DRAFT NCEA-F-0644

July 1999

Review Draft

Guidelines for

Carcinogen Risk Assessment

Risk Assessment Forum

U.S. Environmental Protection Agency

Washington, DC
7/02/99 ii DRAFT

PURPOSE OF THIS DOCUMENT 

The discussion in this document is intended solely as guidance. It is not a regulation, and

does not confer legal rights or impose legal obligations on EPA, States, Tribes, local

governments, regulated entities or any member of the public. 

The predominant guidance provided in this document is for EPA risk assessors to use the 

best science and risk assessment techniques available to them at the time a cancer risk

assessment is conducted. Any final cancer risk assessment may take an approach different from

that described in this document based on factors such as evolving science, the facts of a

particular case, or comments from peer reviewers, the public or others. 
7/02/99 iii DRAFT

GUIDELINES FOR

CARCINOGEN RISK ASSESSMENT

FRL1 [To Be Developed]

2
7/02/99 iv DRAFT

1 CONTENTS

2

3 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

4

5 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

6

7 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

8 1.1. PURPOSE AND SCOPE OF THE GUIDELINES . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

9 1.2. ORGANIZATION AND APPLICATION OF THE GUIDELINES . . . . . . . . . . . . 1-2

10 1.2.1. Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

11 1.2.2. Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

12 1.3. USE OF DEFAULT ASSUMPTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

13 1.3.1. Default Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

14 1.3.2. Major Defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10

15 1.4. CHARACTERIZATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-19

16

17 2. HAZARD ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

18 2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION . . . . . 2-1

19 2.1.1. Analyses of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

20 2.1.2. Presentation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

21 2.2. ANALYSIS OF TUMOR DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

22 2.2.1. Human Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

23 2.2.1.1. Types of Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

24 2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic Studies . . . . . 2-4

25 2.2.1.3. Criteria for Causality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

26 2.2.1.4. Assessment of Evidence of Carcinogenicity from Human Data . 2-8

27 2.2.2. Animal Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

28 2.2.2.1. Long-Term Carcinogenicity Studies . . . . . . . . . . . . . . . . . . . . . . . 2-9

29 2.2.2.2. Perinatal Carcinogenicity Studies . . . . . . . . . . . . . . . . . . . . . . . . 2-15

30 2.2.2.3. Other Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16

31 2.2.3. Structural Analogue Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17

32 2.3. ANALYSIS OF OTHER KEY DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17

33 2.3.1. Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17

34 2.3.2. Structure-Activity Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
CONTENTS (continued)

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1 2.3.3. Comparative Metabolism and Toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . 2-19

2 2.3.4. Toxicological and Clinical Findings .............................. 2-20

3 2.3.5. Events Relevant to Mode of Carcinogenic Action . . . . . . . . . . . . . . . . . . . 2-21

4 2.3.5.1. Direct DNA Reactive Effects ........................... 2-21

5 2.3.5.2. Indirect DNA Effects or Other Effects on Genes/Gene 

6 Expression .......................................... 2-22

7 2.3.5.3. Experimental Considerations in Evaluating Data

8 on Precursor Events .................................. 2-24

9 2.3.5.4. Judging Data ........................................ 2-24

10 2.4. BIOMARKER INFORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-25

11 2.5. MODE OF ACTION-GENERAL CONSIDERATIONS AND FRAMEWORK 

12 FOR ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26

13 2.5.1. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26

14 2.5.2. Evaluating a Postulated Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28

15 2.5.3. Framework for Evaluating a Postulated Carcinogenic 

16 Mode(s) of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29

17 2.5.3.1. Content of the Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-30

18 2.6. WEIGHT-OF-EVIDENCE EVALUATION FOR POTENTIAL HUMAN

19 CARCINOGENICITY .............................................. 2-34

20 2.6.1. Weight-of-Evidence Analysis ................................... 2-35

21 2.6.2. Descriptors for Summarizing Weight of Evidence ................... 2-43

22 2.7. TECHNICAL HAZARD CHARACTERIZATION ........................ 2-45

23 2.8. WEIGHT-OF-EVIDENCE NARRATIVE ............................... 2-47

24

25 3. DOSE-RESPONSE ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

26 3.1. HUMAN STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

27 3.2. MODE OF ACTION AND DOSE-RESPONSE APPROACH . . . . . . . . . . . . . . . . 3-2

28 3.3. DOSE-RESPONSE ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

29 3.3.1. Modeling the Overall Process--Biologically Based Models . . . . . . . . . . . . . 3-4

30 3.3.2. Analysis in the Range of Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

31 3.3.2.1. Applying Information About Key Events . . . . . . . . . . . . . . . . . . . 3-5

32 3.3.2.2. Procedures for Analysis in the Range of Observation of Animal

33 Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
CONTENTS (continued)

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1 3.3.2.3. Point of Departure for Extrapolation from Observed 

2 Animal Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

3 3.3.3. Analysis in the Range of Extrapolation--Default Procedures . . . . . . . . . . . . 3-9

4 3.3.3.1. Linear Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

5 3.3.3.2. Nonlinear Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

6 3.3.3.3. Linear and Nonlinear Extrapolations . . . . . . . . . . . . . . . . . . . . . 3-14

7 3.3.3.4. Use of Toxicity Equivalence Factors and Relative Potency

8 Estimates ........................................... 3-14

9 3.4. RESPONSE DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

10 3.5. DOSE DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17

11 3.5.1. Interspecies Adjustment of Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

12 3.5.2. Adjustment of Dose for Children ................................ 3-18

13 3.5.3. Toxicokinetic Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

14 3.5.4. Route-to-Route Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

15 3.5.5. Dose Averaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-22

16 3.6. DISCUSSION OF UNCERTAINTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-23

17 3.7. TECHNICAL DOSE-RESPONSE CHARACTERIZATION . . . . . . . . . . . . . . . . 3-24

18

19 4. TECHNICAL EXPOSURE CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

20

21 5. RISK CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

22 5.1. PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

23 5.2. APPLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2

24 5.3. PRESENTATION OF RISK CHARACTERIZATION SUMMARY . . . . . . . . . . . 5-3

25 5.4. CONTENT OF RISK CHARACTERIZATION SUMMARY . . . . . . . . . . . . . . . . 5-3

26 6. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

27

28 APPENDIX A. WEIGHT-OF-EVIDENCE NARRATIVES . . . . . . . . . . . . . . . . . . . . . . . . . A-1

29

30 APPENDIX B. RESPONSES TO THE NATIONAL ACADEMY OF SCIENCES 

31 NATIONAL RESEARCH COUNCIL REPORT SCIENCE AND JUDGMENT IN RISK

32 ASSESSMENT (NRC, 1994) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

33
CONTENTS (continued)

7/02/99 vii DRAFT

1 APPENDIX C. CASE STUDY EXAMPLES FOR HAZARD EVALUATION . . . . . . . . . . . C-1

2

3 APPENDIX D. CASE STUDY EXAMPLES FOR MODE-OF-ACTION EVALUATION . D-1

4

5 APPENDIX E. NONLINEAR DOSE-RESPONSE: MARGIN-OF-EXPOSURE 

6 ANALYSIS (TO BE DEVELOPED) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

7

8 APPENDIX F. DOSE-RESPONSE ASSESSMENT FOR A CARCINOGEN POSING 

9 HIGHER RISKS AFTER CHILDHOOD EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1

10

11 APPENDIX G. RESPONSE TO COMMENTS ON OTHER SCIENCE ISSUES (TO BE

12 DEVELOPED) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1

13

14
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1 LIST OF TABLES

2

3

4 Table D-1. Thyroid follicular cell tumor incidence in male rats . . . . . . . . . . . . . . . . . . . . D-2

5 Table D-2. Incidence of transitional cell lesions and stones

6 in males from a 2-year SD rat study ............................... D-11

7 Table D-3. Incidence of bladder hyperplasia and stones in male SD rats

8 treated up to 13 weeks .......................................... D-13

9 Table D-4. Reversal of incidence of bladder hyperplasia and stones following

10 8 weeks treatment and 16 weeks recovery ........................... D-15

11 Table D-5. Clinical chemistry values (urine) in male SD rats treated up to 13 weeks . . D-17

12 Table D-6. Summary results of chronic bioassays .............................. D-25

13 Table F-1. Comparison of tumor incidence in male and female Sprague-Dawley rats

14 from 100 hour inhalation exposures to newborn and mature rats .......... . F-6

15 Table F-2 Comparison of tumor incidence in male and female Sprague-Dawley rats

16 From 5-wk newborn exposure and 52-wk later life exposure . . . . . . . . . . . . . . F-7

17 Table F-3 Results of PBPK modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-8
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1 LIST OF FIGURES

2

3

4 Figure 1-1. Risk characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-20

5 Figure 2-1. Factors for weighing human evidence ............................... 2-36

6 Figure 2-2. Factors for weighing animal evidence ............................... 2-38

7 Figure 2-3. Factors for weighing other key evidence ............................. 2-40

8 Figure 2-4. Factors for weighing totality of evidence ............................ 2-42

9 Figure 3-1. Graphical presentation of data and extrapolation ....................... 3-8
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1. INTRODUCTION

1.1. PURPOSE AND SCOPE OF THE GUIDELINES

1 These guidelines revise and replace United States Environmental Protection Agency

2 (EPA) Guidelines for Carcinogen Risk Assessment published in 51 FR 33992, September 24,

3 1986. The guidelines provide EPA staff and decision makers with guidance for developing and

4 using risk assessments. They also provide basic information to the public about the Agency's risk

5 assessment methods. These guidelines are used with other risk assessment guidelines that the

6 Agency has developed, such as the Mutagenicity Risk Assessment Guidelines (U.S. EPA, 1986c).

7 and the Exposure Assessment Guidelines (U.S. EPA, 1992a). Consideration of other Agency

8 guidance documents is particularly important when procedures for evaluating specific target organ

9 effects have been developed (e.g., assessment of thyroid follicular cell tumors (U.S. EPA,

10 1998a)), or when there is a concern for a particular sensitive subpopulation for which the Agency

11 has developed guidance, for example, EPA Guidelines for Developmental Toxicity Risk

12 Assessment (U.S. EPA, 1991d). These guidelines discuss hazards to children that may result

13 from exposures during preconception, prenatal, or postnatal development to sexual maturity. 

14 Similar guidelines exist for Reproductive Toxicant Risk Assessment (U.S. EPA, 1996c) and for

15 Neurotoxicity Risk Assessment (U.S. EPA, 1998c). All of these guidelines should be consulted

16 when conducting a risk assessment in order to insure that information from studies on

17 carcinogenesis and other health effects are considered together in the overall characterization of

18 risk. This is particularly true in the case in which a precursor effect to tumor is also a precursor

19 or endpoint of other health effects and is used in dose-response assessment. The overall

20 characterization of risk will be the basis for carrying out assessments of instances in which fetuses,

21 infants, or children are at risk or disproportionately affected by economically significant Agency

22 actions. Characterization for the latter purpose is outlined in the Agency guidance by the Office

23 of Children’s Health Protection to carry out E.O. 13045, “Protection of Children From

24 Environmental Health Risks and Safety Risks” issued on April 21, 1997.

25 The guidelines encourage both regularity in procedures to support consistency in scientific

26 components of Agency decision making and innovation to remain up-to-date in scientific thinking.

27 In balancing these goals, the Agency relies on established scientific peer review processes (EPA,

28 1998b). The guidelines incorporate basic principles and science policies based on evaluation of

29 the currently available information. As more is discovered about carcinogenesis, the need will

30 arise to make appropriate changes in risk assessment guidance. The Agency will revise these

31 guidelines when extensive changes are due. In the interim, the Agency will issue special reports,
1

The term "agent" refers generally to any chemical substance, mixture, or physical or biological

entity being assessed, unless otherwise noted (See sec. 1.2.2 for a note on radiation.).

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1 after appropriate peer review, to supplement and update guidance on single topics, (e.g., U.S.

2 EPA, 1991b). The incorporation of new, peer-reviewed scientific understanding and data in an

3 assessment is always consistent with the purposes of these guidelines. 

