, inhibition of programmed cell death,
receptor activation, and immunosuppression. These precursor effects are
discussed below.
2.3.5.1. Direct DNA Effects. Because cancer is the result of
multiple genetic
[[Page 17979]]
defects in genes controlling proliferation and tissue homeostasis
(Vogelstein et al., 1988), the ability of an agent to affect DNA is of
obvious importance. It is well known that many carcinogens are
electrophiles that interact directly with DNA, resulting in DNA damage
and adducts, and subsequent mutations (referred to in these guidelines
as direct DNA effects) that are thought to contribute to the
carcinogenic process (Shelby and Zeiger, 1990; Tinwell and Ashby,
1991). Thus, studies of these phenomena continue to be important in the
assessment of cancer hazard. The EPA has published testing guidelines
for detecting the ability of agents to affect DNA or chromosomes (EPA,
1991a). Information on agents that induce mutations in animal germ
cells also deserves attention; several human carcinogens have been
shown to be positive in rodent tests for the induction of genetic
damage in both somatic and germ cells (Shelby, 1995).
2.3.5.2. Secondary DNA Effects. Similarly of interest are secondary
mechanisms that either increase mutation rates or the number of
dividing cells. An increase in mutations might be due to cytotoxic
exposures causing regenerative proliferation or mitogenic influences,
either of which could result in clonal expansion of initiated cells
(Cohen and Ellwein, 1990). An agent might interfere with the enzymes
involved in DNA repair and recombination (Barrett and Lee, 1992). Also,
programmed cell death (apoptosis) can potentially be blocked by an
agent, thereby permitting replication of damaged cells. For example,
peroxisome proliferators may act by suppressing apoptosis pathways
(Shulte-Hermann et al., 1993; Bayly et al., 1994). An agent may also
generate reactive oxygen species that produce oxidative damage to DNA
and other important macromolecules that become important elements of
the carcinogenic process (Kehrer, 1993; Clayson et al., 1994; Chang et
al., 1988). Damage to certain critical DNA repair genes or other genes
(e.g., the p53 gene) may result in genomic instability, which
predisposes cells to further genetic alterations and increases the
probability of neoplastic progression independent of any exogenous
agent (Harris and Hollstein, 1993; Levine, 1994).
The loss or gain of chromosomes (i.e., aneuploidy) is an effect
that can result in genomic instability (Fearon and Vogelstein, 1990;
Cavenee et al., 1986). Although the relationship between induced
aneuploidy and carcinogenesis is not completely established, several
carcinogens have been shown to induce aneuploidy (Gibson et al., 1995;
Barrett, 1992). Agents that cause aneuploidy interfere with the normal
process of chromosome segregation and lead to chromosomal losses,
gains, or aberrations by interacting with the proteins (e.g.,
microtubules) needed for chromosome movement.
2.3.5.3. Nonmutagenic and Other Effects. A failure to detect DNA
damage and mutation induction in several test systems suggests that a
carcinogenic agent may act by another mode of action.
It is possible for an agent to alter gene expression
(transcriptional, translational, or post-translational modifications)
by means not involving mutations (Barrett, 1995). For example,
perturbation of DNA methylation patterns may cause effects that
contribute to carcinogenesis (Jones, 1986; Goodman and Counts, 1993;
Holliday, 1987). Overexpression of genes by amplification has been
observed in certain tumors (Vainio et al., 1992). Other mechanisms may
involve cellular reprogramming through hormonal mechanisms or receptor-
mediated mechanisms (Ashby et al., 1994; Barrett, 1992).
Gap-junctional intercellular communication is widely believed to
play a role in tissue and organ development and in the maintenance of a
normal cellular phenotype within tissues. A growing body of evidence
suggests that chemical interference with gap-junctional intercellular
communication is a contributing factor in tumor development; many
carcinogens have been shown to inhibit this communication. Thus, such
information may provide useful mechanistic data in evaluating cancer
hazard (Swierenga and Yamasaki, 1992; Yamasaki, 1995).
Both cell death and cell proliferation are mandatory for the
maintenance of homeostasis in normal tissue. The balance between the
two directly affects the survival and growth of initiated cells, as
well as preneoplastic and tumor cell populations (i.e., increase in
cell proliferation or decrease in cell death) (Bellamy et al., 1995;
Cohen and Ellwein, 1990, 1991; Cohen et al., 1991). In studies of
proliferative effects, distinctions should be made between mitogenesis
and regenerative proliferation (Cohen and Ellwein, 1990, 1991; Cohen et
al., 1991). In applying information from studies on cell proliferation
and apoptosis to risk assessment, it is important to identify the
tissues and target cells involved, to measure effects in both normal
and neoplastic tissue, to distinguish between apoptosis and necrosis,
and to determine the dose that affects these processes.
2.3.5.4. Criteria for Judging Mode of Action. Criteria that are
applicable for judging the adequacy of mechanistically based data
include the following:
mechanistic relevance of the data to carcinogenicity,
number of studies of each endpoint,
consistency of results in different test systems and
different species,
similar dose response relationships for tumor and mode of
action-related effects,
tests conducted in accordance with generally accepted
protocols, and
degree of consensus and general acceptance among
scientists regarding interpretation of the significance and specificity
of the tests.
Although important information can be gained from in vitro test
systems, a higher level of confidence is generally given to data that
are derived from in vivo systems, particularly those results that show
a site concordance with the tumor data.
2.4. Biomarker Information
Various endpoints can serve as biological markers of events in
biological systems or samples. In some cases, these molecular or
cellular effects (e.g., DNA or protein adducts, mutation, chromosomal
aberrations, levels of thyroid stimulating hormone) can be measured in
blood, body fluids, cells and tissues to serve as biomarkers of
exposure in both animals and humans (Callemen et al., 1978; Birner et
al., 1990). As such, they can do the following:
act as an internal surrogate measure of chemical dose,
representing as appropriate, either recent (e.g., serum concentration)
or accumulated (e.g., hemoglobin adducts) exposure,
help identify doses at which elements of the carcinogenic
process are operating,
aid in interspecies extrapolations when data are available
from both experimental animal and human cells, and
under certain circumstances, provide insights into the
possible shape of the dose response curve below levels where tumor
incidences are observed (e.g., Choy, 1993).
Genetic and other findings (like changes in proto-oncogenes and
tumor suppressor genes in preneoplastic and neoplastic tissue or
possibly measures of endocrine disruption) can indicate the potential
for disease and as such serve as biomarkers of effect. They, too, can
be used in different ways:
The spectrum of genetic changes in proliferative lesions
and tumors
[[Page 17980]]
following chemical administration to experimental animals can be
determined and compared with those in spontaneous tumors in control
animals, in animals exposed to other agents of varying structural and
functional activities, and in persons exposed to the agent under study.
They may provide a linkage to tumor response.
They may help to identify subpopulations of individuals
who may be at an elevated risk for cancer, e.g., cytochrome P450 2D6/
debrisoquine sensitivity for lung cancer (Caporaso et al., 1989) or
inherited colon cancer syndromes (Kinzler et al., 1991; Peltomaki et
al., 1993).
As with biomarkers of exposure, it may be justified in
some cases to use these endpoints for dose response assessment or to
provide insight into the potential shape of the dose response curve at
doses below those at which tumors are induced experimentally.
In applying biomarker data to cancer assessment (particularly
assessments based on epidemiologic data), one should consider the
following:
routes of exposure
exposure to mixtures
time after exposure
sensitivity and specificity of biomarkers
dose response relationships.
2.5. Mode of Action--Implications for Hazard Characterization and Dose
Response
The interaction of the biology of the organism and the chemical
properties of the agent determine whether there is an adverse effect.
Thus, mode of action analysis is based on physical, chemical, and
biological information that helps to explain critical events in an
agent's influence on development of tumors. The entire range of
information developed in the assessment is reviewed to arrive at a
reasoned judgment. An agent may work by more than one mode of action
both at different sites and at the same tumor site. It is felt that at
least some information bearing on mode of action (e.g., SAR, screening
tests for mutagenicity) is present for most agents undergoing
assessment of carcinogenicity, even though certainty about exact
molecular mechanisms may be rare.
Inputs to mode of action analysis include tumor data in humans,
animals, and among structural analogues as well as the other key data.
The more complete the data package and generic knowledge about a given
mode of action, the more confidence one has and the more one can
replace or refine default science policy positions with relevant
information. Making reasoned judgments is generally based on a data-
rich source of chemical, chemical class, and tumor type-specific
information. Many times there will be conflicting data and gaps in the
information base; one must carefully evaluate these uncertainties
before reaching any conclusion.
Some of the questions that need to be addressed include the
following:
Has a body of data been developed on the agent that fits
with a generally accepted mode of action?
Has the mode of action been published and gained general
scientific acceptance through peer-reviewed research or is it still
speculative?
Is the mode of action consistent with generally agreed-
upon principles and understanding of carcinogenesis?
Is the mode of action reasonably anticipated or assumed,
in the absence of specific data, to operate in humans? How is this
question influenced by information on comparative uptake, metabolism,
and excretion patterns across animals and humans?
Do humans appear to be more or less sensitive to the mode
of action than are animals?
Does the agent affect DNA, directly or indirectly?
Are there important determinants in carcinogenicity other
than effects on DNA, such as changes in cell proliferation, apoptosis,
gene expression, immune surveillance, or other influences?
In making decisions about potential modes of action and the
relevance of animal tumor findings to humans (Ashby et al., 1990), very
often the results of chronic animal studies may give important clues.
Some of the important factors to review include the following:
tumor types, e.g., those responsive to endocrine
influence, those produced by reactive carcinogens (Ashby and Tennant,
1991),
number of tumor sites, sexes, studies, and species
affected or unaffected (Tennant, 1993),
influence of route of exposure; spectrum of tumors; local
or systemic sites,
target organ or system toxicity, e.g., urinary chemical
changes associated with stone formation, effects on immune
surveillance,
presence of proliferative lesions, e.g., hepatic foci,
hyperplasias,
progression of lesions from preneoplastic to benign to
malignant with dose and time,
ratio of malignant to benign tumors as a function of dose
and time,
time of appearance of tumors after commencing exposure,
tumors invading locally, metastasizing, producing death,
tumors at sites in laboratory animals with high or low
spontaneous historical incidence,
biomarkers in tumor cells, both induced and spontaneous,
e.g., DNA or protein adducts, mutation spectra, chromosome changes,
oncogene activation, and
shape of the dose response in the range of tumor
observation, e.g., linear vs. profound change in slope.
Some of the myriad of ways that information from chronic animal
studies influences mode of action judgments include the following.
Multisite and multispecies tumor effects are often associated with
mutagenic agents. Tumors restricted to one sex/species may suggest an
influence restricted to gender, strain, or species. Late onset of
tumors that are primarily benign or are at sites with a high historical
background incidence or show reversal of lesions on cessation of
exposure may point to a growth-promoting mode of action. The
possibility that an agent may act differently in different tissues or
have more than one mode of action in a single tissue must also be kept
in mind.
Simple knowledge of sites of tumor increase in rodent studies can
give preliminary clues as to mode of action. Experience at the National
Toxicology Program (NTP) indicates that substances that are DNA
reactive and produce gene mutations may be unique in producing tumors
in certain anatomical sites, while tumors at other sites may arise from
both mutagenic or nonmutagenic influences (Ashby and Tennant, 1991;
Huff et al., 1991).
Effects on tumor sites in rodents and other mode of action
information has been explored for certain agents (Alison et al., 1994;
Clayson, 1989; ECETOC, 1991; MacDonald et al., 1994; McClain, 1994;
Tischer et al., 1991; ILSI, 1995; Cohen and Ellwein, 1991; FASEB, 1994;
Havu et al., 1990; U.S. EPA, 1991; Li et al., 1987; Grasso and Hinton,
1991; Larson et al., 1994; IARC, 1990; Jack et al., 1983; Stitzel et
al., 1989; Ingram and Grasso, 1991; Bus and Popp, 1987; Prahalada et
al., 1994; Yamada et al., 1994; Hill et al., 1989; Burek et al., 1988).
The selection of a dose response extrapolation procedure for cancer
risk estimation considers mode of action information. When information
is extensive and there is considerable certainty in a given mode of
action, a biologically based or case-specific model that incorporates
data on processes involved is preferred. Obviously, use of such a model
requires
[[Page 17981]]
the existence of substantial data on component parameters of the mode
of action, and judgments on its applicability must be made on a case-
by-case basis.
In the absence of information to develop a biologically based or
case-specific model, understanding of mode of action should be employed
to the extent possible in deciding upon one of three science policy
defaults: Low-dose linear extrapolation, nonlinear, and both
procedures. The overall choice of the default(s) depends upon weighing
the various inputs and deciding which best reflect the mode of action
understanding. A rationale accompanies whichever default or defaults
are chosen.
A default assumption of linearity is appropriate when the evidence
supports a mode of action of gene mutation due to DNA reactivity or
supports another mode of action that is anticipated to be linear. Other
elements of empirical data may also support an inference of linearity,
e.g., the background of human exposure to an agent might be such that
added human exposure is on the linear part of a dose response curve
that is sublinear overall. The default assumption of linearity is also
appropriate as the ultimate default when evidence shows no DNA
reactivity or other support for linearity, but neither is it sufficient
evidence of a nonlinear mode of action to support a nonlinear
procedure.
A default assumption of nonlinearity is appropriate when there is
no evidence for linearity and sufficient evidence to support an
assumption of nonlinearity and a nonlinear procedure. The mode of
action may lead to a dose response relationship that is nonlinear, with
response falling much more quickly than linearly with dose, or being
most influenced by individual differences in sensitivity.
Alternatively, the mode of action may theoretically have a threshold,
e.g., the carcinogenicity may be a secondary effect of toxicity that is
itself a threshold phenomenon.
Both linear and nonlinear procedures may be used in particular
cases. If a mode of action analysis finds substantial support for
differing modes of action for different tumor sites, an appropriate
procedure is used for each. Both procedures may also be appropriate to
discuss implications of complex dose response relationships. For
example, if it is apparent that an agent is both DNA reactive and is
highly active as a promotor at high doses, and there are insufficient
data for modeling, both linear and nonlinear default procedures may be
needed to decouple and consider the contribution of both phenomena.
2.6. Weight of Evidence Evaluation for Potential Human Carcinogenicity
A weight of evidence evaluation is a collective evaluation of all
pertinent information so that the full impact of biological
plausibility and coherence are adequately considered. Identification
and characterization of human carcinogenicity is based on human and
experimental data, the nature, advantages and limitations of which have
been discussed in the preceding sections.
The subsequent sections outline: (1) the basics of weighing
individual lines of evidence and combining the entire body of evidence
to make an informed judgment, (2) classification descriptors of cancer
hazard, and (3) some case study examples to illustrate how the
principles of guidance can be applied to arrive at a classification.
2.6.1. Weight of Evidence Analysis
Judgment about the weight of evidence involves considerations of
the quality and adequacy of data and consistency of responses induced
by the agent in question. The weight of evidence judgment requires
combined input of relevant disciplines. Initial views of one kind of
evidence may change significantly when other information is brought to
the interpretation. For example, a positive animal carcinogenicity
finding may be diminished by other key data; a weak association in
epidemiologic studies may be bolstered by consideration of other key
data and animal findings. Factors typically considered are illustrated
in figures below. Generally, no single weighing factor on either side
determines the overall weight. The factors are not scored mechanically
by adding pluses and minuses; they are judged in combination.
Human Evidence. Analyzing the contribution of evidence from a body
of human data requires examining available studies and weighing them in
the context of well-accepted criteria for causation (see section
2.2.1). A judgment is made about how closely they satisfy these
criteria, individually and jointly, and how far they deviate from them.
Existence of temporal relationships, consistent results in independent
studies, strong association, reliable exposure data, presence of dose-
related responses, freedom from biases and confounding factors, and
high level of statistical significance are among the factors leading to
increased confidence in a conclusion of causality.
Generally, the weight of human evidence increases with the number
of adequate studies that show comparable results on populations exposed
to the same agent under different conditions. The analysis takes into
account all studies of high quality, whether showing positive
associations or null results, or even protective effects. In weighing
positive studies against null studies, possible reasons for
inconsistent results should be sought, and results of studies that are
judged to be of high quality are given more weight than those from
studies judged to be methodologically less sound. See figure 2-1.
BILLING CODE 6560-50-P
[[Page 17982]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.002
Generally, no single factor is determinative. For example, the
strength of association is one of the causal criteria. A strong
association (i.e., a large relatively risk) is more likely to indicate
causality than a weak association. However, finding of a large excess
risk in a single study must be balanced against the lack of consistency
as reflected by null results from other equally well designed and well
conducted studies. In this situation, the positive association of a
single study may either suggest the presence of chance, bias or
confounding, or reflect different exposure conditions. On the other
hand, evidence of weak but consistent associations across several
studies suggests either causality or the same confounder may be
operating in all of these studies.
Animal Evidence. Evidence from long-term or other carcinogenicity
studies in laboratory animals constitutes the second major class of
information bearing on carcinogenicity. See figure 2-2. As discussed in
section 2.2.2., each relevant study must be reviewed and evaluated as
to its adequacy of design and conduct as well as the statistical
significance and biological relevance of its findings. Factors that
usually increase confidence in the predictivity of animal findings are
those of (1) multiplicity of observations in independent studies; (2)
severity of lesions, latency, and lesion progression; (3) consistency
in observations.
[[Page 17983]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.003
Other Key Evidence. Additional information bearing on the
qualitative assessment of carcinogenic potential may be gained from
comparative pharmacokinetic and metabolism studies, genetic toxicity
studies, SAR analysis, and other studies of an agent's properties. See
figure 2-3. Information from these studies helps to elucidate potential
modes of action and biological fate and disposition. The knowledge
gained supports interpretation of cancer studies in humans and animals
and provides a separate source of information about carcinogenic
potential.
[[Page 17984]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.004
Totality of Evidence. In reaching a view of the entire weight of
evidence, all data and inferences are merged. Figure 2-4 indicates the
generalities. In fact, possible weights of evidence span a broad
continuum that cannot be capsulized. Most of the time the data in
various lines of evidence fall in the middle of the weights represented
in the four figures in this section.
[[Page 17985]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.005
BILLING CODE 6560-50-C
The following section and the weight of evidence narrative
discussed in 2.7.2. provide a way to state a conclusion and capture
this complexity in a consistent way.
2.6.2. Descriptors for Classifying Weight of Evidence
Hazard classification uses three categories of descriptors for
human carcinogenic potential: ``known/likely,'' ``cannot be
determined,'' and ``not likely.'' Each category has associated
subdescriptors to further define the conclusion. The descriptors are
not meant to replace an explanation of the nuances of the biological
evidence, but rather to summarize it. Each category spans a wide
variety of potential data sets and weights of evidence. There will
always be gray areas, gradations, and borderline cases. That is why the
descriptors are presented only in the context of a weight of evidence
narrative whose format is given in section 2.7.2. Using them within a
narrative preserves and presents the complexity that is an essential
part of the hazard classification. Applying a descriptor is a matter of
judgment and cannot be reduced to a formula. Risk managers should
consider the entire range of information included in the narrative
rather than focusing simply on the descriptor.
A single agent may be categorized in more than one way if, for
instance, the agent is likely to be carcinogenic by one route of
exposure but not by another (section 2.3.3).
The descriptors and subdescriptors are standardized and are to be
used consistently from case to case. The discussions below explain
descriptors and subdescriptors which appear in italics, and along with
Appendix A and section 2.6.3, illustrate their use.
``Known/Likely''
This category of descriptors is appropriate when the available
tumor effects and other key data are adequate to convincingly
demonstrate carcinogenic potential for humans; it includes:
Agents known to be carcinogenic in humans based on either
epidemiologic evidence or a combination of epidemiologic and
experimental evidence, demonstrating causality between human exposure
and cancer,
Agents that should be treated as if they were known human
carcinogens, based on a combination of epidemiologic data showing a
plausible causal association (not demonstrating it definitively) and
strong experimental evidence.
Agents that are likely to produce cancer in humans due to
the production or anticipated production of tumors by modes of action
that are relevant or assumed to be relevant to human carcinogenicity.
Modifying descriptors for particularly high or low ranking in the
``known/likely'' group can be applied based on scientific judgment and
experience and are as follows:
Agents that are likely to produce cancer in humans based
on data that are at the high end of the weights of evidence typical of
this group,
Agents that are likely to produce cancer in humans based
on data that are at the low end of the weights of evidence typical of
this group.
``Cannot Be Determined''
This category of descriptors is appropriate when available tumor
effects or other key data are suggestive or conflicting or limited in
quantity and, thus, are not adequate to convincingly demonstrate
carcinogenic potential for humans. In general, further agent specific
and generic research and testing are needed to be able to describe
human carcinogenic potential. The descriptor cannot be determined is
used with a subdescriptor that captures the rationale:
Agents whose carcinogenic potential cannot be determined,
but for which there is suggestive evidence that raises concern for
carcinogenic effects,
Agents whose carcinogenic potential cannot be determined
because the existing evidence is composed of conflicting data (e.g.,
some evidence is suggestive of carcinogenic effects, but other equally
pertinent evidence does not confirm any concern),
Agents whose carcinogenic potential cannot be determined
because there are inadequate data to perform an assessment,
Agents whose carcinogenic potential cannot be determined
because no data are available to perform an assessment.
[[Page 17986]]
``Not Likely''
This is the appropriate descriptor when experimental evidence is
satisfactory for deciding that there is no basis for human hazard
concern, as follows (in the absence of human data suggesting a
potential for cancer effects):
Agents not likely to be carcinogenic to humans because
they have been evaluated in at least two well conducted studies in two
appropriate animal species without demonstrating carcinogenic effects,
Agents not likely to be carcinogenic to humans because
they have been appropriately evaluated in animals and show only
carcinogenic effects that have been shown not to be relevant to humans
(e.g., showing only effects in the male rat kidney due to accumulation
of alpha2u-globulin),
Agents not likely to be carcinogenic to humans when
carcinogenicity is dose or route dependent. For instance, not likely
below a certain dose range (categorized as likely above that range) or
not likely by a certain route of exposure (may be categorized as likely
by another route of exposure). To qualify, agents will have been
appropriately evaluated in animal studies and the only effects show a
dose range or route limitation or a route limitation is otherwise shown
by empirical data.
Agents not likely to be carcinogenic to humans based on
extensive human experience that demonstrates lack of effect (e.g.,
phenobarbital).
2.6.3. Case Study Examples
This section provides examples of substances that fit the three
broad categories described above. These examples are based on available
information about real substances and are selected to illustrate the
principles for weight-of-evidence evaluation and the application of the
classification scheme.
These case studies show the interplay of differing lines of
evidence in making a conclusion. Some particularly illustrate the role
that ``other key data'' can play in conclusions.
Example 1: ``Known Human Carcinogen''--Route-Dependent/Linear
Extrapolation
Human Data
Substance 1 is an aluminosilicate mineral that exists in nature
with a fibrous habit. Several descriptive epidemiologic studies have
demonstrated very high mortality from malignant mesothelioma, mainly
of the pleura, in three villages in Turkey, where there was a
contamination of this mineral and where exposure had occurred from
birth. Both sexes were equally affected and at an unusually young
age.
Animal Data
Substance 1 has been studied in a single long-term inhalation
study in rats at one exposure concentration that showed an extremely
high incidence of pleural mesothelioma (98% in treated animals
versus 0% in concurrent controls). This is a rare malignant tumor in
the rat and the onset of tumors occurred at a very early age (as
early as 1 year of age). Several studies involving injection into
the body cavities of rats or mice (i.e., pleural or peritoneal
cavities) also produced high incidences of pleural or peritoneal
mesotheliomas. No information is available on the carcinogenic
potential of substance 1 in laboratory animals via oral and dermal
exposures.
