&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
Draft
Revisions to the
Guidelines for
Carcinogen Risk
Assessment
EPA/600/BP-92/003
August 1994
External Review Draft
Review
Draft
(Do Not
Cite or
Quote)
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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EPA/600/BP-92/003
August 1994
External Review Draft
DRAFT REVISIONS TO THE
GUIDELINES FOR C ARCINOGEN RISK ASSESSMENT
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by
the U.S. Environmental Protection Agency and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its technical accuracy and
policy implications.
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C.
Printed on Recycled Paper
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DISCLAIMER
This document is an external draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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Workshop on Revising the Guidelines for Carcinogen
Risk Assessment
AGENCY: U.S. Environmental Protection Agency.
ACTION: Notice of Meeting.
SUMMARY: 'This notice announces a workshop sponsored by the U.S.
Environmental Protection Agency's (EPA's) Office of Health and
Environmental Assessment (OHEA) and Risk Assessment Forum. OHEA
and the Risk Assessment Forum will convene a panel of experts to
review a draft document entitled, Draft Revisions to the
Guidelines for Carcinogen Risk Assessment (External Review Draft,
August 1994). Discussion will focus on the proposed revisions to,
the Agency's cancer risk assessment guidelines.
DATES: The workshop will be held Monday, September 12, 1994,
through Wednesday, September 14, 1994. The meeting will begin at
S-:30_a,,m._ ^and end at ,5:00 ,p,m. on Monday,, and Tuesday, and wi3-3-
begin at 9:00 a.m. and end at noon on Wednesday. Members of the
public may attend as observers.
ADDRESSES: The meeting will be held at the Hyatt Regency Hotel,
1800 Presidential Street, Reston, Virginia 22090, Tel: 703/709-
1234.
Eastern Research Group, Inc., an EPA contractor, is
providing logistical support for the workshop. To attend the
workshop as an observer, contact Eastern Research Group, Inc.,
110 Hartwell Avenue, Lexington, Massachusetts 02173, Tel:
617/674-7374 by August 31, 1994. Space is limited, so please
register early.
FOR FURTHER INFORMATION CONTACT: For technical inquiries,
contact Dr. Harry.Teitelbaum, U.S. Environmental Protection
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Cancer Guidelines Workshop Page 2 of 3
Agency (8101), 401 M Street, S.W., Washington, DC 20460, Tel:
202/260-6743.
SUPPLEMENTARY INFORMATIONS In 1986, EPA published Guidelines for
Carcinogen Risk Assessment (51 FR 33992; September 24, 1986).
Scientific advances in both risk assessment and carcinogenesis
have led EPA and the broader scientific community to new
perspectives on cancer risk assessment and related new
perspectives on EPA's Cancer Risk Assessment Guidelines.
As part of its efforts to update and revise the 1986
Guidelines, the Agency will hold a workshop in Reston, Virginia,
on September 12-14, 1994, to discuss the major changes
contemplated for these guidelines. At this meeting, experts on
cancer risk assessment and its associated sciences will comment
on and discuss a working draft for revising the 1986 cancer risk
assessment guidelines. The workshop aims to produce
recommendations to the Agency on the use of default assumptions
and wider application of mechanistic data in cancer risk
assessment, among other things.
To obtain a single copy of the draft document (paper or Word
Perfect 5.1 disk), interested parties should contact the ORD
publications office by telephone or FAX, CERI, U.S. Environmental
Protection Agency, 26 West Martin Luther King Drive, Cincinnati,
OH 45268, Tel: (513) 569-7562, FAX (513). 569-7566. Please
provide your name and mailing address, the document title: Draft
Revisions to the Guidelines for Carcinogen Risk Assessment
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Cancer Guidelines Workshop Page 3 of 3
(External Review Draft, August 1994), and EPA document number
EPA/600/BP-92/003 (for paper) or EPA/600/BP-92/003a (for disk)
Date
T
Acting Assistant Administrator
for Research and Development
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CONTENTS
Authors, Contributors, and Reviewers vii
1. INTRODUCTION 1
1.1. PURPOSE AND SCOPE OF THE GUIDELINES 1
1.2. ORGANIZATION AND .APPLICATION OF THE GUIDELINES ........ 1
1.2.1. Organization i
1.2.2. Application 2
1.3. OVERVIEW OF CANCER PROCESSES 3
1.4. USE OF DEFAULT ASSUMPTIONS 8
2. HAZARD ASSESSMENT 9
2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION 9
2.2. ANALYSIS OF TUMOR DATA 11
2.2.1. Human Data 11
2.2.1.1. Types of Studies 11
2.2.1.2. Study Design and Conduct 12
2.2.1.3. Analysis of Causality 15
2.2.1.4. Conclusions 16
2.2.2. Animal Data 17
2.2.2.1. Long-Term Studies 17
2.2.2.2. Special Studies 21
2.2.2.3. Significance for Human Hazard 21
2.3. .ANALYSIS OF OTHER KEY EVIDENCE , 23
2.3.1. Physicochemical Properties 23
2.3.2. Structure-Activity Relationships (SAR) 24
2.3.3. Metabolism and Pharmacokinetics 25
2.3.4. Short-Term Studies , 26
2.3.4.1. Genotoxicity Information 27
2.3.4.2. Other Short-Term Test Information 28
2.3.4.3. Biomarker Information 30
2.3.4.4. Confidence in Conclusions 30
2.4. MODE OF ACTION-IMPLICATIONS FOR HAZARD
CHARACTERIZATION AND DOSE-RESPONSE 31
2.5. HAZARD CHARACTERIZATION 33
2.5.1. Carcinogenic Potential for Humans 33
in
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CONTENTS (continued)
2.5.2. Conditions of Expression 34
2.5.3. Descriptions of Weight of Evidence 35
2.5.4. Hazard Narrative 37
3. DOSE-RESPONSE ASSESSMENT 38
3.1. RESPONSE DATA 39
3.2. DOSE DATA 41
3.2.1. Basic Analyses 42
3.2.2. Pharmacokinetic Analyses 43
3.2.3. Additional Considerations for Dose in Human Studies 44
3.3. SELECTION OF QUANTITATIVE APPROACH 45
3.3.1. Analysis in the Range of Observation 45
3.3.2. Extrapolation 46
3.3.3. Issues for Analysis of Human Studies 48
3.3.4. Use of Toxicity Equivalence Factors and Relative Potency Estimates . 49
3.4. DOSE-RESPONSE CHARACTERIZATION 50
4. EXPOSURE ASSESSMENT AND CHARACTERIZATION 52
5. RISK CHARACTERIZATION 53
5.1. PURPOSE 53
5.2. APPLICATION 54
5.3. CONTENT 54
5.3.1. Presentation 54
5.3.2. Strengths and Weaknesses 55
REFERENCES 56
APPENDIX A 68
IV
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
This external review draft was prepared by a Technical Panel of the Risk Assessment
Forum, Office of Research and Development (ORD).
AUTHORS
Jeanette Wiltse
Richard Hill
Vanessa Vu
Arnold Kuzmack
ORD/Office of Health and Environmental Assessment
Office of Prevention, Pesticides, and Toxic Substances
Office of Prevention, Pesticides, and Toxic Substances
Office of Water
CONTRIBUTORS AND REVIEWERS
To be provided in the next draft.
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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 decision makers with guidance and perspectives
necessary 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 consistency of procedures to support consistency in
scientific components of Agency decision making 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 hi 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 and 1994) provide information and general principles
about risk assessment. Risk assessment utilizes available scientific information bearing on
the properties of an agent and its effects hi 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 decision making on environmental protection. The 1983 and 1994 NRC documents
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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. The risk assessment questions addressed in these guidelines are:
For hazard-Can the agent1 present a carcinogenic hazard to humans, and, if so,
under what circumstances?
For dose-responseAt what levels of exposure might effects occur?
For exposure-What are the conditions of human exposure?
For riskWhat is the character of the risk? How well do data support conclusions
about the nature and extent of the risk?
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 land 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 decision making. Risk assessments support
decisions, but, to maintain their integrity as decision-making tools, 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 hi studying environmental problems hi various media. The guidelines
1The term "agent" refers generally to any chemical substance, mixture, or physical or
biological entity being assessed, unless otherwise noted.
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apply to these various situations, in appropriate detail given the scope and depth of the
particular assessment.
When adopted by the Agency, the guidelines will apply prospectively to new
assessments and to revisions of previous assessments prompted by new data that may alter
previous conclusions.
1.3. OVERVIEW OF CANCER PROCESSES
The following discussion provides background for considering different ways in which
agents may be factors in carcinogenicity. The picture will change 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 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 growth and differentiation of cells.
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; Kaplan et al., 1991; Sidransky et al., 1991;
Salomon et al., 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; Kaplan, et al. 1991; Hollstein et al., 1991; Herschman,
1991; Strausfeld et al., 1991; Forsburg and Nurse, 1991).
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1 Adult tissues, even those composed of rapidly replicating cells, maintain a constant
2 size and cell number (Nunez et al., 1991) by balancing three cell fates: (1) continued
3 replication; (2) differentiation to take on specialized functions; or (3) programmed cell death
4 (apoptosis) (Raff, 1992; Mailer, 1991; Naeve et al., 1991; Schneider et al., 1991; Harris,
5 1990). Neoplastic growth through clonal expansion can result from somatic mutations that
6 inactivate control over cell fate (Kakizuka et al., 1991; deThe et al., 1991; Sidransky et al.,
7 1992; Nowell, 1976). ^
8 Cancers may also be thought of as diseases of the cell cycle. For example, genetic
9 diseases that cause failure of cells to repair DNA damage prior to cell replication predispose
10 people to cancer. These changes are also frequently found in tumor cells in sporadic
11 cancers. These changes appear to be particularly involved at points in cell replication called
12 "checkpoints" where DNA synthesis or mitosis is normally stopped until DNA damage is
13 repaired or cell death induced (Tobey, 1975). A cell that bypasses a checkpoint may acquire
14 a heritable growth advantage. Similar effects on the cell cycle occur when mitogens such as
15 hormones or growth factors stimulate cell growth. Rapid replication in response to tissue
16 injury may also lead to unrepaired DNA damage that is a risk factor for carcinogenesis.
17 Normally a cell's fate is determined by a timed sequence of biochemical signals.
18 Signal transduction in the cell involves chemical signals that bind to receptors, generating
19 further signals in a pathway whose target in many cases is control of transcription of a
20 specific set of genes (Hunter, 1991; Cantley et al., 1991; Collum and Alt, 1990). Cells are
21 subject to growth signals from the same and distant tissues, e.g., endocrine tissues (Schuller,
22 1991) . In addition to hormones produced by endocrine tissues, numerous soluble
23 polypeptide growth factors have been identified that control normal growth and
24 differentiation (Cross and Dexter, 1991; Wellstein et al., 1990). The cells responsive to a
25 particular growth factor are those that express transmembrane receptors that specifically bind
26 the growth factor.
27 Solid tumors develop in stages operationally defined as initiation, promotion, and
28 progression (see, for example, Pilot and Dragan, 1991). These terms, which were coined in
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1 the context of specific experimental designs, are used for convenience in discussing concepts,
2 but they refer to complex events that are not completely understood. During initiation, the
3 cell acquires a genetic change that confers a potential growth advantage. During promotion,
4 clpnal expansion of this altered cell occurs. Later, during progression, a series of genetic
5 and other biological events both enhance the growth advantage of the cells and enlist normal
6 host processes to support tumor development, and cells develop the ability to invade locally
7 and metastasize distally, taking on the characteristics of malignancy. Many endogenous and
8 exogenous factors are known to participate in the process as a whole. These include specific
9 genetic predispositions or variations in ability to detoxify agents, medical history (Harris,
10 1989; Nebreda et al., 1991), infections, chemicals, ionizing radiation, hormones and growth
11 factors, and immune suppression. Several such risk factors likely work together to cause
12 individual human cancers.
13 A cell that has been transformed, acquiring the potential to establish a line of cells
14 that grow to a tumor, will probably realize that potential only rarely. The process of
15 tumorigenesis in animals and humans is a multistep one (Bouk, 1990; Fearon and Vogelstein,
16 1990; Hunter, 1991; Kumar et al., 1990; Sukumar, 1989; Sukumar, 1990), and normal
17 physiological processes appear to be arrayed against uncontrolled growth of a transformed
18 cell (Weinberg, 1989). Powerful inhibition by signals from contact with neighboring normal
19 cells is one known barrier (Zhang et al., 1992). Another is the immune system (at least for
20 viral infection). How a cell with tumorigenic potential acquires additional properties that are
21 necessary to enable it to overcome these and other inhibitory processes is a subject of
22 ongoing research. For known human carcinogens studied thus far, there is an often decades-
23 long latency between exposure to carcinogenic agents and development of tumors (Fidler and
24 Radinsky, 1990; Tanaka et al., 1991; Thompson et al., 1989). This latency is also typical of
25 development of tumors in individuals with genetic diseases that make them cancer prone
26 (Meyn, 1993; Srivastava et al., 1990).
27 The importance of genetic mutation in the carcinogenic process calls for special
28 attention to assessing agents that cause such mutations. Much of the screening and testing of
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1 agents for carcinogenic potential has been driven by the idea of identifying this mode of
2 action. Cognizance of and emphasis on other modes of action such as ones that act at the
3 level of growth signalling within or between cells, through cell receptors, or that indirectly
4 cause genetic change, comes from more recent research. There are not yet standardized tests
5 for many modes of action, but pertinent information may be available in individual cases.
6 Agents of differing characteristics influence cancer development: inorganic and
7 organic, naturally occurring and synthetic, of inanimate or animate origin, endogenous or
8 exogenous, dietary and non-dietary. The means by which these agents act to influence
9 carcinogenesis are variable, and reasoned hazard assessment requires consideration of the
10 multiple ways chemicals influence cells in experimental systems and in humans. Agents
11 exert genotoxic effects either by interacting directly with DNA or by indirect means through
12 intermediary substances (e.g., reactive oxygen species) or processes. Most DNA interactive
13 chemicals are electrophilic or can become electrophilic when metabolically activated.