4 1.2. ORGANIZATION AND APPLICATION OF THE GUIDELINES

5 1.2.1. Organization

6 Publications of the Office of Science and Technology Policy (OSTP, 1985) and the

7 National Research Council (NRC, 1983, 1994) provide information and general principles about

8 risk assessment. Risk assessment uses available scientific information on the properties of an

agent1 9 and its effects in biological systems to provide an evaluation of the potential for harm as a

10 consequence of environmental exposure. The 1983 and 1994 NRC documents organize risk

11 assessment information into four areas: hazard identification, dose-response assessment,

12 exposure assessment, and risk characterization. This structure appears in these guidelines, which

13 additionally emphasize characterization of evidence and conclusions in each part of the

14 assessment. In particular, the guidelines adopt the approach of the NRC's 1994 report in adding a

15 dimension of characterization to the hazard identification step. Added to the identification of

16 hazard is an evaluation of the conditions under which its expression is anticipated. The risk

17 assessment questions addressed in these guidelines are:

18 C For hazard--Can the agent present a carcinogenic hazard to humans, and if so,

19 under what circumstances?

20 C For dose-response--At what levels of exposure might effects occur?

21 C For exposure--What are the conditions of human exposure?

22 C For risk--What is the character of the risk? How well do data support conclusions

23 about the nature and extent of the risk?

24 1.2.2. Application

25 The guidelines apply within the framework of policies provided by applicable EPA statutes

26 and do not alter such policies. The guidelines cover assessment of available data. They do not

27 imply that one kind of data or another is prerequisite for regulatory action concerning any agent. 

28 Risk management applies directives of regulatory legislation, which may require consideration of

29 potential risk, or solely hazard or exposure potential, along with social, economic, technical, and
7/02/99 1-3 DRAFT

1 other factors in decision making. Risk assessments support decisions, but to maintain their

2 integrity as decision making tools, they are not influenced by consideration of the social or

3 economic consequences of regulatory action.

4 The assessment of risk from radiation sources is based on continuing examination of

5 human data by the National Academy of Sciences/National Research Council in its series of

6 numbered reports: “Biological Effects of Ionizing Radiation”. While the general principles of

7 these guidelines apply to radiation risk assessments, their details are most focused on other kinds

8 of agents. They do not attempt to guide the ongoing conduct of radiation risk assessment.

9 Not every EPA assessment has the same scope or depth. Agency staff often conduct

10 screening-level assessments for priority-setting or separate assessments of hazard or exposure for

11 ranking purposes or to decide whether to invest resources in collecting data for a full assessment. 

12 Moreover, a given assessment of hazard and dose-response may be used with more than one

13 exposure assessment that may be conducted separately and at different times as the need arises in

14 studying environmental problems in various media. The guidelines apply to these various

15 situations in appropriate detail given the scope and depth of the particular assessment. For

16 example, a screening assessment may be based almost entirely on structure-activity relationships

17 and default assumptions. As more data become available, assessments can replace or modify

18 default assumptions accordingly. These guidelines do not require that all of the kinds of data

19 covered here be available for either assessment or decision making. The level of detail of an

20 assessment is a matter of Agency management discretion regarding applicable decision making

21 needs.

22 1.3. USE OF DEFAULT ASSUMPTIONS

23 The National Research Council, in its 1983 report on the science of risk assessment (NRC,

24 1983), recognized that default assumptions are necessarily made in risk assessments where gaps

25 exist in general knowledge or in available data for a particular agent. These default assumptions

26 are inferences based on general scientific knowledge of the phenomena in question and are also

27 matters of policy concerning the appropriate way to bridge uncertainties that concern potential

28 risk to human health (or, more generally, to environmental systems) from the agent under

29 assessment. 

30 EPA's 1986 guidelines for cancer risk assessment (EPA, 1986b) were developed to be

31 responsive to the principles of the 1983 NRC report. The guidelines contained a number of

32 default assumptions. They also encouraged research and analysis that would lead to new risk

33 assessment methods and data and anticipated that these would replace defaults. The 1986
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1 guidelines did not explicitly discuss how to depart from defaults. 

2 In its 1994 report on risk assessment, the NRC supported continued use of default

3 assumptions (NRC, 1994). The NRC report thus validated a central premise of the approach to

4 risk assessment that EPA had evolved in preceding years--the making of science policy inferences

5 to bridge gaps in knowledge--while at the same time recommending that EPA develop more

6 systematic and transparent guidelines to inform the public of the default inferences EPA uses in

7 practice. It recommended that the EPA review and update the 1986 guidelines in light of

8 evolving scientific information and experience in practice in applying those guidelines, and that the

9 EPA explain the science and policy considerations underlying current views as to the appropriate

10 defaults and provide general criteria to guide preparers and reviewers of risk assessments in

11 deciding when to depart from a default. 

12 1.3.1. Default Assumptions

13 The 1994 NRC report contains several recommendations regarding flexibility and the use

14 of default options:

15 C EPA should continue to regard the use of default options as a reasonable way to

16 deal with uncertainty about underlying mechanisms in selecting methods and

17 models for use in risk assessment.

18 C EPA should explicitly identify each use of a default option in risk assessments.

19 C EPA should clearly state the scientific and policy basis for each default option.

20 C The Agency should consider attempting to give greater formality to its criteria for

21 a departure from default options in order to give greater guidance to the public and

22 to lessen the possibility of ad hoc, undocumented departures from default options

23 that would undercut the scientific credibility of the Agency's risk assessments. At

24 the same time, the Agency should be aware of the undesirability of having its

25 guidelines evolve into inflexible rules.

26 C EPA should continue to use the Science Advisory Board and other expert bodies. 

27 In particular, the Agency should continue to make the greatest possible use of peer

28 review, workshops, and other devices to ensure broad peer and scientific

29 participation to guarantee that its risk assessment decisions will be based on the

30 best science available through a process that allows full public discussion and peer

31 participation by the scientific community.

32 In the 1983 report (p. 28), NAS defined the use of "inference options" (default options) as

33 a means to bridge inherent uncertainties in risk assessment. These options exist when the
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1 assessment encounters either "missing or ambiguous information on a particular substance" or

2 "gaps in current scientific theory." Since there is no instance in which a set of data on an agent or

3 exposure is complete, all risk assessments must use general knowledge and policy guidance to

4 bridge data gaps. Animal toxicity data are used, for example, to substitute for human data

5 because we do not test human beings. The report described the components of risk assessment in

6 terms of questions encountered during analysis for which inferences must be made. The report

7 noted (p. 36) that many components ". . . lack definitive scientific answers, that the degree of

8 scientific consensus concerning the best answer varies (some are more controversial than others),

9 and that the inference options available for each component differ in their degree of conservatism. 

10 The choices encountered in risk assessment rest, to various degrees, on a mixture of scientific fact

11 and consensus, on informed scientific judgment, and on policy determinations (the appropriate

12 degree of conservatism). . . ." The report did not note that the mix varies significantly from case

13 to case. For instance, a question that arises in hazard identification is how to use experimental

14 animal data when the routes of exposure differ between animals and humans. A spectrum of

15 inferences could be made: The most protective, or risk adverse one is that effects in animals from

16 one route may be seen in humans by another route. An intermediate one is a conditional inference

17 that such translation of effects will be assumed if the agent is absorbed by humans through the

18 second route. A nonprotective one that no inference is possible and the agent's effects in animals

19 must be tested by the second route. The choice of an inference, as the report observed, comes

20 from more than scientific thinking alone. While the report focused mainly on the idea of

21 conservatism of public health as a science policy rationale for making the choice, it did not

22 evaluate other considerations. 

23 These revised guidelines retain the use of default assumptions as recommended in the

24 1994 report. Since the primary goal of EPA actions is public health protection and that,

25 accordingly, as an Agency policy, the defaults used in the absence of scientific data to the contrary

26 have been chosen to be health protective. The defaults described below remain public health

27 conservative when applied in combination in risk assessment, however, any individual default 

28 may not constitute the most conservative position vis-a-vis that position. To do so would lead to 

29 risk assessments that far exceed the actual risks and this would not be in keeping with the

30 principles discussed in the NAS 1994 report. 

31 In addition, the guidelines reflect evaluation of experience in practice in applying defaults

32 and departing from them in individual risk assessments conducted under the 1986 guidelines. The

33 application and departure from defaults and the principles to be used in these judgments have been

34 matters of debate among practitioners and reviewers of risk assessments. The guidelines here are
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1 intended to be both explicit and more flexible than in the past concerning the basis for making

2 departures from defaults, recognizing that expert judgment and peer review are essential elements

3 of the process. 

4 In response to the recommendations of the 1994 report, these guidelines call for

5 identification of the default assumptions used within assessments and for highlighting significant

6 issues about defaults within characterization summaries of component analyses in assessment

7 documents. As to the use of peer review to aid in making judgments about applying or departing

8 from defaults, we agree with the NRC recommendation. The Agency has long made use of

9 workshops, peer review of documents and guidelines, and consultations as well as formal peer

10 review by the Science Advisory Board (SAB). In 1998, the Administrator of EPA published a

11 peer review guidance for EPA scientific work products that increases the amount of peer review

12 for risk assessments as well as other work, continuing a series of guidance actions in response to

13 the NRC report and to SAB recommendations (U.S. EPA, 1994b, 1997b, 1998b).

14 The 1994 NRC report recommended that EPA should consider adopting principles or

15 criteria that would give greater formality and transparency to decisions to depart from defaults. 

16 The report named several possible criteria for such principles (p. 7): ". . . [P]rotecting the public

17 health, ensuring scientific validity, minimizing serious errors in estimating risks, maximizing

18 incentives for research, creating an orderly and predictable process, and fostering openness and

19 trustworthiness. There might be additional relevant criteria. . . ." The report indicated, however,

20 that the committee members had not reached consensus on a single criterion to address the key

21 issue of how much certainty or proof a risk assessor must have in order to justify departing from a

22 default. Appendix N of the report contains two presentations of alternative views held by some

23 committee members on this issue. One view, known as "plausible conservatism," suggested that

24 departures from defaults should not be made unless new information improves the understanding

25 of a biological process to the point that relevant experts reach consensus that the protective

26 default assumption concerning that process is no longer plausible. The same criterion was

27 recommended where the underlying scientific mechanism is well understood, but where a default

28 is used to address missing data. In this case, the default should not be replaced with case-specific

29 data unless it is the consensus of relevant experts that the proffered data make the default

30 assumption no longer plausible. Another view, known as the "maximum use of scientific

31 information" approach, acknowledged that the initial choice of defaults should be protective, but

32 argued that conservatism should not be a factor in determining whether to depart from the default

33 in favor of an alternate biological theory or alternate data. According to this view, it should not

34 be necessary to reach expert consensus that the default assumption had been rendered implausible;
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1 it should be sufficient that risk assessors find the alternate approach more plausible than the

2 default. 

3 The EPA is not adopting a general list of formal decision criteria in the sense of a checklist

4 applicable to departures from defaults. It would not be helpful to generate a checklist of uniform

5 criteria. Risk assessments are highly variable in content and purpose. Screening assessments may

6 be purposely "worst case" in their default assumptions to eliminate problems from further

7 investigation. Subsequent risk assessments based on a fuller data set can discard worst-case

8 default assumptions in favor of plausibly protective assumptions and progressively replace or

9 modify the latter with data. No uniform checklist will fit all cases or all kinds of data. Moreover,

10 some departures from defaults are controversial, some are not. Generic checklists would likely

11 become more a source of rote discussion than of enlightenment about the process. 

12 Nonetheless, for one issue, the EPA has adopted principles to give greater formality and

13 transparency to decisions to depart from defaults. The EPA has developed a framework for

14 evaluating a postulated mode of action which appears in section 2.5, below. The use of mode of

15 action information to make decisions about human relevance of animal data, to assist in

16 identifying sensitive subpopulations, and to decide upon approaches to high dose to low dose

17 extrapolation in dose-response assessment is a fundamental part of these guidelines. The

18 framework of section 2.5. contains principles derived from Bradford Hill criteria for considering

19 causation in human epidemiologic studies and is meant to weigh the question whether empirical

20 data support a mode of action that is proposed in a particular case.