Other Key Data
Information on the physical and chemical properties of substance
1 indicates that it is highly respirable to humans and laboratory
rodents. It is highly insoluble and is not likely to be readily
degraded in biological fluid.
No information is available on the deposition, translocation,
retention, lung clearance, and excretion of the substance after
inhalation exposure or ingestion. Lung burden studies have shown the
presence of elevated levels of the substance in lung tissue samples
of human cases of pleural mesotheliomas from contaminated villages
compared with control villages.
No data are available on genetic or related effects in humans.
The substance has been shown to induce unscheduled DNA synthesis in
human cells in vitro and transformation and unscheduled DNA
synthesis in mouse cells.
The mechanisms by which this substance causes cancer in humans
and animals are not understood, but appear to be related to its
unique physical, chemical, and surface properties. Its fiber
morphology is similar to a known group of naturally occurring
silicate minerals that have been known to cause respiratory cancers
(including pleural mesothelioma) from inhalation exposure and
genetic changes in humans.
Evaluation
Human evidence is judged to establish a causal link between
exposure to substance 1 and human cancer. Even though the human
evidence does not satisfy all criteria for causality, this judgment
is based on a number of unusual observations: large magnitude of the
association, specificity of the association, demonstration of
environmental exposure, biological plausibility, and coherence based
on the entire body of knowledge of the etiology of mesothelioma.
Animal evidence demonstrates a causal relationship between
exposure and cancer in laboratory animals. Although available data
are not optimal in terms of design (e.g., the use of single dose,
one sex only), the judgment is based on the unusual findings from
the only inhalation experiment in rats (i.e., induction of an
uncommon tumor, an extremely high incidence of malignant neoplasms,
and onset of tumors at an early age). Additional evidence is
provided by consistent results from several injection studies
showing an induction of the same tumors by different modes of
administration in more than one species.
Other key data, while limited, support the human and animal
evidence of carcinogenicity. It can be inferred from human and
animal data that this substance is readily deposited in the
respiratory airways and deep lung and is retained for extended
periods of time since first exposure. Information on related fibrous
substances indicates that the modes of action are likely mediated by
the physical and chemical characteristics of the substance (e.g.,
fiber shape, high aspect ratio, a high degree of insolubility in
lung tissues).
Insufficient data are available to evaluate the human
carcinogenic potential of substance 1 by oral exposure. Even though
there is no information on its carcinogenic potential via dermal
uptake, it is not expected to pose a carcinogenic hazard to humans
by that route because it is very insoluble and is not likely to
penetrate the skin.
Conclusion
It is concluded that substance 1 is a known human carcinogen by
inhalation exposure. The weight of evidence of human carcinogenicity
is based on (a) exceptionally increased incidence of malignant
mesothelioma in epidemiologic studies of environmentally exposed
human populations; (b) significantly increased incidence of
malignant mesothelioma in a single inhalation study in rats and in
several injection studies in rats and mice; and (c) supporting
information on related fibrous substances that are known to cause
cancer via inhalation and genetic damage in exposed mammalian and
human mesothelial cells. The human carcinogenic potential of
substance 1 via oral exposure cannot be determined on the basis of
insufficient data. It is not likely to pose a carcinogenic hazard to
humans via dermal uptake because it is not anticipated to penetrate
the skin.
The mode of action of this substance is not understood. In
addition to this uncertainty, dose response information is lacking
for both human and animal data. Epidemiologic studies contain
observations of significant excess cancer risks at relatively low
levels of environmental exposure. The use of linear extrapolation in
a dose response relationship assessment is appropriate as a default
since mode of action data are not available.
Example 2: ``As If Known Human Carcinogen''--Any Exposure Conditions/
Linear Extrapolation
Human Data
Substance 2 is an alkene oxide. Several cohort studies of
workers using substance 2 as a sterilant have been conducted. In the
largest and most informative study, mortality from lymphatic and
hematopoietic cancer was marginally elevated, but a significant
trend was found, especially for lymphatic leukemia and non-Hodgkin's
lymphoma, in relation to estimated cumulative exposure to the
substance. Nonsignificant excesses of lymphatic and hematopoietic
cancer were
[[Page 17987]]
found in three other smaller studies of sterilization personnel.
In one cohort study of chemical workers exposed to substance 2
and other agents, mortality rate from lymphatic and hematopoietic
cancer was elevated, but the excess was confined to a small subgroup
with only occasional low-level exposure to substance 2. Six other
studies of chemical workers are considered more limited due to a
smaller number of deaths. Four studies found an excess of lymphatic
and hematopoietic cancer (which were significant in two); no
increase in mortality rate was observed in the other two studies.
Animal Data
Substance 2 was studied in an oral gavage study in rats.
Treatment of substance 2 resulted in a dose-dependent increased
incidence in forestomach tumors that were mainly squamous-cell
carcinomas.
Substance 2 was also studied in two inhalation studies in mice
and two inhalation studies in rats. In the first mouse study, dose-
dependent increases in combined benign and malignant tumors at
several tissue sites were induced in mice of both sexes (lung tumors
and tumors of the Harderian gland in each sex, and uterine
adenocarcinomas, mammary carcinomas, and malignant lymphomas in
females). In a second study--a screening study for pulmonary tumors
in mice--inhalation exposure to substance 2 resulted in a dose-
dependent increase in lung tumors. In the two inhalation studies in
rats, increased incidences of mononuclear-cell leukemia and brain
tumors were induced in exposed animals of each sex; increased
incidences of peritoneal tumors in the region of the testis and
subcutaneous fibromas were induced in exposed male rats.
Substance 2 induced local sarcomas in mice following
subcutaneous injection. No tumors were found in a limited skin
painting study in mice.
Other Key Data
Substance 2 is a flammable gas at room temperature. The gaseous
form is readily taken up in humans and rats, and in aqueous solution
it can penetrate human skin. Studies in rats indicate that, once
absorbed, substance 2 is uniformly distributed throughout the body.
It is eliminated metabolically by hydrolysis and by conjugation with
glutathione. The ability to form glutathione conjugate varies across
animal species, with the rat being most active, followed by mice and
rabbits.
Substance 2 is a directly acting alkylating agent. It has been
shown to form adducts with hemoglobin in both humans and animals and
with DNA in animals. The increased frequency of hemoglobin adducts,
which have been used as markers of internal dose, has been found to
correlate with the level and cumulative exposure to substance 2.
Significant increases in chromosomal aberrations and sister
chromatid exchanges in peripheral lymphocytes and induction of
micronuclei in the bone marrow cells have been observed in exposed
workers.
Substance 2 also induced chromosomal aberrations and sister
chromatid exchanges in peripheral lymphocytes of monkeys exposed in
vivo. It also induced gene mutation, specific locus mutation, sister
chromatid exchanges, chromosomal aberrations, micronuclei, dominant
lethal mutations, and heritable translocation in rodents exposed in
vivo. In human cells in vitro, it induced sister chromatid
exchanges, chromosomal aberrations, and unscheduled DNA synthesis.
Similar genetic and related effects were observed in rodent cells in
vitro and in nonmammalian systems.
Evaluation
Available epidemiologic studies, taken together, suggest that a
causal association between exposure to substance 2 and elevated risk
of cancer is plausible. This judgment is based on small but
consistent excesses of lymphatic and hematopoietic cancer in the
studies of sterilization workers. Interpretation of studies of
chemical workers is difficult because of possible confounding
exposures. Nevertheless, findings of elevated risks of cancer at
similar sites in chemical workers support the findings in studies of
sterilization workers. Additional support is provided by
observations of DNA damage in the same tissue in which elevated
cancer was seen in exposed workers.
Extensive evidence indicates that substance 2 is carcinogenic to
laboratory animals. Positive results were consistently observed in
all well-designed and well-conducted studies. Substance 2 causes
dose-related increased incidences of tumors at multiple tissue sites
in rats and mice of both sexes by two routes of exposure (oral and
inhalation). The only dermal study that yielded a nonpositive
finding is considered of limited quality.
Other key data significantly add support to the potential
carcinogenicity of substance 2. There is strong evidence of
heritable mutations of exposed rodents and mutagenicity and
clastogenicity both in vivo and in vitro. These findings are
reinforced by observations of similar genetic damage in exposed
workers. Additional support is based on SAR analysis that indicates
that substance 2 is a highly DNA-reactive agent. Structurally
related chemicals, i.e., low-molecular-weight epoxides, also exhibit
carcinogenic effects in laboratory animals.
Conclusion
Substance 2 should be considered as if it were a known human
carcinogen by all routes of exposure. The weight of evidence of
human carcinogenicity is based on (a) consistent evidence of
carcinogenicity in rats and mice by oral and inhalation exposure;
(b) epidemiologic evidence suggestive of a causal association
between exposure and elevated risk of lymphatic and hematopoietic
cancer; (c) evidence of genetic damage in blood lymphocytes and bone
marrow cells of exposed workers; (d) mutagenic effects in numerous
in vivo and in vitro test systems; (e) membership in a class of DNA-
reactive compounds that have been shown to cause carcinogenic and
mutagenic effects in animals; and (f) ability to be absorbed by all
routes of exposure, followed by rapid distribution throughout the
body.
Although the exact mechanisms of carcinogenic action of
substance 2 are not completely understood, available data strongly
indicate a mutagenic mode of action. Linear extrapolation should be
assumed in dose response assessment.
Example 3: ``Likely Human Carcinogen''--Any Exposure Conditions/Linear
Extrapolation
Human Data
Substance 3 is a brominated alkane. Three studies have
investigated the cancer mortality of workers exposed to this
substance. No statistically significant increase in cancer at any
site was found in a study of production workers exposed to substance
3 and several other chemicals. Elevated cancer mortality was
reported in a much smaller study of production workers. An excess of
lymphoma was reported in grain workers who may have had exposure to
substance 3 and other chemical compounds. These studies are
considered inadequate due to their small cohort size; lack of, or
poorly characterized, exposure concentrations; or concurrent
exposure of the cohort to other potential or known carcinogens.
Animal Data
The potential carcinogenicity of substance 3 has been
extensively studied in an oral gavage study in rats and mice of both
sexes, two inhalation studies of rats of different strains of both
sexes, an inhalation study in mice of both sexes, and a skin
painting study in female mice.
In the oral study, increased incidences of squamous-cell
carcinoma of the forestomach were found in rats and mice of both
sexes. Additionally, there were increased incidences of liver
carcinomas in female rats, hemangiosarcomas in male rats, and
alveolar/bronchiolar adenoma of the lung of male and female mice.
Excessive toxicity and mortality were observed in the rat study,
especially in the high-dose groups, which resulted in early
termination of study, and similar time-weighted average doses for
the high- and low-treatment groups.
In the first inhalation study in rats and mice, increased
incidences of carcinomas and adenocarcinomas of the nasal cavity and
hemangiosarcoma of the spleen were found in exposed animals of each
species of both sexes. Treated female rats also showed increased
incidences of alveolar/bronchiolar carcinoma of the lung and mammary
gland fibroadenomas. Treated male rats showed an increased incidence
of peritoneal mesothelioma. In the second inhalation study in rats
(single exposure only), significantly increased incidences of
hemangiosarcoma of the spleen and adrenal gland tumors were seen in
exposed animals of both sexes. Additionally, increased incidences of
subcutaneous mesenchymal tumors and mammary gland tumors were
induced in exposed male and female rats, respectively.
Lifetime dermal application of substance 3 to female mice
resulted in significantly increased incidences of skin papillomas
and lung tumors.
Several chemicals structurally related to substance 3 are also
carcinogenic in rodents. The spectrum of tumor responses induced by
related substances was similar to those seen with substance 3 (e.g.,
forestomach, mammary gland, lung tumors).
[[Page 17988]]
Other Key Data
Substance 3 exists as a liquid at room temperature and is
readily absorbed by ingestion, inhalation, and dermal contact. It is
widely distributed in the body and is eliminated in the urine mainly
as metabolites (e.g., glutathione conjugate).
Substance 3 is not itself DNA-reactive, but is biotransformed to
reactive metabolites as inferred by findings of its covalent binding
to DNA and induction of DNA strand breaks, both in vivo and in
vitro. Substance 3 has been shown to induce sister chromatid
exchanges, mutations, and unscheduled DNA synthesis in human and
rodent cells in vitro. Reverse and forward mutations have been
consistently produced in bacterial assays and in vitro assays using
eukaryotic cells. Substance 3, however, did not induce dominant
lethal mutations in mice or rats, or chromosomal aberrations or
micronuclei in bone marrow cells of mice treated in vivo.
Evaluation
Available epidemiologic data are considered inadequate for an
evaluation of a causal association of exposure to the substance and
excess of cancer mortality due to major study limitations.
There is extensive evidence that substance 3 is carcinogenic in
laboratory animals. Increased incidences of tumors at multiple sites
have been observed in multiple studies in two species of both sexes
with different routes of exposure. It induces tumors both at the
site of entry (e.g., nasal tumors via inhalation, forestomach tumors
by ingestion, skin tumor with dermal exposure) and at distal sites
(e.g., mammary gland tumors). Additionally, it induced tumors at the
same sites in both species and sexes via different routes of
exposure (e.g., lung tumors). With the exception of the oral study
in which the employed doses caused excessive toxicity and mortality,
the other studies are considered adequately designed and well
conducted. Overall, given the magnitude and extent of animal
carcinogenic responses to substance 3, coupled with similar
responses to structurally related substances, these animal findings
are judged to be highly relevant and predictive of human responses.
Other key data, while not very extensive, are judged to be
supportive of carcinogenic potential. Substance 3 has consistently
been shown to be mutagenic in mammalian cells, including human
cells, and nonmammalian cells; thus, mutation is likely a mode of
action for its carcinogenic activity. However, the possible
involvement of other modes of action has not been fully
investigated. Furthermore, induction of genetic changes from in vivo
exposure to substance 3 has not been demonstrated.
Conclusion
Substance 3 is likely to be a human carcinogen by any route of
exposure. In comparison with other agents designated as likely human
carcinogens, the overall weight of evidence for substance 3 puts it
at the high end of the grouping.
The weight of evidence of human carcinogenicity is based on
animal evidence and other key evidence. Human data are inadequate
for an evaluation of human carcinogenicity. The overall weight of
evidence is based on (a) extensive animal evidence showing induction
of increases of tumors at multiple sites in both sexes of two rodent
species via three routes of administration relevant to human
exposure; (b) tumor data of structural analogues exhibiting similar
patterns of tumors in treated rodents; (c) in vitro evidence for
mutagenic effects in mammalian cells and nonmammalian systems; and
(d) its ability to be absorbed by all routes of exposure followed by
rapid distribution throughout the body.
Some uncertainties are associated with the mechanisms of
carcinogenicity of substance 3. Although there is considerable
evidence indicating that mutagenic events could account for
carcinogenic effects, there is still a lack of adequate information
on the mutagenicity of substance 3 in vivo in animals or humans.
Moreover, alternative modes of action have not been explored.
Nonetheless, available data indicate a likely mutagenic mode of
action. Linear extrapolation should be assumed in dose response
assessment.
Example 4: ``Likely Human Carcinogen''--All Routes/Linear and Nonlinear
Extrapolation
Human Data
Substance 4 is a chlorinated alkene solvent. Several cohort
studies of dry cleaning and laundry workers exposed to substance 4
and other solvents reported significant excesses of mortality due to
cancers of the lung, cervix, esophagus, kidney, bladder, lymphatic
and hematopoietic system, colon, or skin. No significant cancer
risks were observed in a subcohort of one these investigations of
dry cleaning workers exposed mainly to substance 4. Possible
confounding factors such as smoking, alcohol consumption, or low
socioeconomic status were not considered in the analyses of these
studies.
A large case-control study of bladder cancer did not show any
clear association with dry cleaning. Several case-control studies of
liver cancer identified an increased risk of liver cancer with
occupational exposure to organic solvents. The specific solvents to
which workers were exposed and exposure levels were not identified.
Animal Data
The potential carcinogenicity of substance 4 has been
investigated in two long-term studies in rats and mice of both sexes
by oral administration and inhalation.
Significant increases in hepatocellular carcinomas were induced
in mice of both sexes treated with substance 4 by oral gavage. No
increases in tumor incidence were observed in treated rats.
Limitations in both experiments included control groups smaller than
treated groups, numerous dose adjustments during the study, and
early mortality due to treatment-related nephropathy.
In the inhalation study, there were significantly increased
incidences of hepatocellular adenoma and carcinoma in exposed mice
of both sexes. In rats of both sexes, there were marginally
significant increased incidences of mononuclear cell leukemia (MCL)
when compared with concurrent controls. The incidences of MCL in
control animals, however, were higher than historical controls from
the conducting laboratory. The tumor finding was also judged to be
biologically significant because the time to onset of tumor was
decreased and the disease was more severe in treated than in control
animals. Low incidences of renal tubular cell adenomas or
adenocarcinomas were also observed in exposed male rats. The tumor
incidences were not statistically significant but there was a
significant trend.
Other Key Data
Substance 4 has been shown to be readily and rapidly absorbed by
inhalation and ingestion in humans and laboratory animals.
Absorption by dermal exposure is slow and limited. Once absorbed,
substance 4 is primarily distributed to and accumulated in adipose
tissue and the brain, kidney, and liver. A large percentage of
substance 4 is eliminated unchanged in exhaled air, with urinary
excretion of metabolites comprising a much smaller percentage. The
absorption and distribution profiles of substance 4 are similar
across species including humans.
Two major metabolites (trichloroacetic acid (TCA), and
trichloroethanol), which are formed by a P-450-dependent mixed-
function oxidase enzyme system, have been identified in all studied
species, including humans. There is suggestive evidence for the
formation of an epoxide intermediate based on the detection of two
other metabolites (oxalic acid and trichloroacetyl amide). In
addition to oxidative metabolism, substance 4 also undergoes
conjugation with glutathione. Further metabolism by renal beta-
lyases could lead to two minor active metabolites (trichlorovinyl
thiol and dichlorothiokente).
Toxicokinetic studies have shown that the enzymes responsible
for the metabolism of substance 4 can be saturated at high
exposures. The glutathione pathway was found to be a minor pathway
at low doses, but more prevalent following saturation of the
cytochrome P-450 pathway. Comparative in vitro studies indicate that
mice have the greater capacity to metabolize to TCA than rats and
humans. Inhalation studies also indicate saturation of oxidative
metabolism of substance 4, which occurs at higher dose levels in
mice than in rats and humans. Based on these findings, it has been
postulated that the species differences in the carcinogenicity of
substance 4 between rats and mice may be related to the differences
in the metabolism to TCA and glutathione conjugates.
Substance 4 is a member of the class of chlorinated organics
that often cause liver and kidney toxicity and carcinogenesis in
rodents. Like many chlorinated organics, substance 4 itself does not
appear to be mutagenic. Substance 4 was generally negative in in
vitro bacterial systems and in vivo mammalian systems. However, a
minor metabolite formed in the kidney by the glutathione conjugation
pathway has been found to be a strong mutagen.
The mechanisms of induced carcinogenic effects of substance 4 in
rats and mice are not completely understood. It has been
[[Page 17989]]
postulated that mouse liver carcinogenesis is related to liver
peroxisomal proliferation and toxicity of the metabolite TCA.
Information on whether or not TCA induces peroxisomal proliferation
in humans is not definitive. The induced renal tumors in male rats
may be related either to kidney toxicity or the activity of a
mutagenic metabolite. The mechanisms of increases in MCL in rats are
not known.
Evaluation
Available epidemiologic studies, taken together, provide
suggestive evidence of a possible causal association between
exposure to substance 4 and cancer incidence in the laundry and dry
cleaning industries. This is based on consistent findings of
elevated cancer risks in several studies of different populations of
dry cleaning and laundry workers. However, each individual study is
compromised by a number of study deficiencies including small
numbers of cancers, confounding exposure to other solvents, and poor
exposure characterization. Others may interpret these findings
collectively as inconclusive.
There is considerable evidence that substance 4 is carcinogenic
to laboratory animals. It induces tumors in mice of both sexes by
oral and inhalation exposure and in rats of both sexes via
inhalation. However, due to incomplete understanding of the mode of
mechanism of action, the predictivity of animal responses to humans
is uncertain.
Animal data of structurally related compounds showing common
target organs of toxicity and carcinogenic effects (but lack of
mutagenic effects) provide additional support for the
carcinogenicity of substance 4. Comparative toxicokinetic and
metabolism information indicates that the mouse may be more
susceptible to liver carcinogenesis than rats and humans. This may
indicate differences of the degree and extent of carcinogenic
responses, but does not detract from the qualitative weight of
evidence of human carcinogenicity. The toxicokinetic information
also indicates that oral and inhalation are the major routes of
human exposure.
Conclusion
Substance 4 is likely to be carcinogenic to humans by all routes
of exposure. The weight of evidence of human carcinogenicity is
based on: (a) Demonstrated evidence of carcinogenicity in two rodent
species of both sexes via two relevant routes of human exposure; (b)
the substance's similarity in structure to other chlorinated
organics that are known to cause liver and kidney toxicity and
carcinogenesis in rodents; (c) suggestive evidence of a possible
association between exposure to the substance in the laundry and dry
cleaning industries and increased cancer incidence; and (d) human
and animal data indicating that the substance is absorbed by all
routes of exposure.
In comparison with other agents designated as likely
carcinogens, the overall weight of evidence places it the lower end
of the grouping. This is because there is a lack of good evidence
that observed excess cancer risk in exposed workers is due solely to
substance 4. Moreover, there is considerable scientific uncertainty
about the human significance of certain rodent tumors associated
with substance 4 and related compounds. In this case, the human
relevance of the animal evidence of carcinogenicity relies on the
default assumption.
Overall, there is not enough evidence to give high confidence in
a conclusion about any single mode of action; it appears that more
than one is plausible in different rodent tissues. Nevertheless, the
lack of mutagenicity of substance 4 and its general growth-promoting
effect on high background tumors as well as its toxicity toward
mouse liver and rat kidney tissue support the view that the
predominant mode is growth-promoting rather than mutagenic. A
mutagenic contribution to carcinogenicity due to a metabolite cannot
be ruled out. The dose response assessment should, therefore, adopt
both default approaches, nonlinear and linear extrapolations. The
latter approach is very conservative since it likely overestimates
risk at low doses in this case, and is primarily useful for
screening analyses.
Example 5: ``Likely/Not Likely Human Carcinogen''--Range of Dose
Limited, Margin-of-Exposure Extrapolation
Human Data
Substance 5 is a metal-conjugated phosphonate. No human tumor or
toxicity data exist on this chemical.
Animal Data
Substance 5 caused a statistically significant increase in the
incidence of urinary bladder tumors in male, but not female, rats at
30,000 ppm (3%) in the diet in a long-term study. Some of these
animals had accompanying urinary tract stones and toxicity. No
bladder tumors or adverse urinary tract effects were seen in two
lower dose groups (2,000 and 8,000 ppm) in the same study. A chronic
dietary study in mice at doses comparable to those in the rat study
showed no tumor response or urinary tract effects. A 2-year study in
dogs at doses up to 40,000 ppm showed no adverse urinary tract
effects.
Other Key Data
Subchronic dosing of rats confirmed that there was profound
development of stones in the male bladder at doses comparable to
those causing cancer in the chronic study, but not at lower doses.