14 Electrophilic molecules may bind covalentiy to DNA to form adducts and this may lead to
15 depurination, depyrimidation or produce DNA strand breaks; such lesions can be converted
16 to mutations with a round of DNA synthesis and cell division. Other DNA-interactive
17 chemicals may cause the same result by intercalation into the DNA helix. Still other
18 chemicals may methylate DNA, changing gene expression. Non-DNA-reactive chemicals
19 produce genotoxic effects by many different processes. They may affect spindle formation
20 or chromosome proteins, interfere with normal growth control mechanisms, or affect
21 enzymes involved with ensuring the fidelity of DNA synthesis (e.g., topoisomerase),
22 recombination, or repair.
23 The "classical" chemical carcinogens in laboratory rodent studies are agents that
24 consistently produce gene mutations and structural chromosome aberrations in short-term
25 tests. A large data base reveals that these mutagenic substances commonly produce tumors
26 at multiple sites and in multiple species (Ashby and Tennant, 1991). Most of the carcinogens
27 identified in human studies, aside from hormones, are also gene or structural chromosome
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1 mutagens (Tennant and Ashby, 1991). Most of these compounds or their metabolites contain
2 electrophilic moieties that react with DMA.
3 Numerical chromosome aberrations, gene amplification and the loss of gene
4 heterozygosity are also found in animal and human tumor cells that may arise from initiating
5 events or during progression. There is reason to believe that accumulation of additional
6 genetic changes is favored by selection in the evolution of tumor cells because they confer
7 additional growth advantages. Exogenous agents may function at any stage of carcinogenesis
8 (Barrett, 1993). Some aberrations may arise as a consequence of genomic instability arising
9 from tumor suppressor gene mutation, e.g., p53 (Harris and Hollstein, 1993). The frequent
10 observation in tumor cells that both of a pair of homologous chromosomes have identical
11 mutation spectra in tumor suppressor genes suggests an ongoing, endogenous process of gene
12 conversion. Currently, there is a paucity of routine test methods to screen for events such as
13 gene conversion or gene amplification, and knowledge regarding the ability of particular
14 agents of environmental interest to induce them is, for the most part, wanting. Work is
15 underway to characterize, measure, and evaluate their significance (Bianchi et al., 1991;
16 Cowell et al., 1991; see Schwab, 1992; Travis et al., 1991).
17 Several kinds of mechanistic studies aid in risk assessment. Comparison of DNA
18 lesions in tumor cells taken from humans to the lesions that a tumorigenic agent causes in
19 experimental systems, can permit inferences about the association of exposure to the agent
20 and an observed human effect (Vahakangas et al., 1992; Hollstein et al., 1991; Hayward et
21 al., 1991). An agent that is observed to cause mutations experimentally may be inferred to
22 have potential for carcinogenic activity (U.S. EPA, 1991a). If such an agent is shown to be
23 carcinogenic in animals, the inference that its mode of action is through mutagenicity is
24 strong. A carcinogenic agent that is not mutagenic in experimental systems, but is mitogenic
25 or affects hormonal levels or causes toxic injury followed by compensatory growth may be
26 inferred to have effects on growth signal transduction or to have secondary carcinogenic
27 effects. The strength of these inferences depends in each case on the nature and extent of all
28 the available data.
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1 Differing modes of action at the molecular level have different dose-response
2 implications for the activity of agents. The carcinogenic activity of a direct-acting mutagen
3 should be a function of the probability of its reaching and reacting with DNA. The activity
4 of an agent that interferes at the level of signal pathways with many potential receptor targets
5 should be a function of multiple reactions. The activity of an agent that acts by causing
6 toxicity followed by compensatory growth should be a function of the toxicity.
7
8 1.4. USE OF DEFAULT ASSUMPTIONS
9 The National Research Council (NRC), in its 1983 report on the science of risk
10 assessment (NRC, 1983), recognized that default assumptions are necessarily made in risk
11 assessments when gaps in general knowledge or in data available for a particular agent are
12 encountered. These default assumptions are inferences based on general scientific knowledge
13 of the phenomena in question and are also matters of policy. In its 1994 report on risk
14 assessment, the NRC supported continued use of default assumptions (NRC, 1994). The
15 1994 report recommended that the EPA explain the science and policy underlying the
16 defaults and provide general criteria for departing from them. (Descriptions of major
17 defaults and their rationales appear in the responses to comments accompanying this
18 document. NOTE: the responses will appear with the Federal Register proposal of revisions
19 and do not accompany this review draft.)
20 Under these guidelines, it is the policy of the EPA to continue to use default
21 assumptions. Instead of considering them to be positions from which departure is justified, it
22 is the policy of these guidelines that default assumptions are indeed default positions, and that
23 they are not used unless supported by the circumstances of a particular case. In each case in
24 which the risk assessor decides that it is appropriate to use a default, the use is explained and
25 the lack or inadequacy of data justifying resort to a default is described.
26 Throughout these guidelines, major defaults are noted in the context of risk
27 assessment subjects. Most major defaults covered in the guidelines arise as a consequence of
28 either using animal toxicity data as a model for human toxicity or in the extrapolation of
8
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observed dose-response relationships to lower dose levels associated with environmental -
exposures of interest. Other default assumptions may be applied in practice that are not
within the scope of coverage of the guidelines. When these are applied, they are similarly
explained. An example of such a default is use of an exposure factor such as food or water
consumption, typical of an adult in the United States. Such factors are often taken from
published compendia that contain numerous exposure factors based on general population
data. These factors are used to apply to specific populations.
2. HAZARD ASSESSMENT
2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION
Hazard Assessment
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. The ingredients of hazard
assessment are reviews of a variety of kinds of data that may range from observations of
tumors to analysis of structure-activity relationships. The purpose of the assessment is not
simply to assemble these separate reviews; 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 of the agent, and implications of these for human hazard and dose-response evaluation.
For example, interpretation of studies on human effects considers structure-activity
relationships and experimental data on animal effects. Similarly, interpretation of animal
tumors considers other relevant data such as effects at the cellular level, comparative
metabolism, and non-cancer toxicity. The reliability of conclusions comes from the
combined strength and coherence of inferences appropriately drawn from all of the available
evidence.
If studies reveal that an agent affects carcinogenic processes under one set of
circumstances, the next question is whether one can infer that the effects will occur under
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1 other or all circumstances. For instance, are effects seen in one species likely to occur in
2 another species? Are effects that occur by one route and level of exposure likely to occur by
3 another route and level? It would take an extraordinary amount of work to empirically
4 address every question involved in these extrapolations. Realistically, hazard assessments
5 must address these issues with the data that are practicably obtainable, combined with
6 inferences drawn from a basic understanding of biological and chemical processes relevant to
7 carcinogenesis and to exposure. Extrapolation of responses from one species to another and
8 from high to low dose can be much stronger if there is information on the mode of action of
9 the agent. Such information can enable greater concentration on particular lines of evidence.
10 To the extent data permit, hazard assessment addresses the mode of action question both as a
11 part of identifying human hazard potential and as an initial step in considering appropriate
12 approaches to dose-response assessment.
13
14 Hazard Characterization
15 The hazard characterization develops conclusions on the questions of human hazard
16 potential and circumstances of its expression. Developing conclusions involves integrating
17 the salient outcomes of analyses of all of the data. This, in turn, involves considering the
18 inferences drawn from data, their strengths and weaknesses in light of the uncertainties of the
19 available data and the state of current scientific knowledge. Presentation of the results of
20 hazard characterization includes:
21 summarizing the evaluations of hazard data,
22 expressing the reasoning from the data to conclusions, and
23 explaining significant strengths or limitations of the conclusions given the limits of
24 available data and knowledge.
25 The discussions that follow cover analysis of tumor data, both animal and human, and
26 analysis of other key evidence about properties and effects that relate to carcinogenic
27 potential. The'discussion covers aspects of evidence pertinent to mode of action and how
28 mode of action can influence thinking about the dose-response relationship. The last part of
10
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Section 2 describes hazard characterization. It discusses the characterization, how the
likelihood of carcinogenic hazard is categorized, and the use of a hazard narrative as a
summary.
2.2. ANALYSIS OF TUMOR DATA
Evidence of carcinogenicity comes from finding tumor increases in humans or in
laboratory animals exposed to a given agent or from finding tumors following exposure to
structural analogues to the compound under review. The significance of the observed or
anticipated tumor effects is evaluated in reference to all of the other key evidence 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. (The use of the term "tumor" here is generic, meaning malignant
neoplasms or a combination of malignant and corresponding benign neoplasms.)
2.2.1. Human Data
2.2.1.1. Types of Studies
Human data may come from studies that select and compare exposed and unexposed
(or less-exposed) populations or from investigations of cases in specific settings, e.g.,
workplace or clinical. Formal studies include cohort, case-control, proportionate ratio,
clinical trial, and correlational studies. Each has well known strengths and weaknesses that
affect interpretation of results (Kelsey et al., 1986; Lilienfeld and Lilienfeld, 1979; Mausner
and Kramer, 1985; Rothman, 1986). Reports of cancer cases can identify associations
particularly when there are unique features such as association with an uncommon tumor
(e.g., vinyl chloride and angiosarcoma or diethylstilbestrol and clear cell carcinoma of the
vagina).
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1 2.2.1.2. Study Design and Conduct
2 In cancer epidemiologic studies, unlike laboratory experiments, the exposures of the
3 study and the control (referent) populations are not controlled. Because most studies are
4 retrospective, and cancer effects are latent for many years, the exposure estimates are usually
5 a reconstruction of what exposures may have been many years in the past. Moreover, the
6 fact that most human cancers have more than one potential cause creates both design and
7 analytical problems of distinguishing among possible causes for an observed effect. These
8 features mean that drawing causal inferences depends on an analysis of many factors. The
9 following discussions cover factors included in an analysis of human data:
10
11 Population Issues
12 The ideal comparison would be between two populations that differ only in exposure
13 to the agent in question. Since this is seldom the case, it is important to identify any sources
14 of bias that are inherent in a study's design or data collection methods. Bias can arise from
15 several sources, including non-comparability between populations of factors such as general
16 health (McMichael, 1976), diet, lifestyle or geographic location; differences in the way case
17 and control individuals recall past events; differences in data collection that result in unequal
18 ascertainment of health effects in the populations; and unequal follow-up of individuals.
19 Both acceptance of studies for assessment and judgment of their strength or weakness
20 depends on identifying sources of bias and their effects on results.
21
22 Exposure Issues
23 Questions to address about exposure are: What can one reliably conclude about the
24 level, duration, route, and frequency of exposure of individuals in one population as
25 compared to another? How sensitive are study results to uncertainties in these parameters?
26 Surrogates are often used to reconstruct exposure parameters when historical
27 measurements are not available. These may take the form of attributing exposures to job
28 classifications in a workplace or to broader occupational or geographic groupings. Use of
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surrogates carries a potential for misclassification in that individuals may be counted in the
incorrect exposure groups. 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 since exposure estimation
is the most critical, but weakest, part of a retrospective study.
Biological markers of exposure or effect 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 (Callemen et al.,
1986; 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.
In deciding whether there is an association between health effects and exposure to an
agent, the evaluation gives studies with more precise and specific exposure estimates greater
weight.
Confounding Factors
A confounding variable is a risk factor, independent of the putative agent, that is
distributed unequally among the exposed and unexposed populations. 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 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
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1 data on smoking status among individuals in the study population, an examination of the
2 possible contribution of cigarette smoking to increased lung cancer risk may be based on
3 information from other sources such as the American Cancer Society's longitudinal studies
4 (Hammand, 1966; Garfmkel and Silverberg, 1991). The effectiveness of adjustments
5 contributes the ability to draw inferences from a study.
6 Different studies involving exposure to an agent may have different confounding
7 factors. If consistent increases in cancer risk are observed across a collection of studies with
8 randomly distributed confounding factors, the inference that the agent under investigation
9 was the etiologic factor is strong, even though complete adjustment for confounding factors
10 cannot be made and no single study supports a strong inference.
11 Biological evidence on confounding factors, such as animal data, structure-activity
12 relationships, and metabolism data, is analyzed. The analysis compares such evidence on the
13 agent under study with evidence on identified confounders to test the plausibility of
14 associating observed human effects either with the agent or with confounding factors.
15
16 Sensitivity
17 Sensitivity, or the ability of a study to detect real effects, is a function of several
18 factors. Greater size of the study population(s) (sample size) increases sensitivity as does
19 greater exposure (levels and duration) of the population members. Because of the often
20 lengthy latency period in cancer development, sensitivity also depends on whether adequate
21 time has elapsed since exposure began for effects to occur. A unique feature that can be
22 ascribed to effects of a particular agent (such as a tumor type that is seen only rarely in the
23 absence of the agent) can increase sensitivity by permitting separation of bias and
24 confounding factors from real effects. Similarly, a biomarker particular to the agent can
25 permit these distinctions. These are all factors to explore in statistical analysis of the data.
26
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2.2.1.3. Analysis of Causality
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 includes 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.
Statistical methods for examining several studies in combination are commonly termed
"meta-analysis." Meta-analysis evaluates whether study results differ randomly from the
hypothesis of no effect (Mann, 1990). If an effect is not present, the observed results ought
to appear to be randomly distributed and cancel each other when studies are combined.
Meta-analysis is an appropriate tool to use if several studies are available. Before applying
meta-analysis, several issues are addressed to decide if the analysis will be meaningful.
These include: controlling for bias and confounding prior to combining studies; establishing
and meeting appropriate criteria for study inclusion; assigning weights to individual studies;
arid anticipating possible publication and aggregation bias (Greenland, 1987; Peto, 1992).
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 modelled 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.