21 The guidelines use a combination of principles and process in the application of and

22 departure from default assumptions. The framework of default assumptions allows risk

23 assessment to proceed when current scientific theory or available case-specific data do not

24 provide firm answers in a particular case, as the 1983 NRC report outlined. Some of the default

25 assumptions bridge large gaps in fundamental knowledge which will be filled by basic research on

26 the causes of cancer and on other biological processes, rather than by agent-specific testing. 

27 Other default assumptions bridge smaller data gaps that can feasiblely be filled for a single agent,

28 such as whether a metabolic pathway in test animals is like (default) or unlike that in humans. 

29 The decision to use a default, or not, is a choice considering available information on an

30 underlying scientific process and agent-specific data, depending on which kind of default it is. 

31 Generally, if a gap in basic understanding exists, or if agent-specific data are missing, the default is

32 used without pause. If data are present, their evaluation may reveal inadequacies that also lead to

33 use of the default. If data support a plausible alternative to the default, but no more strongly than

34 they support the default, both the default and its alternative are carried through the assessment
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1 and characterized for the risk manager. If the alternative to the default are strongly supported by

2 data, the alternative may be used in place of the default. These guidelines provide a framework

3 for making such decisions. Note that, as discussed above, there is a spectrum of difficulty in

4 replacing default positions with empirical data. In the case of showing a mode of action, there is

5 need for extensive experimentation to support an hypothesis as to mode of action for a specific

6 tumor response, including coverage of the issue whether other modes of action are plausible. 

7 Note that screening assessments may appropriately use "worst case" inferences to

8 determine if, even under those conditions, risk is low enough that a problem can be eliminated

9 from further consideration.

10 Scientific peer review, peer consultative workshops and similar processes are the principal

11 ways determining the strength of thinking and generally accepted views within the scientific

12 community about the application of and departure from defaults and about judgments concerning

13 the plausibility and persuasiveness of data in a particular case. 

14 The discussion of major defaults below together with the explicit discussion of the choice

15 of inferences within the assessment and the processes of peer review and peer consultation (U.S.

16 EPA, 1998b) will serve the several goals stated in the 1994 NRC report. One is to encourage

17 research, since results of research efforts will be considered. Another is to allow timely decision

18 making, when time is a constraint, by supporting completion of the risk assessment using defaults

19 as needed. Another is to be flexible, using new science as it develops. Finally, the use of public

20 processes of peer consultation and peer review will ensure that discipline of thought is maintained

21 to support trust in assessment results.

22 There is no one set of rules for making the judgment of whether a data analysis is both

23 biologically plausible and persuasive as applied to the case at hand. Two criteria that apply in

24 these guidelines are that the underlying scientific principle has been generally accepted within the

25 scientific community and that supportive experiments are available that test the application of the

26 principle to the agent under review. For example, mutagenicity through reactivity with DNA has

27 been generally accepted as a carcinogenic influence for many years. This acceptance, together

28 with evidence of such mutagenicity in experiments on an agent, provides plausible and persuasive

29 support for the inference that mutagenicity is a mode of action for the agent.

30 Judgments about plausibility and persuasiveness of analyses vary according to the

31 scientific nature of the default. An analysis of data may replace a default or modify it. An

32 illustration of the former is development of EPA science policy on the issue of the relevance for

33 humans of male rat kidney neoplasia involving "2u-globulin (U.S. EPA, 1991b). The 1991 EPA

34 policy gives guidance on the kind of experimental findings that demonstrate whether the 
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1 "2u-globulin mechanism is present and responsible for carcinogenicity in a particular case. 

2 Before this policy guidance was issued, the default assumption was that neoplasia in question was

3 relevant to humans and indicated the potential for hazard to humans. A substantial body of data

4 was developed by public and private research groups as a foundation for the view that the "2u5 globulin induced response was not relevant to humans. These studies first addressed the "2u6 globulin mechanism in the rat and whether this mechanism has a counterpart in the human being,

7 both were large research efforts. The resulting data presented difficulties; some reviewers were

8 concerned that the mechanism in the rat appeared to be understood only in outline, not in detail,

9 and felt that the data were insufficient to show the lack of a counterpart mechanism in humans. It

10 was particularly difficult to support a negative such as the nonexistence of a mechanism in humans

11 because so little is known about what the mechanisms are in humans. Despite these concerns, in

12 its 1991 policy guidance, EPA concluded that the "2u-globulin induced response in rats should be

13 regarded as not relevant to humans (i.e., as not indicating human hazard). 

14 One conclusion from the development and peer review of this policy is that if the default

15 concerns an inherently complex biological question such as mode of action, large amounts of

16 work will be required to replace the default. A second is that "proof" in the strict sense of having

17 proved a negative is neither reasonable nor required. Rather the alternative may displace the

18 default when it is supported by clear and convincing evidence and is generally accepted in peer

19 review. The issue of relevance may not always be so difficult. It would be an experimentally

20 easier task, for example, to determine whether carcinogenesis in an animal species is due to a

21 metabolite of the agent in question that is not produced in humans. 

22 When scientific processes are understood but case-specific data are missing, defaults can 

23 be constructed to be modified by experimental data, even if data do not suffice to replace them

24 entirely. For example, the approaches adopted in these guidelines for scaling dose from

25 experimental animals to humans are constructed to be either modified or replaced as data become

26 available on toxicokinetic parameters for the particular agent being assessed. Similarly, the

27 selection of an approach or approaches for dose-response assessment is based on a series of

28 decisions that consider the nature and adequacy of available data in choosing among alternative

29 modeling and default approaches. 

30 The 1994 NRC report notes (p. 6) that "[a]s scientific knowledge increases, the science

31 policy choices made by the Agency and Congress should have less impact on regulatory decision

32 making. Better data and increased understanding of biological mechanisms should enable risk

33 assessments that are less dependent on protective default assumptions and more accurate as
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1 predictions of human risk." Undoubtedly, this is the trend as scientific understanding increases. 

2 However, some gaps in knowledge and data will doubtless continue to be encountered in

3 assessment of even data-rich cases, and it will remain necessary for risk assessments to continue

4 using defaults within the framework set forth here. 

5 1.3.2. Major Defaults

6 This discussion covers the major default assumptions commonly employed in a cancer risk

7 assessment and adopted in these guidelines. They are predominantly inferences necessary to use

8 data observed under empirical conditions to estimate events and outcomes under environmental

9 conditions. Several inferential issues arise when effects seen in a subpopulation of humans or

10 animals are used to infer potential effects in the population of environmentally exposed humans. 

11 Several more inferential issues arise in extrapolating the exposure-effect relationship observed

12 empirically to lower-exposure environmental conditions. The following issues cover the major

13 default areas. Typically, an issue has some sub-issues; they are introduced here, but are discussed

14 in greater detail in later sections. 

15 C Is the presence or absence of effects observed in a human population predictive of

16 effects in another exposed human population?

17 C Is the presence or absence of effects observed in an animal population predictive of

18 effects in exposed humans?

19 C How do metabolic pathways relate across species? Among different age groups,

20 between sexes in humans?

21 C How do toxicokinetic processes relate across species? Among different age groups,

22 between sexes in humans?

23 C What is the correlation of the observed dose-response relationship to the relationship

24 at lower doses?

25 1.3.2.1. Is the Presence or Absence of Effects Observed in a Human Population Predictive of

26 Effects in Another Exposed Human Population?

27 When cancer effects in exposed humans are attributed to exposure to an exogenous

28 agent, the default assumption is that such data are predictive of cancer in any other exposed

29 human population. Studies either attributing cancer effects in humans to exogenous agents or

30 reporting no effects are often studies of occupationally exposed humans. By sex, age, and general

31 health, workers are not representative of the general population exposed environmentally to the

32 same agents. In such studies there is no opportunity to observe those who are likely to be under
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1 represented, e.g., fetuses, infants and children, women, or people in poor health, who may

2 respond differently from healthy workers. Therefore, it is understood that this assumption could

3 still underestimate the response of certain human subpopulations. (NRC, 1993a, 1994). 

4 There is not enough knowledge yet to form a basis for any generally applicable, qualitative

5 or quantitative inference to compensate for this knowledge gap. In these guidelines, this problem

6 is left to analysis in individual cases, to be attended to with further general guidance as future

7 research and information allow. When information on a sensitive subpopulation exists, it will be

8 used. For example, an agent such as diethylstilbestrol (DES)causes a rare form of vaginal cancer

9 (clear-cell adenocarcinoma) (Herbst, 1971) in about 1 per thousand of adult women whose

10 mothers were exposed during pregnancy (Hatch et al., 1998). When cancer effects are not found

11 in an exposed human population, this information by itself is not generally sufficient to conclude

12 that the agent poses no carcinogenic hazard to this or other populations of potentially exposed

13 humans including sensitive subpopulations. This is because epidemiologic studies usually have

14 low power to detect and attribute responses, and typically evaluate cancer potential in a restricted

15 population (e.g., by age, occupation, etc.). The topic of susceptibility and variability is addressed

16 further in the discussion of quantitative default assumptions about dose-response relationships

17 below.

18 1.3.2.2. Is the Presence or Absence of Effects Observed in an Animal Population Predictive of

19 Effects in Exposed Humans?

20 The default assumption is that positive effects in animal cancer studies indicate that the

21 agent under study can have carcinogenic potential in humans. Thus, if no adequate human data

22 are present, positive effects in animal cancer studies are a basis for assessing the carcinogenic

23 hazard to humans. This assumption is a public health conservative policy, and it is both

24 appropriate and necessary given that we do not test for carcinogenicity in humans. The

25 assumption is supported by the fact that nearly all of the agents known to cause cancer in humans

26 are carcinogenic in animals in tests with adequate protocols (IARC, 1994; Tomatis et al., 1989;

27 Huff, 1994). Moreover, almost one-third of human carcinogens were identified subsequent to

28 animal testing (Huff, 1993). Further support is provided by research on the molecular biology of

29 cancer processes, which has shown that the mechanisms of control of cell growth and

30 differentiation are remarkably homologous among species and highly conserved in evolution. 

31 Nevertheless, the same research tools that have enabled recognition of the nature and

32 commonality of cancer processes at the molecular level also have the power to reveal differences

33 and instances in which animal responses are not relevant to humans (Linjinsky, 1993; U.S.
2

Understanding an agent’s “mode of action” means understanding the general sequence of events

by which it causes effects on cell growth control that result in cancer. “Mode of action” is used

rather than “mechanism of action” which is a term that implies complete knowledge of the steps

of carcinogenesis at the molecular level, a level of understanding that currently does not exist for

any agent. 

7/02/99 1-12 DRAFT

EPA,1991b). Under these guidelines, available mode of action2 1 information is studied for its

2 implications in both hazard and dose-response assessment and its effect on default assumptions.

3 There may be instances in which the use of an animal model would identify a hazard in

4 animals that is not truly a hazard in humans (e.g., the "2u-globulin association with renal

5 neoplasia in male rats (U.S. EPA, 1991b)). The extent to which animal studies may yield false

6 positive indications for humans is a matter of scientific debate. To demonstrate that a response in

7 animals is not relevant to any human situation, adequate data to assess the relevancy issue must be

8 available.

9 The default assumption is that effects seen at the highest dose tested are appropriate for

10 assessment, but it is necessary that the experimental conditions be scrutinized. Animal studies

11 are conducted at high doses in order to provide statistical power, the highest dose being one that

12 is minimally toxic (maximum tolerated dose). Consequently, the question often arises whether a

13 carcinogenic effect at the highest dose may be a consequence of cell killing with compensatory

14 cell replication or of general physiological disruption, rather than inherent carcinogenicity of the

15 tested agent. There is little doubt that this may happen in some cases, but skepticism exists

16 among some scientists that it is a pervasive problem (Ames and Gold, 1990; Melnick et al., 1993a;

17 Melnick et al., 1993b; Barrett, 1993). If adequate data demonstrate that the effects are solely the

18 result of excessive toxicity rather than carcinogenicity of the tested agent per se, then the effects

19 may be regarded as not appropriate to include in assessment of the potential for human

20 carcinogenicity of the agent. This is a matter of expert judgment, considering all of the data

21 available about the agent including effects in other toxicity studies, structure-activity relationships,

22 and effects on growth control and differentiation.