Sloughing of the epithelium of the urinary tract accompanied the
stones.
There was a lack of mutagenicity relevant to carcinogenicity. In
addition, there is nothing about the chemical structure of substance
5 to indicate DNA-reactivity or carcinogenicity.
Substance 5 is composed of a metal, ethanol, and a simple
phosphorus-oxygen-containing component. The metal is not absorbed
from the gut, whereas the other two components are absorbed. At high
doses, ethanol is metabolized to carbon dioxide, which makes the
urine more acidic; the phosphorus level in the blood is increased
and calcium in the urine is increased. Chronic testing of the
phosphorus-oxygen-containing component alone in rats did not show
any tumors or adverse effects on the urinary tract.
Because substance 5 is a metal complex, it is not likely to be
readily absorbed from the skin.
Evaluation
Substance 5 produced cancer of the bladder and urinary tract
toxicity in male, but not female rats and mice, and dogs failed to
show the toxicity noted in male rats. The mode of action developed
from the other key data to account for the toxicity and tumors in
the male rats is the production of bladder stones. At high but not
lower subchronic doses in the male rat, substance 5 leads to
elevated blood phosphorus levels; the body responds by releasing
excess calcium into the urine. The calcium and phosphorus combine in
the urine and precipitate into multiple stones in the bladder. The
stones are very irritating to the bladder; the bladder lining is
eroded, and cell proliferation occurs to compensate for the loss of
the lining. Cell layers pile up, and finally, tumors develop. Stone
formation does not involve the chemical per se but is secondary to
the effects of its constituents on the blood and, ultimately, the
urine. Bladder stones, regardless of their cause, commonly produce
bladder tumors in rodents, especially the male rat.
Conclusion
Substance 5, a metal aliphatic phosphonate, is likely to be
carcinogenic to humans only under high-exposure conditions following
oral and inhalation exposure that lead to bladder stone formation,
but is not likely to be carcinogenic under low-exposure conditions.
It is not likely to be a human carcinogen via the dermal route,
given that the compound is a metal conjugate that is readily ionized
and its dermal absorption is not anticipated. The weight of evidence
is based on (a) bladder tumors only in male rats; (b) the absence of
tumors at any other site in rats or mice; (c) the formation of
calcium-phosphorus-containing bladder stones in male rats at high,
but not low, exposures that erode bladder epithelium and result in
profound increases in cell proliferation and cancer; and (d) the
absence of structural alerts or mutagenic activity.
There is a strong mode of action basis for the requirements of
(a) high doses of substance 5, (b) which lead to excess calcium and
increased acidity in the urine, (c) which result in the
precipitation of stones and (d) the necessity of stones for toxic
effects and tumor hazard potential. Lower doses fail to perturb
urinary constituents, lead to stones, produce toxicity, or give rise
to tumors. Therefore, dose response assessment should assume
nonlinearity.
A major uncertainty is whether the profound effects of substance
5 may be unique to the rat. Even if substance 5 produced stones in
humans, there is only limited evidence that humans with bladder
stones develop cancer. Most often human bladder stones are either
passed in the urine or lead to symptoms resulting in their removal.
However, since one cannot totally dismiss the male rat findings,
some hazard
[[Page 17990]]
potential may exist in humans following intense exposures. Only
fundamental research could illuminate this uncertainty.
Example 6: ``Cannot Be Determined''--Suggestive Evidence
Human Data
Substance 6 is an unsaturated aldehyde. In a cohort study of
workers in a chemical plant exposed to a mixture of chemicals with
substance 6 as a minor component, an elevated risk of cancer than
was expected was reported. This study is considered inadequate
because of multiple exposures, small cohort, and poor exposure
characterization.
Animal Data
Substance 6 was tested for potential carcinogenicity in a
drinking water study in rats, an inhalation study in hamsters, and a
skin painting study in mice. No significant increases in tumors were
observed in male rats treated with substance 6 at three dose levels
in drinking water. However, a significant increase of adrenal
cortical adenomas was found in the only treated female dose group
administered a dose equivalent to the high dose of males. This study
used a small number of animals (20 per dose group).
No significant finding was detected in the inhalation study in
hamsters. This study is inadequate due to the use of too few
animals, short duration of exposure, and inappropriate dose
selection (use of a single exposure that was excessively toxic as
reflected by high mortality).
No increase in tumors was induced in the skin painting study in
mice. This study is of inadequate design for carcinogenicity
evaluation because of several deficiencies: small number of animals,
short duration of exposure, lack of reporting about the sex and age
of animals, and purity of test material.
Substance 6 is structurally related to lowmolecularweight
aldehydes that generally exhibit carcinogenic effects in the
respiratory tracts of laboratory animals via inhalation exposure.
Three skin painting studies in mice and two subcutaneous injection
studies of rats and mice were conducted to evaluate the carcinogenic
potential of a possible metabolite of substance 6 (identified in
vitro). Increased incidences of either benign or combined benign and
malignant skin tumors were found in the dermal studies. In the
injection studies of rats and mice, increased incidences of local
sarcomas or squamous cell carcinoma were found at the sites of
injection. All of these studies are limited by the small number of
test animals, the lack of characterization of test material, and the
use of single doses.
Other Key Data
Substance 6 is a flammable liquid at room temperature. Limited
information on its toxicokinetics indicates that it can be absorbed
by all routes of exposure. It is eliminated in the urine mainly as
glutathione conjugates. Substance 6 is metabolized in vitro by rat
liver and lung microsomal preparations to a dihydroxylated aldehyde.
No data were available on the genetic and related effects of
substance 6 in humans. It did not induce dominant lethal mutations
in mice. It induced sister chromatid exchanges in rodent cells in
vitro. The mutagenicity of substance 6 is equivocal in bacteria. It
did not induce DNA damage or mutations in fungi.
Evaluation
Available human data are judged inadequate for an evaluation of
any causal relationship between exposure to substance 6 and human
cancer.
The carcinogenic potential of substance 6 has not been
adequately studied in laboratory animals due to serious deficiencies
in study design, especially the inhalation and dermal studies. There
is some evidence of carcinogenicity in the drinking water study in
female rats. However, the significance and predictivity of that
study to human response are uncertain since the finding is limited
to occurrence of benign tumors, one sex, and at the high dose only.
Additional suggestion for animal carcinogenicity comes from
observation that a possible metabolite is carcinogenic at the site
of administration. This metabolite, however, has not been studied in
vivo. Overall, the animal evidence is judged to be suggestive for
human carcinogenicity.
Other key data, taken together, do not add significantly to the
overall weight of evidence of carcinogenicity. SAR analysis
indicates that substance 6 would be DNA-reactive. However,
mutagenicity data are inconclusive. Limited in vivo data do not
support a mutagenic effect. While there is some evidence of DNA
damage in rodent cells in vitro, there is either equivocal or no
evidence of mutagenicity in nonmammalian systems.
Conclusion
The human carcinogenicity potential of substance 6 cannot be
determined on the basis of available information. Both human and
animal data are judged inadequate for an evaluation. There is
evidence suggestive of potential carcinogenicity on the basis of
limited animal findings and SAR considerations. Data are not
sufficient to judge whether there is a mode of carcinogenic action.
Additional studies are needed for a full evaluation of the potential
carcinogenicity of substance 6. Hence, dose response assessment is
not appropriate.
Example 7: ``Not Likely Human Carcinogen''--Appropriately Studied
Chemical in Animals Without Tumor Effects
Human Data
Substance 7, a plant extract, has not been studied for its toxic
or carcinogenic potential in humans.
Animal Data
Substance 7 has been studied in four chronic studies in three
rodent species. In a feeding study in rats, males showed a
nonsignificant increase in benign tumors of the parathyroid gland in
the high-dose group, where the incidence in concurrent controls
greatly exceeded the historical control range. Females demonstrated
a significant increase in various subcutaneous tumors in the low-
dose group, but findings were not confirmed in the high-dose group,
and there was no dose response relationship. These effects were
considered as not adding to the evidence of carcinogenicity. No
tumor increases were noted in a second adequate feeding study in
male and female rats. In a mouse feeding study, no tumor increases
were noted in dosed animals. There was some question as to the
adequacy of the dosing; however it was noted that in the mouse 90-d
subchronic study, a dose of twice the high dose in the chronic study
led to significant decrements in body weight. In a hamster study
there were no significant increases in tumors at any site. No
structural analogues of substance 7 have been tested for cancer.
Other Key Data
There are no structural alerts that would suggest that substance
7 is a DNA-reactive compound. It is negative for gene mutations in
bacteria and yeast, but positive in cultured mouse cells. Tests for
structural chromosome aberrations in cultured mammalian cells and in
rats are negative; however, the animals were not tested at
sufficiently high doses. Substance 7 binds to proteins of the cell
division spindle; therefore, there is some likelihood for producing
numerical chromosome aberrations, an endpoint that is sometimes
noted in cancers. In sum, there is limited and conflicting
information concerning the mutagenic potential of the agent.
The compound is absorbed via oral and inhalation exposure but
only poorly via the skin.
Evaluation
The only indication of a carcinogenic effect comes from the
finding of benign tumors in male rats in a single study. There is no
confirmation of a carcinogenic potential from dosed females in that
study, in males and females in a second rat study, or from mouse and
hamster studies.
There is no structural indication that substance 7 is DNA-
reactive, there is inconsistent evidence of gene mutations, and
chromosome aberration testing is negative. The agent binds to cell
division spindle proteins and may have the capacity to induce
numerical chromosome anomalies. Further information on gene
mutations and in vivo structural and numerical chromosome
aberrations may be warranted.
Conclusion
Substance 7 is not likely to be carcinogenic to humans via all
relevant routes of exposure. This weight of evidence judgment is
largely based on the absence of significant tumor increases in
chronic rodent studies. Adequate cancer studies in rats, mice, and
hamsters fail to show any carcinogenic effect; a second rat study
showed an increase in benign tumors at a site in dosed males, but
not females.
2.7. Presentation of Results
The results of the hazard assessment are presented in the form of
an overall technical hazard characterization. Additionally, a weight of
evidence narrative is used when the conclusion as to carcinogenic
potential needs to be
[[Page 17991]]
presented separately from the overall characterization.
2.7.1. Technical Hazard Characterization
The hazard characterization has two functions. First, it presents
results of the hazard assessment and an explanation of how the weight
of evidence conclusion was reached. It explains the potential for human
hazard, anticipated attributes of its expression, and mode of action
considerations for dose response. Second, it contains the information
needed for eventual incorporation into a risk characterization
consistent with EPA guidance on risk characterization (U.S. EPA, 1995).
The characterization qualitatively describes the conditions under
which the agent's effects may be expressed in human beings. These
qualitative hazard conditions are ones that are observable in the
toxicity data without having done either quantitative dose response or
exposure assessment. The description includes how expression is
afffected by route of exposure and dose levels and durations of
exposure.
The discussion of limitations of dose as a qualitative aspect of
hazard addresses the question of whether reaching a certain dose range
appears to be a precondition for a hazard to be expressed; for example,
when carcinogenic effects are secondary to another toxic effect that
appears only when a certain dose level is reached. The assumption is
made that an agent that causes internal tumors by one route of exposure
will be carcinogenic by another route, if it is absorbed by the second
route to give an internal dose. Conversely, if there is a route of
exposure by which the agent is not absorbed (does not cross an
absorption barrier; e.g., the exchange boundaries of skin, lung, and
digestive tract through uptake processes) to any significant degree,
hazard is not anticipated by that route. An exception to the latter
statement would be when the site of contact is also the target tissue
of carcinogenicity. Duration of exposure may be a precondition for
hazard if, for example, the mode of action requires cytotoxicity or a
physiologic change, or is mitogenicity, for which exposure must be
sustained for a period of time before effects occur. The
characterization could note that one would not anticipate a hazard from
isolated, acute exposures. The above conditions are qualitative ones
regarding preconditions for effects, not issues of relative absorption
or potency at different dose levels. The latter are dealt with under
dose response assessment (section 3), and their implications can only
be assessed after human exposure data are applied in the
characterization of risk.
The characterization describes conclusions about mode of action
information and its support for recommending dose response approaches.
The hazard characterization routinely includes the following in
support of risk characterization:
a summary of results of the assessment,
identification of the kinds of data available to support
conclusions and explanation of how the data fit together, highlighting
the quality of the data in each line of evidence, e.g., tumor effects,
short-term studies, structure-activity relationships), and highlighting
the coherence of inferences from the different kinds of data,
strengths and limitations (uncertainties) of the data and
assessment, including identification of default assumptions invoked in
the face of missing or inadequate data,
identification of alternative interpretations of data that
are considered equally plausible,
identification of any subpopulations believed to be more
susceptible to the hazard than the general population,
conclusions about the agent's mode of action and
recommended dose response approaches,
significant issues regarding interpretation of data that
arose in the assessment. Typical ones may include:
--determining causality in human studies,
--dosing (MTD), background tumor rates, relevance of animal tumors to
humans,
--weighing studies with positive and null results, considering the
influence of other available kinds of evidence,
--drawing conclusions based on mode of action data versus using a
default assumption about the mode of action.
2.7.2. Weight of Evidence Narrative
The weight of evidence narrative summarizes the results of hazard
assessment employing the descriptors defined in section 2.6.1. The
narrative (about two pages in length) explains an agent's human
carcinogenic potential and the conditions of its expression. If data do
not allow a conclusion as to carcinogenicity, the narrative explains
the basis of this determination. An example narrative appears below.
More examples appear in Appendix A.
The items regularly included in a narrative are:
name of agent and Chemical Abstracts Services number, if
available,
conclusions (by route of exposure) about human
carcinogenicity, using a standard descriptor from section 2.6.1,
summary of human and animal tumor data on the agent or its
structural analogues, their relevance, and biological plausibility,
other key data (e.g., structure-activity data,
toxicokinetics and metabolism, short-term studies, other relevant
toxicity or clinical data),
discussion of possible mode(s) of action and appropriate
dose response approach(es),
conditions of expression of carcinogenicity, including
route, duration, and magnitude of exposure.
Example Narrative
Aromatic Compound
CAS# XXX
CANCER HAZARD SUMMARY
Aromatic compound (AR) is known to be carcinogenic to humans by
all routes of exposure.
The weight of evidence of human carcinogenicity is based on (a)
consistent evidence of elevated leukemia incidence in studies of
exposed workers and significant increases of genetic damage in bone
marrow cells and blood lymphocytes of exposed workers; (b)
significantly increased incidence of cancer in both sexes of several
strains of rats and mice; (c) genetic damage in bone marrow cells of
exposed rodents and effects on intracellular signals that control
cell growth.
AR is readily absorbed by all routes of exposure and rapidly
distributed throughout the body. The mode of action of AR is not
understood. A dose response assessment that assumes linearity of the
relationship is recommended as a default.
SUPPORTING INFORMATION
Data include numerous human epidemiologic and biomonitoring
studies, long-term bioassays, and other data on effects of AR on
genetic material and cell growth processes. The key epidemiologic
studies and animal studies are well conducted and reliable. The
other data are generally of good quality also.
Human Effects
Numerous epidemiologic and case studies have reported an
increased incidence or a causal relationship associating exposure to
AR and leukemia. Among the studies are five for which the design and
performance as well as follow-up are considered adequate to
demonstrate the causal relationship. Biomonitoring studies of
exposed workers have found dose-related increases in chromosomal
aberrations in bone marrow cells and blood lymphocytes.
Animal Effects
AR caused increased incidence of tumors in various tissues in
both sexes of several rat and mouse strains. AR also caused
chromosomal aberrations in rabbits, mice, and rats--as it does in
humans.
[[Page 17992]]
Other Key Data
AR itself is not DNA-reactive and is not mutagenic in an array
of test systems both in vitro and in vivo. Metabolism of AR yields
several metabolites that have been separately studied for effects on
carcinogenic processes. Some have mutagenic activity in test systems
and some have other effects on cell growth controls inside cells.
MODE OF ACTION
No rodent tumor precisely matches human leukemia in pathology.
The closest parallel is a mouse cancer of blood-forming tissue.
Studies of the effects of AR at the cell level in this model system
are ongoing. As yet, the mode of action of AR is unclear, but most
likely the carcinogenic activity is associated with one or a
combination of its metabolites. It is appropriate to apply a linear
approach to the dose response assessment pending a better
understanding because: (a) genetic damage is a typical effect of AR
exposure in mammals and (b) metabolites of AR produce mutagenic
effects in addition to their other effects on cell growth controls;
AR is a multitissue carcinogen in mammals suggesting that it is
affecting a common controlling mechanism of cell growth.
3. Dose Response Assessment
Dose response assessment first addresses the relationship of dose
2 to the degree of response observed in an experiment or human
study. When environmental exposures are outside of the range of
observation, extrapolations are necessary in order to estimate or
characterize the dose relationship (ILSI, 1995). In general, three
extrapolations may be made: from high to low doses, from animal to
human responses, and from one route of exposure to another.
---------------------------------------------------------------------------
\2\ For this discussion, ``exposure'' means contact of an agent
with the outer boundary of an organism. ``Applied dose'' means the
amount of an agent presented to an absorption barrier and available
for absorption. ``Internal dose'' means the amount crossing an
absorption barrier (e.g., the exchange boundaries of skin, lung, and
digestive tract) through uptake processes. ``Delivered dose'' for an
organ or cell means the amount available for interaction with that
organ or cell (U.S. EPA, 1992a).
---------------------------------------------------------------------------
The dose response assessment proceeds in two parts. The first is
assessment of the data in the range of empirical observation. This is
followed by extrapolations either by modeling, if there are sufficient
data to support a model, or by a default procedure based as much as
possible on information about the agent's mode of action. The following
discussion covers the assessment of observed data and extrapolation
procedures, followed by sections on analysis of response data and
analysis of dose data. The final section discusses dose response
characterization.
3.1. Dose Response Relationship
In the discussion that follows, reference to ``response'' data
includes measures of tumorigenicity as well as other responses related
to carcinogenicity. The other responses may include effects such as
changes in DNA, chromosomes, or other key macromolecules, effects on
growth signal transduction, induction of physiological or hormonal
changes, effects on cell proliferation, or other effects that play a
role in the process. Responses other than tumorigenicity may be
considered part of the observed range in order either to extend the
tumor dose response analysis or to inform it. The nontumor response or
responses also may be used in lieu of tumor data if they are considered
to be a more informative representation of the carcinogenic process for
an agent (see section 3.2).
3.1.1. Analysis in the Range of Observation
Biologically Based and Case-Specific Models. A biologically based
model is one whose parameters are calculated independently of curve-
fitting of tumor data. If data are sufficient to support a biologically
based model specific to the agent and the purpose of the assessment is
such as to justify investing resources supporting use, this is the
first choice for both the observed tumor and related response data and
for extrapolation below the range of observed data in either animal or
human studies. Examples are the two-stage models of initiation plus
clonal expansion and progression developed by Moolgavkar and Knudson
(1981) and Chen and Farland (1991). Such models require extensive data
to build the form of the model as well as to estimate how well it
conforms with the observed carcinogenicity data. Theoretical estimates
of process parameters, such as cell proliferation rates, are not used
to enable application of such a model (Portier, 1987).
Similarly preferred as a first choice are dose response models
based on general concepts of mode of action and data on the agent. For
a case-specific model, model parameters and data are obtained from
studies on the agent.
In most cases, a biologically based or case-specific model will not
be practicable, either because the necessary data do not exist or the
decisions that the assessment are to support do not justify or permit,
the time and resources required. In these cases, the analysis proceeds
using curve-fitting models followed by default procedures for
extrapolation, based, to the extent possible, on mode of action and
other biological information about the agent. These methods and
assumptions are described below.
Curve-Fitting and Point of Departure for Extrapolation. Curve-
fitting models are used that are appropriate to the kind of response
data in the observed range. Any of several models can be used; e.g.,
the models developed for benchmark dose estimation for noncancer
endpoints may be applied (Barnes et al., 1995).
For some data sets, particularly those with extreme curvature, the
impact of model selection can be significant. In these cases, the
choice is rationalized on biological grounds as possible. In other
cases, the nature of the data or the way it is reported will suggest
other types of models; for instance, when longitudinal data on tumor
development are available, time to tumor or survival models may be
necessary and appropriate to fit the data.
A point of departure for extrapolation is estimated. This is a
point that is either a data point or an estimated point that can be
considered to be in the range of observation, without any significant
extrapolation. The LED10--the lower 95% confidence limit on a dose
associated with 10% extra risk--is such a point and is the standard
point of departure, adopted as a matter of science policy to remain as
consistent and comparable from case to case as possible.3 It is
also a comparison point for noncancer endpoints (U.S. EPA, 1991f). The
central estimate of the ED10 also may be appropriate for use in
relative hazard and potency ranking.
---------------------------------------------------------------------------
\3\ It is appropriate to report the central estimate of the
ED10, the upper and lower 95% confidence limits, and a
graphical representation of model fit.
---------------------------------------------------------------------------
For some data sets, a choice of point of departure other than the
LED10 may be appropriate. For example, if the observed response is
below the LED10, then a lower point may be a better choice.
Moreover, some forms of data may not be amenable to curve-fitting
estimation, but to estimation of a ``low-'' or ``no-observable-adverse-
effect level'' (LOAEL, NOAEL) instead, e.g., certain continuous data.
The rationale supporting the use of the LED10 is that a 10%
response is at or just below the limit of sensitivity of discerning a
significant difference in most long-term rodent studies. The lower
confidence limit on dose is used to appropriately account for
experimental uncertainty (Barnes et al., 1995) and for consistency with
the ``benchmark dose'' approach for noncancer assessment; it does not
provide information about human
[[Page 17993]]
variability. In laboratory studies of cancer or noncancer endpoints,
the level of dose at which increased incidence of effects can be
detected, as compared to controls, is a function of the size of the
sample (e.g., number of animals), dose spacing, and other design
aspects. In noncancer assessment, the dose at which significant effects
are not observed is traditionally termed the NOAEL. This is not, in
fact, a level of zero effect. The NOAEL in most study protocols is
about the same as an LED5 or LED10--the lower 95% confidence
limit on a dose associated with a 5% or 10% increased effect (Faustman
et al., 1994; Haseman, 1983). Adopting parallel points of departure for
cancer and noncancer assessment is intended to make discussion and
comparison of the two kinds of assessment more comparable because of
their similar science and science policy bases and similar analytic
approaches.
Analysis of human studies in the observed range is designed case by
case, depending on the type of study and how dose and response are
measured in the study. In some cases the agent may have discernible
interactive effects with another agent (e.g., asbestos and smoking),
making possible estimation of contribution of the agent and others as
risk factors. Also, in some cases, estimation of population risk in
addition, or in lieu of, individual risk may be appropriate.
3.1.2. Analysis in the Range of Extrapolation
Extrapolation to lower doses is usually necessary, and in the
absence of a biologically based or case-specific model, is based on one
of the three default procedures described below. The Agency has adopted
these three procedures as a matter of science policy based on current
hypotheses of the likely shapes of dose response curves for differing
modes of action. The choice of the procedure to be used in an
individual case is a judgment based on the agent's modes of action.
Linear. A default assumption of linearity is appropriate when the
evidence supports a mode of action of gene mutation due to DNA
reactivity or supports another mode of action that is anticipated to be
linear. Other elements of empirical support may also support an
inference of linearity, e.g., the background of human exposure to an
agent might be such that added human exposure is on the linear part of
a dose response curve that is sublinear overall. The default assumption
of linearity is also appropriate as the ultimate science policy default
when evidence shows no DNA reactivity or other support for linearity,
but neither does it show sufficient evidence of a nonlinear mode of
action to support a nonlinear procedure.