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1 Temporal relationship: The disease occurs within a biologically reasonable time after
2 initial exposure. This feature must be present if causality is to be considered. The
3 initial period of exposure to the agent is the accepted starting point in most
4 epidemiologic studies.
5 Consistency: Associations occur in several independent studies of a similar exposure
6 in different populations, or associations occur consistently for different subgroups in
7 the same study.
8 Magnitude of the association: A causal relationship is more credible when the risk
9 estimate is large and precise (narrow confidence intervals).
10 Biological gradient: The risk ratio increases with increasing exposure or dose. A
11 strong dose-response relationship across several categories of exposure, latency, and
12 duration is supportive for causality given that confounding is unlikely to be correlated
13 with exposure. The absence of a dose-response relationship is not by itself evidence
14 against a causal relationship.
15 Specificity of the association: The likelihood of a causal interpretation is increased if
16 an exposure produces a specific effect (one or more tumor types also found in other
17 studies) or if a given effect has a unique exposure.
18 Biological plausibility: The association makes sense in terms of biological
19 knowledge. Information is considered from animal toxicology, pharmacokinetics,
20 structure-activity relationship analysis, and short-term studies of the agent's influence
21 on events in the carcinogenic process considered.
22 Coherence: The cause-and-effect interpretation is in logical agreement with what is
23 known about the natural history and biology of the disease, i.e., the entire body of
24 knowledge about the agent.
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26 2.2.1.4. Conclusions
27 Critical evaluation of each human study includes the exposure-effect relationship,
28 exposure assessment, selection and comparison of groups, sample size, and handling of
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latency, confounders, and bias as discussed under section 2.2.1.2. The weight of a study in
providing inferences about human carcinogenicity depends upon this evaluation, the statistical
confidence in conclusions, and biological plausibility of findings. Greater plausibility and
stronger inferences come from meeting criteria for causality. The plausibility of an
exposure-effect finding can be increased or decreased by other relevant findings such as
evidence of structure-activity relationships with other agents, studies of mode of action,
understanding of metabolic pathways, and animal toxicology findings.
The above considerations of the studies available are integrated in a summary of
results.
2.2.2. Animal Data
Various kinds of whole animal test systems are currently used or are under
development for evaluating potential carcinogenicity. Standardized, long-term studies
involving chronic exposure for most of the lifespan of an animal are generally accepted for
evaluation of tumor effects (Tomatis et al., 1989; Rail, 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 assaying agents for specific modes of action.
2.2.2.1. Long-Term Studies
Current standardized long-term studies in rodents test at least 50 animals in each of
three treatment group and in the control group usually for 2 years (US EPA, 1983a,b,c).
Other, similar protocols have been and continue to be used by many laboratories. Studies
are examined for production of tumors and preneoplastic lesions, as well as for toxicity
reactions that may give clues to the mode of action of the agent in producing tumors.
Analyses of study results are by dose, sex, species and route of exposure. Analysis of
toxicity covers both its potential role in carcinogenicity and its separate potential for causing^
effects in humans. The assessment may identify general toxicity as a more important end
point for consideration than carcinogenicity.
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1 All available studies of tumor effects in whole animals are considered, at least
2 preliminarily. The analysis discards studies judged to be wholly inadequate in protocol or
3 conduct and carries forward the remainder. Studies for analysis include both those that are
4 optimal in terms of current protocols and conduct, and studies whose adequacy is limited in
5 some respect. Limitations may be in aspects such as duration, dosing, or sensitivity. Such
6 limited studies can contribute as their deficiencies permit.
7 Evaluation of tumor effects includes both biological significance and statistical
8 significance (Haseman, 1984, 1985, 1990).
9
10 Statistical Significance of Animal Responses
11 Statistical analysis of a long-term study considers each tumor type separately. Benign
12 and malignant lesions of the same cell type, usually within a single tissue or organ, are
13 combined (McConnell, 1986). Trend tests and pairwise comparison tests are the
14 recommended tests for determining whether chance, rather than a treatment-related effect, is
15 a plausible explanation for an apparent increase in tumor incidence. A trend test such as the
16 Cochran-Armitage test (Snedecor and Cochran, 1967) asks whether the results in all dose
17 groups together increase as dose increases. By convention, a trend is statistically significant
18 if the probability that an effect seen would have occurred by chance is 5% or less
19 (p<0.05). A pairwise comparison test such as the Fisher exact test (Fisher, 1932) asks
20 whether an incidence in one dose group is increased over the control group. Again, by
21 convention, a statistically significant comparison is one for which p<0.05 that the increased
22 incidence is due to chance. Significance in either kind of test is sufficient to reject the
23 hypothesis that chance accounts for the result. A statistically significant response may or
24 may not be biologically significant and vice versa.
25 The standard comparison is tumor effect in dosed animals as compared with
26 concurrent control animals. Additional insights about both statistical and biological
27 significance can come from an examination of historical control data (Haseman et al., 1984).
28 If such data are used, the discussion needs to address several issues that affect comparability
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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. It is most desirable to compare historical and current data from the same
laboratory and supplier, gathered closely in time. Always, the discussion of results covers
issues of comparability.
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 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.
Evaluation of Animal Responses
Observation of tumor effects under varying circumstances (e.g., test species or strain,
route of administration, sex, replication in different laboratories) lends support to
significance of findings for animal carcinogenicity. Progression of lesions observed in
subchronic studies or in the chronic study, greater ratio of malignant to benign tumors, dose-
response trend, or reduced latency of neoplastic lesions are among the findings that also lend
support. Finding a very uncommon tumor type suggests biological significance even if the
increase in incidence is not statistically significant, particularly if the increase is dose-related.
The high dose in long-term studies is generally selected to provide maximum
statistical ability to detect treatment-related effects. Two or more additional doses,
reasonably spaced, are at some fraction of the high dose. The high dose, in EPA
requirements, is one that, in an animal lifetime, produces some toxic effects without either
unduly affecting mortality from effects other than cancer, or producing significant adverse
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1 effects on the animals' health (see generally NRC, 1993b). Dose selections for long-term
2 studies are based on results of subchronic studies along with any available information on the
3 disposition, metabolism, and pharmacokinetics of the test agent. The aims in selecting dose
4 levels are both adequate sensitivity and the opportunity to observe tumor effects specific to
5 the agent. Failure to reach an adequately high dose reduces the sensitivity of the study. On
6 the other hand, excessive general toxicity, or toxicity in a target tissue for tumor effects,
7 raises the question whether tumor effects are specific to the agent or are nonspecific effects
8 secondary to the toxicity. There is no completely satisfactory answer to the possible conflict
9 between these aims. For instance, reducing the high dose to avoid any toxicity would reduce
10 the sensitivity of a protocol that is at best able to detect an increase of tumor incidence of
11 10%, if there is no spontaneous background incidence (Haseman, 1983). Reliable detection
12 of a 1 % increase would require using thousands of animals, which is not a feasible option.
13 Any of the following treatment-related findings may indicate that other, noncancer
14 toxicity may confound results of the cancer studies:
15 A 10%, or greater, increase in mortality due to toxicity
16 Significant toxicity manifested by a decrement in body weight greater than 10 to 15%
17 (not due to reduced food consumption from impalatability)
18 Significant toxicity manifested by clinical signs, hematological or chemistry measures,
19 or changes in organ weight, morphology, and histopathology that may interact with
20 the carcinogenic process or obscure interpretation of the results
21 Absence of tumor effects in an adequately sensitive study that is well conducted is accepted
22 as a negative finding. Studies with undue effects on mortality or health that show no tumor
23 effects are accepted as negative, if lower doses are appropriately spaced and mortality does
24 not remove too many animals from the study. Studies of inadequate sensitivity may be used
25 to bound the range of potential risk. Studies that show tumor effects only at doses with
26 undue effects on mortality or health are compromised and may or may not contribute to the
27 analysis, depending on interpretation in the context of other study results and other lines of
28 evidence. The effect of toxicity is examined, even when it is not excessive. In all cases, the
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strength of inferences to be drawn about human carcinogenic potential from the statistical, and
biological significance of animal tumor effects is judged in conjunction with all other
available data.
2.2.2.2. Special Studies
Specialized short-term studies often use protocols that screen for effects, usually
preneoplastic effects, in a single tissue. The selected tissue is, in a sense, the test system.
Certain systems of this kind are thought to be particularly informative about carcinogenic
potential of defined chemical classes or agents, or modes of action (reviewed in Vainio et al.,
1992). Other experimental systems such as strain A mice that are bred to be particularly
sensitive may also provide some information. Nevertheless, limitations in experimental
protocol, such as short duration, limited histology, or lack of complete development of
tumors, make results from these studies correspondingly limited in their contribution to
assessment. Their results are appropriate for identifying subjects of further research and for
considering the effects seen in context with other evidence, especially regarding potential
i
modes of action. Transgenic animals carrying introduced proto-oncogenes, or activated
oncogenes or tumor suppressor genes are promising experimental systems for probing mode
of action questions when experience that supports their interpretation is in place. The
strength of inferences that such studies support rests on their contribution to the congruity of
evidence about an agent.
2.2.2.3. Significance for Human Hazard
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.
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1 Several kinds of observations from animal studies can contribute to the judgment
2 whether animal responses indicate a significant carcinogenic hazard to humans. Significance
3 is a function of the number of factors present and, for a factor such as malignancy, the
4 severity of the observed pathology. The following observations add significance:
5 uncommon tumor types
6 tumors at multiple sites
7 tumors by more than one route of administration
8 tumors in multiple species, strains, or both sexes
9 progression of lesions from preneoplastic to benign to malignant
10 reduced latency of neoplastic lesions
11 metastases
12 unusual magnitude of tumor response
13 proportion of malignant tumors
14 dose-related increases
15 These features or their absence also may provide useful suggestions about a possible
*
16 mode(s) of action of an agent. For example, multisite and multispecies effects are often
17 associated with genotoxic agents. Effects restricted to one sex may suggest an influence
18 connected with gender-related biological differences such as hormonal status. Late onset of
19 tumors that are primarily benign, or reversal of lesions on cessation of exposure, may point
20 to a growth-promoting mode of action. Similarly, a general increase in incidence of a
21 tumor with a high spontaneous background with no increase in malignancy, may indicate a
22 growth-promoting activity. The possibility that an agent may act differently in different
23 tissues or have more than one mode of action in a single tissue is kept in mind.
24 Site concordance of tumor effects between animals and humans is an issue to be
25 considered in each case. Thus far, there is evidence that growth control mechanisms at the
26 level of the cell are homologous among mammals, but there is no evidence that these
27 mechanisms are site concordant. Moreover, agents observed to produce tumors in both
28 humans and animals have produced tumors either at the same (e.g., vinyl chloride) or
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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.
Relevance of tumor responses in nonhuman species to human hazard is an integral
judgment in analysis of bioassay results. The assumption of relevance is less appropriate
when a body of evidence supports a mode of action in animals that would not be seen in
humans under the same conditions of exposure. The Agency will undertake analyses of
relevance issues as needed in reports to be published from time to time (e.g., U.S. EPA,
1991b).
2.3. ANALYSIS OF OTHER KEY EVIDENCE
Certain structural, chemical, and biological attributes of an agent provide key
information about its potential to cause or influence carcinogenic events. These attributes
along with comparative studies between species also provide clues as to the potential mode
of carcinogenic action and the potential dose-response relationship. The following sections
provide guidance for analyses of these attributes.
2.3.1. Physicochemical Properties
Physicochemical properties affect an agent's absorption, tissue distribution
(bioavailability), biotransformation, or degradation in the body. These dispositions are
important determinants of hazard potential. 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 that 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 to decide whether they would facilitate such reactions.
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1 2.3.2. Structure-Activity Relationships (SAR)
2 Results of SAR analysis may strengthen or weaken the concern for an agent's
3 carcinogenic potential, depending on confidence in the SAR analysis. SAR analysis supports
4 evaluation of carcinogenic potential of both tested and untested chemicals.
5 Currently, SAR analysis is most useful for chemicals that are believed to produce
6 carcinogenesis, at least initially, through covalent interaction with DNA (i.e., DNA-reactive,
7 mutagenic, electrophilic, or proelectrophilic chemicals) (Ashby and Tennant, 1991). The
8 following parameters are useful in comparing an agent to its structural analogues and
9 congeners that have tumor effects and effects on related biological processes such as receptor
10 binding, genotoxicity, and general toxicity (Woo and Arcos, 1989):
11 nature and reactivity of the electrophilic moiety or moieties present
12 potential to form electrophilic reactive intermediate(s) through chemical,
13 photochemical, or metabolic activation
14 contribution of the carrier molecule to which the electrophilic moiety(ies) is attached
15 physicochemical properties (e.g., physical state, solubility, octanol-water partition
16 coefficient, half-life in aqueous solution)
17 structural and substructural features (e.g., electronic, stearic, molecular geometric)
18 metabolic pattern (e.g., metabolic pathways and activation and detoxification ratio)
19 possible exposure route(s) of the agent
20 Suitable SAR analysis of non-DNA-reactive chemicals and of DNA-reactive chemicals
21 that do not appear to bind covalentiy to DNA requires knowledge or postulation of the
22 probable mode(s) of action of closely related carcinogenic structural analogues (e.g.,
23 receptor-mediated, cytotoxicity related). Examination of the physicochemical and
24 biochemical properties of the agent may then allow one to assess the likelihood of its activity
25 by that mode of action.
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2.3.3. Metabolism and F'harmacokinetics
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, to support identification of active metabolites, to
identify changes in distribution and metabolic pathway or pathways over a dose range and
between species, and to make comparisons among different routes of exposure.
If 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 determination of tissue dosimetry, species-to-species
extrapolation of dose, and route-to-route extrapolation (Connolly and Andersen, 1991; see
section 3.2.2.). Without such data, it is necessary to assume that pharmacokinetic and
metabolic processes are qualitatively comparable between species if this appears generally
reasonable.