23 When cancer effects are not found in well-conducted animal cancer studies in two or

24 more appropriate species and other information does not support the carcinogenic potential of

25 the agent, these data provide a basis for concluding that the agent is not likely to possess human

26 carcinogenic potential, in the absence of human data to the contrary. This default assumption

27 about lack of cancer effects has limitations. It is recognized that animal studies (and

28 epidemiologic studies as well) have very low power to detect cancer effects. Detection of a 10%
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1 tumor incidence is generally the limit of power with standard protocols for animal studies (with

2 the exception of rare tumors that are virtually markers for a particular agent, e.g., angiosarcoma

3 caused by vinyl chloride). In some situations, the tested animal species may not be predictive of

4 effects in humans; for example, arsenic shows only minimal or no effect in animals, while it is

5 clearly positive in humans. Therefore, it is important to consider other information as well; 

6 absence of mutagenic activity or absence of carcinogenic activity among structural analogues, can

7 increase the confidence that negative results in animal studies indicate a lack of human hazard. 

8 Another limitation is that standard animal study protocols are not yet available for effectively

9 studying perinatal effects. The potential for effects on the very young generally must be

10 considered separately. Perinatal studies accomplished by modification of existing adult bioassay

11 protocols need to be required in special circumstances under existing Agency policy (U.S. EPA,

12 1997a,b)

13 The default assumption is that target organ concordance is not a prerequisite for

14 evaluating the implications of animal study results for humans. Target organs of carcinogenesis

15 for agents that cause cancer in both animals and humans are most often concordant at one or

16 more sites (Tomatis et al., 1989; Huff, 1994). However, concordance by site is not uniform. 

17 The mechanisms of control of cell growth and differentiation are concordant among species, but

18 there are marked differences among species in the way control is managed in various tissues. For

19 example, in humans, mutations of the tumor suppressor genes p53 and retinoblastoma are

20 frequently observed genetic changes in tumors. These tumor suppressor genes are also observed

21 to be operating in some rodent tissues, but other growth control mechanisms predominate in other

22 rodent tissues. Thus, an animal response may be due to changes in a control that are relevant to

23 humans, but appear in animals in a different way. However, it is appropriate under these

24 guidelines to consider the influences of route of exposure, metabolism, and, particularly, some

25 modes of action that may either support or not support target organ concordance between animals

26 and humans. When data allow, these influences are considered in deciding whether the default

27 remains appropriate in individual instances (NRC, 1994, p. 121). Another exception to the basic

28 default of not assuming site concordance exists in the context of toxicokinetic modeling. Site

29 concordance is inherently assumed when these models are used to estimate delivered dose in

30 humans based on animal data.

31 The default is to include benign tumors observed in animal studies in the assessment of

32 animal tumor incidence if they have the capacity to progress to the malignancies with which they

33 are associated. This default is consistent with the approach of the National Toxicology Program

34 and the International Agency for Research on Cancer and is somewhat more protective of public
7/02/99 1-14 DRAFT

1 health than not including benign tumors in the assessment. This treats the benign and malignant

2 tumors as representative of related responses to the test agent (McConnell et al., 1986), which is

3 scientifically appropriate. Nonetheless, in assessing findings from animal studies, a greater

4 proportion of malignancy is weighed more heavily than a response with a greater proportion of

5 benign tumors. Greater frequency of malignancy of a particular tumor type in comparison with

6 other tumor responses observed in an animal study is also a factor to be considered in selecting

7 the response to be used in dose-response assessment.

8 Benign tumors that are not observed to progress to malignancy are assessed on a case9 by-case basis. There is a range of possibilities for their overall significance. They may deserve

10 attention because they are serious health problems even though they are not malignant; for

11 instance, benign tumors may be a health risk because of their effect on the function of a target

12 tissue such as the brain. They may be significant indicators of the need for further testing of an

13 agent if they are observed in a short term test protocol, or such an observation may add to the

14 overall weight of evidence if the same agent causes malignancies in a long term study. 

15 Knowledge of the mode of action associated with a benign tumor response may aid in the

16 interpretation of other tumor responses associated with the same agent.

17 1.3.2.3. How Do Metabolic Pathways Relate Across Species? Among different age groups,

18 between sexes in humans?

19 The default assumption is that there is a similarity of the basic pathways of metabolism

20 and the occurrence of metabolites in tissues in regard to the species-to-species extrapolation of

21 cancer hazard and risk. If comparative metabolism studies were to show no similarity between

22 the tested species and humans and a metabolite(s) were the active form, there would be less

23 support for an inference that the animal response(s) relates to humans. In other cases, parameters

24 of metabolism may vary quantitatively between species; this becomes part of deciding on an

25 appropriate human equivalent dose based on animal studies, optimally in the context of a

26 toxicokinetic model. While the basic pathways are assumed to be the same among humans, the

27 presence of polymorphisms and the maturation of the pathways in infants needs to be considered. 

28 The active form of an agent may be present to differing degrees, or completely absent, which may

29 result in greater or lesser risk for subpopulations.
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1 1.3.2.4. How Do Toxicokinetic Processes Relate Across Species? Among different age

2 groups, between sexes in humans?

3 A major issue is how to estimate human equivalent doses in extrapolating from animal

4 studies. As a default for oral exposure, a human equivalent dose for adults is estimated from

5 data on another species by an adjustment of animal applied oral dose by a scaling factor of body

6 weight to the 0.75 power. This adjustment factor is used because it represents scaling of metabolic

7 rate across animals of different size. Because the factor adjusts for a parameter that can be

8 improved on and brought into more sophisticated toxicokinetic modeling, when such data become

9 available, the default assumption of 0.75 power can be refined or replaced. The same factor is

10 used for children because it is slightly more protective than using children’s body weight (see

11 section 1.3.5.2). 

12 For inhalation exposure, a human equivalent dose for adults is estimated by default

13 methodologies that provide estimates of lung deposition and of internal dose. The methodologies

14 can be refined to more sophisticated forms with data on toxicokinetic and metabolic parameters of

15 the specific agent. This default assumption, like the one with oral exposure, is selected in part

16 because it lays a foundation for incorporating better data. Because of the differences for infants

17 and children, for gases and aerosols, an adjustment is made for their breathing rate and their

18 body weight. For inhaled particles, the adjustment does not take into account the different size

19 and spacing of airways of children and adults; this difference could result in children and adults

20 retaining particles with a different size distribution and different toxicologic properties. To reduce

21 this uncertainty, EPA is developing a default dosimetry model for children that is based on

22 children's inhalation parameters. The use of information to improve dose estimation from applied

23 to internal to delivered dose is encouraged, including use of toxicokinetic modeling instead of any

24 default, where data are available.

25 The processes of absorption, distribution, and elimination have important differences

26 among infants, adults, and older adults, e.g., infants tend to absorb metals through the gut more

27 rapidly and more efficiently than older children or adults (Calabrese, 1986). Renal elimination is

28 also not as efficient in infants. While these processes reach adult compentency at about the time

29 of weaning, they may have important implications, particularly when the dose-response

30 relationship for an agent is considered to be nonlinear and there is an exposure scenario

31 disproportionately affecting infants, because in these cases the magnitude of dose is more

32 pertinent than the usual approach in linear extrpolation, of averaging dose across a lifetime.

33 Efficiency of intestinal absorption in older adults tends to be generally less overall for most

34 chemicals. Another notable difference is that, post-weaning (about one year), children have a
7/02/99 1-16 DRAFT

1 higher metabolic rate than adults (Renwick, 1999) and may toxify or detoxify agents at a

2 correspondingly higher rate.. 

3

4 For a route-to-route of exposure extrapolation, the default assumption is that an agent

5 that causes internal tumors by one route of exposure will be carcinogenic by another route if it is

6 absorbed by the second route to give an internal dose. This is a qualitative assumption and is

7 considered to be public health conservative. The rationale is that for internal tumors an internal

8 dose is significant no matter what the route of exposure. Additionally, the metabolism of the

9 agent will be qualitatively the same for an internal dose. The issue of quantitative extrapolation of

10 the dose-response relationship from one route to another is addressed case by case. Quantitative

11 extrapolation is complicated by considerations such as first-pass metabolism, but is approachable

12 with empirical data. Adequate data are necessary to demonstrate that an agent will act differently

13 by one route versus another route of exposure. 

14

15 1.3.2.5. What Is the Correlation of the Observed Dose-Response Relationship to the

16 Relationship at Lower Doses?

17 If sufficient data are available, a biologically based model for both the observed range and

18 extrapolation below that range may be used. While no standard biologically based models are in

19 existence, one may be developed if extensive data exist in a particular case and the purpose of the

20 assessment justifies the investment of resources needed. The default procedure for the observed

21 range of data, when a biologically based model is not used, is to use a curve-fitting model for

22 incidence data.

23 In the absence of data supporting a biologically based model for extrapolation outside of

24 the observed range, the choice of approach is based on the view of mode of action of the agent

25 arrived at in the hazard assessment. 

26 The basic default is to assume linearity and use a linear default approach when the mode

27 of action information is supportive of linearity or mode of action is not understood. The linear

28 approach is used when a view of the mode of action indicates a linear response, for example,

29 when a conclusion is made that an agent directly causes alterations in DNA, a kind of interaction

30 that not only theoretically requires one reaction, but also is likely to be additive to ongoing,

31 spontaneous gene mutation. Other kinds of activity may have linear implications, e.g., linear rate32 limiting steps, that support a linear procedure also. The linear approach is to draw a straight line

between a point of departure from observed data, generally, as a default, the LED10 33 , and the

34 origin (zero incremental dose, zero incremental response). Other points of departure may be
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more appropriate for certain data sets; these may be used instead of the LED10 1 . This approach is

generally considered to be public health protective. The LED10 2 is the lower 95% limit on a dose

3 that is estimated to cause a 10% response. This level is chosen to account (protectively) for

4 experimental variability. Additionally, it is chosen because it rewards experiments with better

5 designs in regard to number of doses and dose spacing, since these generally will have narrower

6 confidence limits. It is also an appropriate representative of the lower end of the observed range

7 because the limit of detection of studies of tumor effect is about 10%.

8 The linear default is thought to generally provide an upper bound calculation of potential

9 risk at low doses. e.g., a 1/100,000 to 1/1,000,000 risk; the straight line approach gives numerical

10 results about the same as a linearized multistage procedure. This upper bound is thought to be

11 public health conservative at low doses for the range of human variability considering the typical

12 Agency target range for risk management of 1/1,000,000 to 1/10,000, although it may not

13 completely do so (Bois et al., 1995) if pre-existing disease or genetic constitution place a

14 percentage of the population at risk from any exposure above zero to xenobiotics, natural or

15 manmade. The question of what may be the actual variability in human sensitivity is one that the

16 1994 NRC report discussed as did the 1993 NRC report on pesticides in children and infants. The

17 NRC has recommended research on the question, and the EPA and other agencies are conducting

18 such research. Given the current state of knowledge, the EPA will assume that the linear default

19 procedure adequately accounts for human variability unless there is case-specific information for a

20 given agent that indicates a particularly sensitive subpopulation, in which case the special

21 information will be used.

22 When adequate data on mode of action show that linearity is not plausible, and provide

23 sufficient evidence to support a nonlinear mode of action for the general population and any

24 subpopulations of concern, the default changes to a different approach-- a margin of exposure

25 analysis--which assumes that nonlinearity is more reasonable. The departure point is again

generally the LED10 26 when incidence data are modeled. When the data available are continuous

27 data such as blood levels of hormones or organ weight, a NOAEL/LOAEL procedure is typically

28 used since modeling approaches for deriving a point of departure from continuous data are not yet

29 available. Until these modeling approaches are developed and adopted, continuous data and data

30 sets that are a mixture of incidence and continuous data can be examined by the NOAEL/LOAEL

31 procedure. In the nonlinear approach, the margin that exists between a human exposure of interest

32 and the point of departure is examined for adequacy to protect public health. A margin of

33 exposure analysis may be used as the basis to consider the protectiveness of a possible

34 environmental criterion for regulation or to judge whether an existing exposure might present risk.
3

A “key event” is an empirically observed precursor consistent with a mode of action.