For linear extrapolation, a straight line is drawn from the point
of departure to the origin--zero dose, zero response (Flamm and
Winbush, 1984; Gaylor and Kodell, 1980; Krewski et al., 1984). This
approach is generally conservative of public health, in the absence of
information about the extent of human variability in sensitivity to
effects. When a linear extrapolation procedure is used, the risk
characterization summary displays the degree of extrapolation that is
being made from empirical data and discusses its implications for the
interpretation of the resulting quantitative risk estimates.
Nonlinear. A default assumption of nonlinearity is appropriate when
there is no evidence for linearity and sufficient evidence to support
an assumption of nonlinearity. The mode of action may lead to a dose
response relationship that is nonlinear, with response falling much
more quickly than linearly with dose, or being most influenced by
individual differences in sensitivity. Alternatively, the mode of
action may theoretically have a threshold, e.g., the carcinogenicity
may be a secondary effect of toxicity or of an induced physiological
change (see example 5, section 2.6.3) that is itself a threshold
phenomenon.
As a matter of science policy under this analysis, nonlinear
probability functions are not fitted to the response data to
extrapolate quantitative low-dose risk estimates because different
models can lead to a very wide range of results, and there is currently
no basis, generally, to choose among them. Sufficient information to
choose leads to a biologically based or case-specific model. In cases
of nonlinearity, the risk is not extrapolated as a probability of an
effect at low doses. A margin of exposure analysis is used, as
described below, to evaluate concern for levels of exposure. The margin
of exposure is the LED10 or other point of departure divided by
the environmental exposure of interest. The EPA does not generally try
to distinguish between modes of action that might imply a ``true
threshold'' from others with a nonlinear dose response relationship.
Except in unusual cases where extensive information is available, it is
not possible to distinguish between these empirically.
The environmental exposures of interest, for which margins of
exposure are estimated, may be actual or projected future levels. The
risk manager decides whether a given margin of exposure is acceptable
under applicable management policy criteria. The risk assessment
provides supporting information to assist the decisionmaker.
The EPA often conducts margin of exposure analyses to accompany
estimates of reference doses or concentrations (RfD, RfC) for noncancer
endpoints.4 The procedure for a margin of exposure analysis for a
response related to carcinogenicity is operationally analogous, the
difference being that a threshold of cancer response is not necessarily
presumed. If, in a particular case, the evidence indicates a threshold,
as in the case of carcinogenicity being secondary to another toxicity
that has a threshold, the margin of exposure analysis for the toxicity
is the same as is done for a noncancer endpoint, and an RfD or RfC for
that toxicity also may be estimated and considered in cancer
assessment.
---------------------------------------------------------------------------
\4\ An RfD or RfC is an estimate with uncertainty spanning
perhaps an order of magnitude of daily exposure to the human
population (including sensitive subgroups) that is anticipated to be
without appreciable deleterious effects during a lifetime. It is
arrived at by dividing empirical data on effects by uncertainty
factors that consider inter- and intraspecies variability, extent of
data on all important chronic exposure toxicity endpoints, and
availability of chronic as opposed to subchronic data.
---------------------------------------------------------------------------
The analogy between margin of exposure analysis for noncancer and
cancer responses begins with the analogy of points of departure; for
both it is an effect level, either LED10 or other point (presented
as a human equivalent dose or concentration), as data support. For
cancer responses, when animal data are used, the point of departure is
a human equivalent dose or concentration arrived at by interspecies
dose adjustment or toxicokinetic analysis. It is likely that many of
the margin of exposure analyses for cancer will be for responses other
than tumor incidence. This is because the impetus for considering a
carcinogenic agent to have a nonlinear dose response will be a
conclusion that there is sufficient evidence to support that view, and
this evidence will often be information about a response that is a
precursor to tumors.
To support a risk manager's consideration of the margin of
exposure, information is provided in a risk assessment about current
understanding of the phenomena that may be occurring as dose (exposure)
decreases substantially below the observed data. The goal is to provide
as much information as possible about the risk reduction that
accompanies lowering of exposure. To this end, some important points to
address include:
[[Page 17994]]
The slope of the observed dose response relationship at
the point of departure and its uncertainties and implications for risk
reduction associated with exposure reduction (a shallow slope suggests
less reduction than a steep slope),
The nature of the response used for the dose response
assessment,
The nature and extent of human variability in sensitivity
to the phenomena involved,
Persistence of the agent in the body,
Human sensitivity to the phenomena as compared with
experimental animals.
As a default assumption for two of these points, a factor of no
less than 10-fold each may be employed to account for human variability
and for interspecies differences in sensitivity when humans may be more
sensitive than animals. When humans are found to be less sensitive than
animals, a default factor of no smaller than a 1/10 fraction may be
employed to account for this. If any information about human
variability or interspecies differences is available, it is used
instead of the default or to modify it as appropriate. In the case of
analysis based on human studies, obviously, interspecies differences
are not a factor. It should be noted that the dose response
relationship and inter- or intraspecies variability in sensitivity are
independent. That is, reduction of dose reduces risk; it does not
change variability. To support consideration of acceptability of a
margin of exposure by the risk manager, the assessment considers all of
the hazard and dose response factors together; hence, the factors for
inter- and intraspecies differences alone are not to be considered a
default number for an acceptable margin of exposure. (See Section
1.3.2.5.)
It is appropriate to provide a graphical representation of the data
and dose response modeling in the observed range, also showing exposure
levels of interest to the decisionmaker. (See figure 3-1.) In order to
provide a frame of reference, by way of comparison, a straight line
extrapolation may be displayed to show what risk levels would be
associated with decreasing dose, if the dose response were linear. If
this is done, the clear accompanying message is that, in this case of
nonlinearity, the response falls disproportionately with decreasing
dose.
BILLING CODE 6560-50-P
[[Page 17995]]
[GRAPHIC] [TIFF OMITTED] TN23AP96.006
BILLING CODE 6560-50-C
[[Page 17996]]
Linear and Nonlinear. Both linear and nonlinear procedures may be
used in particular cases. If a mode of action analysis finds
substantial support for differing modes of action for different tumor
sites, an appropriate procedure is used for each. Both procedures may
also be appropriate to discuss implications of complex dose response
relationships. For example, if it is apparent that an agent is both DNA
reactive and is highly active as a promotor at high doses, and there
are insufficient data for modeling, both linear and nonlinear default
procedures may be needed to decouple and consider the contribution of
both phenomena.
3.1.3. Use of Toxicity Equivalence Factors and Relative Potency
Estimates
A toxicity equivalence factor (TEF) procedure is one used to derive
quantitative dose response estimates for agents that are members of a
category or class of agents. TEFs are based on shared characteristics
that can be used to order the class members by carcinogenic potency
when cancer bioassay data are inadequate for this purpose (U.S. EPA,
1991c). The ordering is by reference to the characteristics and potency
of a well-studied member or members of the class. Other class members
are indexed to the reference agent(s) by one or more shared
characteristics to generate their TEFs. The TEFs are usually indexed at
increments of a factor of 10. Very good data may permit a smaller
increment to be used. Shared characteristics that may be used are, for
example, receptor-binding characteristics, results of assays of
biological activity related to carcinogenicity, or structure-activity
relationships.
TEFs are generated and used for the limited purpose of assessment
of agents or mixtures of agents in environmental media when better data
are not available. When better data become available for an agent, its
TEF should be replaced or revised. Criteria for constructing TEFs are
given in U.S. EPA (1991b). The criteria call for data that are adequate
to support summing doses of the agents in mixtures. To date, adequate
data to support use of TEF's has been found in only one class of
compounds (dioxins) (U.S. EPA, 1989a).
Relative potencies can be similarly derived and used for agents
with carcinogenicity or other supporting data. These are conceptually
similar to TEFs, but they are less firmly based in science and do not
have the same level of data to support them. They are used only when
there is no better alternative.
The uncertainties associated with both TEFs and relative potencies
are explained whenever they are used.
3.2. Response Data
Response data for analysis include tumor incidence data from human
or animal studies as well as data on other responses as they relate to
an agent's carcinogenicity, such as effects on growth control processes
or cell macromolecules or other toxic effects. Tumor incidence data are
ordinarily the basis of dose response assessment, but other response
data can augment such assessment or provide separate assessments of
carcinogenicity or other important effects.
Data on carcinogenic processes underlying tumor effects may be used
to support biologically based or case-specific models. Other options
for such data exist. If confidence is high in the linkage of a
precursor effect and the tumor effect, the assessment of tumor
incidence may be extended to lower dose levels by linking it to the
assessment of the precursor effect (Swenberg et al., 1987). Even if a
quantitative link is not appropriate, the assessment for a precursor
effect may provide a view of the likely shape of the dose response
curve for tumor incidence below the range of tumor observation (Cohen
and Ellwein, 1990; Choy, 1993). If responses other than tumor incidence
are regarded as better representations of the carcinogenicity of the
agent, they may be used in lieu of tumor responses. For example, if it
is concluded that the carcinogenic effect is secondary to another toxic
effect, the dose response for the other effect will likely be more
pertinent for risk assessment. As another example, if disruption of
hormone activity is the key mode of action of an agent, data on hormone
activity may be used in lieu of tumor incidence data.
If adequate positive human epidemiologic response data are
available, they provide an advantageous basis for analysis since
concerns about interspecies extrapolation do not arise. Adequacy of
human exposure data for quantification is an important consideration in
deciding whether epidemiologic data are the best basis for analysis in
a particular case. If adequate exposure data exist in a well-designed
and well-conducted epidemiologic study that detects no effects, it may
be possible to obtain an upper-bound estimate of the potential human
risk to provide a check on plausibility of available estimates based on
animal tumor or other responses, e.g., do confidence limits on one
overlap the point estimate of the other?
When animal studies are used, response data from a species that
responds most like humans should be used if information to this effect
exists. If this is unknown and an agent has been tested in several
experiments involving different animal species, strains, and sexes at
several doses and different routes of exposure, all of the data sets
are considered and compared, and a judgment is made as to the data to
be used to best represent the observed data and important biological
features such as mode of action. Appropriate options for presenting
results include:
Use of a single data set,
Combining data from different experiments (Stiteler et
al., 1993; Vater et al., 1993),
Showing a range of results from more than one data set,
Showing results from analysis of more than one
statistically significant tumor response based on differing modes of
action,
Representing total response in a single experiment by
combining animals with statistically significant tumors at more than
one site, or
A combination of these options.
The approach judged to best represent the data is presented with
the rationale for the judgment, including the biological and
statistical considerations involved. The following are some points to
consider:
Quality of study protocol and execution,
Proportion of malignant neoplasms,
Latency of onset of neoplasia,
Number of data points to define the relationship of dose
and response,
Background incidence in test animal,
Differences in range of response among species, sexes,
strains,
Most sensitive responding species, and
Availability of data on related precursor events to tumor
development.
Analyses of carcinogenic effects other than tumor incidence are
similarly presented and evaluated for their contribution to a best
judgment on how to represent the biological data for dose response
assessment.
3.3. Dose Data
Whether animal experiments or epidemiologic studies are the sources
of data, questions need to be addressed in arriving at an appropriate
measure of dose for the anticipated environmental exposure. Among these
are:
Whether the dose is expressed as an environmental
concentration, applied dose, or delivered dose to the target organ,
[[Page 17997]]
Whether the dose is expressed in terms of a parent
compound, one or more metabolites, or both,
The impact of dose patterns and timing where significant,
Conversion from animal to human doses, where animal data
are used, and
The conversion metric between routes of exposure where
necessary and appropriate.
In practice, there may be little or no information on the
concentration or identity of the active form at a target; being able to
compare the applied and delivered doses between routes and species is
the ideal, but is rarely attained. Even so, the objective is to use
available data to obtain as close to a measure of internal or delivered
dose as possible.
The following discussion assumes that the analyst will have data of
varying detail in different cases about toxicokinetics and metabolism.
Discussed below are approaches to basic data that are most frequently
available, as well as approaches and judgments for improving the
analysis based on additional data. The estimation of dose in human
studies is tailored to the form of dose data available.
3.3.1. Interspecies Adjustment of Dose
When adequate data are available, the doses used in animal studies
can be adjusted to equivalent human doses using toxicokinetic
information on the particular agent. The methods used should be
tailored to the nature of the data on a case-by-case basis. In rare
cases, it may also be possible to make adjustments based on
toxicodynamic considerations. In most cases, however, there are
insufficient data available to compare dose between species. In these
cases, the estimate of human equivalent dose is based on science policy
default assumptions. The defaults described below are modified or
replaced whenever better comparative data on toxicokinetic or metabolic
relationships are available. The availability and discussion of the
latter also may permit reduction or discussion of uncertainty in the
analysis.
For oral exposure, the default assumption is that delivered doses
are related to applied dose by a power of body weight. This assumption
rests on the similarities of mammalian anatomy, physiology, and
biochemistry generally observed across species. This assumption is more
appropriate at low applied dose concentrations where sources of
nonlinearity, such as saturation or induction of enzyme activity, are
less likely to occur. To derive an equivalent human oral dose from
animal data, the default procedure is to scale daily applied doses
experienced for a lifetime in proportion to body weight raised to the
0.75 power (W0.75). Equating exposure concentrations in parts per
million units for food or water is an alternative version of the same
default procedure because daily intakes of these are in proportion to
W0.75. The rationale for this factor rests on the empirical
observation that rates of physiological processes consistently tend to
maintain proportionality with W0.75. A more extensive discussion
of the rationale and data supporting the Agency's adoption of this
scaling factor is in U.S. EPA, 1992b. Information such as blood levels
or exposure biomarkers or other data that are available for
interspecies comparison are used to improve the analysis when possible.
The default procedure to derive an human equivalent concentration
of inhaled particles and gases is described in U.S. EPA (1994) and
Jarabek (1995a,b). The methodology estimates respiratory deposition of
inhaled particles and gases and provides methods for estimating
internal doses of gases with different absorption characteristics. The
method is able to incorporate additional toxicokinetics and metabolism
to improve the analysis if such data are available.
3.3.2. Toxicokinetic Analyses
Physiologically based mathematical models are potentially the most
comprehensive way to account for toxicokinetic processes affecting
dose. Models build on physiological compartmental modeling and attempt
to incorporate the dynamics of tissue perfusion and the kinetics of
enzymes involved in metabolism of an administered compound.
A comprehensive model requires the availability of empirical data
on the carcinogenic activity contributed by parent compound and
metabolite or metabolites and data by which to compare kinetics of
metabolism and elimination between species. A discussion of issues of
confidence accompanies presentation of model results (Monro, 1992).
This includes considerations of model validation and sensitivity
analysis that stress the predictive performance of the model. When a
delivered dose measure is used in animal to human extrapolation of dose
response data, the assessment should discuss the confidence in the
assumption that the toxicodynamics of the target tissue(s) will be the
same in both species. Toxicokinetic data can improve dose response
assessment by accounting for sources of change in proportionality of
applied to internal or delivered dose at various levels of applied
dose. Many of the sources of potential nonlinearity involve saturation
or induction of enzymatic processes at high doses. An analysis that
accounts for nonlinearity (for instance, due to enzyme saturation
kinetics) can assist in avoiding overestimation or underestimation of
low dose response otherwise resulting from extrapolation from a
sublinear or supralinear part of the experimental dose response curve
(Gillette, 1983). Toxicokinetic processes tend to become linear at low
doses, an expectation that is more robust than low-dose linearity of
response (Hattis, 1990). Accounting for toxicokinetic nonlinearities
allows better description of the shape of the curve at relatively high
levels of dose in the range of observation, but cannot determine
linearity or nonlinearity of response at low dose levels (Lutz, 1990a;
Swenberg et al., 1987).
Toxicokinetic modeling results may be presented as the preferred
method of estimating human equivalent dose or in parallel discussion
with default assumptions depending on relative confidence in the
modeling.
3.3.3. Route-to-Route Extrapolation
Judgments frequently need to be made about the carcinogenicity of
an agent through a route of exposure different than the one in the
underlying studies. For example, exposures of interest may be through
inhalation of an agent tested primarily through animal feeding studies
or through ingestion of an agent that showed positive results in human
occupational studies from inhalation exposure.
Route-to-route extrapolation has both qualitative and quantitative
aspects. For the qualitative aspect, the assessor weighs the degree to
which positive results through one route of exposure in human or animal
studies support a judgment that similar results would have been
observed in appropriate studies using the route of exposure of
interest. In general, confidence in making such a judgment is
strengthened when the tumor effects are observed at a site distant from
the portal of entry and when absorption through the route of exposure
of interest is similar to absorption via the tested routes. In the
absence of contrary data, the qualitative default assumption is that,
if the agent is absorbed by a route to give an internal dose, it may be
carcinogenic by that route. (See section 2.7.1.)
When a qualitative extrapolation can be supported, quantitative
extrapolation may still be problematic in the absence of adequate data.
The differences in biological processes among routes of
[[Page 17998]]
exposure (oral, inhalation, dermal) can be great because of, for
example, first-pass effects and differing results from different
exposure patterns. There is no generally applicable method for
accounting for these differences in uptake processes in quantitative
route-to-route extrapolation of dose response data in the absence of
good data on the agent of interest. Therefore, route-to-route
extrapolation of dose data relies on a case-by-case analysis of
available data. When good data on the agent itself are limited, an
extrapolation analysis can be based on expectations from physical and
chemical properties of the agent, properties and route-specific data on
structurally analogous compounds, or in vitro or in vivo uptake data on
the agent. Route-to-route uptake models may be applied if model
parameters are suitable for the compound of interest. Such models are
currently considered interim methods; further model development and
validation is awaiting the development of more extensive data (see
generally, Gerrity and Henry, 1990). For screening or hazard ranking,
route-to-route extrapolation may be based on assumed quantitative
comparability as a default, as long as it is reasonable to assume
absorption by compared routes. When route-to-route extrapolation is
used, the assessor's degree of confidence in both the qualitative and
quantitative extrapolation should be discussed in the assessment and
highlighted in the dose response characterization.
3.3.4. Dose Averaging
The cumulative dose received over a lifetime, expressed as lifetime
average daily dose, is generally considered an appropriate default
measure of exposure to a carcinogen (Monro, 1992). 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.
While this is a reasonable default assumption based on theoretical
considerations, departures from it are expected. Another approach is
needed in some cases, such as when dose-rate effects are noted (e.g.,
formaldehyde). Cumulative dose may be replaced, as appropriate and
justified by the data, with other dose measures. In such cases,
modifications to the default assumption are made to take account of
these effects; the rationale for the selected approach is explained.
In cases where a mode of action or other feature of the biology has
been identified that has special dose implications for sensitive
subpopulations (e.g., differential effects by sex or disproportionate
impacts of early-life exposure), these are explained and are recorded
to guide exposure assessment and risk characterization. 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.
These issues are explored and pointed out for attention in the exposure
assessment and risk characterization.
3.4. Discussion of Uncertainties
The exploration of significant uncertainties in data for dose and
response and in extrapolation procedures is part of the assessment. The
presentation distinguishes between model uncertainty and parameter
uncertainty. Model uncertainty is an uncertainty about a basic
biological question. For example, a default, linear dose response
extrapolation may have been made based on tumor and other key evidence
supporting the view that the model for an agent's mode of action is a
DNA-reactive process. Discussion of the confidence in the extrapolation
is appropriately done qualitatively or by showing results for
alternatives that are equally plausible. It is not useful, for example,
to conduct quantitative uncertainty analysis running multiple forms of
linear models. This would obviate the function of the policy default.
Parameter uncertainties deal with numbers representing statistical
or analytical measures of variance or error in data or estimates.
Uncertainties in parameters are described quantitatively, if
practicable, through sensitivity analysis and statistical uncertainty
analysis. With the recent expansion of readily available computing
capacity, computer methods are being adapted to create simulated
biological data that are comparable with observed information. These
simulations can be used for sensitivity analysis, for example, to
analyze how small, plausible variations in the observed data could
affect dose response estimates. These simulations can also provide
information about experimental uncertainty in dose response estimates,
including a distribution of estimates that are compatible with the
observed data. Because these simulations are based on the observed
data, they cannot assist in evaluating the extent to which the observed
data as a whole are idiosyncratic rather than typical of the true
situation. If quantitative analysis is not possible, significant
parameter uncertainties are described qualitatively. In either case,
the discussion highlights uncertainties that are specific to the agent
being assessed, as distinct from those that are generic to most
assessments.
Estimation of the applied dose in a human study has numerous
uncertainties such as the exposure fluctuations that humans experience
compared with the controlled exposures received by animals on test. In
a prospective cohort study, there is opportunity to monitor exposure
and human activity patterns for a period of time that supports
estimation of applied dose (U.S. EPA, 1992a). In a retrospective study,
exposure may be based on monitoring data but is often based on human
activity patterns and levels reconstructed from historical data,
contemporary data, or a combination of the two. Such reconstruction is
accompanied by analysis of uncertainties considered with sensitivity
analysis in the estimation of dose (Wyzga, 1988; U.S. EPA, 1986a).
These uncertainties can also be assessed for any confounding factor for
which a quantitative adjustment of dose response data is made (U.S.
EPA, 1984).
3.5. Technical Dose Response Characterization
As with hazard characterization, the dose response characterization
serves the dual purposes of presenting a technical characterization of
the assessment results and supporting the risk characterization.
The characterization presents the results of analyses of dose data,
of response data, and of dose response. When alternative approaches are
plausible and persuasive in selecting dose data, response data, or
extrapolation procedures, the characterization follows the alternative
paths of analysis and presents the results. The discussion covers the
question of whether any should be preferred over others because it (or
they) better represents the available data or corresponds to the view
of the mechanism of action developed in the hazard assessment. The
results for different tumor types by sex and species are provided along
with the one(s) preferred. Similarly, results for responses other than
tumor incidence are shown if appropriate.
Numerical dose response estimates are presented to one significant
figure. Numbers are qualified as to whether they represent central
tendency or upper bounds and whether the method used is inherently more
likely to overestimate or underestimate (Krewski et al., 1984).
In cases where a mode of action or other feature of the biology has
been identified that has special implications
[[Page 17999]]
for early-life exposure, differential effects by sex, or other concerns
for sensitive subpopulations, these are explained. Similarly, any
expectations that high dose-rate exposures may alter the risk picture
for some portion of the population are described. These and other
perspectives are recorded to guide exposure assessment and risk
characterization. Whether the lifetime average daily dose or another
measure of dose should be considered for differing exposure scenarios
is discussed.
Uncertainty analyses, qualitative or quantitative if possible, are
highlighted in the characterization.
The dose response characterization routinely includes the
following, as appropriate for the data available:
Identification of the kinds of data available for analysis
of dose and response and for dose response assessment,
Results of assessment as above,
Explanation of analyses in terms of quality of data
available,
Selection of study/response and dose metric for
assessment,
Discussion of implications of variability in human
susceptibility, including for susceptible subpopulation,
Applicability of results to varying exposure scenarios--
issues of route of exposure, dose rate, frequency, and duration,
Discussion of strengths and limitations (uncertainties) of
the data and analyses that are quantitative as well as qualitative, and
Special issues of interpretation of data, such as:
--Selecting dose data, response data, and dose response approach(es),
--Use of meta-analysis,
--Uncertainty and quantitative uncertainty analysis.
4. Technical Exposure Characterization
Guidelines for exposure assessment of carcinogenic and other agents
are published (U.S. EPA, 1992a) and are used in conjunction with these
cancer risk assessment guidelines. Presentation of exposure descriptors
is a subject of discussion in EPA risk characterization guidance (U.S.