Adequate metabolism and pharmacokinetic data can be applied toward the following
as data permit. Confidence in conclusions is greatest 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 el 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
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1 identify issues in comparing species and formulating dose-response .assessment
2 approaches.
3 Identifying changes in pharmacokinetics and metabolic pathways with increases in
4 dose. These changes may result in the formation and accumulation of toxic products
5 following saturation of detoxification enzymes. These studies have an important role
6 in providing a rationale for dose selection in carcinogenicity studies. In addition, they
7 may be important in estimating a dose over a range of high to low exposure for the
8 purpose of dose-response assessment.
9 Determining bioavailability via different routes of exposure by analyzing uptake
10 processes under various exposure conditions. This analysis supports identification of
11 hazard for untested routes. In addition, use of physicochemical data (e.g., octanol-
12 water partition coefficient information) can support an inference about the likelihood
13 of dermal absorption (Flynn, 1990).
14 In all of these areas, attempts are made to clarify and describe as much as possible
15 the variability to be expected because of differences in species, sex, age, and route of
16 exposure. Utilization of pharmacokinetic information takes into account that there may be
17 subpopulations of individuals who are particularly vulnerable to the effects of an agent
18 because of pharmacokinetic or metabolic differences (genetically or environmentally
19 determined).
20
21 2.3.4. Short-Term Studies
22 Results from short-term in vitro and in vivo studies of effects that relate to
23 carcinogenic events are useful in the interpretation of epidemiological and animal data on
24 tumor effects. They are often the primary source of information about possible modes of
25 action.
26 A large number of short-term assays exist for examining biological activities relevant
27 to the carcinogenic process (e.g., mutagenesis, tumor promotion, aberrant intercellular
28 communication, increased cell proliferation, malignant transformation, immunosuppression).
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These assess a variety of end points; confidence in the inferences to be drawn from results of
each assay varies according to experience with the test system and its interpretation. Both
the relevance of tested end points to potential carcinogenicity and confidence in interpretation
are issues explicitly evaluated.
2.3.4.1. Genotoxicity Information
"Genotoxicity" is an inclusive term that refers to effects of agents that interact with or
alter the genome. "Mutagenicity" constitutes the part of genotoxicity associated with
heritable changes in DNA structure or content. Mutations, including changes in DNA
sequence and gene amplification, and structural and numerical chromosome aberrations are
attributes of tumor cells and of predisposing genetic diseases. Recent studies on oncogenes
provide additional evidence for the linkage between mutation and cancer (Bishop, 1991).
Activation of proto oncogenes to oncogenes can be triggered, for example, by point
mutations, DNA insertions, or chromosomal translocation (Bishop, 1991). In addition, the
inactivation of tumor suppressor genes can occur by chromosomal deletion or loss or by
mitotic recombination (Bishop, 1989; Varmus, 1989; Stanbridge and Cavenee, 1989).
Mutagenic endpoints are, therefore, of obvious interest.
The process by which an agent brings about mutations is also of interest, in part
because different processes have different implications for the dose-response relationship.
Agents may react directly with DNA as evidenced by DNA adducts, strand breakage, and
intercalation between bases. Alternatively, they may react with other cell components, with
DNA damage as an indirect consequence, e.g., interfere with spindle formation or with
repair or recombination processes. Different numbers of chemical reactions during critical
periods of the cell cycle are inherent in direct versus indirect processes, with implications for
linearity or nonlinearities in the dose-response relationship.
The EPA has published testing protocols and guidelines for detection of mutagenicity
(U.S. EPA, 199la). A method to portray data graphically is the genetic activity profile
methodology (Garrett et al., 1984; Waters et al., 1988).
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1 A higher level of confidence that an agent is a mutagen is assigned to agents that
2 consistently induce sequence or structural changes in DNA in a number of test systems.
3 Although important information can be gained from in vitro assays, a higher level of
4 confidence is given to a data set that includes in vivo data because many agents require
5 metabolic conversion to an active intermediate for biological activity. Metabolic activation
6 systems can be incorporated into in vitro assays, but they may not mimic mammalian
7 metabolic activation and inactivation. If available, human genetic toxicity end points relevant
8 to carcinogenesis are important in vivo data.
9 Because mutagenic carcinogens have been observed to induce tumors across species
10 and at multiple sites, evidence of both mutagenicity and tumor responses in multiple species
11 or sexes significantly increases concern for the human carcinogenic potential of an agent.
12 Agents that induce mutations in the germinal cells of animals are similarly of concern.
13 About 30 agents, many of which are carcinogenic to humans, have been shown to induce
14 germ cell mutations in animals and may be anticipated to do so in somatic cells. Absence of
15 mutagenicity in several test systems, preferably including in vivo tests for genotoxicity in the
16 tumor target tissue (e.g., DNA alkylation, unscheduled DNA synthesis) suggests that another
17 mode of action may be important (Ashby, J., chapters in Vainio et al., 1992). The absence
18 of evidence of mutagenicity in an adequate data base and the lack of responses in a chronic
19 rodent bioassay gives reasonable confidence that an agent is not a human cancer hazard
20 unless there is human evidence to the contrary.
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22 2.3.4.2. Other Short-Term Test Information
23 Nongenotoxic agents are also known to affect carcinogenesis. For example,
24 hormones and growth factors or agents that mimic them alter DNA functions that are critical
25 in the cell cycle. Other agents may cause cell toxicity which results in compensatory cell
26 replication or selectively promotes growth of initiated cells. Dose-response relationships for
27 some agents may be tied to their cell toxicity.
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The relationship of dose and response is more complex for agents that affect the
function of cell cycle controls, encountering issues of human variability in endocrine and
exocrine balance and background exposures (U.S. EPA, 1994).
Clues about nongenotoxic modes of action can come from tests for increased cell
proliferation, cell transformation, aberrant intercellular communication, receptor mediated
effects, or changes in gene transcription.
Cell proliferation plays a key role at each stage in the carcinogenic process, and it is
well established that increased rates of cell proliferation are associated with increased cancer
risk. An agent may produce cellular toxicity with compensatory cell proliferation or act as a
mitogen directly or indirectly (e.g., by affecting endocrine function). Increased risk may be
due to bypassing DNA repair checkpoints or to interference with apoptosis or differentiation
resulting in an increase in the number of proliferating cells at risk of spontaneous and
induced genetic damage. Evidence for an increased rate of cell division may be determined
by measuring the mitotic index, or by supplying a specific DNA precursor to cells (e.g., 3H-
thymidine or bromodeoxyuridine) and counting the percentage of cells that have incorporated
the precursor into replicating DNA, or by immunodetection of proliferation-specific antigens.
These analyses can be carried out in vitro, or during prechronic animal studies, or as part of
long-term animal studies.
Assays for measuring perturbation of gap-junctional intercellular communication may
provide an indication of carcinogenicity, especially promotional activity (Yamasaki, 1990).
Cell transformation assays have been widely used for studying mechanistic aspects of
chemical carcinogenesis because in vitro cell transformation is considered to be relevant to
the in vivo carcinogenic process. Both of these kinds of assays have been proposed as tests
for identifying nongenotoxic carcinogens (Swierenga, S.H.H. and Yamasaki, H., chapter in
Vainioetal., 1992)
There is as yet a paucity of standard tests for effects specifically on the cell cycle,
although as the fields of cell cycle and cancer research merge, more cell cycle study methods
may become available for screening agents for such effects.
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1 2.3.4.3. Biomarker Information
2 Several genotoxicity end points and other measurements can serve as biological
3 markers of events in biological systems or samples. In some cases, these molecular or
4 cellular effects (e.g., DNA or protein adducts, structural chromosomal aberrations, levels of
5 thyroid stimulating hormone) can be measured in blood, body fluids, and cancer target
6 organs to serve as biomarkers of exposure (Callemen et al., 1978; Birner et al., 1990). As
7 such they can do the following:
8 act as an internal, surrogate measure of chemical dose,
9 help identify doses at which elements of the carcinogenic process are operating,
10 aid in interspecies extrapolations when data are available from experimental animal
11 and human cells, and
12 under certain circumstances, provide insights into the shape of the dose-response
13 curve below levels where tumor incidences are observed (e.g., Choy, 1993).
14 Genetic and other findings (like changes in proto-oncogenes and tumor suppressor
15 genes in neoplastic tissue or measures of endocrine disruption) can indicate the potential for
16 disease and as such serve as biomarkers of effect. They, too, can be used in different ways:
17 The spectrum of genetic changes in tumors following chemical administration to
18 experimental animals can be determined and compared to those in spontaneous tumors
19 in control animals, in animals exposed to other agents, and in persons exposed to the
20 agent under study.
21 As with biomarkers of exposure, it may be justified in some cases to use these
22 endpoints to provide insight into the shape of the dose-response curve at doses below
23 those at which tumors are induced.
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25 2.3.4.4. Confidence in Conclusions
26 All data from short-term studies become part of the overall evaluation. It is
27 recognized that agents will not typically produce uniform responses in all short-term tests.
28 The reviewer is often left with a mix of responses in different tests. To the extent data
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1 permit, the reviewer makes judgments as to genotoxic and nongenotoxic effects, the degree
2 to which the effects may influence observed tumor formation, and the influence of effects in
3 judging human hazard potential and dose-response implications. Considerations and elements
4 of confidence in such judgments include, but are not limited to, the following:
5 spectrum of endpoints relevant to carcinogenicity
6 number of studies of each endpoint
7 consistency of results in different test systems and different species
8 in vivo as well as in vitro observations
9 reproducibility of results in a test system
10 existence of a dose-response relationship for effects
11 , tests conducted in accordance with generally accepted protocols .
12 degree of consensus and general experience among scientists regarding interpretation
13 of the significance and specificity of the tests
14 At the end of the evaluation, when conclusions are offered, the conclusions are accompanied
15 by a statement of the confidence supported by the data base.
16
17 2.4. MODE OF ACTION-IMPLICATIONS FOR HAZARD CHARACTERIZATION
18 AND DOSE-RESPONSE
19 The analysis of the entire range of data reviewed in the assessment of tumor effects
20 studies and other key evidence is the support for arriving at a judgment about the potential
21 mode(s) of action of an agent. The purpose for analyzing the mode of action is to make a
22 reasoned judgment about the ways agents appear to be producing carcinogenic effects. This
23 judgment affects the ways of characterizing hazards to humans and means of evaluating
24 potential dose-response relationships. Given the gaps in the general understanding of
25 carcinogenesis, these analyses and judgments do not lay out detailed molecular mechanisms.
26 Nevertheless, commonly available data support general views as to physical, chemical, and
27 biological factors that appear to be influencing the carcinogenic process and are
28 acknowledged by scientists as playing a role. For hazard characterization, these analyses
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1 address the relevance of the animal tumor model to humans and the conditions of expression
2 of potential hazard (see, for example, Appendix A, Narratives 1 and 4). For dose-response
3 assessment, these analyses help to guide the development of biologically based models and,
4 in the absence of detailed information, to select among different default techniques for low-
5 dose extrapolation.
6 The analysis reviews all the relevant information on the agent including observed
7 tumor responses and other key evidence. The entire list of factors to be considered in this
8 analysis would be beyond the scope of these guidelines to list. Some of the important ones
9 include the following:
10 number of tumor sites, sexes, studies, and species affected or unaffected
11 nature of tumor sites, e.g., responsive to endocrine influence or not
12 route of exposure effects
13 target organ or systemic effects (e.g., target organ toxicity, urinary chemical changes
14 associated with stones, effects on immune surveillance)
15 progression of lesions from preneoplastic to benign to malignant
16 early or late appearance of tumors after exposure
17 proportion of malignant to benign tumors
18 tumors invading locally, metastasizing, producing death
19 tumors at sites in laboratory animals with high or low spontaneous historical incidence
20 biomarker of effect in tumor cells, such as mutation spectra (like or unlike
21 spontaneous tumors), DNA or protein adducts, chromosome changes
22 shape of the dose response in the range of tumor observation
23 structural relationship of the agent or its metabolites to mutagenic carcinogens
24 structural relationship of the agent or its metabolites to agents that are non-DNA-
25 reactive, or influence other processes (e.g., hormone disruption)
26 genotoxic effects
27 effects on cell proliferation (e.g., mitogenic effects, effects on differentiation or
28 apoptosis, response to toxicity)
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effects on intercellular communication
evidence of initiating or promoting activity in two-stage assays in vitro or in vivo
Dose-response default positions include low-dose linear extrapolation, nonlinear, and
both procedures. Both procedures are warranted when (a) tumors at different sites appear to
have different modes of action (see, for example, Appendix A, Narrative 4), (b) there is
evidence in support of both default procedures (see, for example, Appendix A, Narrative 5),
and (c) there is considerable uncertainty as to potential mode of action (see, for example,
Appendix A, Narratives 3 and 6).
Many combinations of data elements are possible. For example, a data set that might
lead to a conclusion that a linear dose-response default is reasonable might include: tumors
at multiple sites, sexes, and species, with structural alerts and good evidence of DNA-
reactivity in short-term tests (e.g., Appendix A, Narrative 7). Finding that there are tumor
responses in two species at sites that have high spontaneous rates of tumor incidence, the
effect at each site being to accelerate appearance of tumors without altering the spontaneous
proportion of benign to malignant tumors coupled with finding no structural alerts or
evidence of genotoxicity in extensive short-term testing, would support a nonlinear dose-
response default procedure.
2.5. HAZARD CHARACTERIZATION
2.5.1. Carcinogenic Potential for Humans
The hazard characterization summarizes and evaluates the analyses of pertinent data
concerning carcinogenic hazard potential and applies them to make a conclusion as to
carcinogenicity to human beings. The conclusion regards both the general population and
subpopulations identified as particularly sensitive. Evaluation addresses the significant
strengths and weaknesses of existing data. It also explains the reasoning that supports data
interpretation, including the use of default assumptions, and weighs the coherence of
implications of different kinds of data. The central purpose of evaluation is to explore and
explain how the available empirical evidence may relate to biological activity in humans.