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1 A sufficient basis to support this nonlinear procedure will include data on responses that

are key events3 2 integral to the carcinogenic process. This means that the point of departure

3 mostly will be from these precursor response data, e.g., hormone levels, mitogenic effects, rather

4 than tumor incidence data. 

5 The mode of action may have specific implications to be considered for risk potential of

6 certain exposure scenarios. For instance, stimulus of cell growth through hormonal or other signal

7 disruption or as a result of damage from toxicity are reversible if the exposure is for a short time

8 since homeostasis brings a return to normal levels after cessation of exposure. Another feature of

9 a specific exposure scenario may be the exposure of a sensitive subpopulation. If the population

10 exposed in a particular scenario is wholly or largely composed of a subpopulation for whom

11 evidence indicates a special sensitivity to the agent’s mode of action, an adequate margin of

12 exposure would be larger than for general population exposure.

13 When the mode of action information indicates that the dose-response may be adequately

14 described by both a linear and a nonlinear approach, then the default is to present both the

15 linear and margin of exposure analyses. An assessment may use both linear and nonlinear

16 approaches if linearity is not plausible and nonlinearity has support, but a mode of action is not

17 defined, or different responses are thought to result from different modes of action or a response

18 appears to be very different at high and low doses due to influence of separate modes of action. 

19 The results may be needed for assessment of combined risk from agents with common modes of

20 action.

21 A default assumption is made that cumulative dose received over a lifetime, expressed as

22 a lifetime average daily dose, is an appropriate measure of dose. This assumes that a high dose

23 of such an agent received over a shorter period of time is equivalent to a low dose spread over a

24 lifetime. This is thought to be a relatively public health protective assumption and has empirical

25 support (Monro, 1992). An example of effects of short-term, high exposure that results in

26 subsequent cancer development is treatment of cancer patients with certain chemotherapeutic

27 agents. An example of cancer from long-term exposure to an agent of relatively low potency is

28 smoking. When sufficient information is available indicating that the carcinogenic mode of action

29 supports a nonlinear dose-response approach, a different approach may be used. Such an

30 approach includes considering the margin of exposure that exists between exposure and the point

31 of departure from the observed data range. In these cases, short-term exposure estimates (several

32 days to several months may be more appropriate than the lifetime average daily dose. In these
7/02/99 1-19 DRAFT

1 cases both agent concentration and duration are likely to be important, because such effects are

2 generally observed to be reversible at cessation of very short-term exposure.

3 1.4. CHARACTERIZATIONS

4 The risk characterization process first summarizes findings on hazard, dose-response, and

5 exposure characterizations, then develops an integrative analysis of the whole risk case. It ends in

6 a non technical Risk Characterization Summary. The Risk Characterization Summary is a

7 presentation for risk managers who may or may not be familiar with the scientific details of cancer

8 assessment. It also provides information for other interested readers. The initial steps in the risk

9 characterization process are to make building blocks in the form of characterizations of the

10 assessments of hazard, dose-response, and exposure. The individual assessments and

11 characterizations are then integrated to arrive at risk estimates for exposure scenarios of interest. 

12 As part of the characterization process, explicit evaluations will be made of the hazard and risk

13 potential for susceptible populations, including children (U.S EPA 1995a,b). There are two

14 reasons for individually characterizing the hazard, dose-response, and exposure assessments. One

15 is that they are often done by different people than those who do the integrative analyses. The

16 second is that there is very often a lapse of time between the conduct of hazard and dose17 response analyses and the conduct of exposure assessment and integrative analysis. Thus, it is

18 necessary to capture characterizations of assessments as the assessments are done to avoid the

19 need to go back and reconstruct them. Finally, frequently a single hazard assessment is used by

20 several programs for several different exposure scenarios. Figure 1-2 shows the relationships of

21 analyses. The figure does not necessarily correspond to the number of documents involved; there

22 may be one or several. "Integrative analysis" is a generic term. At EPA, the documents of

23 various programs that contain integrative analyses have other names such as the "Staff Paper" that

24 discusses air quality criteria issues. In the following sections, the elements of this figure are

25 discussed.
7/02/99 1-20 DRAFT

Figure 1-1. Risk Characterization
1

“Mode” of action is contrasted with “mechanism” of action, which implies a more detailed,

molecular description of key processes and events than is meant by mode of action.

7/02/99 2-1 DRAFT

2. HAZARD ASSESSMENT

1 2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION

2 2.1.1. Analyses of Data

3 The purpose of hazard assessment is to review and evaluate data pertinent to two

4 questions: (1) whether an agent may pose a carcinogenic hazard to human beings and (2) under

5 what circumstances an identified hazard may be expressed (NRC, 1994, p. 142). Hazard

6 assessment is composed of analyses of a variety of data that may range from observations of

7 tumor responses to analysis of structure-activity relationships. The purpose of the assessment is

8 not simply to assemble these separate evaluations; its purpose is to construct a total case analysis

9 examining the biological story the data reveal as a whole about carcinogenic effects, mode of

10 action, and implications of these for human hazard and dose-response evaluation. Weight-of11 evidence conclusions come from the combined strength and coherence of inferences appropriately

12 drawn from all of the available evidence. To the extent that data permit, hazard assessment

13 addresses the question of mode of action as both an initial step in identifying human hazard

14 potential and as a part of considering appropriate approaches to dose-response assessment.

15 The topics in this chapter include analysis of tumor data, both animal and human, and

16 analysis of other key information about properties and effects that relate to carcinogenic potential. 

17 The chapter addresses how information can be used to evaluate potential modes of action. It also

provides guidance on performing a weight-of-evidence evaluation.4 18

19

20 2.1.2. Presentation of Results

21 Presentation of the results of hazard assessment follows Agency guidance as discussed in

22 Section 2.7. The results are presented in a technical hazard characterization that serves as a

23 support to later risk characterization. It includes:

24

25 C a summary of the evaluations of hazard data,

26 C the rationales for its conclusions, and

27 C an explanation of the significant strengths or limitations of the conclusions.

28

29 Another presentation feature is the use of a weight-of-evidence narrative that includes
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1 both a conclusion about the weight-of-evidence of carcinogenic potential and a summary of the

2 data on which the conclusion rests. This narrative is a brief summary that replaces the

3 alphanumerical classification system used in EPA’s previous guidelines.

4

5 2.2. ANALYSIS OF TUMOR DATA

6 Evidence of carcinogenicity comes from finding tumor increases in humans or laboratory

7 animals exposed to a given agent, or from finding tumors following exposure to structural

8 analogues to the compound under review. The significance of observed or anticipated tumor

9 effects is evaluated in reference to all the other key data on the agent. This section contains

10 guidance for analyzing human and animal studies to decide whether there is an association

11 between exposure to an agent or a structural analogue and occurrence of tumors. Note that the

12 use of the term “tumor” here is generic, meaning malignant neoplasms or a combination of

13 malignant and corresponding benign neoplasms.

14 Observation of only benign neoplasia may or may not have significance. Benign tumors

15 that are not observed to progress to malignancy are assessed on a case-by-case basis. There is a

16 range of possibilities for their overall significance. They may deserve attention because they are

17 serious health problems even though they are not malignant; for instance, benign tumors may be a

18 health risk because of their effect on the function of a target tissue such as the brain. They may be

19 significant indicators of the need for further testing of an agent if they are observed in a short20 term test protocol, or such an observation may add to the overall weight of evidence if the same

21 agent causes malignancies in a long-term study. Knowledge of the mode of action associated with

22 a benign tumor response may aid in the interpretation of other tumor responses associated with

23 the same agent. In other cases, observation of a benign tumor response alone may have no

24 significant health hazard implications when other sources of evidence show no suggestion of

25 carcinogenicity.

26

27 2.2.1. Human Data

28 Human data may come from epidemiologic studies or case reports. Epidemiology is the

29 study of the distributions and causes of disease within human populations. The goals of cancer

30 epidemiology are to identify differences in cancer risk between different groups in a population or

31 between different populations, and then to determine the extent to which these differences in risk

32 can be attributed causally to specific exposures to exogenous or endogenous factors. 

33 Epidemiologic data are extremely useful in risk assessment because they provide direct evidence

34 that a substance produces cancer in humans, thereby avoiding the problem of species-to-species
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1 inference. Thus, when available human data are extensive and of good quality, they are generally

2 preferable over animal data and should be given greater weight in hazard characterization and

3 dose-response assessment, although both are utilized.

4 Null results from a single epidemiologic study cannot prove the absence of carcinogenic

5 effects because they can arise either from being truly negative or from inadequate statistical

6 power, inadequate design, imprecise estimates, or confounding factors. However, null results

7 from a well-designed and well-conducted epidemiologic study that contains usable exposure data

8 can help to define upper limits for the estimated dose of concern for human exposure if the overall

9 weight of the evidence indicates that the agent is potentially carcinogenic in humans.

10 Epidemiology can also complement experimental evidence in corroborating or clarifying

11 the carcinogenic potential of the agent in question. For example, observations from epidemiologic

12 studies that elevated cancer incidence occurs at sites corresponding to those at which laboratory

13 animals experience increased tumor incidence can strengthen the weight of evidence of human

14 carcinogenicity. On the other hand, strong nonpositive epidemiologic data alone or in conjunction

15 with compelling mechanistic information can lend support to a conclusion that animal responses

16 may not be predictive of a human response. Furthermore, the advent of biochemical or molecular

17 epidemiology may help improve understanding of the mechanisms of human carcinogenesis.

18

19 2.2.1.1. Types of Studies

20 The major types of cancer epidemiologic studies are analytical studies and descriptive or

21 correlation studies. Each study type has well-known strengths and weaknesses that affect

22 interpretation of results as summarized below (Kelsey et al., 1986; Lilienfeld and Lilienfeld, 1979;

23 Mausner and Kramer, 1985; Rothman, 1986).

24 Analytical epidemiologic studies are most useful for identifying an association between

25 human exposure and adverse health effects. Analytical study designs include case-control studies

26 and cohort studies. In case-control studies, groups of individuals with (cases) and without

27 (controls) a particular disease are identified and compared to determine differences in exposure. 

28 In cohort studies, a group of “exposed” and “nonexposed” individuals are identified and studied

29 over time to determine differences in disease occurrence. Cohort studies can either be performed

30 prospectively, or retrospectively from historical records.

31 Descriptive or correlation epidemiologic studies (sometimes called ecological studies)

32 examine differences in disease rates among populations in relation to age, gender, race, and

33 differences in temporal or environmental conditions. In general, these studies can only identify

34 patterns or trends in disease occurrence over time or in different geographical locations, but
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1 cannot ascertain the causal agent or degree of exposure. These studies, however, are often very

2 useful for generating hypotheses for further research.

3 Biochemical or molecular epidemiologic studies are studies in which laboratory methods

4 are incorporated in analytical investigations. The application of techniques for measuring cellular

5 and molecular alterations due to exposure to specific environmental agents may allow conclusions

6 to be drawn about the mechanisms of carcinogenesis. The use of biological biomarkers in

7 epidemiology may improve assessment of exposure and internal dose.

8 Case reports describe a particular effect in an individual or group of individuals who were

9 exposed to a substance. These reports are often anecdotal or highly selected in nature and are of

10 limited use for hazard assessment. However, reports of cancer cases can identify associations,

11 particularly when there are unique features such as an association with an uncommon tumor (e.g.,

12 vinyl chloride and angiosarcoma or diethylstilbestrol and clear-cell carcinoma of the vagina).

13

14 2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic Studies

15 Criteria for assessing the adequacy of epidemiologic studies are well recognized. 

16 Characteristics that are desirable in these studies include (1) clear articulation of study objectives

17 or hypothesis; (2) proper selection and characterization of the exposed and control groups; (3)

18 adequate characterization of exposure; (4) sufficient length of follow-up for disease occurrence;

19 (5) valid ascertainment of the causes of cancer morbidity and mortality; (6) proper consideration

20 of bias and confounding factors; (7) adequate sample size to detect an effect; (8) clear, well21 documented, and appropriate methodology for data collection and analysis; (9) adequate response

22 rate and methodology for handling missing data; and (10) complete and clear documentation of

23 results. Ideally, these conditions should be satisfied, where appropriate, but rarely can a study

24 meet all of them. No single criterion determines the overall adequacy of a study. The following

25 discussions highlight the major factors included in an analysis of epidemiologic studies.