EPA, 1995). The exposure characterization is a technical
characterization that presents the assessment results and supports risk
characterization.
The characterization provides a statement of purpose, scope, level
of detail, and approach used in the assessment, identifying the
exposure scenario(s) covered. It estimates the distribution of
exposures among members of the exposed population as the data permit.
It identifies and compares the contribution of different sources and
routes and pathways of exposure. Estimates of the magnitude, duration,
and frequency of exposure are included as available monitoring or
modeling results or other reasonable methods permit. The strengths and
limitations (uncertainties) of the data and methods of estimation are
made clear.
The exposure characterization routinely includes the following, as
appropriate and possible for the data available:
Identification of the kinds of data available,
Results of assessment as above,
Explanation of analyses in terms of quality of data
available,
Uncertainty analyses as discussed in Exposure Assessment
Guidelines, distinguishing uncertainty from variability, and
Explanation of derivation of estimators of ``high end'' or
central tendency of exposure and their appropriate use.
5. Risk Characterization
5.1. Purpose
The risk characterization process includes an integrative analysis
followed by a presentation in a Risk Characterization Summary, of the
major results of the risk assessment. The Risk Characterization Summary
is a nontechnical discussion that minimizes the use of technical terms.
It is an appraisal of the science that supports the risk manager in
making public health decisions, as do other decisionmaking analyses of
economic, social, or technology issues. It also serves the needs of
other interested readers. The summary is an information resource for
preparation of risk communication information, but being somewhat
technical, is not itself the usual vehicle for communication with every
audience.
The integrative analysis brings together the assessments and
characterizations of hazard, dose response, and exposure to make risk
estimates for the exposure scenarios of interest. This analysis is
generally much more extensive than the Risk Characterization Summary.
It may be peer-reviewed or subject to public comment along with the
summary in preparation for an Agency decision. The integrative analysis
may be titled differently by different EPA programs (e.g., ``Staff
Paper'' for criteria air pollutants), but it typically will identify
exposure scenarios of interest in a decisionmaking and present risk
analyses associated with them. Some of the analyses may concern
scenarios in several media, others may examine, for example, only
drinking water risks. It also may be the document that contains
quantitative analyses of uncertainty.
The values supported by a risk characterization throughout the
process are transparency in environmental decisionmaking, clarity in
communication, consistency in core assumptions and science policies
from case to case, and reasonableness. While it is appropriate to err
on the side of protection of health and the environment in the face of
scientific uncertainty, common sense and reasonable application of
assumptions and policies are essential to avoid unrealistic estimates
of risk (U.S. EPA, 1995). Both integrative analyses and the Risk
Characterization Summary present an integrated and balanced picture of
the analysis of the hazard, dose response, and exposure. The risk
analyst should provide summaries of the evidence and results and
describe the quality of available data and the degree of confidence to
be placed in the risk estimates. Important features include the
constraints of available data and the state of knowledge, significant
scientific issues, and significant science and science policy choices
that were made when alternative interpretations of data existed (U.S.
EPA, 1995). Choices made about using default assumptions or data in the
assessment are explicitly discussed in the course of analysis, and if a
choice is a significant issue, it is highlighted in the summary.
5.2. Application
Risk characterization is a necessary part of generating any Agency
report on risk, whether the report is preliminary to support allocation
of resources toward further study or comprehensive to support
regulatory decisions. In the former case, the detail and sophistication
of the characterization are appropriately small in scale; in the latter
case, appropriately extensive. Even if a document covers only parts of
a risk assessment (hazard and dose response analyses for instance), the
results of these are characterized.
Risk assessment is an iterative process that grows in depth and
scope in stages from screening for priority-making, to preliminary
estimation, to fuller examination in support of complex regulatory
decisionmaking. Default assumptions are used at every stage because no
database is ever complete, but they are predominant at screening stages
and are used less as more data are gathered and incorporated at later
stages. Various provisions in EPA-administered statutes require
decisions
[[Page 18000]]
based on findings that represent all stages of iteration. There are
close to 30 provisions within the major statutes that require decisions
based on risk, hazard, or exposure assessment. For example, Agency
review of premanufacture notices under section 5 of the Toxic
Substances Control Act relies on screening analyses, while requirements
for industry testing under section 4 of that Act rely on preliminary
analyses of risk or simply of exposure. At the other extreme, air
quality criteria under the Clean Air Act rest on a rich data collection
required by statute to undergo reassessment every few years. There are
provisions that require ranking of hazards of numerous pollutants--by
its nature a screening level of analysis--and other provisions that
require a full assessment of risk. Given this range in the scope and
depth of analyses, not all risk characterizations can or should be
equal in coverage or depth. The risk assessor must carefully decide
which issues in a particular assessment are important to present,
choosing those that are noteworthy in their impact on results. For
example, health effect assessments typically rely on animal data since
human data are rarely available. The objective of characterization of
the use of animal data is not to recount generic issues about
interpreting and using animal data. Agency guidance documents cover
these. Instead, the objective is to call out any significant issues
that arose within the particular assessment being characterized and
inform the reader about significant uncertainties that affect
conclusions.
5.3. Presentation of Risk Characterization Summary
The presentation is a nontechnical discussion of important
conclusions, issues, and uncertainties that uses the hazard, dose-
response, exposure, and integrative analyses for technical support. The
primary technical supports within the risk assessment are the hazard
characterization, dose response characterization, and exposure
characterization described in this guideline. The risk characterization
is derived from these. The presentation should fulfill the aims
outlined in the purpose section above.
5.4. Content of Risk Characterization Summary
Specific guidance on hazard, dose response, and exposure
characterization appears in previous sections. Overall, the risk
characterization routinely includes the following, capturing the
important items covered in hazard, dose response, and exposure
characterization.
Primary conclusions about hazard, dose response, and
exposure, including equally plausible alternatives,
Nature of key supporting information and analytic methods,
Risk estimates and their attendant uncertainties,
including key uses of default assumptions when data are missing or
uncertain,
Statement of the extent of extrapolation of risk estimates
from observed data to exposure levels of interest (i.e., margin of
exposure) and its implications for certainty or uncertainty in
qualtifying risk,
Significant strengths and limitations of the data and
analyses, including any major peer reviewers' issues,
Appropriate comparison with similar EPA risk analyses or
common risks with which people may be familiar, and
Comparison with assessment of the same problem by another
organization.
Appendix A
This appendix contains several general illustrations of weight
of evidence narratives. In addition, after narrative #5 is an
example of a briefing summary format.
NARRATIVE #1 Chlorinated Alkene
CAS# XXX
CANCER HAZARD SUMMARY
Chlorinated alkene (cl-alkene) is likely to be carcinogenic to
humans by all routes of exposure. The weight of evidence of human
carcinogenicity of cl-alkene is based on (a) findings of
carcinogenicity in rats and mice of both sexes by oral and
inhalation exposures; (b) its similarity in structure to other
chlorinated organics that are known to cause liver and kidney
damage, and liver and kidney tumors in rats and mice; (c) suggestive
evidence of a possible association between cl-alkene exposure of
workers in the laundry and dry cleaning industries and increased
cancer risk in a number of organ systems; and (d) human and animal
data indicating that cl-alkene is absorbed by all routes of
exposure.
In comparison with other agents designated as likely
carcinogens, the overall weight of evidence for cl-alkene places it
at the low end of the grouping. This is because one cannot attribute
observed excess cancer risk in exposed workers solely to cl-alkene.
Moreover, there is considerable scientific uncertainty about the
human significance and relevance of certain rodent tumors associated
with exposure to cl-alkene and other chlorinated organics, but
insufficient evidence about mode of action for the animal tumors.
Hence, the human relevance of the animal evidence of carcinogenicity
relies on a default assumption of relevance.
There is no clear evidence about the mode of action for each
tumor type induced in rats and mice. Available evidence suggests
that cl-alkene induces cancer mainly by promoting cell growth rather
than via direct mutagenic action, although a mutagenic mode of
action for rat kidney tumors cannot be ruled out. The dose response
assessment should, therefore, adopt both default approaches,
nonlinear and linear. It is recognized that the latter approach
likely overestimates risk at low doses if the mode of action is
primarily growth-promoting. This approach, however, may be useful
for screening analyses.
SUPPORTING INFORMATION
Human Data
A number of epidemiologic studies of dry cleaning and laundry
workers that have reported elevated incidences of lung, cervix,
esophagus, kidney, blood and lymphoid cancers. Many of these studies
are confounded by co-exposure to other petroleum solvents, making
them limited for determining whether the observed increased cancer
risks are causally related to cl-alkene. The only investigation of
dry cleaning workers with no known exposure to other chemicals did
not evaluate other confounding factors such as smoking, alcohol
consumption, and low socioeconomic status to exclude the possible
contribution of these factors to cancer risks.
Animal Data
The carcinogenic potential of cl-alkene has been adequately
investigated in two chronic studies in two rodent species, the first
study by gavage and the second study by inhalation. Cl-alkene is
carcinogenic in the liver in both sexes of mice when tested by
either route of exposure. It causes marginally increased incidences
of mononuclear cell leukemia (MCL) in both sexes of rats and low
incidences of a rare kidney tumor in male rats by inhalation. No
increases in tumor incidence were found in rats treated with cl-
alkene by gavage. This rat study was considered limited because of
high mortality of the animals.
Although cl-alkene causes increased incidences of tumors at
multiple sites in two rodent species, controversy surrounds each of
the tumor endpoints concerning their relevance and/or significance
to humans (see discussion under Mode of Action).
Other Key Data
Cl-alkene is a member of a class of chlorinated organics that
often cause liver and kidney toxicity and carcinogenesis in rodents.
Like many chlorinated hydrocarbons, cl-alkene itself is tested
negative in a battery of standard genotoxicity tests using bacterial
and mammalian cells systems including human lymphocytes and
fibroblast cells. There is evidence, however, that a minor
metabolite generated by an enzyme found in rat kidney tissue is
mutagenic. This kidney metabolite has been hypothesized to be
related to the development of kidney tumors in the male rat. This
metabolic pathway appears to be operative in the human kidney.
Human data indicate that cl-alkene is readily absorbed via
inhalation but to a much lesser extent by skin contact. Animal data
show that cl-alkene is absorbed well by the oral route.
[[Page 18001]]
MODE OF ACTION
The mechanisms of cl-alkene-induced mouse liver tumors are not
completely understood. One mechanism has been hypothesized to be
mediated by a genotoxic epoxide metabolite generated by enzymes
found in the mouse liver, but there is a lack of direct evidence in
support of this mechanism. A more plausible mechanism that still
needs to be further defined is related to liver peroxisomal
proliferation and toxicity by TCA (trichloroacetic acid), a major
metabolite of cl-alkene. However, there are no definitive data
indicating that TCA induces peroxisomal proliferation in humans.
The mechanisms by which cl-alkene induces kidney tumors in male
rats are even less well understood. The rat kidney response may be
related to either kidney toxicity or the activity of a mutagenic
metabolite of the parent compound.
The human relevance of cl-alkene-induced MCL in rats is unclear.
The biological significance of marginally increased incidences of
MCL has been questioned by some, since this tumor occurs
spontaneously in the tested rat strain at very high background
rates. On the other hand, it has been considered by others to be a
true finding because there was a decreased time to onset of the
disease and the disease was more severe in treated as compared with
untreated control animals. The exact mechanism by which cl-alkene
increases incidences of MCL in rats is not known.
Overall, there is not enough evidence to give high confidence in
a conclusion about any single mode of action; it would appear that
more than a single mode operates in different rodent tissues. The
apparent lack of mutagenicity of cl-alkene itself and its general
growth-promoting effect on high background tumors as well as its
toxicity toward mouse liver and rat kidney tissue support the view
that its predominant mode of action is cell growth promoting rather
than mutagenic. A mutagenic contribution to the renal
carcinogenicity due to a metabolite cannot be entirely ruled out.
NARRATIVE #2
Unsaturated Aldehyde
CAS# XXX
CANCER HAZARD SUMMARY
The potential human hazard of unsaturated aldehyde (UA) cannot
be determined, but there are suggestive data for carcinogenicity.
The evidence on carcinogenicity consists of (a) data from an
oral animal study showing a response only at the highest dose in
female rats, with no response in males and (b) the fact that other
low-molecular-weight aldehydes have shown tumorigenicity in the
respiratory tract after inhalation. The one study of UA effects by
the inhalation route was not adequately performed. The available
evidence is too limited to describe human carcinogenicity potential
or support dose response assessment.
SUPPORTING INFORMATION
Human Data
An elevated incidence of cancer was reported in a cohort of
workers in a chemical plant who were exposed to a mixture of
chemicals including UA as a minor component. The study is considered
inadequate because of the small size of the cohort studied and the
lack of adequate exposure data.
Animal Data
In a long-term drinking water study in rats, an increased
incidence of adrenal cortical adenomas was found in the highest-
dosed females. No other significant finding was made. The oral rat
study was well conducted by a standard protocol. In a 1-year study
in hamsters at one inhalation dose, no tumors were seen. This study
was inadequate due to high mortality and consequent short duration.
The chemical is very irritating and is a respiratory toxicant in
mammals. The animal data are too limited for conclusions to be
drawn.
Structural Analogue Data
UA's structural analogues, formaldehyde and acetaldehyde, both
have carcinogenic effects on the rat respiratory tract.
Other Key Data
The weight of results of mutagenicity tests in bacteria, fungi,
fruit flies, and mice result in an overall conclusion of not
mutagenic; UA is lethal to bacteria to a degree that makes testing
difficult and test results difficult to interpret. The chemical is
readily absorbed by all routes.
MODE OF ACTION
Data are not sufficient to judge whether there is a carcinogenic
mode of action.
NARRATIVE #3
Alkene Oxide
CAS# XXX
CANCER HAZARD SUMMARY
Alkene oxide (AO) should be dealt with as if it were a known
human carcinogen by all routes of exposure. Several studies in
workers, when considered together, suggest an elevated risk of
leukemia and lymphoma after long-term exposure to AO, even though no
single study conclusively demonstrates that AO caused the cancer. In
addition, animal cancer and mutagenicity studies as well as short-
term tests of mutagenicity have strongly consistent results that
support a level of concern equal to having conclusive human studies.
The weight of evidence of human carcinogenicity is based on (a)
consistent evidence of carcinogenicity of AO in rats and mice by
both oral and inhalation exposure; (b) studies in workers that taken
together suggest elevated risk of leukemia and lymphoma to workers
exposed to AO and show genetic damage in blood lymphocytes in
exposed workers; (c) mutagenic effects in numerous test systems and
heritable gene mutations in animals; and (d) membership in a class
of DNA-reactive compounds that are regularly observed to cause
cancer in animals.
Due to its ready absorption by all routes of exposure and rapid
distribution throughout the body, AO is expected to pose a risk by
any route of exposure. The strong evidence of a mutagenic mode of
action supports dose response assessment that assumes linearity of
the relationship.
SUPPORTING INFORMATION
Human Data
Elevated risks of lymphatic cancer and cancer of blood-forming
tissue have been reported in exposed workers in several studies. The
interpretation of the studies separately is complicated by exposures
to other agents in each so there is no single study that
demonstrates that AO caused the effects; nevertheless, several of
the studies together are considered suggestive of AO carcinogenicity
because they consistently show cancer elevation in the same tissues.
Biomonitoring studies of exposed workers find DNA damage in blood
lymphocytes and the degree of DNA damage correlates with the level
and duration of AO exposure. Finding this damage in the same tissue
in which elevated cancer was seen in workers adds further weight to
the positive suggestion from the worker cancer studies. The human
data are from well-conducted studies.
Animal Data
AO causes cancer in multiple tissue sites in rats and mice of
both sexes by oral and inhalation exposure. The database is more
extensive than usual and the studies are good. The observation of
multisite, multispecies carcinogenic activity by an agent is
considered to be very strong evidence and is often the case with
highly mutagenic agents. There are also good studies showing that AO
causes heritable germ cell mutations in mice after inhalation
exposure--a property that is very highly correlated with
carcinogenicity.
Structural Analogue Data
Organic epoxides are commonly found to have carcinogenic effects
in animals, particularly the low-molecular-weight ones.
Other Key Data
The structure and DNA reactivity of AO support potential
carcinogenicity. Both properties are highly correlated with
carcinogenicity. Positive mutagenicity tests in vitro and in vivo
add to this support and are reinforced by observation of similar
genetic damage in exposed workers.
AO is experimentally observed to be readily absorbed by all
routes and rapidly distributed through the body.
MODE OF ACTION
All of the available data are strongly supportive of a mutagenic
mode of action, with a particular human target in lymphatic and
blood-forming tissue. The current scientific consensus is that there
is virtually complete correspondence between ability of an agent to
cause heritable germ cell mutations, as AO does, and
carcinogenicity. All of this points to a mutagenic mode of action
and supports assuming linearity of the dose response relationship.
NARRATIVE #4
Bis-benzenamine
CAS# XXX
CANCER HAZARD SUMMARY
This chemical is likely to be carcinogenic to humans by all
routes of exposure. Its
[[Page 18002]]
carcinogenic potential is indicated by (a) tumor and toxicity
studies on structural analogues, which demonstrate the ability of
the chemical to produce thyroid follicular cell tumors in rats and
hepatocellular tumors in mice following ingestion and (b) metabolism
and hormonal information on the chemical and its analogues, which
contributes to a working mode of action and associates findings in
animals with those in exposed humans. In comparison with other
agents designated as likely carcinogens, the overall weight of
evidence for this chemical places it at the lower end of the
grouping. This is because there is a lack of tumor response data on
this agent itself.
Biological information on the compound is contradictory in terms
of how to quantitate potential cancer risks. The information on
disruption on thyroid-pituitary status argues for using a margin of
exposure evaluation. However, the chemical is an aromatic amine, a
class of agents that are DNA-reactive and induce gene mutation and
chromosome aberrations, which argues for low-dose linearity.
Additionally, there is a lack of mode of action information on the
mouse liver tumors produced by the structural analogues, also
pointing toward a low-dose linear default approach. In recognition
of these uncertainties, it is recommended to quantitate tumors using
both nonlinear (to place a lower bound on the risks) and linear (to
place an upper bound on the risks) default approaches. Given the
absence of tumor response data on the chemical per se, it is
recommended that tumor data on close analogues be used to possibly
develop toxicity equivalent factors or relative potencies.
Overall, this chemical is an inferential case for potential
human carcinogenicity. The uncertainties associated with this
assessment include (1) the lack of carcinogenicity studies on the
chemical, (2) the use of tumor data on structural analogues, (3) the
lack of definitive information on the relevance of thyroid-pituitary
imbalance for human carcinogenicity, and (4) the different potential
mechanisms that may influence tumor development and potential risks.
SUPPORTING INFORMATION
Human Data
Worker exposure has not been well characterized or quantified,
but recent medical monitoring of workers exposed over a period of
several years has uncovered alterations in thyroid-pituitary
hormones (a decrease in T3 and T4 and an increase in TSH) and
symptoms of hypothyroidism. A urinary metabolite of the chemical has
been monitored in workers, with changes in thyroid and pituitary
hormones noted, and the changes were similar to those seen in an
animal study.
Animal Data
The concentration of the urinary metabolite in rats receiving
the chemical for 28 days was within twofold of that in exposed
workers, a finding associated with comparable changes in thyroid
hormones and TSH levels. In addition, the dose of the chemical given
to rats in this study was essentially the same as that of an
analogue that had produced thyroid and pituitary tumors in rats. The
human thyroid responds in the same way as the rodent thyroid
following short-term, limited exposure. Although it is not well
established that thyroid-pituitary imbalance leads to cancer in
humans as it does in rodents, information in animals and in exposed
humans suggests similar mechanisms of disrupting thyroid-pituitary
function and the potential role of altered TSH levels in leading to
thyroid carcinogenesis.
Structural Analogue Data
This chemical is an aromatic amine, a member of a class of
chemicals that has regularly produced carcinogenic effects in
rodents and gene and structural chromosome aberrations in short-term
tests. Some aromatic amines have produced cancer in humans.
Close structural analogues produce thyroid follicular cell
tumors in rats and hepatocellular tumors in mice following
ingestion. The thyroid tumors are associated with known
perturbations in thyroid-pituitary functioning. These compounds
inhibit the use of iodide by the thyroid gland, apparently due to
inhibition of the enzyme that synthesizes the thyroid hormones (T3,
T4). Accordingly, blood levels of thyroid hormones decrease, which
induce the pituitary gland to produce more TSH, a hormone that
stimulates the thyroid to produce more of its hormones. The thyroid
gland becomes larger due to increases in the size of individual
cells and their proliferation and upon chronic administration,
tumors develop. Thus, thyroid tumor development is significantly
influenced by disruption in the thyroid-pituitary axis.
Other Key Data
The chemical can be absorbed by the oral, inhalation, and dermal
routes of exposure.
MODE OF ACTION
Data on the chemical and on structural analogues indicate the
potential association of carcinogenesis with perturbation of
thyroid-pituitary homeostasis. Structural analogues are genotoxic,
thus raising the possibility of different mechanisms by which this
chemical may influence tumor development.
NARRATIVE #5
Brominated Alkane (BA)
CAS# XXX
CANCER HAZARD SUMMARY
Brominated alkane (BA) is likely to be a human carcinogen by all
routes of exposure. The weight of evidence for human carcinogenicity
is at the high end of agents in the ``likely'' group. Findings are
based on very extensive and significant experimental findings that
include (a) tumors at multiple sites in both sexes of two rodent
species via three routes of administration relevant to human
exposure, (b) close structural analogues that produce a spectrum of
tumors like BA, (c) significant evidence for the production of
reactive BA metabolites that readily bind to DNA and produce gene
mutations in many systems including cultured mammalian and human
cells, and (d) two null and one positive epidemiologic study; in the
positive study, there may have been exposure to BA. These findings
support a decision that BA might produce cancer in exposed humans.
In comparison to other agents considered likely human carcinogens,
the overall weight of evidence for BA puts it near the top of the
grouping. Given the agent's mutagenicity, which can influence the
carcinogenic process, a linear dose-response extrapolation is
recommended.
Uncertainties include the lack of adequate information on the
mutagenicity of BA in mammals or humans in vivo, although such
effects would be expected.
SUPPORTING INFORMATION
Human Data
The information on the carcinogenicity of BA from human studies
is inadequate. Two studies of production workers have not shown
significant increases in cancer from exposure to BA and other
chemicals. An increase in lymphatic cancer was reported in a
mortality study of grain elevator workers who may have been exposed
to BA (and other chemicals).
Animal Data
BA produced tumors in four chronic rodents studies. Tumor
increases were noted in males and females of rats and mice following
oral dermal and inhalation exposure (rat--oral and two inhalation,
mouse--oral and dermal). It produces tumors both at the site of
application (e.g., skin with dermal exposure) and at sites distal to
the portal of entry into the body (e.g., mammary gland) following
exposure from each route. Tumors at the same site were noted in both
sexes of a species (blood vessel), both species (forestomach) and
via different routes of administration (lung). Some tumors developed
after very short latency, metastasized extensively, and produced
death, an uncommon findings in rodents. The rodent studies were well
designed and conducted except for the oral studies, in which the
doses employed caused excessive toxicity and mortality. However,
given the other rodent findings, lower doses would also be
anticipated to be carcinogenic.
Structural Analogue Data
Several chemicals structurally related to BA are also
carcinogenic in rodents. Among four that are closest in structure,
tumors like those seen for BA were often noted (e.g., forestomach,
mammary, lung), which helps to confirm the findings for BA itself.