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No single kind of evidence can reveal the whole biological story otherwise told by
bringing together all of the available information. Moreover, the kinds of evidence that are
the most telling are different from case to case. Some illustrations of this are well known:
Metabolism information~the presence or absence of a single enzymewas the key to
explaining why certain aromatic amines cause bladder cancer in dogs and humans, but not in
rodents. The activity and high levels of a small protein, alpha 2u globulin, in male rats was
the key to finding that a particular kidney tumor in these animals has no relevant parallel in
humans. Appendix A contains several examples highlighting key roles for other kinds, of
information:
Narrative 4 illustrates that data on an agent's homology in biological effects and
chemical structure to a carcinogenic analogue can support the conclusion that the
agent is likely to be carcinogenic, although there are no tumor effects data on the
agent itself.
Narrative 1 illustrates how information on physiological perturbation and physical
irritation can explain mode of action sufficiently to define dose as a limiting factor on
potential expression of carcinogenic effects.
Narrative 6 shows that information on absorption and toxicity can lead to different
conclusions about carcinogenic potential by different routes of exposure.
2.5.2. Conditions of Expression
A characterization qualitatively describes the conditions under which the agent's
effects may be expressed in human beings. The description includes routes of exposure and
dose levels and durations of exposure.
Discussion of approaches to quantitative assessment of internal or delivered dose
according to route of exposure appears in section 3 below. Qualitative characterization
describes whether the route of exposure is likely to be a limiting factor on expression of
effects. The supporting data include studies hi which effects of the agent or analogues on
animals or humans have been observed by differing routes, or physical-chemical properties,
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or pharmacokinetics studies. For certain agents there may be enough known about properties
of their class to make confident conclusions regarding absorption by different routes. For
others, more data on the specific agent may be necessary as conclusions may be less
confident, or not possible, without such information. Discussion of these issues is part of the
characterization.
Relationship of dose level, pattern, and duration of exposure to effects can
theoretically take many forms. Hazard assessment examines all of the kinds of evidence to
see what processes or events in carcinogenesis a specific agent appears to affect. This
information is used to relate the mode of action in general terms to the approach to
quantitative dose-response assessment. The mode of action may imply a linear or a non-
linear dose-response relationship, or a threshold of dose below which effects will not occur
in an individual. The implications of available information about human variability in
sensitivity or with respect to threshold effects are important elements. These implications
relate to both the qualitative characterization of hazard and the quantitative assessment of
dose-response.
2.5.3. Descriptions of Weight of Evidence
The hazard narrative, described in the following subsection, presents the weight of
evidence in terms of likelihood of human carcinogenicity using descriptors. The descriptors
are not meant to replace an explanation of the nuances of the biological evidence, but rather
to summarize it. Each descriptor spans a wide variety of data sets and weights of evidence.
There will always be gray areas, gradations, and borderline cases. The narrative preserves
and presents this complexity, which is an essential part of the hazard characterization.
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
focus simply on the descriptor.
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Note that one agent may fit more than one descriptor, if, for instance, the agent were
likely to be carcinogenic by one route of exposure and not by another. Use of the
descriptors is illustrated hi Appendix A.
The descriptions below are intended only as guidance and provide some typical
examples; the examples are not exhaustive or comprehensive.
"Likely" or "Known"
These descriptors are appropriate when the evidence provides a reasonable assurance
of carcinogenic potential for human beings and supports proceeding with the risk assessment.
"Likely" is the descriptive term generally used. "Known" is used when the weight of
evidence gives especially high assurance because either an association between
carcinogenicity and a specific route of exposure is drawn from human data, or conclusions
from other kinds of data give confidence that is equal to having human data.
"Cannot be determined"
"Cannot be determined" is appropriate whenever support for a conclusion about
carcinogenic potential for human beings is not sufficient to proceed with the assessment.
Where appropriate, the narrative explains the situation. Examples may include the
following, among others.
The evidence raises a concern for carcinogenic effects, but falls short of supporting a
conclusion about the likelihood of effects. The narrative provides a summary of the
research or testing needed to explore the issue further. The added descriptor, "testing
candidate" is appropriate in these cases to flag the agent for attention by testing
programs.
The data are inadequate to perform an assessment.
The information is inconclusive or conflicting, e.g., some evidence is suggestive of
carcinogenic effects, but other equally pertinent evidence does not confirm any
concern.
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"Not Likely"
"Not likely" is appropriate whenever evidence about an agent generally, or about a
condition of exposure, is satisfactory for deciding that there is not a basis for concern.
Again, the narrative explains the conclusion. Examples may include the following:
The agent has been adequately characterized empirically, and the conclusion is
negative, or the only positive data are not considered relevant to human beings.
Adequate empirical characterization generally includes well conducted, long-term
animal studies on an agent or its structural analogue with consistent findings from
analysis of other key evidence.
The evidence shows that under certain conditions of exposure, no expression of
carcinogenic effects is anticipated. The agent's carcinogenic potential is categorized
as "not likely" for those conditions, e.g., a route of exposure or a defined dose level.
The agent has been adequately characterized empirically, and the only positive
indication of effects was seen under experimental circumstances that are implausible
for raising an environmental concern, e.g., injection of a polymer.
2.5.4. Hazard Narrative
A narrative summarizes the results of hazard characterization. The narrative,
optimally two pages or less in length, explains an agent's human carcinogenic potential and
the conditions of its expression. If data do not allow a conclusion, the narrative explains this
determination. Examples of narratives appear in Appendix A below as guidance for format
and content. The items regularly included are these:
name of agent and Chemical Abstracts Services number, if available
a brief identification of the kinds of data available
conclusions (by route of exposure) about human carcinogenicity, described as
"known" or " likely," "not likely," or "cannot be determined"
summary of tumor data, human or animal, on the agent and/or its structural
analogues, their relevance, and biological plausibility
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1 Other key evidence, e.g., structure-activity data, pharmacokinetics and metabolism,
2 short-term studies, other relevant toxicity data
3 effect of route of exposure
4 discussion of possible mode(s) of action and appropriate dose-response approaches)
5 strengths and weaknesses of the evidence and assumptions significant to the case
6 The following items may be added if justified:
7 depiction of the evidence as particularly strong or weak as compared to other cases.
8 a description of one or more research approaches or tests that can be anticipated to
9 resolve a critical question identified in the assessment.
10
11 3. DOSE-RESPONSE ASSESSMENT
12
13 Dose-response assessment addresses both the relationship of dose2 to degree of
14 response observed empirically and the nature of this relationship at environmental exposure
15 levels of interest below the range of observation. Analysis of responses includes tumor
16 responses in human and animal studies and the agent's effects on macromolecules involved in
17 growth control or other toxic effects that may play a role in carcinogenesis. Analyses of
18 dose use available metabolism and pharmacokinetic data to identify appropriate measures of
19 applied dose and, as data permit, improve the analysis by identifying measures of internal or
20 delivered dose. If empirical data are from animal studies, the analysis extrapolates animal
21 doses to human equivalent doses. Depending on the availability of data, dose-response
22 assessments are carried out for any effect associated with an agent's carcinogenicity to assess
23 potential for effects from environmental exposure.
24 2For this discussion, "exposure" means contact of an agent with the outer boundary of an
25 organism. "Applied dose" means the amount of an agent presented to an absorption barrier
26 and available for absorption. "Internal dose" means the amount crossing an absorption
27 barrier (e.g., the exchange boundaries of skin, lung, and digestive tract) through uptake
28 processes. "Delivered dose" for an organ or cell means the amount available for interaction
29 with that organ or cell (U.S. EPA, 1992a).
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1 3.1. RESPONSE DATA
2 Response data of interest for analysis include tumor incidence data from human or
3 animal studies. They also include data on other responses as they relate to an agent's
4 carcinogenic effects, for instance, effects on growth control processes or cell macromolecules
5 or other toxic effects. Tumor incidence data is ordinarily the basis of dose-response
6 assessment, but availability of other response data can augment such assessment or provide
7 separate assessments of other important effects. Data on carcinogenic processes underlying
8 tumor effects may be used to support biologically based models. If confidence is high in the
9 linkage of a precursor effect and the tumor effect, the assessment of tumor incidence may be
10 extended to lower dose levels by linking it to the assessment of the precursor effect
11 (Swenberg et al., 1987). Linking analyses may not be appropriate; even so, the assessment
12 for a precursor effect may provide a view of the likely shape of the dose-response curve for
13 tumor incidence below the range of tumor observation (Cohen1 and Ellwein, 1990; Choy,
14 1993). The effects of an agent on cell macromolecules may be used as markers of
15 carcinogenic effect for which a separate dose-response assessment may be done, or such
16 effects may provide markers of exposure in support of dose analysis. In some cases, the
17 dose-response relationship for a response other than tumor incidence may be more useful
18 than tumor incidence to assess potential effects associated with environmental exposure. For
19 example, if it is concluded that the carcinogenic effect is secondary to another toxic effect,
20 the dose-response for the other effect will likely be more pertinent for risk assessment. The
21 dose-response relationship for a precursor effect may also be more useful, for example,
22 disruption of hormone activity when this is considered the key mode of action of an agent.
23 These kinds of supplementary analyses are presented and evaluated whenever data are
24 available to support them.
25 If adequate positive human epidemiologic data are available, they provide an
26 advantageous basis for analysis since concerns about interspecies extrapolation do not arise.
27 Adequacy of human exposure data for quantification is an important consideration in deciding
28 whether epidemiologic data are the optimal basis for analysis in a particular case. Positive
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1 data are analyzed to estimate response to environmental exposure in the observed range.
2 (U.S. EPA, 1992a). Extrapolation to lower environmental exposure ranges is carried out, as
3 needed. Analysis of associated responses as described in the paragraph above supplements
4 analyses of human tumor data as well as those of animal tumor data. If adequate exposure
5 data exist in a well-designed and well-conducted epidemiologic study that detects no effects,
6 it may be possible to obtain an upper-bound estimate of the potential human risk to provide a
7 check on plausibility of available estimates based on animal tumor or other responses.
8 When animal studies are used, response data from a species that responds most like
9 humans should be used, if information to this effect exists. If this is unknown, and an agent
10 has been tested in several experiments involving different animal species, strains, and sexes
11 at several doses and different routes of exposure, all of the data sets are considered and
12 compared, and a judgment is made as to the data to be used to best represent the observed
13 tumor incidence data Snd important biological features such as mode of action. Appropriate
14 options for presenting results include use of a single data set, combining data from different
15 experiments (Stiteler et al., 1993; Vater et ah, 1993), showing a range of results from more
16 than one data set, showing results from analysis of more than one tumor response based on
17 differing modes of action, representing total response in a single experiment by combining
18 animals with tumors, or a combination of these options. The approach judged to best
19 represent the data is presented with the rationale for the judgment, including the biological
20 and statistical considerations involved. The following are some points to consider:
21 high quality of study protocol and execution
22 malignancy of neoplasms
23 latency of onset of neoplasia,
24 number of data points to define the relationship of dose and response
25 background incidence in test animal
26 most sensitive responding species
27 data on a related effect (e.g., DNA adduct formation) are available to augment the
28 tumor effect data
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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.
3.2. 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 and, in case of
animal data, a measure of dose appropriate to humanshuman equivalent dosethat is
matched to the anticipated route of environmental exposure. Among these are: (1) Is the
parent compound, a metabolite, or both, active in the process? and (2) How well do data at
hand measure available dose and support pharmacokinetic modeling of internal or delivered
dose?
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 reach a measure of internal or delivered dose if possible.
Even if pharmacokinetic and metabolic data are sufficient to derive a measure of
delivered dose to the target, the dose-response relationship is also affected by kinetics of
reactions at the target (pharmacodynamics) and by other steps in the development of
neoplasia. With few exceptions, these processes are currently undefined, and this is a
limitation to the ultimate aim of dose analysis which is to have a measure of effective dose to
the target.
The following discussion assumes that the analyst will have data of varying detail in
different cases about pharmacokinetics 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.
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1 3.2.1. Basic Analyses
2 When there are insufficient data available to compare dose between species, the
3 default assumption is that delivered doses through oral exposure are related to applied dose
4 by a power of body weight. This assumption rests on the similarities of mammalian
5 anatomy, physiology, and biochemistry generally observed across species. This assumption
6 is more appropriate at low applied dose concentrations where sources of nonlinearity, such as
7 saturation or induction of enzyme activity, are less likely to occur. To derive a human
8 equivalent oral dose from animal data, the default procedure is to scale daily applied doses
9 experienced for a lifetime in proportion to body weight raised to the 3/4 power (W3/4).
10 Equating exposure concentrations in parts per million units for food or water is an alternative
11 version of the same default procedure because daily intakes of these are in proportion to
12 \y3/4. The rationale for this factor rests on the empirical observation that rates of
13 physiological processes consistently tend to maintain proportionality with W3/4. A more
14 extensive discussion of the rationale and data supporting the Agency's adoption of this
15 scaling factor can be found in (U.S. EPA, 1992b). Information such as blood levels or
16 exposure biomarkers that are available for interspecies comparison is used to improve the
17 analysis when possible.
18 The default procedure to derive a human equivalent concentration of inhaled particles
19 and gases is described in U.S. EPA (in press) and Jarabek (in press). The methodology
20 estimates respiratory deposition of inhaled particles and gases and provides methods for
21 estimating internal doses of gases with different absorption characteristics. The method is
22 able to incorporate additional pharmacokinetics and metabolism to improve the analysis if
23 such data are available.
24 The differences in biological processes among routes of exposure (oral, inhalation,
25 dermal) can be great because of, for example, first pass effects and differing results from
26 different exposure patterns. There is no generally applicable method for accounting for these
27 differences in uptake processes in quantitative route-to-route extrapolation of dose-response
28 data in the absence of good data on the agent of interest. Therefore, route-to-route
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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 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).