26

27 Population Issues

28 The ideal comparison would be between two populations that differ only in exposure to

29 the agent in question. Because this is seldom the case, it is important to identify sources of bias

30 inherent in a study’s design or data collection methods. Bias can arise from several sources,

31 including noncomparability between populations of factors such as general health (McMichael,

32 1976), diet, lifestyle, or geographic location; differences in the way case and control individuals

33 recall past events; differences in data collection that result in unequal ascertainment of health

34 effects in the populations; and unequal follow-up of individuals. Both acceptance of studies for
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1 assessment and judgment of their strengths or weaknesses depend on identifying their sources of

2 bias and the effects on study results.

3

4

5 Exposure Issues

6 For epidemiologic data to be useful in determining whether there is an association between

7 health effects and exposure to an agent, there must be adequate characterization of exposure

8 information. In general, greater weight should be given to studies with more precise and specific

9 exposure estimates.

10 Questions to address about exposure are: What can one reliably conclude about the level,

11 duration, route, and frequency of exposure of individuals in one population as compared with

12 another? How sensitive are study results to uncertainties in these parameters?

13 Actual exposure measurements are not available for many retrospective studies. 

14 Therefore, surrogates are often used to reconstruct exposure parameters. These may involve

15 attributing exposures to job classifications in a workplace or to broader occupational or

16 geographic groupings. Use of surrogates carries a potential for misclassification in that

17 individuals may be placed in an incorrect exposure group. Misclassification generally leads to

18 reduced ability of a study to detect differences between study and referent populations.

19 When either current or historical monitoring data are available, the exposure evaluation

20 includes consideration of the error bounds of the monitoring and analytic methods and whether

21 the data are from routine or accidental exposures. The potentials for misclassification and

22 measurement errors are amenable to both qualitative and quantitative analysis. These are essential

23 analyses for judging a study’s results because exposure estimation is the most critical part of a

24 retrospective study.

25 Biological markers potentially offer excellent measures of exposure (Hulka and Margolin,

26 1992; Peto and Darby, 1994). Validated markers of exposure such as alkylated hemoglobin from

27 exposure to ethylene oxide (van Sittert et al., 1985) or urinary arsenic (Enterline et al., 1987) can

28 greatly improve estimates of dose. Markers closely identified with effects promise to greatly

29 increase the ability of studies to distinguish real effects from bias at low levels of relative risk

30 between populations (Taylor et al., 1994; Biggs et al., 1993) and to resolve problems of

31 confounding risk factors.

32

33 Confounding Factors

34 Because epidemiologic studies are mostly observational, it is not possible to guarantee the
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1 control of confounding variables, which may affect the study outcome. A confounding variable is

2 a risk factor, independent of the putative agent, that is distributed unequally among the exposed

3 and unexposed populations (e.g., smoking habits, lifestyle). Adjustment for possible confounding

4 factors can occur either in the design of the study (e.g., matching on critical factors) or in the

5 statistical analysis of the results. The influence of a potential confounding factor is limited by the

6 effect of the exposure of interest. For example, a twofold effect of an exposure requires that the

7 confounder effect be at least as big. The latter may not be possible owing to the presentation of

8 the data or because needed information was not collected during the study. In this case, indirect

9 comparisons may be possible. For example, in the absence of data on smoking status among

10 individuals in the study population, an examination of the possible contribution of cigarette

11 smoking to increased lung cancer risk may be based on information from other sources such as

12 the American Cancer Society’s longitudinal studies (Hammand, 1966; Garfinkel and Silverberg,

13 1991). The effectiveness of adjustments contributes to the ability to draw inferences from a

14 study.

15 Different studies involving exposure to an agent may have different confounding factors. 

16 If consistent increases in cancer risk are observed across a collection of studies with different

17 confounding factors, the inference that the agent under investigation was the etiologic factor is

18 strengthened, even though complete adjustment for confounding factors cannot be made and no

19 single study supports a strong inference.

20 It also may be the case that the agent of interest is a risk factor in conjunction with another

21 agent. This relationship may be revealed in a collection of studies such as in the case of asbestos

22 exposure and smoking.

23

24 Sensitivity

25 Sensitivity, or the ability of a study to detect real effects, is a function of several factors. 

26 Greater size of the study population(s) (sample size) increases sensitivity, as does greater

27 exposure (levels and duration) of the population members. Because of the often long latency

28 period in cancer development, sensitivity also depends on whether adequate time has elapsed

29 since exposure began for effects to occur. A unique feature that can be ascribed to the effects of

30 a particular agent (such as a tumor type that is seen only rarely in the absence of the agent) can

31 increase sensitivity by permitting separation of bias and confounding factors from real effects. 

32 Similarly, a biomarker particular to the agent can permit these distinctions. Statistical re-analyses

33 of data, particularly an examination of different exposure indices, can give insight on potential

34 exposure-response relationships. These are all factors to explore in statistical analysis of the data.
7/02/99 2-7 DRAFT

1 Statistical Considerations

2 The analysis applies appropriate statistical methods to ascertain whether or not there is

3 any significant association between exposure and effects. A description of the method or methods

4 should include the reasons for their selection. Statistical analyses of the potential effects of bias or

5 confounding factors are part of addressing the significance of an association, or lack of one, and

6 whether a study is able to detect any effect.

7 The analysis augments examination of the results for the whole population with

8 exploration of the results for groups with comparatively greater exposure or time since first

9 exposure. This may support identifying an association or establishing a dose-response trend. 

10 When studies show no association, such exploration may apply to determining an upper limit on

11 potential human risk for consideration alongside results of animal tumor effects studies.

12

13 Combining Statistical Evidence Across Studies

14 Meta-analysis is a means of comparing and synthesizing studies dealing with similar health

15 effects and risk factors. It is intended to introduce consistency and comprehensiveness into what

16 otherwise might be a more subjective review of the literature. When utilized appropriately, meta17 analysis can enhance understanding of associations between sources and their effects that may not

18 be apparent from examination of epidemiologic studies individually. Whether to conduct a meta19 analysis depends on several issues. These include the importance of formally examining sources

20 of heterogeneity, the refinement of the estimate of the magnitude of an effect, and the need for

21 information beyond that provided by individual studies or a narrative review. Meta-analysis may

22 not be useful in some circumstances. These include when the relationship between exposure and

23 disease is obvious without a more formal analysis; when there are only a few studies of the key

24 health outcomes; when there is insufficient information from available studies related to disease,

25 risk estimate, or exposure classification; or when there are substantial confounding or other biases

26 that cannot be adjusted for in the analysis (Blair et al., 1995; Greenland, 1987; Peto, 1992).

27

28 2.2.1.3. Criteria for Causality

29 A causal interpretation is enhanced for studies to the extent that they meet the criteria

30 described below. None of the criteria is conclusive by itself, and the only criterion that is essential

31 is the temporal relationship. These criteria are modeled after those developed by Bradford Hill in

32 the examination of cigarette smoking and lung cancer (Rothman, 1986), and they need to be

33 interpreted in the light of all other information on the agent being assessed.

34
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1 C Temporal relationship: The development of cancers requires certain latency

2 periods, and while latency periods vary, existence of such periods is generally

3 acknowledged. Thus, the disease has to occur within a biologically reasonable

4 time after initial exposure. This feature must be present if causality is to be

5 considered.

6 C Consistency: Associations occur in several independent studies of a similar

7 exposure in different populations, or associations occur consistently for different

8 subgroups in the same study. This feature usually constitutes strong evidence for a

9 causal interpretation when the same bias or confounding is not also duplicated

10 across studies.

11 C Magnitude of the association: A causal relationship is more credible when the risk

12 estimate is large and precise (narrow confidence intervals).

13 C Biological gradient: The risk ratio (i.e., the ratio of the risk of disease or death

14 among the exposed to the risk of the unexposed) increases with increasing

15 exposure or dose. Statistical significance is important, and a strong dose-response

16 relationship across several categories of exposure, latency, and duration is

17 supportive for causality, given that confounding is unlikely to be correlated with

18 exposure. The absence of a dose-response relationship, however, is not by itself

19 evidence against a causal relationship.

20 C Specificity of the association: The likelihood of a causal interpretation is increased

21 if an exposure produces a specific effect (one or more tumor types also found in

22 other studies) or if a given effect has a unique exposure.

23 C Biological plausibility: The association makes sense in terms of biological

24 knowledge. Information is considered from animal toxicology, toxicokinetics,

25 structure-activity relationship analysis, and short-term studies of the agent’s

26 influence on events in the carcinogenic process considered.

27 C Coherence: The cause-and-effect interpretation is in logical agreement with what

28 is known about the natural history and biology of the disease, i.e., the entire body

29 of knowledge about the agent.

30

31 2.2.1.4. Assessment of Evidence of Carcinogenicity from Human Data

32 In the evaluation of carcinogenicity based on epidemiologic studies, it is necessary to

33 critically evaluate each study for confidence in findings and conclusions as discussed under

34 Section 2.2.1.2. All studies that are properly conducted, whether yielding positive or null results,
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1 or even suggesting protective carcinogenic effects, should be considered in assessing the totality

2 of the human evidence. Although a single study may be indicative of a cause-effect relationship,

3 confidence in inferring a causal relationship is increased when several independent studies are

4 concordant in showing the association, when the association is strong, and when other criteria for

5 causality are also met. Conclusions about the overall evidence for carcinogenicity from available

6 studies in humans should be summarized along with a discussion of strengths or limitations of the

7 conclusions.

8

9 2.2.2. Animal Data

10 Various whole-animal test systems are currently used or are under development for

11 evaluating potential carcinogenicity. Cancer studies involving chronic exposure for most of the

12 lifespan of an animal are generally accepted for evaluation of tumor effects (Tomatis et al., 1989;

13 Rall, 1991; Allen et al., 1988; but see Ames and Gold, 1990). Other studies of special design are

14 useful for observing formation of preneoplastic lesions or tumors or investigating specific modes

15 of action. Their applicability is made on a case-by-case basis.

16

17 2.2.2.1. Long-Term Carcinogenicity Studies

18 The objective of long-term carcinogenesis bioassays is to determine the potential

19 carcinogenic hazard and dose-response relationships of the test agent. Carcinogenicity rodent

20 studies are designed to examine the production of tumors as well as preneoplastic lesions and

21 other indications of chronic toxicity that may provide evidence of treatment-related effects and

22 insights into the way the test agent produces tumors. Current standardized carcinogenicity

23 studies in rodents test at least 50 animals per sex per dose group in each of three treatment groups

24 and in a concurrent control group, usually for 18 to 24 months, depending on the rodent species

25 tested (OECD, 1981; U.S. EPA, 1983a-c). The high dose in long-term studies is generally

26 selected to provide the maximum ability to detect treatment-related carcinogenic effects while not

27 compromising the outcome of the study through excessive toxicity or inducing inappropriate

28 toxicokinetics (e.g., overwhelming absorption or detoxification mechanisms). The purpose of two

29 or more lower doses is to provide some information on the shape of the dose-response curve. 

30 Similar protocols have been and continue to be used by many laboratories worldwide.

31 All available studies of tumor effects in whole animals are considered, at least

32 preliminarily. The analysis discards studies judged to be wholly inadequate in protocol, conduct,

33 or results. Criteria for the technical adequacy of animal carcinogenicity studies have been

34 published and should be used as guidance to judge the acceptability of individual studies (NTP,
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1 1984; OSTP, 1985). Care is taken to include studies that provide some evidence bearing on

2 carcinogenicity or that help interpret effects noted in other studies, even if they have some

3 limitations of protocol or conduct. Such limited, but not wholly inadequate, studies can

4 contribute as their deficiencies permit. The findings of long-term rodent bioassays are always

5 interpreted in conjunction with results of prechronic studies along with metabolism toxicokinetic

6 metabolism studies and other pertinent information, if available. Evaluation of tumor effects

7 requires consideration of both biological and statistical significance of the findings (Haseman,

8 1984, 1985, 1990, 1995). The following sections highlight the major issues in the evaluation of

9 long-term carcinogenicity studies.