In sum, all of the tumor findings help to establish animal
carcinogenicity and support potential human carcinogenicity for BA.
Other Key Data
BA itself is not reactive, but from its structure it was
expected to be metabolized to reactive forms. Extensive metabolism
studies have confined this presumption and have demonstrated
metabolites that bind to DNA and cause breaks in the DNA chain.
These lesions are readily converted to gene mutations in bacteria,
fungi, higher plants, insects and mammalian and human cells in
culture. There are only a limited number of reports on the induction
of chromosome aberrations in mammals and humans; thus far they are
negative.
[[Page 18003]]
MODE OF ACTION
Human carcinogens often produce cancer in multiple sites of
multiple animal species and both sexes and are mutagenic in multiple
test systems. BA satisfies these findings. It produces cancer in
males and females of rats and mice. It produces gene mutations in
cells across all life forms--plants, bacteria and animals--including
mammals and humans. Given the mutagenicity of BA exposure and the
multiplicity and short latency of BA tumor induction, it is
reasonable to use a linear approach for cancer dose-response
extrapolation.
BRIEFING SUMMARY
----------------------------------------------------------------------------------------------------------------
Designation or
Route(s) Class rationale Dose response
----------------------------------------------------------------------------------------------------------------
All.................................. Likely................. High end............... Default-linear.
----------------------------------------------------------------------------------------------------------------
Basis for classification/dose response
1. Human data: Two studies of production workers show no
increase in cancer (one had a small sample size; the other had mixed
chemical exposures). An increase in lymphatic cancer is seen among
grain elevator workers who may have been exposed to other chemicals.
2. Animal data: BA produces tumors at multiple sites in male and
female rats and mice following oral, dermal, and inhalation
exposure. Tumors are seen at the site of administration and distally
and are often consistent across sex, species, and route of
administration; some develop early, metastasize, and cause death.
3. Structural analogue data: Close analogues produce some of the
same tumors as are seen with BA.
4. Other key data: BA is metabolized to a reactive chemical that
binds DNA and produces gene mutations in essentially every test
system including cultured human cells.
5. Mode of action: Like most known human carcinogens, BA is
mutagenic in most test systems.
6. Hazard classification/uncertainties: There is a rich database
on BA demonstrating its potential ability to cause tumors in humans,
including (a) multiple animal tumors, (b) by appropriate routes of
exposure, (c) a mode of action relevant to human carcinogenicity,
and (d) some information in humans. Together they lead to a
designation near the high end of the likely human carcinogen class.
7. Dose response: Given the anticipated mode of action, a linear
default dose response relationship should be assumed.
Appendix B
This appendix contains responses to the National Academy of
Sciences National Research Council report Science and Judgment in Risk
Assessment (NRC, 1994).
Recommendations of the National Academy of Sciences National
Research Council
In 1994, the National Academy of Sciences published a report
Science and Judgment in Risk Assessment. The 1994 report was written
by a Committee on Risk Assessment of Hazardous Air Pollutants formed
under the Academy's Board on Environmental Studies and Toxicology,
Commission on Life Sciences, National Research Council. The report
was called for under Section 112(o)(1)(A,B) of the Clean Air Act
Amendments of 1990, which provided for the EPA to arrange for the
Academy to review:
risk assessment methodology used by the EPA to
determine the carcinogenic risk associated with exposure to
hazardous air pollutants from source categories and subcategories
subject to the requirements of this section and
improvements in such methodology.
Under Section 112(o)(2)(A,B), the Academy was to consider the
following in its review:
the techniques used for estimating and describing the
carcinogenic potency to humans of hazardous air pollutants and
the techniques used for estimating exposure to
hazardous air pollutants (for hypothetical and actual maximally
exposed individuals as well as other exposed individuals).
To the extent practicable, the Academy was also to review
methods of assessing adverse human health effects other than cancer
for which safe thresholds of exposure may not exist [Section
112(o)(3)]. The Congress further provided that the EPA Administrator
should consider, but need not adopt, the recommendations in the
report and the views of the EPA Science Advisory Board with respect
to the report. Prior to the promulgation of any standards under
Section 112(f), the Administrator is to publish revised guidelines
for carcinogenic risk assessment or a detailed explanation of the
reasons that any recommendations contained in the report will not be
implemented [Section 112(o)(6)].
The following discussion addresses the recommendations of the
1994 report that are pertinent to the EPA cancer risk assessment
guidelines. Guidelines for assessment of exposure, of mixtures, and
of other health effects are separate EPA publications. Many of the
recommendations were related to practices specific to the exposure
assessment of hazardous air pollutants, which are not covered in
cancer assessment guidelines. Recommendations about these other
guidelines or practices are not addressed here.
Hazard Classification
The 1994 report contains the following recommendation about
classifying cancer hazard:
The EPA should develop a two-part scheme for
classifying evidence on carcinogenicity that would incorporate both
a simple classification and a narrative evaluation. At a minimum,
both parts should include the strength (quality) of the evidence,
the relevance of the animal model and results to humans, and the
relevance of the experimental exposures (route, dose, timing, and
duration) to those likely to be encountered by humans.
The report also presented a possible matrix of 24 boxes that
would array weights of evidence against low, medium, or high
relevance, resulting in 24 codes for expressing the weight and
relevance.
These guidelines adopt a set of descriptors and subdescriptors
of weight of evidence in three categories: ``known/likely,''
``cannot be determined,'' and ``not likely,'' and a narrative for
presentation of the weight of evidence findings. The descriptors are
used within the narrative. There is no matrix of alphanumerical
weight of evidence boxes.
The issue of an animal model that is not relevant to humans has
been dealt with by not including an irrelevant response in the
weighing of evidence, rather than by creating a weight of evidence
then appending a discounting factor as the NRC scheme would do. The
issue is more complex than the NRC matrix makes apparent. Often the
question of relevance of the animal model applies to a single tumor
response, but one encounters situations in which there are more
tumor responses in animals than the questioned one. Dealing with
this complexity is more straightforward if it is done during the
weighing of evidence rather than after as in the NRC scheme.
Moreover, the same experimental data are involved in deciding on the
weight of evidence and the relevance of a response. It would be
awkward to go over the same data twice.
In recommending that the relevance of circumstances of human
exposure also be taken into account, the NRC appears to assume that
all of the actual conditions of human exposure will be known when
the classification is done. This is not the case. More often than
not, the hazard assessment is applied to assessment of risks
associated with exposure to different media or environments at
different times. In some cases, there is no priority to obtaining
exposure data until the hazard assessment has been done. The
approach of these guidelines is to characterize hazards as to
whether their expression is intrinsically limited by route of
exposure or by reaching a particular dose range based strictly on
toxicological and other biological features of the agent. Both the
use of descriptors and the narrative specifically capture this
information. Other aspects of appropriate application of the hazard
and dose response assessment to particular human exposure scenarios
are dealt with in the characterization of the dose response
assessment, e.g., the applicability of the dose response assessment
to scenarios with differing frequencies and durations.
The NRC scheme apparently intended that the evidence would be
weighed, then given a low, medium, or high code for some combination
of relevance of the animal response, route of exposure, timing,
duration, or frequency. The 24 codes contain none of this specific
information, and in fact, do not communicate what the conclusion is
about. To make the codes communicate the information apparently
intended would require some multiple of the 24 in the NRC scheme. As
the number of codes increases, their utility for communication
decreases.
Another reason for declining to use codes is that they tend to
become outdated as research reveals new information that was not
contemplated when they were adopted. This has been the case with the
classification system under the EPA, 1986 guidelines.
Even though these guidelines do not adopt a matrix of codes, the
method they provide
[[Page 18004]]
of using descriptors and narratives captures the information the NRC
recommended as the most important, and in the EPA's view, in a more
transparent manner.
Dose Response
The 1994 report contains the following recommendations
about dose response issues:
EPA should continue to explore, and when
scientifically appropriate, incorporate toxicokinetic models of the
link between exposure and biologically effective dose (i.e., dose
reaching the target tissue).
Despite the advantages of developing consistent risk
assessments between agencies by using common assumptions (e.g.,
replacing surface area with body weight to the 0.75 power), EPA
should indicate other methods, if any, that would be more accurate.
EPA should continue to use the linearized multistage
model as a default option but should develop criteria for
determining when information is sufficient to use an alternative
extrapolation model.
EPA should continue to use as one of its risk
characterization metrics upper-bound potency estimates of the
probability of developing cancer due to lifetime exposure. Whenever
possible, this metric should be supplemented with other descriptions
of cancer potency that might more adequately reflect the uncertainty
associated with the estimates.
EPA should adopt a default assumption for differences
in susceptibility among humans in estimating individual risks.
In the analysis of animal bioassay data on the
occurrence of multiple tumor types, the cancer potencies should be
estimated for each relevant tumor type that is related to exposure
and the individual potencies should be summed for those tumors.
The use of toxicokinetic models is encouraged in these
guidelines with discussion of appropriate considerations for their
use. When there are questions as to whether such a model is more
accurate in a particular case than the default method for estimating
the human equivalent dose, both alternatives may be used. It should
be noted that the default method for inhalation exposure is a
toxicokinetic model.
The rationale for adopting the oral scaling factor of body
weight to the 0.75 power has been discussed above in the explanation
of major defaults. The empirical basis is further explored in U.S.
EPA, 1992b. The more accurate approach is to use a toxicokinetic
model when data become available or to modify the default when data
are available as encouraged under these guidelines. As the U.S. EPA,
1992b discussion explores in depth, data on the differences among
animals in response to toxic agents are basically consistent with
using a power of 1.0, 0.75, or 0.66. The Federal agencies chose the
power of 0.75 for the scientific reasons given in the previous
discussion of major defaults; these were not addressed specifically
in the NRC report. It was also considered appropriate, as a matter
of policy, for the agencies to agree on one factor. Again, the
default for inhalation exposure is a model that is constructed to
become better as more agent-specific data become available.
The EPA proposes not to use a computer model such as the
linearized multistage model as a default for extrapolation below the
observed range. The reason is that the basis for default
extrapolation is a theoretical projection of the likely shape of the
curve considering mode of action. For this purpose, a computer model
looks more sophisticated than a straight line extrapolation, but is
not. The extrapolation will be by straight line as explained in the
explanation of major defaults. This was also recommended by workshop
reviewers of a previous draft of these guidelines (U.S. EPA, 1994b).
In addition, a margin of exposure analysis is proposed to be used in
cases in which the curve is thought to be nonlinear, based on mode
of action. In both cases, the observed range of data will be modeled
by curve fitting in the absence of supporting data for a
biologically based or case-specific model.
The result of using straight line extrapolation is thought to be
an upper bound on low-dose potency to the human population in most
cases, but as discussed in the major defaults section, it may not
always be. Exploration and discussion of uncertainty of parameters
in curve-fitting a model of the observed data or in using a
biologically based or case-specific model is called for in the dose
response assessment and characterization sections of these
guidelines.
The issue of a default assumption for human differences in
susceptibility has been addressed under the major defaults
discussion in section 1.3 with respect to margin of exposure
analysis. The EPA has considered but decided not to adopt a
quantitative default factor for human differences in susceptibility
when a linear extrapolation is used. In general, the EPA believes
that the linear extrapolation is sufficiently conservative to
protect public health. Linear approaches (both LMS and straight line
extrapolation) from animal data are consistent with linear
extrapolation on the same agents from human data (Goodman and
Wilson, 1991; Hoel and Portier, 1994). If actual data on human
variability in sensitivity are available they will, of course, be
used.
In analyzing animal bioassay data on the occurrence of multiple
tumor types, these guidelines outline a number of biological and
other factors to consider. The objective is to use these factors to
select response data (including nontumor data as appropriate) that
best represent the biology observed. As stated in section 3 of the
guidelines, appropriate options include use of a single data set,
combining data from different experiments, showing a range of
results from more than one data set, showing results from analysis
of more than one tumor response based on differing modes of action,
representing total response in a single experiment by combining
animals with tumors, or a combination of these options. The approach
judged to best represent the data is presented with the rationale
for the judgment, including the biological and statistical
considerations involved. The EPA has considered the approach of
summing tumor incidences and decided not to adopt it. While multiple
tumors may be independent, in the sense of not arising from
metastases of a single malignancy, it is not clear that they can be
assumed to represent different effects of the agent on cancer
processes. In this connection, it is not clear that summing
incidences provides a better representation of the underlying
mode(s) of action of the agent than combining animals with tumors or
using another of the several options noted above. Summing incidences
would result in a higher risk estimate, a step that appears
unnecessary without more reason.
Risk Characterization
When EPA reports estimates of risk to decisionmakers
and the public, it should present not only point estimates of risk,
but also the sources and magnitudes of uncertainty associated with
these estimates.
Risk managers should be given characterizations of risk
that are both qualitative and quantitative, i.e., both descriptive
and mathematical.
EPA should consider in its risk assessments the limits
of scientific knowledge, the remaining uncertainties, and the desire
to identify errors of either overestimation or underestimation.
In part as a response to these recommendations, the
Administrator of EPA issued guidelines for risk characterization and
required implementation plans from all programs in EPA (U.S. EPA,
1995). The Administrator's guidance is followed in these cancer
guidelines. The assessments of hazard, dose response, and exposure
will all have accompanying technical characterizations covering
issues of strengths and limitations of data and current scientific
understanding, identification of defaults utilized in the face of
gaps in the former, discussions of controversial issues, and
discussions of uncertainties in both their qualitative, and as
practicable, their quantitative aspects.
Appendix C
Overview of Cancer Processes
The following picture is changing as research reveals more about
carcinogenic processes. Nevertheless, it is apparent that several
general modes of action are being elucidated from direct reaction
with DNA to hormonal or other growth-signaling processes. While the
exact mechanism of action of an agent at the molecular level may not
be clear from existing data, the available data will often provide
support for deducing the general mode of action. Under these
guidelines, using all of the available data to arrive at a view of
the mode of action supports both characterization of human hazard
potential and assessment of dose response relationships.
Cancers are diseases of somatic mutation affecting cell growth
and differentiation. The genes that control cell growth, programmed
cell death, and cell differentiation are critical to normal
development of tissues from embryo to adult metazoan organisms.
These genes continue to be critical to maintenance of form and
function of tissues in the adult (e.g., Meyn, 1993) and changes in
them are essential elements of carcinogenesis (Hsu et al., 1991;
Kakizuka et al., 1991; Bottaro et al., 1991; Sidransky et al., 1991;
Salomon et al.,
[[Page 18005]]
1990; Srivastava et al., 1990). The genes involved are among the
most highly conserved in evolution as evidenced by the great
homology of many of them in DNA sequence and function in organisms
as phylogenetically distant as worms, insects, and mammals (Auger et
al., 1989a, b; Hollstein et al., 1991; Herschman, 1991; Strausfeld
et al., 1991; Forsburg and Nurse, 1991).
Mutations affecting three general categories of genes have been
implicated in carcinogenesis. Over 100 oncogenes have been found in
human and animal tumors that act as dominant alleles, whereas there
are about 10 known tumor suppressor genes that are recessive in
action. The normal alleles of these genes are involved with control
of cell division and differentiation; mutated alleles lead to a
disruption in these functions. The third class are mutator genes
that predispose the genome to enhanced mutagenic events that
contribute further to the carcinogenic process.
Adult tissues, even those that are composed of rapidly
replicating cells, maintain a constant size and cell number (Nunez
et al., 1991) by balancing three cell fates: (1) continued
replication, (2) differentiation to take on specialized functions,
or (3) programmed cell death (apoptosis) (Raff, 1992; Maller, 1991;
Naeve et al., 1991; Schneider et al., 1991; Harris, 1990).
Neoplastic growth through clonal expansion can result from somatic
mutations that inactivate control over cell fate (Kakizuka et al.,
1991; deThe et al., 1991; Sidransky et al., 1992; Nowell, 1976).
Cancers may also be thought of as diseases of the cell cycle.
For example, genetic diseases that cause failure of cells to repair
DNA damage prior to cell replication predispose people to cancer.
These changes are also frequently found in tumor cells in sporadic
cancers. These changes appear to be particularly involved at points
in cell replication called ``checkpoints'' where DNA synthesis or
mitosis is normally stopped until DNA damage is repaired or cell
death induced (Tobey, 1975). A cell that bypasses a checkpoint may
acquire a heritable growth advantage. Similar effects on the cell
cycle occur when mitogens such as hormones or growth factors
stimulate cell growth. Rapid replication in response to tissue
injury may also lead to unrepaired DNA damage that is a risk factor
for carcinogenesis.
Normally a cell's fate is determined by a timed sequence of
biochemical signals. Signal transduction in the cell involves
chemical signals that bind to receptors, generating further signals
in a pathway whose target in many cases is control of transcription
of a specific set of genes (Hunter, 1991; Cantley et al., 1991;
Collum and Alt, 1990). Cells are subject to growth signals from the
same and distant tissues, e.g., endocrine tissues (Schuller, 1991).
In addition to hormones produced by endocrine tissues, numerous
soluble polypeptide growth factors have been identified that control
normal growth and differentiation (Cross and Dexter, 1991; Wellstein
et al., 1990). The cells responsive to a particular growth factor
are those that express transmembrane receptors that specifically
bind the growth factor.
Solid tumors develop in stages operationally defined as
initiation, promotion, and progression (see, for example, Pitot and
Dragan, 1991). These terms, which were coined in the context of
specific experimental designs, are used for convenience in
discussing concepts, but they refer to complex events that are not
completely understood. During initiation, the cell acquires a
genetic change that confers a potential growth advantage. During
promotion, clonal expansion of this altered cell occurs. Later,
during progression, a series of genetic and other biological events
both enhance the growth advantage of the cells and enlist normal
host processes to support tumor development and cells develop the
ability to invade locally and metastasize distally, taking on the
characteristics of malignancy. Many endogenous and exogenous factors
are known to participate in the process as a whole. These include
specific genetic predispositions or variations in ability to
detoxify agents, medical history (Harris, 1989; Nebreda et al.,
1991), infections, exposure to chemicals or ionizing radiation,
hormones and growth factors, and immune suppression. Several such
risk factors likely work together to cause individual human cancers.
A cell that has been transformed, acquiring the potential to
establish a line of cells that grow to a tumor, will probably
realize that potential only rarely. The process of tumorigenesis in
animals and humans is a multistep one (Bouk, 1990; Fearon and
Vogelstein, 1990; Hunter, 1991; Kumar et al., 1990; Sukumar, 1989;
Sukumar, 1990) and normal physiological processes appear to be
arrayed against uncontrolled growth of a transformed cell (Weinberg,
1989). Powerful inhibition by signals from contact with neighboring
normal cells is one known barrier (Zhang et al., 1992). Another is
the immune system (at least for viral infection). How a cell with
tumorigenic potential acquires additional properties that are
necessary to enable it to overcome these and other inhibitory
processes is a subject of ongoing research. For known human
carcinogens studied thus far, there is an often decades-long latency
between exposure to carcinogenic agents and development of tumors
(Fidler and Radinsky, 1990; Tanaka et al., 1991; Thompson et al.,
1989). This latency is also typical of tumor development in
individuals with genetic diseases that make them prone to cancer
(Meyn, 1993; Srivastava et al., 1990).
The importance of genetic mutation in the carcinogenic process
calls for special attention to assessing agents that cause such
mutations. Heritable genetic defects that predispose humans to
cancer are well known and the number of identified defects is
growing. Examples include xeroderma pigmentosum (DNA repair defect)
and Li Fraumeni and retinoblastoma (both are tumor suppressor gene
mutations). Much of the screening and testing of agents for
carcinogenic potential has been driven by the idea of identifying
this mode of action. Cognizance of and emphasis on other modes of
action such as ones that act at the level of growth signalling
within or between cells, through cell receptors, or that indirectly
cause genetic change, comes from more recent research. There are not
yet standardized tests for many modes of action, but pertinent
information may be available in individual cases.
Agents of differing characteristics influence cancer
development: inorganic and organic, naturally occurring and
synthetic, of inanimate or animate origin, endogenous or exogenous,
dietary and nondietary. The means by which these agents act to
influence carcinogenesis are variable, and reasoned hazard
assessment requires consideration of the multiple ways that
chemicals influence cells in experimental systems and in humans.
Agents exert mutagenic effects either by interacting directly with
DNA or by indirect means through intermediary substances (e.g.,
reactive oxygen species) or processes. Most DNA-reactive chemicals
are electrophilic or can become electrophilic when metabolically
activated. Electrophilic molecules may bind covalently to DNA to
form adducts, and this may lead to depurination, depyrimidation, or
produce DNA strand breaks; such lesions can be converted to
mutations with a round of DNA synthesis and cell division. Other
DNA-interactive chemicals may cause the same result by intercalation
into the DNA helix. Still other chemicals may methylate DNA,
changing gene expression. Non-DNA-reactive chemicals produce
genotoxic effects by many different processes. They may affect
spindle formation or chromosome proteins, interfere with normal
growth control mechanisms, or affect enzymes involved with ensuring
the fidelity of DNA synthesis (e.g., topoisomerase), recombination,
or repair.
The ``classical'' chemical carcinogens in laboratory rodent
studies are agents that consistently produce gene mutations and
structural chromosome aberrations in short-term tests. A large
database reveals that these mutagenic substances commonly produce
tumors at multiple sites and in multiple species (Ashby and Tennant,
1991). Most of the carcinogens identified in human studies, aside
from hormones, are also gene or structural chromosome mutagens
(Tennant and Ashby, 1991). Most of these compounds or their
metabolites contain electrophilic moieties that react with DNA.
Numerical chromosome aberrations, gene amplification, and the
loss of gene heterozygosity are also found in animal and human tumor
cells and may arise from initiating events or during progression.
There is reason to believe that accumulation of additional genetic
changes is favored by selection in the evolution of tumor cells
because they confer additional growth advantages (Hartwell and
Kastan, 1994). Exogenous agents may function at any stage of
carcinogenesis (Barrett, 1993). Some aberrations may arise as a
consequence of genomic instability arising from tumor suppressor
gene mutation, e.g., p53 (Harris and Hollstein, 1993). The frequent
observation in tumor cells that both of a pair of homologous
chromosomes have identical mutation spectra in tumor suppressor
genes suggests an ongoing, endogenous process of gene conversion.
Currently, there is a paucity of routine test methods to screen for
events such as gene conversion or gene amplification and knowledge
regarding the
[[Page 18006]]
ability of particular agents of environmental interest to induce
them is, for the most part, wanting. Work is under way to
characterize, measure, and evaluate their significance (Travis et
al., 1991).
Several kinds of mechanistic studies aid in risk assessment.
Comparison of DNA lesions in tumor cells taken from humans with the
lesions that a tumorigenic agent causes in experimental systems can
permit inferences about the association of exposure to the agent and
an observed human effect (Vahakangas et al., 1992; Hollstein et al.,
1991; Hayward et al., 1991). An agent that is observed to cause
mutations experimentally may be inferred to have potential for
carcinogenic activity (U.S. EPA, 1991a). If such an agent is shown
to be carcinogenic in animals, the inference that its mode of action
is through mutagenicity is strong. A carcinogenic agent that is not
mutagenic in experimental systems but is mitogenic or affects
hormonal levels or causes toxic injury followed by compensatory
growth may be inferred to have effects on growth signal transduction
or to have secondary carcinogenic effects. The strength of these
inferences depends in each case on the nature and extent of all the
available data.