3.2.2. Pharmacokinetic Analyses
Physiologically based mathematical models are potentially the most comprehensive
way to account for pharmacokinetic 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. Another assumption made when a delivered dose measure is used
in animal-to-human extrapolation of dose-response data is that the pharmacodynamics of the
target tissue(s) will be the same in both species. This assumption is discussed, and
confidence in accepting it is considered in presenting results.
Pharmacokinetic data can improve dose-response assessment by accounting for
sources of change in proportionality of applied to internal or de21ivered 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 over-
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1 estimation or under-estimation of low dose-response otherwise resulting from extrapolation
2 from a sublinear or supralinear part of the experimental dose-response curve. (Gillette,
3 1983). Pharmacokinetic processes tend to become linear at low doses, an expectation that is
4 more robust than low dose linearity of response (Hattis, 1990). Accounting for
5 pharmacokinetic nonlinearities allows better description of the shape of the curve at higher
6 levels of dose, but cannot determine linearity or nonlinearity of response at low dose levels
7 (Lutz, 1990; Swenberg etal., 1987).
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9 3.2.3. Additional Considerations for Dose in Human Studies
10 The applied dose in a human study has uncertainties because of the exposure
11 fluctuations that humans experience compared with the controlled exposures received by
12 animals on test. In a prospective cohort study, there is opportunity to monitor exposure and
13 human activity patterns for a period of time that supports estimation of applied dose (U.S.
14 EPA, 1992a). In a retrospective cohort study, exposure may be based on monitoring data,
15 but is often based on human activity patterns and levels reconstructed from historical data,
16 contemporary data, or a combination of the two. Such reconstruction is accompanied by
17 analysis of uncertainties considered with sensitivity analysis in the estimation of dose
18 (Wyzga, 1988; U.S. EPA, 1986a). These uncertainties can also be assessed for any
19 confounding factor, for which a quantitative adjustment of dose-response data is made (U.S.
20 EPA, 1984).
21 Exposure levels of groups of people in the study population often are represented by
22 an average when they are actually distributed in a range. The full extent of data are
23 analyzed and portrayed in the dose-response analysis when possible (U.S. EPA, 1986a).
24 The cumulative dose of an agent is commonly used when modeling human data. This
25 can be done, as in animal studies, with a default assumption in the absence of data that
26 support a different dose surrogate. Given data of sufficient quality, dose rate or peak
27 exposure can be used as an alternative surrogate to cumulative dose.
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3.3. SELECTION OF QUANTITATIVE APPROACH
The goal in choosing an approach is to achieve the closest possible correspondence
between the approach and the view of the agent's mode(s) of action. For this purpose, it is
appropriate to analyze the dose-response of observed tumor incidence and other carcinogenic
effects as discussed above, in section 3.1. Pharmacokinetic analysis or interspecies scaling or
other appropriate methods are used to derive human-equivalent measures of the animal-
administered dose. If the hazard assessment describes more than one mode of action as
plausible and persuasive given the data available, corresponding alternative approaches for
dose-response analysis are considered.
3.3.1. Analysis in the Range of Observation
The analysis first addresses responses in the observed range. The responses include
tumor incidence as well as other responses relevant to carcinogenicity. The latter may
enlighten judgment about extrapolation or extend the range of observation (see section
3.3.1.).
If data are sufficient to support a biologically based model specific to the agent, this
is the first choice for both the observed tumor data and for extrapolation to the range of
environmental exposures of interest. Dose-response models based on general concepts of a
mode of action are next in amount of information required. For a specific agent, model
parameters are obtained from laboratory 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 values for parameters, e.g., theoretical cell proliferation rates, are not used
to enable application of such a model (Portier, 1987). If data are not available for such a
model to be applied to observed tumor incidence data, a multistage model is appropriate for
curve-fitting as a default approach (Zeise et al., 1987), unless characteristics of the data
make another model more appropriate. For instance, when longitudinal data on tumor
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1 development are available, time-to-tumor or survival models may be appropriate and
2 necessary to fit the data. Analyses of responses other than tumor effects are individually
3 designed and used to inform the extrapolation approach or used in lieu of tumor data if they
4 better portray the biology.
5 The ED10 (dose associated with a 10% response) of the observed dose-response for
6 tumor incidence (or another effect related to carcinogenesis) or is identified. This level is
7 adopted as a matter of Agency policy in order to remain consistent and comparable from case
8 to case; the rationale supporting this choice is that 10% is the limit of sensitivity of most
9 studies of tumor incidence. Determination of the ED10 is a matter of judgment. Because
10 statistical considerations (e.g., the number and spacing of dose levels, sample sizes, and the
11 precision and accuracy of dose measurements) can affect the precision of model estimates,
12 upper and lower confidence limits are stated with the ED,0. The divergence between upper
13 and lower confidence bounds provides a sense of the precision with which the modeling can
14 make projections in that range. The ED10 provides a point of departure for extrapolation of
15 tumor incidence data (or a related effect) and is an estimate that can be used for comparison
16 with similar analyses of the observed range of noncancer effects of an agent (U.S. EPA,
17 199 If) or to support hazard ranking among carcinogenic agents. It is also employed in the
18 dose-response and risk characterizations as a basis for describing the magnitude of
19 extrapolation needed to reach the range of environmental exposure of interest.
20
21 3.3.2. Extrapolation
22 If a biologically based or mode of action-based model has been used to portray the
23 observed data and confidence in the model is good, it may be extended to the level of
24 environmental exposure of interest. The reliability of the model is appropriately examined
25 by considering the point at which the upper and lower confidence bound on the curve diverge
26 to an unacceptable extent. If data are insufficient to support this extension, extrapolation
27 relies on a default procedure reflecting the general mode(s) of action supported by the
28 available biological information. The Agency adopts the three default procedure options
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described below as matters of policy based on current theory of the likely shapes of dose-
response curves for differing modes of action. If a carcinogenic agent acts by accelerating
the same carcinogenic process that leads to the background occurrence of cancer, the added
effect on the population at low doses marginally above background level is expected to be
linear. Above background level, the population response may continue to be linear (e.g., in
the case of an agent acting directly on DNA), or be nonlinear reflecting complex
physiological processes or events caused by multiple reactions (e.g., numerical chromosomal
changes following reactions with spindle proteins, interrupting spindle formation). If the
agent acts by mode of action with no endogenous counterpart, a population response
threshold may exist (Crump et al., 1976; Peto, 1978; Hoel, 1980; Lutz, 1990). The Agency
reviews each assessment as to the evidence on mode of action and other biological or
statistical evidence that indicates the suitability of a particular procedure. In all cases, a
rationale is included to justify the use of the selected procedure.
Linear
If the mode of action being considered leads to an expected linear low dose-tumor
incidence relationship, a straight line is drawn from the EDi0 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, particularly in the absence of
information about the extent of human variability in sensitivity to effects.
Nonlinear
The mode of action being considered may project that the dose-response relationship
is nonlinear or is most influenced by individual differences in sensitivity. In this case, a
model incorporating nonlinearity may be used to provide estimates of the proportion of the
population at risk for specific doses of interest, e.g., 1/1000, 1/10000 lifetime risk levels. If
an appropriate model is not available, a margin of exposure analysis may be used as
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1 described below. The margin of exposure is the EDJO divided by the environmental exposure
2 of interest.
3 The mode of action may be one that involves a population threshold, e.g., the
4 carcinogenicity may be a secondary effect of toxicity that is itself a threshold phenomenon.
5 In these cases, the risk is not extrapolated as a probability of an effect at low doses.
6 Instead, a margin of exposure presentation is made in the risk characterization. The
7 environmental exposures of interest may be actual levels, projected levels after control, or
8 potential environmental standards. The risk manager decides whether a given margin of
9 exposure is adequate under applicable management policy criteria. The risk assessment
10 provides supporting information to assist the decision maker. Analysis to support
11 consideration of the margin of exposure conveys as much as possible about the anticipated
12 dose-response relationship in humans. This includes consideration of comparative sensitivity
13 of animals and humans, where animal data are the observed data, and consideration of human
14 variability in sensitivity to the putative mode(s) of action.
15
16 Both Linear and Nonlinear
17 Both linear and nonlinear procedures may be used in a particular case. If differing
18 modes of action appear to be involved in effects at different target tissues, the tissues may be
19 dealt with separately by linear and nonlinear procedures. If a mode of action analysis finds
20 equal support for modes of action with differing implications, both linear and nonlinear
21 procedures may be used.
22 If a dose-response assessment relies on data for an endpoint other than cancer, for
23 example, endocrine disruption, the assessment is individually designed and a rationale is
24 presented. The margin of exposure procedure is used when appropriate.
25
26 3.3.3. Issues for Analysis of Human Studies
27 Issues and uncertainties arising in dose-response assessment based on epidemiological
28 studies are analyzed in each case. Several sources of uncertainty need to be addressed in the
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dose-response analysis. Consideration needs to be given to the data on the exposure and
mortality experience of the study population and of the population that will represent the
background incidence of the neoplasm(s) involved. In this area, there are potentials for
mistakes or uncertainty in the data or adjustments to the data concerning the occurrence or
level of exposure of the population members, mortality experience of a population,
incomplete follow-up of individuals, exposure (or not) of individuals to confounding causes,
or consideration of latency of response. These are assessed by analyzing the sensitivity of
dose-response study results to errors where data permit. Other kinds of uncertainty can
occur because of small sample size which can magnify the effects of misclassification or
change assumptions about statistical distribution that underlie tests of statistical significance
(Wyzga, 1988). These uncertainties are discussed. Where possible, analyses of the
sensitivity of results to the potential variability in the data in these areas are performed.
The suitability of various available mathematical procedures for quantifying risk
attributed to exposure to the study agent is discussed. These methods (e.g., absolute risk,
relative risk, excess additive risk) account differently for duration of exposure and
background risk, and one or more can be used in the analysis as data permit. The use of
several of these methods is encouraged when they can be used appropriately in order to gain
perspectives on study results.
3.3.4. 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 characteristic 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.
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1 Shared characteristics that may be used are, for example, receptor-binding characteristics,
2 results of assays of biological activity related to carcinogenicity or structure-activity
3 relationships.
4 TEFs are generated and used for the limited purpose of assessment of agents or
5 mixtures of agents in environmental media when better data are not available. When better
6 data become available for an agent, its TEF should be replaced or revised. Criteria for
7 constructing TEF's are given in U.S. EPA (1991b). The criteria call for data that are
8 adequate to support summing doses of the agents in mixtures.
9 Relative potencies can be similarly derived and used for agents with carcinogenicity
10 or other supporting data. The criteria for these basically the same as for TEF's with the
11 exception that data of a quality that will support summing doses of agents in a mixture are
12 not required.
13 The uncertainties associated with both TEF's and relative potencies are explained
14 whenever they are used.
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16 3.4. DOSE-RESPONSE CHARACTERIZATION
17 The conclusions of dose-response analysis are presented in a characterization section.
18 Because alternative approaches may be plausible and persuasive in selecting dose data,
19 response data, or extrapolation procedures, the characterization presents the judgments made
20 in such selections. The results for the approach or approaches chosen are presented with a
21 rationale for the one (or more than one if they are equally supported) that is considered to
22 best represent the available data and best correspond to the view of the mechanism of action
23 developed in the hazard assessment.
24 The exploration of significant uncertainties in data for dose and response and in
25 extrapolation procedures is part of the characterization. A distinction is made in the
26 presentation between model uncertainty and measurement uncertainty. Model uncertainty is
27 an uncertainty about a basic biological question. For example, a linear dose-response
28 extrapolation may have been made based on tumor and other key evidence supporting the
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view that an agent's mode of action is through mutagenicity. Discussion of the confidence in
this approach is appropriately done qualitatively, it is not amenable to useful quantitative
uncertainty analysis. Measurement uncertainties deal with numbers representing statistical or
analytical measures of variance or error in data or estimates. Uncertainties in measurement
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 measurement uncertainties are described qualitatively. In either case, the
discussion highlights those uncertainties that are specific to the agent being assessed, as
distinct from those that are generic to most assessments.
Numerical dose-response estimates are presented to one significant figure and
qualified as to whether they represent central tendency or statistical upperbounds, and
whether the method used is Inherently more likely to over-estimate, or under-estimate
(Krewski etal., 1984).
In cases where a mode of action or other feature of the biology has been identified
that has special implications 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. Special problems arise when the human exposure situation of concern
suggests exposure regimens, e.g., route and dosing schedule that are substantially different
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1 from those used in the relevant animal studies. The cumulative dose received over a
2 lifetime, expressed as average daily exposure prorated over a lifetime, is generally
3 considered an appropriate measure of exposure to a carcinogen particularly for an agent that
4 acts by damaging DNA, if consistent with available data. The assumption is made that a
5 high dose of a carcinogen received over a short period of time is equivalent to a
6 corresponding low dose spread over a lifetime; this is based on theoretical considerations.
7 This approach becomes more problematic as the exposures in question become more intense
8 but less frequent, or when there is evidence that the agent acts by a mode of action involving
9 dose-rate effects. These issues are explored and pointed out for attention in the exposure
10 assessment and risk characterization.
11
12 4. EXPOSURE ASSESSMENT AND CHARACTERIZATION
13
14 Guidelines for exposure assessment of carcinogenic and other agents are published in
15 U.S. EPA (1992a) and are to be used in conjunction with these cancer risk assessment
16 guidelines. The exposure characterization is a key part of the exposure assessment; it is the
17 summary explanation of the exposure assessment. Major points of exposure characterization
18 are reiterated here to be considered in the cancer risk assessment context. The
19 characterization
20 provides a statement of purpose, scope, level of detail, and approach used in the
21 assessment;
22 presents the estimates of exposure and dose by pathway and route for individuals,
23 population segments, and populations in a manner appropriate for the intended risk
24 characterization;
25 provides an evaluation of the overall quality of the assessment and the degree of
26 confidence the authors have in the estimates of exposure and dose and the conclusions
27 drawn; and
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communicates the results of exposure assessment to the risk assessor, who can then
use the exposure characterization, along with the characterization of the other risk
assessment elements, to develop a risk characterization.