10

11 Dosing Issues

12 Among the many criteria for technical adequacy of animal carcinogenicity studies is the

13 appropriateness of dose selection. The selection of doses for chronic bioassays requires scientific

14 judgments and must be based on sound toxicologic principles. Dose selection should be made on

15 the basis of relevant toxicologic information from prechronic, mechanistic, and toxicokinetic and

16 mechanistic studies. How well the dose selection is made can be evaluated only after the

17 completion of the bioassay. A scientific rationale for dose selection should be clearly articulated

18 (ILSI, 1997).

19 In order to obtain the most relevant information from a long-term carcinogenicity study, it

20 is important to maximize exposure conditions to the test material. At the same time, there is a

21 need for caution in using excessive high-dose levels that would confound the interpretation of

22 study results to humans. The middle and lowest doses should be selected to characterize the shape

23 of the dose-response curve as much as possible. It is important that the doses are adequately

24 spaced so that the study would provide relevant dose-response data for assessing human hazard

25 and risk. If the testing of potential carcinogenicity is being combined with an evaluation of

26 noncancer chronic toxicity, the study should be designed to include one dose that does not elicit

27 adverse effects.

28 With regard to the appropriateness of the high dose, an adequate high dose would be one

29 that produces some toxic effects without either unduly affecting mortality from effects other than

30 cancer or producing significant adverse effects on the nutrition and health of the test animals

31 (OECD, 1981; NRC, 1993b). If the test agent does not appear to cause any specific target organ

32 toxicity or perturbation of physiological function, an adequate high dose would be one that causes

33 no more than 5%-10% reduction of body weight gain over the lifespan of the animals. The high

34 dose would be considered inadequate if no toxicity is observed. On the other hand, significant
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1 increases in mortality from effects other than cancer generally indicate that an adequate high dose

2 has been exceeded. Other signs of treatment-related toxicity associated with an excessive high

3 dose may include the following: (a) reduction of body weight gain greater than 10%, (b)

4 significant increases in abnormal behavioral and clinical signs, (c) significant changes in

5 hematology or clinical chemistry, (d) saturation of absorption and detoxification mechanisms, or

6 (e) marked changes in organ weight, morphology, and histopathology. It should be noted that

7 practical upper limits have been established to avoid the use of excessively high doses in long8 term carcinogenicity studies of environmental chemicals (e.g., 5% of the test substance in the feed

9 for dietary studies or 1 g/kg of body weight for oral gavage studies [OECD, 1981]).

10 For dietary studies, weight gain reductions should be evaluated as to whether there is a

11 palatability problem or an issue with food efficiency; certainly, the latter is a toxic manifestation. 

12 In the case of inhalation studies with respirable particles, evidence of impairment of normal

13 clearance of particles from the lung should be considered along with other signs of toxicity to the

14 respiratory airways to determine whether the high exposure concentration has been appropriately

15 selected. For dermal studies, evidence of skin irritation may indicate that an adequate high dose

16 has been reached (U.S. EPA, 1989d).

17 Interpretation of carcinogenicity study results is profoundly affected by study exposure

18 conditions, especially by inappropriate dose selection. This is particularly important in studies

19 that are nonpositive for carcinogenicity, since failure to reach a sufficient dose reduces the

20 sensitivity of the studies. A lack of tumorigenic responses at exposure levels that cause significant

21 impairment of animal survival may also not be acceptable. In addition, overt toxicity or

22 inappropriate toxicokinetics due to excessively high doses may result in tumor effects that are

23 secondary to the toxicity rather than directly attributable to the agent.

24 There are several possible outcomes regarding the study interpretation of the significance

25 and relevance of tumorigenic effects associated with exposure or dose levels below, at, or above

26 an adequate high dose. General guidance is given here that should not be taken as prescriptive;

27 for each case, the information at hand is evaluated and a rationale should be given for the position

28 taken.

29

30 C Adequate high dose: If an adequate high dose has been utilized, tumor effects are

31 judged positive or negative depending on the presence or absence of significant tumor

32 incidence increases, respectively.

33 C Excessive high dose: If toxicity or mortality is excessive at the high dose,

34 interpretation depends on the finding of tumors or not.
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1 (a) Studies that show tumor effects only at excessive doses may be compromised

2 and may or may not carry weight, depending on the interpretation in the context

3 of other study results and other lines of evidence. Results of such studies,

4 however, are generally not considered suitable for dose-response extrapolation if

5 it is determined that the mode(s) of action underlying the tumorigenic responses

6 at high doses are not operative at lower doses.

7 (b) Studies that show tumors at lower doses, even though the high dose is excessive

8 and may be discounted, should be evaluated on their own merits.

9 (c) If a study does not show an increase in tumor incidence at a toxic high dose and

10 appropriately spaced lower doses are used without such toxicity or tumors, the

11 study is generally judged as negative for carcinogenicity.

12 C Inadequate high dose: Studies of inadequate sensitivity where an adequate high dose

13 has not been reached may be used to bound the dose range where carcinogenic effects

14 might be expected.

15

16

17 Statistical Considerations

18 The main aim of statistical evaluation is to determine whether exposure to the test agent is

19 associated with an increase of tumor development. Statistical analysis of a long-term study should

20 be performed for each tumor type separately. The incidence of benign and malignant lesions of

21 the same cell type, usually within a single tissue or organ, are considered separately and are

22 combined when scientifically defensible (McConnell et al., 1986).

23 Trend tests and pairwise comparison tests are the recommended tests for determining

24 whether chance, rather than a treatment-related effect, is a plausible explanation for an apparent

25 increase in tumor incidence. A trend test such as the Cochran-Armitage test (Snedecor and

26 Cochran, 1967) asks whether the results in all dose groups together increase as dose increases. A

27 pairwise comparison test such as the Fisher exact test (Fisher, 1950) asks whether an incidence in

28 one dose group is increased over the control group. By convention, for both tests a statistically

29 significant comparison is one for which p <0.05 that the increased incidence is due to chance. 

30 Significance in either kind of test is sufficient to reject the hypothesis that chance accounts for the

31 result. A statistically significant response may or may not be biologically significant and vice

32 versa. The selection of a significance level is a policy choice based on a trade-off between the

33 risks of false positives and false negatives. A significance level of greater or less than 5% is

34 examined to see if it confirms other scientific information. When the assessment departs from a
7/02/99 2-13 DRAFT

1 simple 5% level, this should be highlighted in the risk characterization. A two-tailed test or a one2 tailed test can be used. In either case a rationale is provided.

3 Considerations of multiple comparisons should also be taken into account. Haseman

4 (1983) analyzes typical animal bioassays testing both sexes of two species and concludes that,

5 because of multiple comparisons, a single tumor increase for a species-sex-site combination that is

6 statistically significant at the 1% level for common tumors or 5% for rare tumors corresponds to a

7 7%-8% significance level for the study as a whole. Therefore, animal bioassays presenting only

8 one significant result that falls short of the 1% level for a common tumor must be treated with

9 caution.

10

11 Concurrent and Historical Controls

12 The standard for determining statistical significance of tumor incidence comes from a

13 comparison of tumors in dosed animals as compared with concurrent control animals. Additional

14 insights about both statistical and biological significance can come from an examination of

15 historical control data (Tarone, 1982; Haseman, 1995). Historical control data can add to the

16 analysis, particularly by enabling identification of uncommon tumor types or high spontaneous

17 incidence of a tumor in a given animal strain. Identification of common or uncommon situations

18 prompts further thought about the meaning of the response in the current study in context with

19 other observations in animal studies and with other evidence about the carcinogenic potential of

20 the agent. These other sources of information may reinforce or weaken the significance given to

21 the response in the hazard assessment. Caution should be exercised in simply looking at the

22 ranges of historical responses because the range ignores differences in survival of animals among

23 studies and is related to the number of studies in the database.

24 In analyzing results for uncommon tumors in a treated group that are not statistically

25 significant in comparison to concurrent controls, the analyst can use the experience of historical

26 controls to conclude that the result is in fact unlikely to be due to chance. In analyzing results for

27 common tumors, a different set of considerations comes into play. Generally speaking,

28 statistically significant increases in tumors should not be discounted simply because incidence

29 rates in the treated groups are within the range of historical controls or because incidence rates in

30 the concurrent controls are somewhat lower than average. Random assignment of animals to

31 groups and proper statistical procedures provide assurance that statistically significant results are

32 unlikely to be due to chance alone. However, caution should be used in interpreting results that

33 are barely statistically significant or in which incidence rates in concurrent controls are unusually

34 low in comparison with historical controls.
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1 In cases where there may be reason to discount the biological relevance to humans of

2 increases in common animal tumors, such considerations should be weighed on their own merits

3 and clearly distinguished from statistical concerns.

4 When historical control data are used, the discussion needs to address several issues that

5 affect comparability of historical and concurrent control data. Among these issues are the

6 following: genetic drift in the laboratory strains, differences in pathology examination at different

7 times and in different laboratories (e.g., in criteria for evaluating lesions; variations in the

8 techniques for preparation or reading of tissue samples among laboratories), and comparability of

9 animals from different suppliers. The most relevant historical data come from the same laboratory

10 and same supplier, gathered within 2 or 3 years one way or the other of the study under review;

11 other data should be used only with extreme caution.

12

13 Assessment of Evidence of Carcinogenicity from Long-Term Animal Studies

14 In general, observation of tumor effects under different circumstances lends support to the

15 significance of the findings for animal carcinogenicity. Significance is a function of the number of

16 factors present and, for a factor such as malignancy, the severity of the observed pathology. The

17 following observations add significance to the tumor findings:

18

19 C uncommon tumor types;

20 C tumors at multiple sites;

21 C tumors by more than one route of administration;

22 C tumors in multiple species, strains, or both sexes;

23 C progression of lesions from preneoplastic to benign to malignant;

24 C reduced latency of neoplastic lesions;

25 C metastases;

26 C unusual magnitude of tumor response;

27 C proportion of malignant tumors; and

28 C dose-related increases.

29

30 These guidelines adopt the science policy position that tumor findings in animals indicate

31 that an agent may produce such effects in humans. Moreover, the absence of tumor findings in

32 well-conducted, long-term animal studies in at least two species provides reasonable assurance

33 that an agent may not be a carcinogenic concern for humans. Each of these is a default

34 assumption that may be adopted, when appropriate, after evaluation of tumor data and other key
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1 evidence.

2 Site Concordance

3 Site concordance of tumor effects between animals and humans is an issue to be

4 considered in each case. Thus far, there is evidence that growth control mechanisms at the level

5 of the cell are homologous among mammals, but there is no evidence that these mechanisms are

6 site concordant. Moreover, agents observed to produce tumors in both humans and animals have

7 produced tumors either at the same (e.g., vinyl chloride) or different sites (e.g., benzene) (NRC,

8 1994). Hence, site concordance is not assumed a priori. On the other hand, certain processes

9 with consequences for particular tissue sites (e.g., disruption of thyroid function) may lead to an

10 anticipation of site concordance.

11

12 2.2.2.2. Perinatal Carcinogenicity Studies

13 The objective of perinatal carcinogenesis studies is to determine the carcinogenic potential

14 and dose-response relationships of the test agent in the developing organism. Some investigators

15 have postulated that the age of initial exposure to a chemical carcinogen may influence the

16 carcinogenic response (Vesselinovitch et al., 1979; Rice, 1979; McConnell, 1992). Current

17 standardized long-term carcinogenesis bioassays generally begin dosing animals at 6-8 weeks of

18 age and continue dosing for the lifespan of the animal (18-24 months). This protocol has been

19 modified in some cases to investigate the potential of the test agent to induce transplacental

20 carcinogenesis or to investigate the potential differences following perinatal and adult exposures;

21 but currently there is not a standardized protocol for testing agents for carcinogenic effects

22 following prenatal or early postnatal exposure. 