Differing modes of action at the molecular level have different
dose response implications for the activity of agents. The
carcinogenic activity of a direct-acting mutagen should be a
function of the probability of its reaching and reacting with DNA.
The carcinogenic activity of an agent that interferes at the level
of signal pathways with many potential receptor targets should be a
function of multiple reactions. The carcinogenic activity of an
agent that acts by causing cell toxicity followed by compensatory
growth should be a function of the toxicity.
References
Alison, R.H.; Capen, C.C.; Prentice, D.E. (1994) Neoplastic lesions
of questionable significance to humans. Toxicol. Pathol. 22: 179-
186.
Allen, B.C.; Crump, K.S.; Shipp, A.M. (1988) Correlation between
carcinogenic potency of chemicals in animals and humans. Risk Anal.
8: 531-544.
Ames, B.N.; Gold, L.S. (1990) Too many rodent carcinogens:
mitogenesis increases mutagenesis. Science 249: 970-971.
Anderson, E.; Deisler, P.F.; McCallum, D.; St. Helaire, C.; Spitzer,
H.L.; Strauss, H.; Wilson, J.D.; Zimmerman, R. (1993) Key issues in
carcinogen risk assessment guidelines. Society for Risk Analysis.
Ashby, J.; Tennant, R.W. (1991) Definitive relationships among
chemical structure, carcinogenicity and mutagenicity for 301
chemicals tested by the U.S. NTP. Mutat. Res. 257: 229-306.
Ashby, J.; Tennant, R.W. (1994) Prediction of rodent carcinogenicity
for 44 chemicals: results. Mutagenesis 9: 7-15.
Ashby, J.; Doerrer, N.G.; Flamm, F.G.; Harris, J.E.; Hughes, D.H.;
Johannsen, F.R.; Lewis, S.C.; Krivanek, N.D.; McCarthy, J.F.;
Moolenaar, R.J.; Raabe, G.K.; Reynolds, R.C.; Smith, J.M.; Stevens,
J.T.; Teta, M.J.; Wilson, J.D. (1990) A scheme for classifying
carcinogens. Regul. Toxicol. Pharmacol. 12: 270-295.
Ashby, J.; Brady, A.; Elcombe, C.R.; Elliott, B.M.; Ishmael, J.;
Odum, J.; Tugwood, D.; Keltle, S.; Purchase, I.F.H. (1994)
Mechanistically based human hazard assessment of peroxisome
proliferator-induced hepatocarcinogenesis. Hum. Exper. Toxicol. 13:
1-117.
Auger, K.R.; Carpenter, C.L.; Cantley, L.C.; Varticovski, L. (1989a)
Phosphatidylinositol 3-kinase and its novel product,
phosphatidylinositol 3-phosphate, are present in Saccharomyces
cerevisiae. J. Biol. Chem. 264: 20181-20184.
Auger, K.R.; Sarunian, L.A.; Soltoff, S.P.; Libby, P.; Cantley, L.C.
(1989b) PDGF-dependent tyrosine phosphorylation stimulates
production of novel polyphosphoinositides in intact cells. Cell 57:
167-175.
Barnes, D.G.; Daston, G.P; Evans, J.S.; Jarabek, A.M.; Kavlock,
R.J.; Kimmel, C.A.; Park, C.; Spitzer, H.L. (1995) Benchmark dose
workshop: criteria for use of a benchmark dose to estimate a
reference dose. Regul. Toxicol. Pharmacol. 21: 296-306.
Barrett, J.C. (1992) Mechanisms of action of known human
carcinogens. In: Mechanisms of carcinogenesis in risk
identification. IARC Sci. Pubs. No. 116, Lyon, France: International
Agency for Research on Cancer; 115-134.
Barrett, J.C. (1993) Mechanisms of multistep carcinogenesis and
carcinogen risk assessment. Environ. Health Perspect. 100: 9-20.
Barrett, J.C. (1995) Role of mutagenesis and mitogenesis in
carcinogenesis. Environ. Mutagenesis, in press.
Barrett, J. C.; Lee, T. C. (1992) Mechanisms of arsenic-induced gene
amplification. In: Gene amplification in mammalian cells: A
comprehensive guide (ed. R. E. Kellems), Marcel Dekker, New York:
441-446.
Bayly, A.C.; Roberts, R.A.; Dive, C. (1994) Suppression of liver
cell apoptosis in vitro by the nongenotoxic hepatocarcinogen and
peroxisome proliferator nafenopin. J. Cell. Biol. 125: 197-203.
Bellamy, C.O.C.; Malcomson, R.D.G.; Harrison, D.J.; Wyllie, A.H.
(1995) Cell death in health and disease: The biology and regulation
of apoptosis. Seminars in cancer biology, Apoptosis in oncogenesis
and chemotherapy 6: 3-16.
Bianchi, A.B.; Navone, N.M.; Alda, C.M.; Conti, C.J. (1991)
Overlapping loss of heterozygosity by mitotic recombination on more
chromosome 7F1-ter in skin carcinogenesis. Proc. Nat. Acad. Sci. 88:
7590-7594.
Biggs, P.J.; Warren, W.; Venitt, S.; Stratton, M.R. (1993) Does a
genotoxic carcinogen contribute to human breast cancer? The value of
mutational spectra in unraveling the etiology of cancer. Mutagenesis
8: 275-283.
Birner et al. (1990) Biomonitoring of aromatic amines. III:
Hemoglobin binding and benzidine and some benzidine congeners. Arch.
Toxicol. 64(2): 97-102.
Blair, A.; Burg, J.; Foran, J.; Gibb, H.; Greenland, S.; Morris, R.;
Raabe, G.; Savitz, D.; Teta, J.; Wartenberg, D.; Wong, O.;
Zimmerman, R. (1995) Guidelines for application of meta-analysis in
environmental epidemiology. Regul. Toxicol. Pharmacol. 22: 189-197.
Bois, F.Y.; Krowech, G.; Zeise, L. (1995) Modeling human
interindividual variability in metabolism and risk: the example of
4-aminobiphenyl. 15: 205-213.
Bottaro, D.P.; Rubin, J.S.; Faletto, D.L.; Chan, A.M.L.; Kmieck,
T.E.; Vande Woude, G.F.; Aaronson, S.A. (1991) Identification of the
hepatocyte growth factor receptor as the c-met proto-oncogene
product. Science 251: 802-804.
Bouck, N. (1990) Tumor angiogenesis: the role of oncogenes and tumor
suppressor genes. Cancer Cells 2: 179-183.
Burek, J.D.; Patrick, D.H.; Gerson, R.J. (1988) Weight-of-biological
evidence approach for assessing carcinogenicity. In: Grice, H.C.;
Cimina, J.L., eds. Carcinogenicity. New York, NY: Springer Verlag;
pp. 83-95.
Bus, J.S.; Popp, J.A. (1987) Perspectives on the mechanism of action
of the splenic toxicity of aniline and strucurally related
compounds. Fd. Chem. Toxicol. 25: 619-626.
Callemen, C.J.; Ehrenberg, L.; Jansson, B.; Osterman-Golkar, S.;
Segerback, D.; Svensson, K; Wachtmeister, C.A. (1978) Monitoring and
risk assessment by means of alkyl groups in hemoglobin in persons
occupationally exposed to ethylene oxide. J. Environ. Pathol.
Toxicol. 2: 427-442.
Cantley, L.C.; Auger, K.R.; Carpenter, C.; Duckworth, B.; Graziani,
A.; Kapeller, R.; Soltoff, S. (1991) Oncogenes and signal
transduction. Cell 64: 281-302.
Caporaso, N.; Hayes, R.B.; Dosemeci, M.; Hoover, R.; Ayesh, R.;
Hetzel, M.; Idle, J. (1989) Lung cancer risk, occupational exposure,
and the debrisoquine metabolic phenotype. Cancer Res. 49: 3675-3679.
Cavenee, W.K.; Koufos, A.; Hansen, M. F. (1986) Recessive mutant
genes predisposing to human cancer. Mutation Research 168: 3-14.
Chang, C.C.; Jone, C.; Trosko, J. E.; Peterson, A. R.; Sevanian, A.
(1988) Effect of cholesterol epoxides on the inhibition of
intercellular communication and on mutation induction in Chinese
hamster V79 cells. Mutation Research 206: 471-478.
[[Page 18007]]
Chen, C.; Farland, W. (1991) Incorporating cell proliferation in
quantitative cancer risk assessment: approaches, issues, and
uncertainties. In: Butterworth, B.; Slaga, T.; Farland, W.; McClain,
M., eds. Chemical induced cell proliferation: Implications for risk
assessment. New York, NY: Wiley-Liss; pp. 481-499.
Choy, W.N. (1993) A review of the dose-response induction of DNA
adducts by aflatoxin B2 and its implications to quantitative
cancer-risk assessment. Mutat. Res. 296: 181-198.
Clayson, D.B. (1989) Can a mechanistic rationale be provided for
non-genotoxic carcinogens identified in rodent bioassays? Mutat.
Res. 221: 53-67.
Clayson, D.B.; Mehta, R.; Iverson, F. (1994) Oxidative DNA damage--
The effects of certain genotoxic and operationally non-genotoxic
carcinogens. Mutat. Res. 317: 25-42.
Cogliano, V.J. (1986) The U.S. EPA's methodology for adjusting the
reportable quantities of potential carcinogens. Proceedings of the
7th National Conference on Management of Uncontrollable Hazardous
Wastes (Superfund '86). Washington, DC: Hazardous Wastes Control
Institute, 182-185.
Cohen, S.W.; Ellwein, L.B. (1990) Cell proliferation in
carcinogenesis. Science 249: 1007-1011.
Cohen, S.M.; Ellwein, L.B. (1991) Genetic errors, cell proliferation
and carcinogenesis. Cancer Res. 51: 6493-6505.
Cohen, S.M.; Purtilo, D.T.; Ellwein, L.B. (1991) Pivotal role of
increased cell proliferation in human carcinogenesis. Mod. Pathol.
4: 371-375.
Collum, R.G.; Alt, F.W. (1990) Are myc proteins transcription
factors? Cancer Cells 2: 69-73.
Connolly, R.B.; Andersen, M.E. (1991) Biologically based
pharmacodynamic models: tools for toxicological research and risk
assessment. Ann. Rev. Pharmacol. Toxicol. 31: 503-523.
Cross, M.; Dexter, T. (1991) Growth factors in development,
transformation, and tumorigenesis. Cell 64: 271-280.
D'Souza, R.W.; Francis, W.R.; Bruce, R.D.; Andersen, M.E. (1987)
Physiologically based pharmacokinetic model for ethylene chloride
and its application in risk assessment. In: Pharmacokinetics in risk
assessment. Drinking Water and Health. Vol. 8. Washington, DC:
National Academy Press.
deThe, H.; Lavau, C.; Marchio, A.; Chomienne, C.; Degos, L.; Dejean,
A. (1991) The PML-RAR fusion mRNA generated by the t
(15;17) translocation in acute promyelocytic leukemia encodes a
functionally altered RAR. Cell 66: 675-684.
Enterline, P.E.; Henderson, V.L.; Marsh, G.M. (1987) Exposure to
arsenic. Amer. J. Epidemiol. 125: 929-938.
European Centre for Ecotoxicology and Toxicology of Chemicals
(ECETOC). (1991) Early indicators of non-genotoxic carcinogenesis.
ECETOC Monograph No. 16. Brussels: ECETOC. Printed in Mutat. Res.
248: 211-374.
Faustman, E.M.; Allen, B.C.; Kavlock, R.J.; Kimmel, C.A. (1994)
Dose-response assessment for developmental toxicity. Fund. Appl.
Toxicol. 23: 478-486.
Fearon, E.; Vogelstein, B. (1990) A genetic model for colorectal
tumorigenesis. Cell 61: 959-967.
Federated Association of Societies of Experimental Biology (FASEB)
(1994) Evaluation of evidence for the carcinogenicity of butylated
hydroxyanisole (BHA). Life Sciences Research Office, Bethesda, MD.
Letter from Hamilton Brown (FDA) to John Rice (FASEB), July 28,
1994. Letter from John Rice (FASEB) to Ed Arnold (FDA), August 4,
1994.
Fidler, I.J.; Radinsky, R. (1990) Genetic control of cancer
metastasis. J. Natl. Cancer Inst. 82: 166-168.
Fisher, R.A. (1950) Statistical methods for research workers.
Edinborough, Scotland: Oliver and Boyd.
Flamm, W.G.; Winbush, J.S. (1984) Role of mathematical models in
assessment of risk and in attempts to define management strategy.
Fund. Appl. Toxicol. 4: S395-S401.
Flynn, G.L. (1990) Physicochemical determinants of skin absorption.
In: Gerrity, T.R.; Henry, C.J., eds. Principles of route to route
extrapolation for risk assessment. New York, NY: Elsevier Science
Publishing Co.; pp. 93-127.
Forsburg, S.L.; Nurse, P. (1991) Identification of a G1-type cyclin
pug1+ in the fission yeast Schizosaccharomyces pombe. Nature 351:
245-248.
Garfinkel, L.; Silverberg, E. (1991) Lung cancer and smoking trends
in the United States over the past 25 years. Cancer 41: 137-145.
Gaylor, D.W.; Kodell, R.L. (1980) Linear interpolation algorithm for
low-dose risk assessment of toxic substances. J. Environ. Pathol.
Toxicol. 4: 305-312.
Gerrity, T.R.; Henry, C., eds. (1990) Principles of route to route
extrapolation for risk assessment. New York, NY: Elsevier Science
Publishing Co.
Gibson, D.P.; Aardema, M.J.; Kerckaert, G.A.; Carr, G.J.;
Brauninger, R.M.; LeBoeuf, R.A. (1995) Detection of aneuploidy-
inducing carcinogens in the Syrian hamster embryo (SHE) cell
transformation assay. Mutat. Res.; Genet. Toxicol. 343: 7-24.
Gillette, J.R. (1983) The use of pharmacokinetics in safety testing.
In: Homburger, ed. Safety evaluation and regulation of chemicals 2.
2nd Int. Conf., Cambridge, MA: Karger, Basel; pp. 125-133.
Goldsworthy, T.L.; Hanigan, M.H.; and Pitot, H.C. (1986) Models of
hepatocarcinogenesis in the rat--contrasts and comparisons. CRC
Crit. Rev. Toxicol. 17: 61-89.
Goodman, G.; Wilson, R. (1991) Predicting the carcinogenicity of
chemicals in humans from rodent bioassay data. Environ. Health
Perspect. 94: 195-218.
Goodman, J.I.; Counts, J.L. (1993) Hypomethylation of DNA: A
possible nongenotoxic mechanism underlying the role of cell
proliferation in carcinogenesis. Environ. Health Perspect. 101 Supp.
5: 169-172.
Goodman, J.I.; Ward, J.M.; Popp, J.A.; Klaunig, J.E.; Fox, T.R.
(1991) Mouse liver carcinogenesis: Mechanisms and relevance. Fund.
Appl. Toxicol. 17: 651-665.
Grasso, P.; Hinton, R.H. (1991) Evidence for and possible mechanisms
of non-genotoxic carcinogenesis in rodent liver. Mutat. Res. 248:
271-290.
Greenland, S. (1987) Quantitative methods in the review of
epidemiologic literature. Epidemiol. Rev. 9: 1-29.
Hammand, E.C. (1966) Smoking in relation to the death rates of one
million men and women. In: Haenxzel, W., ed. Epidemiological
approaches to the study of cancer and other chronic diseases.
National Cancer Institute Monograph No. 19. Washington, DC.
Harris, C.C. (1989) Interindividual variation among humans in
carcinogen metabolism, DNA adduct formation and DNA repair.
Carcinogenesis 10: 1563-1566.
Harris, C.C.; Hollstein, M. (1993) Clinical implications of the p53
tumor suppressor gene. N. Engl. J. Med. 329: 1318-1327.
Harris, H. (1990) The role of differentiation in the suppression of
malignancy. J. Cell Sci. 97: 5-10.
Hartwell, L.H.; Kastan, M.B. (1994) Cell cycle control and cancer.
Science 266: 1821-1828.
Haseman, J.K. (1983) Issues: a reexamination of false-positive rates
for carcinogenesis studies. Fund. Appl. Toxicol. 3: 334-339.
Haseman, J.K. (1984) Statistical issues in the design, analysis and
interpretation of animal carcinogenicity studies. Environ. Health
Perspect. 58: 385-392.
Haseman, J.K. (1985) Issues in carcinogenicity testing: dose
selection. Fund. Appl. Toxicol. 5: 66-78.
Haseman, J.K. (1990) Use of statistical decision rules for
evaluating laboratory animal carcinogenicity studies. Fund. Appl.
Toxicol. 14: 637-648.
Haseman, J.K. (1995) Data analysis: Statistical analysis and use of
historical control data. Regul. Toxicol. Pharmacol. 21: 52-59.
Haseman, J.K.; Huff, J.; Boorman, G.A. (1984) Use of historical
control data in carcinogenicity studies in rodents. Toxicol. Pathol.
12: 126-135.
Hattis, D. (1990) Pharmacokinetic principles for dose-rate
extrapolation of carcinogenic risk from genetically active agents.
Risk Anal. 10: 303-316.
Havu, N.; Mattsson, H.; Ekman, L.; Carlsson, E. (1990)
Enterochromaffin-like cell carcinoids in the rat gastric mucosa
following long-term administration of ranitidine. Digestion 45: 189-
195.
[[Page 18008]]
Hayward, N.K.; Walker, G.J.; Graham, W.; Cooksley, E. (1991)
Hepatocellular carcinoma mutation. Nature 352: 764.
Hayward, J.J.; Shane, B.S.; Tindall, K.R.; Cunningham, M.L. (1995)
Differential in vivo mutagenicity of the carcinogen-noncarcinogen
pair 2,4- and 2,6-diaminotoluene. Carcinogenesis. In press.
Herschman, H.R. (1991) Primary response genes induced by growth
factors or promoters. Ann. Rev. Biochem. 60: 281-319.
Hill, R.N.; Erdreich, L.S.; Paynter, O.E.; Roberts, P.A.; Rosenthal,
S.L.; Wilkinson, C.F. (1989) Thyroid follicular cell carcinogenesis.
Fund. Appl. Toxicol. 12: 629-697.
Hoel, D.G.; Portier, C.J. (1994) Nonlinearity of dose-response
functions for carcinogenicity. Environ. Health Perspect. 102 Suppl
1: 109-113.
Hoel, D.G.; Haseman, J.K.; Hogam, M.D.; Huff, J.; McConnell, E.E.
(1988) The impact of toxicity on carcinogenicity studies:
Implications for risk assessment. Carcinogenesis 9: 2045-2052.
Holliday, R. (1987) DNA methylation and epigenetic defects in
carcinogenesis. Mutation Research 181: 215-217.
Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C.C. (1991)
p53 mutations in human cancers. Science 253: 49-53.
Hsu, I.C.; Metcaff, R.A.; Sun, T.; Welsh, J.A.; Wang, N.J.; Harris,
C.C. (1991) Mutational hotspot in human hepatocellular carcinomas.
Nature 350: 427-428.
Huff, J.E. (1993) Chemicals and cancer in humans: first evidence in
experimental animals. Environ. Health Perspect. 100: 201-210.
Huff, J.E. (1994) Chemicals causally associated with cancers in
humans and laboratory animals. A perfect concordance. In:
Carcinogenesis. Waalkes, M.P.; Ward, J.M., eds., New York, NY: Raven
Press; pp. 25-37.
Hulka, B.S.; Margolin, B.H. (1992) Methodological issues in
epidemiologic studies using biological markers. Am. J. Epidemiol.
135: 122-129.
Hunter, T. (1991) Cooperation between oncogenes. Cell 64: 249-270.
Ingram, A.J.; Grasso, P. (1991) Evidence for and possible mechanisms
of non-genotoxic carcinogenesis in mouse skin. Mutat. Res. 248: 333-
340.
International Agency for Research on Cancer (IARC). (1990)
Ciclosporin. IARC monographs on the evaluation of carcinogenic risks
to humans. Vol. 50. Lyon, France: IARC; pp. 77-114.
IARC. (1994) IARC monographs on the evaluation of carcinogenic risks
to humans. Vol. 60, Some industrial chemicals. Lyon, France: IARC;
pp. 13-33.
International Life Science Institute (ILSI). (1995) The use of
biological data in cancer risk assessment. In: Olin, S.; Farland,
W.; Park, C.; Rhomberg, L.; Scheuplein, R.; Starr, T.; Wilson, J.,
eds. Low-dose extrapolation of cancer risks: Issues and
Perspectives. Washington, DC: ILSI Press; pp. 45-60.
Ito, N.; Shirai, T.; and Hasegawa, R. (1992) Medium-term bioassays
for carcinogens. in ``Mechanisms of Carcinogenesis in Risk
Identifications'' (eds., H. Vainio, PN. Magee, DB McGregor and AJ
McMichael), Lyon, International Agency for Research on Cancer, pp.
353-388.
Jack, D.; Poynter, D.; Spurling, N.W. (1983) Beta-adrenoreceptor
stimulants and mesoovarian leiomyomas in the rat. Toxicology 2: 315-
320.
Jarabek, A.M. (1995a) The application of dosimetry models to
identify key processes and parameters for default dose-response
assessment approaches. Toxicol. Lett. 79:171-184.
Jarabek, A.M. (1995b) Interspecies extrapolation based on
mechanistic determinants of chemical disposition. Human Eco. Risk
Assess. 1(5):641-662.
Jones, P.A. (1986) DNA methylation and cancer. Cancer Res. 46: 461-
466.
Kehrer, J.P. (1993) Free radicals as mediators of tissue injury and
disease. Crit. Rev. Toxicol. 23: 21-48.
Kelsey, J.L.; Thompson, W.D.; Evans, A.S. (1986) Methods in
observational epidemiology. New York, NY: Oxford University Press.
Mr. Kinzler, K.W.; Nilbert, M.C.; Su, L.-K.; Vogelstein, B.; Bryan,
T.M.; Levy, D.B.; Smith, K.J.; Preisinger, A.C.; Hedge, P.;
McKechnie, D.; Finniear, R.; Markham, A.; Groffen, J.; Boguski,
M.S.; Altschul, S.J.; Horii, A.; Ando, H.; Miyoshi, Y.; Miki, Y.;
Nishisho, I.; Nakamura, Y. (1991) Identification of FAP locus genes
from chromosome 5q21. Science 253: 661-665.
Kodell, R.L.; Park, C.N. (1995) Linear extrapolation in cancer risk
assessment. ILSI Risk Science Institute: Washington, D.C. In press.
Kraus, A.L.; Munro, I.C.; Orr, J.C.; Binder, R.L.; LeBoeuf, R.A.;
Williams, G.M. (1995) Benzoyl peroxide: An integrated human safety
assessment for carcinogenicity. Regul. Toxicol. Pharmacol. 21: 87-
107.
Krewski, D.; Brown, C.; Murdoch, D. (1984) Determining ``safe''
levels of exposure: Safety factors of mathematical models. Fund.
Appl. Toxicol. 4: S383-S394.
Krewski, D.; Murdoch, D.J.; Withey, J.R. (1987) The application of
pharmacokinetic data in carcinogenic risk assessment. In:
Pharmacokinetics in risk assessment. Drinking water and health. Vol.
8. Washington, DC: National Academy Press; pp. 441-468.
Kripke, M.L. (1988) Immunoregulation of carcinogenesis: Past,
present, and future. J. Natl. Cancer Inst. 80: 722-727.