In general, the magnitude, duration, and frequency of exposure provide fundamental
information for estimating the concentration of the carcinogen to which the organism is
exposed. These data are generated from monitoring information, modeling results, or
reasoned estimates. The potential for exposure via ingestion, inhalation, and dermal
penetration from relevant sources of exposures, including multiple avenues of intake from the
same source, are appropriate matters to consider in cancer risk assessment.
5. RISK CHARACTERIZATION
5.1. PURPOSE
The risk characterization summarizes and integrates the major results of the risk
assessment in a way that makes them understandable for all interested readers. Since the risk
characterization provides a transition between risk assessment and risk management, one of
its objectives is to be an appraisal of the science that the risk manager can use, along with
other decision making resources, to make public health decisions. A complete
characterization presents the risk assessment as an integrated, and balanced, picture of the
analysis of the hazard, dose response, and exposure. It is the risk analyst's obligation to
communicate not only summaries of the evidence and results, but also perspectives on the
quality of available data and the degree of confidence to be placed in the risk estimates.
These perspectives on the science include explaining the constraints of available data and the
state of knowledge about the phenomena studied. All of this is in aid of a major aim of
characterization which is to explain not only significant scientific issues, but also the
significant science and science policy choices made when alternative interpretations of data
exist.
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1 5.2. APPLICATION
2 A risk characterization is a necessary part of any Agency report on risk, whether the
3 report is a preliminary one prepared to support allocation of resources toward further study
4 or a comprehensive one prepared to support regulatory decisions. In the former case, the
5 detail and sophistication of the characterization are appropriately small in scale, in the latter
6 case, appropriately extensive. Even if only parts of a risk assessment (hazard and dose-
7 response analyses for instance) are covered in a document, the risk characterization matches
8 the extent of coverage of the document.
9
10 5.3. CONTENT
11 Each of the following subjects should be covered in the risk characterization.
12
13 5.3.1. Presentation
14 The presentation of the results of the assessment should fulfill the aims as outlined in
15 the purpose section above. The summary draws from the key points of the individual
16 characterizations of hazard, dose response, and exposure analysis performed separately under
17 these guidelines. The summary integrates these characterizations into an overall risk
18 characterization (AIHC, 1989).
19 The presentation of results clearly explains the numerical estimates of risk. For
20 example, when estimates of individual risk or population risk (incidence) are used, there are
21 several features of such estimates that risk managers need to understand. They include, for
22 instance, whether the numbers represent average exposure circumstances or maximum
23 potential exposure. The size of the population considered to be at risk and the distribution of
24 individuals' risks within the population should be given. When risks to a sensitive
25 subpopulation have been identified and characterized, the explanation covers the special
26 characterization of this population.
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The presentation identifies and explains the significant issues encountered when the
data support alternative interpretation, describes the choice made and the scientific or science
policy rationale for the choice.
5.3.2. Strengths and Weaknesses
The risk characterization summarizes the kinds of data brought together in the
analysis and the reasoning upon which the assessment rests. The description conveys the
major strengths and weaknesses of the assessment that arise from availability of data and the
current limits of understanding of the process of cancer causation. Health risk is a function
of the three elements of hazard, dose response, and exposure. Confidence in the results of a
risk assessment is, thus, a function of confidence in the results of the analyses of each
element. The important issues and interpretations of data are explained, and the risk
manager is given a clear picture of consensus or lack of consensus that exists about
significant aspects of the assessment. In addition, the peer-reviewed conclusions of other
governmental or international bodies are provided for information. Whenever more than one
view of the weight of evidence or dose-response characterization is supported by the data and
the policies of these guidelines, and when choosing between them is difficult, the views are
presented together. If one has been selected over another, the rationale is given; if not, both
are presented as plausible alternative results. If a quantitative uncertainty analysis of data is
appropriate, it is presented in the risk characterization; in any case, qualitative discussion of
important uncertainties is appropriate.
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1 APPENDIX A: EXAMPLES OF NARRATIVES
2
3 Narrative # 1
4 CAS#XXXX
5
6 The main kinds of evidence available for consideration of potential human
7 carcinogenicity of this metal conjugated aliphatic phosphonate are animal studies of
8 tumorigenicity, short-term tests of genotoxicity and animal studies of toxicity. No human
9 data are available. The animal studies provide a reasonable basis for understanding the
10 reasons for carcinogenic effects of the compound in laboratory animals and their
11 applicability for judging potential human carcinogenicity.
12 Metal conjugate caused a statistically significant increase in the incidence of urinary
13 bladder hyperplasia and tumors (urinary bladder transitional cell papillomas and carcinomas)
14 in male, but not female, Charles River CD rats at 30,000 ppm in the diet in a long-term
15 study. Some high dose animals had urinary tract stones. No tumors, hyperplasia or stones
16 were seen in two lower dose groups (2,000 and 8,000 ppm) in the same study. A study in
17 Charles River CD-I male and female mice at similar dietary doses to those hi the rat study
18 showed no tumor response or urinary tract effects. A two-year study in dogs at doses up to
19 40,000 ppm showed no urinary tract effects. A major metabolite of metal conjugate that
20 contains the phosphonate moiety caused no tumor response or urinary tract effects in a well-
21 conducted two-year bioassay hi rats.
22 In a 90-day study hi the same rat strain, observations of urinary tract function and
23 histopathology revealed a pattern of physiological changes hi both males and females at doses
24 in the same range as the chronic bioassay (8,000, 30,000, 50,000 ppm). These included
25 proteinuria, increased blood phosphorus and urinary calcium, diuresis, and a sharp drop in
26 urine pH. The calcium effects and diuresis were seen only at doses at or above 30,000 ppm,
27 and were more pronounced hi the males. Histopathology evaluation revealed a pattern of
28 cytotoxicity hi the urinary tract with hyperplasia hi the urinary bladder that was seen at doses
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at or above 30,000 ppm and significantly more extensive in the males. In males, but not
females, urinary bladder stones containing calcium and phosphorus were observed in
association with bladder hyperplasia. Treatment of male rats for at least eight weeks
followed by a return to normal diet for up to 21 weeks showed significant reversibility in the
frequency of both stones and bladder hyperplasia. A three-generation reproductive study hi
rats showed urinary tract toxicity consistent with the results of the 90-day study: stones,
cytotoxicity, and hyperplasia at 24,000 ppm; no urinary tract effects at 6,000 or 12,000 ppm.
Short-term studies including an in vivo mouse micronucleus test, gene mutation tests
in Salmonella, and DNA damage tests hi yeast indicate that metal conjugate is not genotoxic.
Confidence hi the extent and nature of this data set is considered medium for supporting this
conclusion. Metal conjugate is not hi a structural chemical category characterized as having
carcinogenic effects hi the urinary tract.
The results of the tumorigenicity, toxicity, and genotoxicity studies support a
conclusion that the male rat tumors were the result of high dose toxicity hi the urinary tract.
It is clear that formation of stones hi the male rat caused profound hyperplasia which, in
turn, led to tumor formation. The corollary would be: No stones, no tumors.
It is concluded that metal conjugate is likely to be carcinogenic to humans only under
conditions that repeat the pattern of effects seen in the male rat at high doses and not likely
without those conditions. Likewise, it is unlikely to be a human carcinogen under conditions
hi which bladder stone formation is unlikely to develop. Certain observations suggest that
humans are much less sensitive to this particular carcinogenic process than are male rats.
First, of the several species and sexes studied at high doses, only the male rat responded with
effects hi the urinary tract. Second, in rodents, bladder stones of any composition including
inert materials like glass frequently lead to tumor development. Third, there is little
indication that humans with bladder stones have a corresponding tumor response in the
bladder. As the data support the view that carcinogenicity would be secondary to toxicity of
the chemical, it is recommended that metal conjugate dose-response and human exposure
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analyses of high exposure scenarios be developed to support a margin of exposure approach
to human risk characterization.
Uncertainties include (1) incomplete data on the relationship between stone formation
and neoplasia in the human bladder, (2) whether the observations in the rat are biologically
relevant to humans. Overall, the conclusions drawn about the process of carcinogenesis in
the male rat are strongly based. The inference that this process would occur in humans is
weak.
Narrative # 2
CAS# XXX
The potential for human carcinogenicity of aromatic alkene (ar-alkene) cannot be
determined from the available data. However, evaluation of animal studies and short-term
studies on ar-alkene and its metabolite suggest an hypothesis as to potential carcinogenicity
that requires further research, and the chemical is a testing candidate. Epidemiologic studies
performed on ar-alkene are considered inadequate for evaluation.
In a two-year gavage bioassay hi F344/N rats and B6C3F1 mice, male mice showed a
statistically significant, increased incidence of combined adenomas and carcinomas of the
lung. However, the NTP, sponsor of the bioassay, considered the response equivocal
because of experimental design problems. Two inhalation studies hi Sprague-Dawley female
rats showed increased incidence of mammary tumors which was considered equivocal
because of lack of a dose-response trend and high historical incidence of this response.
Studies hi these rats by gavage or ip injection were negative. The metabolite is rapidly
broken down at the pH of the glandular stomach; thus, no internal dose is expected from
administration by this route.
Ar-alkene is mutagenic hi bacterial test systems only with metabolic activation. Both
positive and negative responses were seen hi mammalian cells in vitro. The metabolite is
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clearly mutagenic in numerous in vitro tests. Both ar-alkene and its metabolite produce
inconsistent results in in vivo test systems.
Ar-alkene is fat soluble and readily absorbed by all exposure routes. It is distributed
generally in the body. The kinetics of conversion of ar-alkene to its metabolite and the
bioavailability of the metabolite have not been well characterized. ,If ar-alkene has
carcinogenic potential, the long-term animal studies and the mutagenicity tests suggest that
this potential will depend on the bioavailability and activity of the metabolite. At present,
the data on these factors are not strong enough to support a conclusion. Depending on the
pharmacokinetics of ar-alkene and its metabolite, carcinogenicity may result from
bioavailability of the metabolite at the site of administration or at internal tissues sites.
Further study, preferably by inhalation, is needed.
Narrative #3
CAS# XXX
Information on this chlorinated cyclic hydrocarbon includes case histories of exposed
humans, tumor findings in the liver of mice but not hi rats, and the absence of genotoxic
effects hi short-term tests.
Some persons with aplastic anemia have reported exposure to the chemical; some of
these people have also developed leukemia. No other information on humans exists as to
potential carcinogenic effects.
Multiple feeding studies in males and females of several strains of mice indicate that
exposure to the chemical leads to hepatocellular tumors. Tumors were not found at other
anatomical sites in mice or at any site in two chronic rat feeding studies.
The chemical is a potent inducer of liver microsomal enzymes at doses associated
with liver cancer in mice. Chemical administration leads to liver enlargement due hi part to
increases in liver cell size; this enlargement persists until tumor development. The extent to
which the chemical leads to increases in cell proliferation has not been established. Some
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1 studies indicate that the chemical may increase the amount of reactive oxygen radicals and
2 lipid peroxidation at high doses. It promotes liver tumors in mice following initiation by a
3 genotoxic agent at doses associated with tumors from exposure to the chemical alone.
4 Testing for genotoxicity has generally led to negative findings, and there are no
5 structural alerts. The chemical does not increase the frequency of gene mutations in bacteria,
6 fungi and algae; there is a questionable increase in cultured mouse lymphoma cells. No or
7 marginal increases in structural aberrations were noted in two studies of cultured Chinese
8 hamster cells and in a study of Syrian hamster bone marrow cells in vivo; there was no
9 increase in sister chromatid exchanges in the cultured hamster cells.
10 Uncertainties include the finding that the only indication of a carcinogenic response
11 comes from the mouse liver, the most common cancer site in rodents and the one that is
12 uniquely positive with many other chlorinated hydrocarbons. Other sites in the mouse and
13 all of them in the rat fail to indicate a carcinogenic potential. There is no significant
14 genotoxic potential or structural indicators of carcinogenicity. Cancer responses are
15 associated with high doses that lead to induction of liver microsomal enzymes and potential
16 reactive oxygen species.
17 It is hard to interpret the carcinogenic findings; there are cancer responses in the
18 mouse liver, but they occur at high doses of this nongenotoxic compound that are associated
19 with demonstrable effects on the liver, doses that are far above anticipated human exposures.
20 Overall, the evidence is on the border between a likely human carcinogen and cannot be
21 determined. Further mechanistic work is needed to differentiate these alternatives. Until
22 such time, it will be assumed that the chemical is likely to be a human carcinogen. Cancer
23 risks should be evaluated by a margin of exposure evaluation.
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Case Study # 4
CAS# XXX
There are no tumor data on this bis-benzenamine. Evaluation of its carcinogenic
potential comes from tumor studies on structural analogues and toxicity information on the
chemical and its analogues. The chemical is considered likely to be carcinogenic to humans
by all routes of exposure. Mechanistic information helps to associate findings in animals
with those in exposed humans.
Close structural analogues produce thyroid follicular cell tumors and hepatocellular
tumors hi rats and mice following ingestion. The thyroid tumors are associated with known
perturbations in thyroid-pituitary functioning. These compounds inhibit the accumulation of
iodide into the thyroid gland, apparently due to inhibition of the enzyme that synthesizes the
thyroid hormones. 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 in rats is significantly influenced t>y disruption in the
thyroid-pituitary axis.