Kumar, R.; Sukumar, S.; Barbacid, M. (1990) Activation of ras
oncogenes preceding the onset of neoplasia. Science 248: 1101-1104.
Larson, J.L.; Wolf, D.C.; Butterworth, B.E. (1994) Induced
cytotoxicity and cell proliferation in the hepatocarcinogenicity of
chloroform in female B6C3F1 mice: comparison of administration by
gavage in corn oil vs. ad libitum in drinking water. Fundam. Appl.
Toxicol. 22: 90-102.
Levine, A.M. (1993) AIDs-related malignancies: The emerging
epidemic. J. Natl. Cancer Inst. 85: 1382-1397.
Levine, P.H.; Hoover, R.N., eds. (1992) The emerging epidemic of
non-Hodgkin's lymphoma: Current knowledge regarding etiological
factors. Cancer Res. 52: 5426s-5574s.
Levine, A. J.; Momand, J.; Finlay, C. A. (1991) The p53 tumour
suppressor gene. Nature 351: 453-456.
Levine, A.J.; Perry, M.E.; Chang, A., et al. (1994) The 1993 Walter
Hubert Lecture: The role of the p53 tumor-suppressor gene in
tumorigenesis. Brit. J. Cancer 69: 409-416.
Li, J.L.; Okada, S.; Hamazaki, S.; Ebina, Y.; Midorikawa, O. (1987)
Subacute nephrotoxicity and induction of renal cell carcinoma in
mice treated with ferric nitrilotriacetate. Cancer Res. 47: 1867-
1869.
Lijinsky, W. (1993) Species differences in carcinogenesis. In Vivo
7: 65-72.
Lilienfeld, A.M.; Lilienfeld, D. (1979) Foundations of epidemiology,
2nd ed. New York, NY: Oxford University Press.
Loeb, L.A. (1991) Mutator phenotype may be required for multistage
carcinogenesis. Cancer Res. 51: 3075-3079.
Lutz, W.K. (1990a) Endogenous genotoxic agents and processes as a
basis of spontaneous carcinogenesis. Mutat. Res. 238: 287-295.
Lutz, W.K. (1990b) Dose-response relationship and low dose
extrapolation in chemical carcinogenesis. Carcinogenesis 11: 1243-
1247.
MacDonald, J.S.; Lankas, G.R.; Morrissey, R.E. (1994) Toxicokinetic
and mechanistic considerations in the interpretation of the rodent
bioassay. Toxicol. Pathol. 22: 124-140.
Maller, J.L. (1991) Mitotic control. Curr. Opin. Cell Biol. 3: 269-
275.
Maronpot, R.R.; Shimkin, M.B.; Witschi, H.P.; Smith, L.H.; and
Cline, J.M. (1986) Strain A mouse pulmonary tumor test results for
chemicals previously tested in National Cancer Institute
carcinogenicity test. J. Natl. Cancer Inst. 76: 1101-1112.
Marsman, D.S.; Popp, J.A. (1994) Biological potential of basophilic
hepatocellular foci and hepatic adenoma induced by the peroxisome
proliferator, Wy-14,643. Carcinogenesis 15: 111-117.
Mausner, J.S.; Kramer, S. (1985) Epidemiology, 2nd ed. Philadelphia:
W.B. Saunders Company.
McClain, R.M. (1994) Mechanistic considerations in the regulation
and classification of chemical carcinogens. In: Kotsonis, F.N.;
Mackey, M.; Hjelle, J., eds. Nutritional toxicology. New York, NY:
Raven Press; pp. 273-304.
McConnell, E.E.; Solleveld, H.A.; Swenberg, J.A.; Boorman, G.A.
(1986) Guidelines for combining neoplasms for evaluation of rodent
carcinogenesis studies. J. Natl. Cancer Inst. 76: 283-289.
McMichael, A.J. (1976) Standardized mortality ratios and the
``healthy worker effect'': scratching beneath the surface. J. Occup.
Med. 18: 165-168.
[[Page 18009]]
Melnick, R.L.; Huff, J.E.; Barrett, J.C.; Maronpot, R.R.; Lucier,
G.; Portier, C.J. (1993a) Cell proliferation and chemical
carcinogenesis: A symposium overview. Molecular Carcinogenesis 7:
135-138.
Melnick, R.L.; Huff, J.E.; Barrett, J.C.; Maronpot, R.R.; Lucier,
G.; Portier, C.J. (1993b) Cell proliferation and chemical
carcinogenesis. Molecular Carcinogeneis 7: 135-138.
Meyn, M.S. (1993) High spontaneous intrachromosomal recombination
rates in ataxia-telangiectasia. Science 260: 1327-1330.
Modrich, P. (1994) Mismatch repair, genetic stability, and cancer.
Science 266: 1959-1960.
Monro, A. (1992) What is an appropriate measure of exposure when
testing drugs for carcinogenicity in rodents? Toxicol. Appl.
Pharmacol. 112: 171-181.
Moolgavkar, S.H.; Knudson, A.G. (1981) Mutation and cancer: A model
for human carcinogenesis. J. Natl. Cancer Inst. 66: 1037-1052.
Morrison, V.; Ashby, J. (1994) A preliminary evaluation of the
performance of the mutaTM mouse (lacZ) and Big BlueTM
(lacI) transgenic mouse mutation assays. Mutagenesis 9: 367-375.
Naeve, G.S.; Sharma, A.; Lee, A.S. (1991) Temporal events regulating
the early phases of the mammalian cell cycle. Curr. Opin. Cell Biol.
3: 261-268.
National Research Council (NRC). (1983) Risk Assessment in the
federal government: Managing the process. Committee on the
Institutional Means for Assessment of Risks to Public Health,
Commission on Life Sciences, NRC. Washington, DC: National Academy
Press.
NRC. (1993a) Pesticides in the diets of infants and children.
Committee on Pesticides in the Diets of Infants and Children,
Commission on Life Sciences, NRC. Washington, DC: National Academy
Press.
NRC. (1993b) Issues in risk Assessment. Committee on Risk Assessment
Methodology, Commission on Life Sciences, NRC. Washington, DC:
National Academy Press.
NRC. (1994) Science and judgment in risk assessment. Committee on
Risk Assessment of Hazardous Air Pollutants, Commission on Life
Sciences, NRC. Washington, DC: National Academy Press.
National Toxicology Program (NTP). (1984) Report of the Ad Hoc Panel
on Chemical Carcinogenesis Testing and Evaluation of the National
Toxicology Program, Board of Scientific Counselors. Washington, DC:
U.S. Government Printing Office. 1984-421-132: 4726.
Nebreda, A.R.; Martin-Zanca, D.; Kaplan, D.R.; Parada, L.F.; Santos,
E. (1991) Induction by NGF of meiotic maturation of xenopus oocytes
expressing the trk proto-oncogene product. Science 252: 558-561.
Nowell, P. (1976) The clonal evolution of tumor cell populations.
Science 194: 23-28.
Nunez, G.; Hockenberry, D.; McDonnell, J.; Sorenson, C.M.;
Korsmeyer, S.J. (1991) Bcl-2 maintains B cell memory. Nature 353:
71-72.
Office of Science and Technology Policy (OSTP). (1985) Chemical
carcinogens: Review of the science and its associated principles.
Federal Register 50: 10372-10442.
Organization for Economic Cooperation and Development (OECD). (1981)
Guidelines for testing of chemicals. Carcinogenicity studies. No.
451. Paris, France.
Peltomaki, P.; Aaltonen, L.A.; Sisonen, P.; Pylkkanen, L.; Mecklin,
J.-P.; Jarvinen, H.; Green, J.S.; Jass, J.R.; Weber, J.L.; Leach,
F.S.; Petersen, G.M.; Hamilton, S.R.; de la Chapelle, A.;
Vogelstein, B. (1993) Genetic mapping of a locus predisposing human
colorectal cancer. Science 260: 810-812.
Peto, J. (1992) Meta-analysis of epidemiological studies of
carcinogenesis. In: Mechanisms of carcinogenesis in risk assessment.
IARC Sci. Pubs. No. 116, Lyon, France: IARC; pp. 571-577.
Peto, J.; Darby, S. (1994) Radon risk reassessed. Nature 368: 97-98.
Pitot, H.; Dragan, Y.P. (1991) Facts and theories concerning the
mechanisms of carcinogenesis. FASEB J. 5: 2280-2286.
Portier, C. (1987) Statistical properties of a two-stage model of
carcinogenesis. Environ. Health Perspect. 76: 125-131.
Prahalada, S.; Majka, J.A.; Soper, K.A.; Nett, T.M.; Bagdon, W.J.;
Peter, C.P.; Burek, J.D.; MacDonald, J.S.; van Zwieten, M.J. (1994)
Leydig cell hyper plasian and adenomas in mice treated with
finasteride, 5-reductase inhibitor: A possible mechanism.
Fund. Appl. Toxicol. 22: 211-219.
Raff, M.C. (1992) Social controls on cell survival and cell death.
Nature 356: 397-400.
Rall, D.P. (1991) Carcinogens and human health: Part 2. Science 251:
10-11.
Rothman, K.T. (1986) Modern epidemiology. Boston: Little, Brown and
Company.
Salomon, D.S.; Kim, N.; Saeki, T.; Ciardiello, F. (1990)
Transforming growth factor --an oncodevelopmental growth
factor. Cancer Cells 2: 389-397.
Schneider, C.; Gustincich, S.; DelSal, G. (1991) The complexity of
cell proliferation control in mammalian cells. Curr. Opin. Cell
Biol. 3: 276-281.
Schuller, H.M. (1991) Receptor-mediated mitogenic signals and lung
cancer. Cancer Cells 3: 496-503.
Schulte-Hermann, R.; Bursch, W.; Kraupp-Grasl, B.; Oberhammer, F.;
Wagner, A.; Jirtle, R. (1993) Cell proliferation and apoptosis in
normal liver and preneoplastic foci. Environ. Health Perspect. 101
(Supp. 5): 87-90.
Shelby, M.D.; Zeiger, E. (1990) Activity of human carcinogens in the
Salmonella and rodent bone-marrow cytogenetics tests. Mutat. Res.
234: 257-261.
Shelby, M.D. (1994) Human germ cell mutations. Environ. Molec.
Mutagen. 23 (Supp. 24): 30-34.
Sidransky, D.; Von Eschenbach, A.; Tsai, Y.C.; Jones, P.;
Summerhayes, I.; Marshall, F.; Paul, M.; Green, P.; Hamilton, P.F.;
Vogelstein, B. (1991) Identification of p53 gene mutations in
bladder cancers and urine samples. Science 252: 706-710.
Sidransky, D.; Mikkelsen, T.; Schwechheimer, K.; Rosenblum, M.L.;
Cavanee, W.; Vogelstein, B. (1992) Clonal expansion of p53 mutant
cells is associated with brain tumor progression. Nature 355: 846-
847.
Sisk, S.C.; Pluta, L.J.; Bond, J.A.; Recio, L. (1994) Molecular
analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice
following inhalation exposure to 1,3-butadiene. Carcinogenesis
15(3): 471-477.
Snedecor, G.W.; Cochran, W.G. (1978) Statistical methods, Sixth ed.
Ames, Iowa: Iowa State University Press; 593 pp.
Srivastava, S.; Zou, Z.; Pirollo, K.; Blattner, W.; Chang, E. (1990)
Germ-line transmission of a mutated p53 gene in a cancer-prone
family with Li-Fraumeni syndrome. Nature 348(6303): 747-749.
Stewart, B.W. (1994) Mechanisms of apoptosis: Integration of
genetic, biochemical, and cellular indicators. J. Natl. Cancer Inst.
86: 1286-1296.
Stiteler, W.H.; Knauf, L.A.; Hertzberg, R.C.; Schoeny, R.S. (1993) A
statistical test of compatibility of data sets to a common dose-
response model. Reg. Tox. Pharmacol. 18: 392-402.
Stitzel, K.A.; McConnell, R.F.; Dierckman, T.A. (1989) Effects of
nitrofurantoin on the primary and secondary reproductive organs of
female B6C3F1 mice. Toxicol. Pathol. 17: 774-781.
Strausfeld, U.; Labbe, J.C.; Fesquet, D.; Cavadore, J.C.; Dicard,
A.; Sadhu, K.; Russell, P.; Dor'ee, M. (1991) Identification of a
G1-type cyclin puc1+ in the fission yeast Schizosaccharomyces pombe.
Nature 351: 242-245.
Sukumar, S. (1989) ras oncogenes in chemical carcinogenesis. Curr.
Top. Microbiol. Immunol. 148: 93-114.
Sukumar, S. (1990) An experimental analysis of cancer: Role of ras
oncogenes in multistep carcinogenesis. Cancer Cells 2: 199-204.
Swenberg, J.A.; Richardson, F.C.; Boucheron, J.A.; Deal, F.H.;
Belinsky, S.A.; Charbonneau, M.; Short, B.G. (1987) High to low dose
extrapolation: Critical determinants involved in the dose-response
of carcinogenic substances. Environ. Health Perspect. 76: 57-63.
Swierenga, S.H.H.; Yamasaki, H. (1992) Performance of tests for cell
transformation and gap junction intercellular communication for
detecting nongenotoxic carcinogenic activity. In: Mechanisms of
carcinogenesis in risk identification. IARC Sci. Pubs. No. 116,
Lyon, France: International Agency for Research on Cancer; pp. 165-
193.
[[Page 18010]]
Tanaka, K.; Oshimura, M.; Kikiuchi, R.; Seki, M.; Hayashi, T;
Miyaki, M. (1991) Suppression of tumorigenicity in human colon
carcinoma cells by introduction of normal chromosome 5 or 18. Nature
349: 340-342.
Tarone, R.E. (1982) The use of historical control information in
testing for a trend in proportions. Biometrics 38: 215-220.
Taylor, J.H.; Watson, M.A.; Devereux, T.R.; Michels, R.Y.;
Saccomanno, G.; Anderson, M. (1994) Lancet 343: 86-87.
Tennant, R.W. (1993) Stratification of rodent carcinogenicity
bioassay results to reflect relative human hazard. Mutat. Res. 286:
111-118.
Tennant, R.W.; Ashby, J. (1991) Classification according to chemical
structure, mutagenicity to Salmonella and level of carcinogenicity
of a further 39 chemicals tested for carcinogenicity by the U.S.
National Toxicology Program. Mutat. Res. 257: 209-277.
Tennant, R.W.; Elwell, M.R.; Spalding, J.W.; Griesemer, R.A. (1991)
Evidence that toxic injury is not always associated with induction
of chemical carcinogenesis. Molec. Carcinogen. 4: 420-440.
Tennant, R.W.; French, J.E.; Spalding, J.W. (1995) Identifying
chemical carcinogens and assessing potential risk in short-term
bioassays using transgenic mouse models. Environ. Health Perspect.
103:942-950.
Thompson, T.C.; Southgate, J.; Kitchener, G.; Land, H. (1989)
Multistage carcinogenesis induced by ras and myc oncogenes in a
reconstituted organ. Cell 56: 917-3183.
Tinwell, H.; Ashby, J. (1991) Activity of the human carcinogen
MeCCNU in the mouse bone marrow mironucleus test. Environ. Molec.
Mutagen. 17: 152-154.
Tischler, A.S.; McClain, R.M.; Childers, H.; Downing, J. (1991)
Neurogenic signals regulate chromaffin cell proliferation and
mediate the mitogenic effect of reserpine in the adult rat adrenal
medulla. Lab. Invest. 65: 374-376.
Tobey, R.A. (1975) Different drugs arrest cells at a number of
distinct stages in G2. Nature 254: 245-247.
Todd, G.C. (1986) Induction of reversibility of thyroid
proliferative changes in rats given an antithyroid compound. Vet.
Pathol. 23: 110-117.
Tomatis, L.; Aitio, A.; Wilbourn, J.; Shuker, L. (1989) Human
carcinogens so far identified. Jpn. J. Cancer Res. 80: 795-807.
Travis, C.C.; McClain, T.W.; Birkner, P.D. (1991)
Diethylnitrosamine-induced hepatocarcinogenesis in rats: A
theoretical study. Toxicol. Appl. Pharmacol. 109: 289-309.
U.S. Environmental Protection Agency. (1983a) Good laboratory
practices standards--toxicology testing. Federal Register 48: 53922.
U.S. Environmental Protection Agency. (1983b) Hazard evaluations:
Humans and domestic animals. Subdivision F. Available from: NTIS,
Springfield, VA; PB 83-153916.
U.S. Environmental Protection Agency. (1983c) Health effects test
guidelines. Available from: NTIS, Springfield, VA; PB 83-232984.
U.S. Environmental Protection Agency. (1984) Estimation of the
public health risk from exposure to gasoline vapor via the gasoline
marketing system. Office of Health and Environmental Assessment,
Washington, DC.
U.S. Environmental Protection Agency. (1986a) Health assessment
document for beryllium. Office of Health and Environmental
Assessment, Washington, DC.
U.S. Environmental Protection Agency. (1986b) Guidelines for
carcinogen risk assessment. Federal Register 51(185):33992-34003.
U.S. Environmental Protection Agency. (1989a) Interim procedures for
estimating risks associated with exposures to mixtures of
chlorinated dibenzo-p-dioxins and -dibenzofurans (CDDs and CDFs) and
1989 update. Risk Assessment Forum, Washington, DC. EPA/625/3-89/
016.
U.S. Environmental Protection Agency. (1989b) Workshop on EPA
guidelines for carcinogen risk assessment. Risk Assessment Forum,
Washington, DC. EPA/625/3-89/015.
U.S. Environmental Protection Agency. (1989c) Workshop on EPA
guidelines for carcinogen risk assessment: use of human evidence.
Risk Assessment Forum, Washington, DC. EPA/625/3-90/017.
U.S. Environmental Protection Agency. (1991a) Pesticide assessment
guidelines: Subdivision F, hazard evaluation, human and domestic
animals. Series 84, Mutagenicity, Addendum 9. Office of Pesticide
Programs, Washington, DC. PB91-158394, 540/09-91-122.
U.S. Environmental Protection Agency. (1991b) Alpha-2u-globulin:
Association with chemically induced renal toxicity and neoplasia in
the male rat. Risk Assessment Forum, Washington, DC. EPA/625/3-91/
019F.
U.S. Environmental Protection Agency. (1991c) Workshop report on
toxicity equivalency factors for polychlorinated biphenyl congeners.
Risk Assessment Forum, Washington, DC. EPA/625/3-91/020.
U.S. Environmental Protection Agency. (1991f) Guidelines for
developmental toxicity risk assessment. Federal Register 56(234):
63798-63826.
U.S. Environmental Protection Agency. (1992a) Guidelines for
exposure assessment. Federal Register 57(104): 22888-22938.
U.S. Environmental Protection Agency. (1992b) Draft report: A cross-
species scaling factor for carcinogen risk assessment based on
equivalence of mg/kg\3/4\/day. Federal Register 57(109): 24152-
24173.
U.S. Environmental Protection Agency. (1992c) Health assessment for
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds
(Chapters 1 through 8). Workshop Review Drafts. EPA/600/AP-92/001a
through 001h.
U.S. Environmental Protection Agency. (1994) Methods for derivation
of inhalation reference concentrations and application of inhalation
dosimetry. Office of Health and Environmental Assessment,
Environmental Criteria and Assessment Office, Research Triangle
Park, NC. EPA/600/8-90/066F.
U.S. Environmental Protection Agency. (1994a) Estimating exposure to
dioxin-like compounds. Office of Health and Environmental
Assessment, Office of Research and Development, Washington, DC.
External Review Draft, 3 vol. EPA/600/6-88/005Ca, Cb, Cc.
U.S. Environmental Protection Agency. (1994b) Report on the workshop
on cancer risk assessment guidelines issues. Office of Research and
Development, Risk Assessment Forum, Washington, DC. EPA/630/R-94/
005a.
U.S. Environmental Protection Agency. (1995) Policy for risk
characterization. Memorandum of Carol M. Browner, Administrator,
March 21, 1995, Washington, D.C.
U.S. Food and Drug Administration (1987) Sponsored compounds in
food-producing animals; criteria and procedures for evaluating the
safety of carcinogenic residues. final rule. 21 CFR Parts 70, 500,
514 and 571.
Vahakangas, K.H.; Samet, J.M.; Metcalf, R.A.; Welsh, J.A.; Bennett,
W.P.; Lane, D.P.; Harris, C.C. (1992) Mutation of p53 and ras genes
in radon-associated lung cancer from uranium miners. Lancet 339:
576-578.
Vainio, H.; Magee, P.; McGregor, D.; McMichael, A.J. (1992)
Mechanisms of carcinogenesis in risk identification. IARC Sci. Pubs.
No. 116. Lyon, France: IARC.
Van Sittert, N.J.; De Jong, G.; Clare, M.G.; Davies, R.; Dean, B.J.;
Wren, L.R.; Wright, A.S. (1985) Cytogenetic, immunological, and
hematological effects in workers in an ethylene oxide manufacturing
plant. Br. J. Indust. Med. 42:19-26.
Vater, S.T.; McGinnis, P.M.; Schoeny, R.S.; Velazquez, S. (1993)
Biological considerations for combining carcinogenicity data for
quantitative risk assessment. Reg. Toxicol. Pharmacol. 18: 403-418.
Vogelstein, B.; Fearon, E. R.; Hamilton, S. R.; Kern, S. E.;
Presinger, A. C.; Leppert, M.; Nakamura, Y.; White, R.; Smits, A. M.
M.; Bos, J. L. (1988) Genetic alterations during colorectal-tumor
development. New England Journal of Medicine 319: 525-532.
Weinberg, R.A. (1989) Oncogenes, antioncogenes, and the molecular
bases of multistep carcinogenesis. Cancer Res. 49: 3713-3721.
[[Page 18011]]
Wellstein, A.; Lupu, R.; Zugmaier, G.; Flamm, S.L.; Cheville, A.L.;
Bovi, P.D.; Basicico, C.; Lippman, M.E.; Kern, F.G. (1990) Autocrine
growth stimulation by secreted Kaposi fibroblast growth factor but
not by endogenous basic fibroblast growth factor. Cell Growth
Differ. 1: 63-71.
Woo, Y.T.; Arcos, J.C. (1989) Role of structure-activity
relationship analysis in evaluation of pesticides for potential
carcinogenicity. In: Ragsdale, N.N.; Menzer, R.E., eds.
Carcinogenicity and pesticides: Principles, issues, and
relationship. ACS Symposium Series No. 414. San Diego: Academic
Press; pp. 175-200.
Wyzga, R.E. (1988) The role of epidemiology in risk assessments of
carcinogens. Adv. Mod. Environ. Toxicol. 15: 189-208.
Yamada, T.; Nakamura, J.; Murakami, M.; Okuno, Y.; Hosokawa, S.;
Matsuo, M.; Yamada, H. (1994) The correlation of serum luteinizing
hormone levels with the induction of Leydig cell tumors in rats by
oxolinic acid. Toxicol. Appl. Pharmacol. 129: 146-154.
Yamasaki, H. (1990) Gap junctional intercellular communication and
carcinogenesis. Carcinogenesis 11: 1051-1058.
Yamasaki, H. (1995) Non-genotoxic mechanisms of carcinogenesis:
Studies of cell transformation and gap junctional intercellular
communication. Toxicol. Lett. 77: 55-61.
Zhang, K.; Papageorge, A.G.; Lowry, D.R. (1992) Mechanistic aspects
of signalling through ras in NIH 3T3 cells. Science 257: 671-674.
[FR Doc. 96-9711 Filed 4-22-96; 8:45 am]
BILLING CODE 6560-50-P