Human and rat thyroid glands respond similarly to short-term exposure. Worker
exposure has not been well characterized or quantified, but recent medical monitoring of
workers exposed over a several year period has uncovered decreases in thyroid hormones,
increases hi TSH and symptoms of hypothyroidism. A urinary metabolite of the chemical
has been monitored in the workers. Its concentration was only about 2-fold lower than that
hi rats receiving the chemical for 28-days, which led to similar changes in thyroid and
pituitary hormones. In addition, the dose of the chemical given to rats hi this study was
essentially the same as that of an analogue that had produced thyroid tumors hi rats.
Although the human thyroid responds as does that of rodents following limited exposure, it is
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1 not well established that thyroid-pituitary imbalance leads to cancer in humans as it does in
2 rodents.
3 The chemical is an aromatic amine, a member of a class of chemicals that has
4 regularly produced carcinogenic effects in rodents; some have produced cancer in humans.
5 Structural analogues are genotoxic; they produce gene mutations in cultured bacteria and
6 mammalian cells and structural chromosome aberrations and DNA damage in mammalian
7 cultured cells; these are characteristics of chemicals that produce cancer hi multiple
8 anatomical sites and species. The genotoxic effects could influence the development of
9 tumors in the thyroid and liver.
10 Given the information at hand on potential thyroid and liver tumors hi rodents among
11 close structural analogues, it is reasonable to conclude that the chemical is likely to be a
12 human carcinogen. Biological information on the compound leads one hi differing directions
13 as to how to quantitate potential cancer risks. The information on disruption on thyroid-
14 pituitary status argues for using a margin of exposure evaluation, whereas that bearing on the
15 chemical's being an aromatic amine and having gene and structural chromosome mutation
16 activity, coupled with a lack of mode of action information on the liver tumors, points
17 toward a low dose linear approach. In recognition of these differences, it is appropriate to
18 quantitate thyroid tumors using both a nonlinear or margin of exposure and linear techniques;
19 liver tumors should be evaluated by the linear approach. Tumor data on the close analogues
20 should be utilized in projections of potential risks from exposure. Given the absence of
21 direct tumor information on the chemical per se, potential risks should only be presented as
22 screening estimates with rough order-of-magnitude values. The chemical can be absorbed by
23 the oral, inhalation and dermal routes of exposure.
24 Uncertainties include (1) the lack of carcinogenicity studies on the chemical, (2) the
25 employment of data on structural analogs, (3) the lack of established information on the
26 relevance of thyroid-pituitary unbalance for human carcinogenicity, and (4) the different
27 potential mechanisms that may influence tumor development and potential risks. Overall,
28 this is a strong inferential case for potential human carcinogenicity.
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Narrative #5
CAS#XXXX
The main evidence available for consideration of potential human carcinogenicity of
chlorinated alkene (cl-alkene) includes animal studies of tumorigenicity, short-term tests of
genotoxicity, and studies of metabolism and toxicity of this and related chlorinated aliphatics.
Epidemiologic studies have been made of workers exposed to cl-alkene, but the studies do
not demonstrate either an association or lack of one between cancer and exposure to cl-
alkene. The chemical is considered likely to be carcinogenic to humans by all routes of
exposure.
Administration of cl-alkene by gavage (corn oil vehicle) or by inhalation to B6C3F1
mice in long-term studies caused statistically significant, dose-related increases in
hepatocellular carcinomas hi both sexes as well as reduced time-to-tumor hi the gavage
studies. Doses were 0, 100, 200 ppm hi the inhalation study; time-weighted average doses
hi the gavage studies were 536 or 1072 mg/kg and 300 or 600 mg/kg to male and female
mice, respectively. In long-term inhalation studies hi F344/N rats (0, 200, 400 ppm)
tubular cell hyperplasia was observed hi male rats and hi one high dose female. Renal tubule
neoplasms were observed in male rats. The response was not statistically significant by
pairwise comparison, but there was a statistically significant, positive dose-response trend.
The historical control incidence of renal tubule tumor in F344/N males is less than 1/2%.
The probability of observing two such rare tumors in 50 animals is less than 0.001. A
second response observed hi the rat study was an increased incidence of mononuclear cell
leukemia. Analyses of the incidence of disease in the study and supplementary analyses of
the progression of the disease showed that there was a statistically significant, increased
incidence in low dose males and females, in high dose males and, marginally, hi high dose
females. In addition, there was a significant increase in extent of the disease hi treated
animals of both sexes and a significant shortening of the tune of disease onset in females. In
long-term studies hi Sprague-Dawley rats by gavage and inhalation, slight, but not
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statistically significant, increases in kidney tumors were noted; there were no observations of
mononuclear cell leukemia. In chronic and subchronic studies, cl-alkene has demonstrated
nephrotoxicity in both rats and mice.
Cl-alkene has been tested in numerous test systems for mutagenic effects; results
indicate that the compound is not genotoxic. Primary metabolites of cl-alkene also test as not
genotoxic. A pathway of metabolism identified in the rat generates a secondary mutagenic
metabolite in the rat kidney at high doses; this pathway is also active in humans. In general,
cl-alkene shares chemical characteristics of related chlorinated ethanes and ethenes in toxicity
and tumorigenicity particularly, but not exclusively, toward the mouse liver and rat kidney,
and in lack of DNA reactivity or mutagenicity in short-term studies. Similarly, the primary
metabolites of these compounds are not notably genotoxic.
More than one mechanism has been proposed by which cl-alkene might cause each
tumor response; available data do not clearly support any of the various mechanistic views.
If a single mechanism accounts for the compound's activity, it is not genotoxicity. The
mononuclear leukemia and liver responses in rodents suggest a general accelerating
influence on underlying neoplastic processes. The kidney response might be associated with
the toxic effects of cl-alkene in the kidney or with mutagenic activity of the secondary
metabolite; the data do not show an answer. As a whole the data do not point to the linearity
at low doses generally expected of mutagenic compounds, nor are the data strong enough to
describe how cl-alkene might have a threshold or non-linear dose response relationship at low
doses. It is recommended that two assessments of dose response and of exposure to cl-
alkene be developed; one using a model that assumes linearity at low dose, a second to
support a margin of exposure approach to human risk characterization.
Uncertainties include (1) how the compound acts to influence carcinogenic processes
in animals, and how it might act in humans, and (2) implications of the data for dose
response assessment.
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Narrative # 6
CAS#XXX
Human and animal studies indicate that this metal is likely to be carcinogenic to
humans by inhalation and not likely to be carcinogenic via oral or dermal exposure.
Absorption of compounds of the metal varies significantly by route of exposure: inhalation
> oral > > dermal. Toxic mechanisms are not established, but interference with essential
metals and complexes with certain proteins may be operative. There is some evidence for
genotoxic effects in cultured cells, but it is not expected that the metal would directly react
with DNA.
Several epidemiologic studies of workers exposed to the metal in the air have been
conducted. Increases in lung and prostatic cancer have been reported in some but not all
studies. Interpretation is hindered by the presence of concomitant confounding workplace
exposures (e.g., arsenic) and influences of smoking.
In the only rodent inhalation study, rats developed significantly increased incidences
of lung cancer. In contrast to these findings, multiple feeding and drinking water studies in
rodents have failed to indicate carcinogenic responses from daily doses up to over 100-times
those in the inhalation study. Increases in testicular interstitial cell tumors have been found
in rodent injection studies of soluble metal salts. These compounds lead to necrosis of the
testicular germ cell epithelium due to changes in the blood supply with corresponding
increases in hormones that stimulate the interstitial cells; such studies are not considered to
be applicable to human exposures.
Compounds of the metal vary significantly in their solubility: the metal and many
salts are very insoluble, whereas a few of the salts are readily soluble. Following inhalation,
about 50% of the metal is absorbed into the body, while only about 5% is absorbed from the
gut (10-fold lower than via inhalation). Dermal absorption is insignificant.
The metal may have genotoxic potential as well -as other toxic properties; both should
be considered in evaluating potential risks of exposure. It competes and interferes with the
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1 functioning of a number of requked metals; it also can bind to certain parts of proteins
2 (sulfhydryl groups) and interfere with their activity. In genotoxicity studies, it is generally
3 negative for gene mutations hi bacteria but positive hi cultured mammalian cells. Although
4 conflicting observations for structural chromosome aberrations have been found in human
5 lymphocytes cultured from exposed workers and hi cell lines exposed in culture, negative
6 findings have been noted hi mouse bone marrow studies. It is not expected that the metal
7 would directly interact with and damage cellular DNA. The evidence does not support a
8 conclusion as to the mode of action; it is recommended that both a linear and nonlinear or
9 margin of exposure approach be taken to dose-response assessment.
10
11 Narrative # 7
12 CAS # XXX
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14 There is strong evidence that short-chain alkene (SCA) is likely to be a human
15 carcinogen via the inhalation pathway. This conclusion is based on an inhalation animal
16 bioassay (involving both sexes of two species) of SCA showing an unambiguous positive
17 responses and is supported by genotoxicity and metabolic data. We can infer that SCA is
18 also likely to be a human carcinogen for the ingestion and dermal pathways. Some human
19 studies were positive, but methodological problems make them inconclusive.
20 In the animal bioassay, rats and mice were exposed hi air on a schedule simulating
21 occupational exposure. Exposure levels were 625 and 1,250 ppm for mice, and 1,000 and
22 8,000 ppm for rats. Statistically significant increases hi tumor rates were observed hi
23 multiple sites hi both sexes of both species. In the case of the mice, early high mortality due
24 to the tumors was observed.
25 SCA and its metabolites are positive in several genotoxicity tests, including mutage-
26 nicity tests hi bacteria with metabolic activation. Several metabolites of SCA are alkylating
27 agents and are directing acting mutagens hi bacterial tests. Moreover, metabolism experi-
28 ments hi both rats and mice using radio-labeled SCA show binding to nucleoproteins and
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DNA. SCA is also structurally related to other compounds which are positive in animal -bio-
assays.
There have been five occupational epidemiology studies involving inhalation exposure
to SCA. Three of them showed statistically significant risks of cancer; however, hi two of
them, workers were also exposed to solvents and the third did not control for smoking. The
two negative studies were flawed by low power and possible bias. Thus, the human studies
considered together are inconclusive.
The direct evidence for carcinogenicity of SCA is based on inhalation exposure.
However, since the observed tumors in the animal bioassay occurred at several sites distant
from the portal of entry, and since the compound can be absorbed through the
gastrointestinal tract and the skin, we assume that it is also likely to be a human carcinogen
through these pathways as well.
Based on pharmacokinetic, metabolism and genotoxicity data, linear extrapolation of
response to low doses is felt to be appropriate. Studies of the pharmacokinetics of SCA hi
rats and mice indicate first-order kinetics below a saturation level. Metabolites of SCA are
genotoxic and are likely to be carcinogenic, and, although the metabolic pathway is
saturable, tumors were observed in the bioassay at exposure levels lower than the saturation
level.
Although the case for carcinogenicity of SCA is very strong, there are some
uncertainties. Since the epidemiological data are inconclusive, we face the usual
uncertainties in extrapolating from animals to humans. We have an unusually strong set of
mechanistic data supporting linear extrapolation, but uncertainties hi extrapolating from high
to low doses remain, as always.
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1 Narrative # 8
2 CAS # XXX
3
4 Organophosphate has been tested in several animal bioassays, one of which produced
5 positive results but was possibly flawed, and others which were negative or provide limited
6 support for those results. Genotoxicity and metabolism data also include some that support a
7 concern about carcinogenicity but do not constitute a consistent pattern. No human data are
8 available. Thus, from the data, one cannot determine the likelihood of human
9 carcinogenicity.
10 Three chronic feeding studies have been performed with rats. In a study with
11 Osborne-Mendel rats, statistically significant increases were observed hi combined benign
12 and malignant kidney tumors in both dose groups for males and hi high-dose females, as
13 compared with pooled controls. A statistically significant trend hi malignant pancreatic
14 tumors and benign thyroid tumors was found hi males only. Concerns were raised in audits
15 of the test facility where this study was performed. In a second study with Sprague-Dawley
16 rats, a non-statistically-significant increase hi benign thyroid tumors was observed in males.
17 In a third study with Wistar rats, a statistically significant increasing trend was observed in
18 benign pancreatic tumors in males.
19 Two studies have been performed with mice. In the first, no excess of tumors was
20 found, but the study was flawed. In the second, statistically significant increases were
21 observed of benign lung tumors in males and of malignant lymphomas in females, both at a
22 low dose, but similar increases were not found at two higher doses.
23 Evaluating the rat data together, we see that the strongest result was observed in the
24 Osborne-Mendel rat study, about which doubts have been raised hi an audit. In the Sprague-
25 Dawley rat study, an increase, although not statistically significant, was observed in the same
26 type of tumor hi the same sex as one of the Osborne-Mendel rat responses. Similarly, in the
27 Wistar rat study, a statistically significant trend was observed hi the same sex and site as one
28 of the Osborne-Mendel rat responses, although only benign tumors were seen in this study.
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The latter two studies have limited weight in reaching a conclusion that the compound is
carcinogenic in humans, since only benign tumors were found, but they tend to confirm the
results of the first study because the tumors were observed hi the same sites. The sort of
problems noted hi the audit of the facility where the Osborne-Mendel rat study was
performed - poor record keeping, possible misdosing of the animals - are more likely to
mask a positive result than to create a false positive, but they do raise a concern about
reliance on the results.
The two studies hi mice can be discounted, the first because of flawed methodology,
the second because the response was seen only at one low dose.
In genotoxicity testing, the compound was found to cause unscheduled DNA
synthesis, but other tests were negative or (hi one case) equivocal. This forms the basis for
some concern, but is not strong enough or consistent enough to contribute significantly to a
judgment on human carcinogenicity. The compound is not structurally related to known
human or animal carcinogens.
In summary, there are one positive, but possibly flawed, animal feeding study and
two other animal studies showing weakly positive results hi the same sites. No human data
are available, and the other data are inconclusive. This set of data as a whole does not
support a determination of human carcinogenic potential.
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3S-U.S. GOVERNMENT PRINTING OFFICE: 1994 - 550-064/80017
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