23 Several cancer bioassay studies have compared adult and perinatal exposures (see

24 McConnell, 1992; U.S. EPA, 1996a). A review of these reveals that perinatal exposure rarely

25 identifies carcinogens that are not found in standard animal bioassays. Exposure that is perinatal

26 sometimes slightly increases the incidence of a given type of tumor. The increase may reflect an

27 increased length of exposure and a higher dose for the developing organism relative to the adult,

28 or an increase in sensitivity in some cases. Additionally, exposure that is perinatal through

29 adulthood sometimes reduces the latency period for tumors to develop in the growing organism

30 (U.S. EPA, 1996a). 

31 Because the perinatal exposure studies done to date provide only marginal additions to

32 knowledge as compared with standard bioassay protocols, EPA evaluates the need for such a

33 study agent-by-agent (U.S. EPA, 1997a,b). Perinatal study data analysis follows the principles

34 discussed above for evaluating other long-term carcinogenicity studies. When differences in
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1 responses in perinatal animals compared to adult animals suggest an increased susceptibility or

2 sensitivity of perinatal or postnatal animals, such as the ones below, a separate evaluation of the

3 response is prepared:

4

5 C a difference in dose-response relationship

6 C presence of different tumor types

7 C an earlier onset of tumors

8 C an increase in the incidence of tumors

9 An illustrative case study appears in Appendix E.

10

11 2.2.2.3. Other Studies

12 Various intermediate-term studies often use protocols that screen for carcinogenic or

13 preneoplastic effects, sometimes in a single tissue. Some involve the development of various

14 proliferative lesions, like foci of alteration in the liver (Goldsworthy et al., 1986). Others use

15 tumor endpoints, like the induction of lung adenomas in the sensitive strain A mouse (Maronpot

16 et al., 1986) or tumor induction in initiation-promotion studies using various organs such as the

17 bladder, intestine, liver, lung, mammary gland, and thyroid (Ito et al., 1992). In these tests, the

18 selected tissue is, in a sense, the test system rather than the whole animal. Important information

19 concerning the steps in the carcinogenic process and mode of action can be obtained from

20 “start/stop” experiments. In these protocols, an agent is given for a period of time to induce

21 particular lesions or effects, then stopped to evaluate the progression or reversibility of processes

22 (Todd, 1986; Marsman and Popp, 1994).

23 Assays in genetically engineered rodents may provide insight into the chemical and gene

24 interactions involved in carcinogenesis (Tennant et al., 1995). These mechanistically based

25 approaches involve activated oncogenes that are introduced (transgenic) or tumor suppressor

26 genes that are deleted (knocked out). If appropriate genes are selected, not only may these

27 systems provide information on mechanisms, but the rodents typically show tumor development

28 earlier than the standard bioassay. Transgenic mutagenesis assays also represent a mechanistic

29 approach for assessing the mutagenic properties of agents as well as developing quantitative

30 linkages between exposure, internal dose, and mutation related to tumor induction (Morrison and

31 Ashby, 1994; Sisk et al., 1994; Hayward et al., 1995). These systems use a stable genomic

32 integration of a lambda shuttle vector that carries a lacI target gene and a lacZ reporter gene.

33 The support that these studies give to a determination of carcinogenicity rests on their

34 contribution to the consistency of other evidence about an agent. For instance, benzoyl peroxide
7/02/99 2-17 DRAFT

1 has promoter activity on the skin, but the overall evidence may be less supportive (Kraus et al.,

2 1995). These studies also may contribute information about mode of action. One needs to

3 recognize the limitations of these experimental protocols such as short duration, limited histology,

4 lack of complete development of tumors, or experimental manipulation of the carcinogenic

5 process that may limit their contribution to the overall assessment. Generally, their results are

6 appropriate as aids in the assessment for interpreting other toxicological evidence (e.g., rodent

7 chronic bioassays), especially regarding potential modes of action. With sufficient validation,

8 these studies may partially or wholly replace chronic bioassays in the future (Tennant et al., 1995).

9

10 2.2.3. Structural Analogue Data

11 For some chemical classes, there is significant information available on the carcinogenicity

12 of analogues, largely in rodent bioassays. Analogue effects are instructive in investigating

13 carcinogenic potential of an agent as well as identifying potential target organs, exposures

14 associated with effects, and potential functional class effects or modes of action. All appropriate

15 studies are included and analyzed, whether indicative of a positive effect or not. Evaluation

16 includes tests in various animal species, strains, and sexes; with different routes of administration;

17 and at various doses, as data are available. Confidence in conclusions is a function of how similar

18 the analogues are to the agent under review in structure, metabolism, and biological activity. This

19 confidence needs to be considered to ensure a balanced position.

20

21 2.3. ANALYSIS OF OTHER KEY DATA

22 The physical, chemical, and structural properties of an agent, as well as data on endpoints

23 that are thought to be critical elements of the carcinogenic process, provide valuable insights into

24 the likelihood of human cancer risk. The following sections provide guidance for analyses of

25 these data.

26

27 2.3.1. Physicochemical Properties

28 Physicochemical properties affect an agent’s absorption, tissue distribution

29 (bioavailability), biotransformation, and degradation in the body and are important determinants

30 of hazard potential (and dose-response analysis). Properties to analyze include, but are not

31 limited to, the following: molecular weight, size, and shape; valence state; physical state (gas,

32 liquid, solid); water or lipid solubility, which can influence retention and tissue distribution; and

33 potential for chemical degradation or stabilization in the body.

34 An agent’s potential for chemical reaction with cellular components, particularly with
7/02/99 2-18 DRAFT

1 DNA and proteins, is also important. The agent’s molecular size and shape, electrophilicity, and

2 charge distribution are considered in order to decide whether they would facilitate such reactions.

3

4 2.3.2. Structure-Activity Relationships

5 Structure-activity relationship (SAR) analyses and models can be used to predict

6 molecular properties, surrogate biological endpoints, and carcinogenicity. Overall, these analyses

7 provide valuable initial information on agents, may strengthen or weaken concern, and are part of

8 the weight of evidence.

9 Currently, SAR analysis is most useful for chemicals and metabolites that are believed to

10 initiate carcinogenesis through covalent interaction with DNA (i.e., DNA-reactive, mutagenic,

11 electrophilic, or proelectrophilic chemicals) (Ashby and Tennant, 1991). For organic chemicals,

12 the predictive capability of SAR analysis combined with other toxicity information has been

13 demonstrated (Ashby and Tennant, 1994). The following parameters are useful in comparing an

14 agent to its structural analogues and congeners that produce tumors and affect related biological

15 processes such as receptor binding and activation, mutagenicity, and general toxicity (Woo and

16 Arcos, 1989):

17 C nature and reactivity of the electrophilic moiety or moieties present;

18 C potential to form electrophilic reactive intermediate(s) through chemical,

19 photochemical, or metabolic activation;

20 C contribution of the carrier molecule to which the electrophilic moiety(ies) is attached;

21 C physicochemical properties (e.g., physical state, solubility, octanol-water partition

22 coefficient, half-life in aqueous solution);

23 C structural and substructural features (e.g., electronic, stearic, molecular geometric);

24 C metabolic pattern (e.g., metabolic pathways and activation and detoxification ratio);

25 and

26 C possible exposure route(s) of the agent.

27

28 Suitable SAR analysis of non-DNA-reactive chemicals and of DNA-reactive chemicals

29 that do not appear to bind covalently to DNA requires knowledge or postulation of the probable

30 mode(s) of action of closely related carcinogenic structural analogues (e.g., receptor-mediated,

31 cytotoxicity-related). Examination of the physicochemical and biochemical properties of the

32 agent may then provide the rest of the information needed in order to make an assessment of the

33 likelihood of the agent’s activity by that mode of action.

34
7/02/99 2-19 DRAFT

1 2.3.3. Comparative Metabolism and Toxicokinetics

2 Studies of the absorption, distribution, biotransformation, and excretion of agents permit

3 comparisons among species to assist in determining the implications of animal responses for

4 human hazard assessment, supporting identification of active metabolites, identifying changes in

5 distribution and metabolic pathway or pathways over a dose range, and making comparisons

6 among different routes of exposure.

7 If extensive data are available (e.g., blood/tissue partition coefficients and pertinent

8 physiological parameters of the species of interest), physiologically based pharmacokinetic models

9 can be constructed to assist in a determination of tissue dosimetry, species-to-species

10 extrapolation of dose, and route-to-route extrapolation (Connolly and Andersen, 1991; see

11 Section 3.2.2). If it is not contrary to available data, it is assumed as a default that toxicokinetic

12 and metabolic processes are qualitatively comparable between species. Discussion of the defaults

13 regarding quantitative comparison and their modifications appears in Chapter 3.

14 The qualitative question of whether an agent is absorbed by a particular route of exposure

15 is important for weight-of-evidence classification, discussed in Section 2.7.1. Decisions whether

16 route of exposure is a limiting factor on expression of any hazard, in that absorption does not

17 occur by a route, are based on studies in which effects of the agent, or its structural analogues,

18 have been observed by different routes, on physical-chemical properties, or on toxicokinetics

19 studies.

20 Adequate metabolism and pharmacokinetic data can be applied toward the following as

21 data permit. Confidence in conclusions is enhanced when in vivo data are available.

22

23 C Identifying metabolites and reactive intermediates of metabolism and determining

24 whether one or more of these intermediates are likely to be responsible for the

25 observed effects. This information on the reactive intermediates will appropriately

26 focus SAR analysis, analysis of potential modes of action, and estimation of internal

27 dose in dose-response assessment (D’Souza et al., 1987; Krewski et al., 1987).

28 C Identifying and comparing the relative activities of metabolic pathways in animals with

29 those in humans as well as different ages. This analysis can provide insights for

30 extrapolating results of animal studies to humans.

31 C Describing anticipated distribution within the body and possibly identifying target

32 organs. Use of water solubility, molecular weight, and structure analysis can support

33 qualitative inferences about anticipated distribution and excretion. In addition,

34 describing whether the agent or metabolite of concern will be excreted rapidly or
7/02/99 2-20 DRAFT

1 slowly or will be stored in a particular tissue or tissues to be mobilized later can

2 identify issues in comparing species and formulating dose-response assessment

3 approaches.

4 C Identifying changes in toxicokinetics and metabolic pathways with increases in dose. 

5 These changes may result in important differences in disposition of the agent or its

6 generation of active forms of the agent between high and low dose levels. These

7 studies play an important role in providing a rationale for dose selection in

8 carcinogenicity studies.

9 C Identifying and comparing metabolic process differences by age, sex, or other

10 characteristic so that sensitive subpopulations can be recognized. For example,

11 metabolic capacity with respect to P450 enzymes in newborn children is extremely

12 limited compared to adults, so that a requirement for metabolic activation of a

13 carcinogen will limit its effect in young, whereas a requirement for metabolic

14 deactivation will result in increased sensitivity of this subpopulation (Cresteil, 1998). 

15 A variety of changes in toxicokinetics and physiology occur from fetal to post16 weaning, to young child. Any of these may make a difference to risk (Renwick, 1998)

17 C Determining bioavailability via different routes of exposure by analyzing uptake

18 processes under various exposure conditions. This analysis supports identification of

19 hazards for untested routes. In addition, use of physicochemical data (e.g., octanol20 water partition coefficient information) can support an inference about the likelihood

21 of dermal absorption (Flynn, 1990).

22

23 In all of these areas, attempts are made to clarify and describe as much as possible the

24 variability to be expected because of differences in species, sex, age, and route of exposure. The

25 analysis takes into account the presence of subpopulations of individuals who are particularly

26 vulnerable to the effects of an agent because of toxicokinetic or metabolic differences (genetically

27 or environmentally determined) (Bois et al., 1995), and is a special emphasis for assessment of

28 risks to children.

29

30

31 2.3.4. Toxicological and Clinical Findings

32 Toxicological findings in experimental animals and clinical observations in humans are an

33 important resource to the cancer hazard assessment. Such findings provide information on

34 physiological effects and effects on enzymes, hormones, and other important macromolecules, as
7/02/99 2-21 DRAFT

1 well as on target organs for toxicity. Given that the cancer process represents defects in terminal

2 differentiation, growth control, and cell death, developmental studies of agents may provide an

3 understanding of the activity of an agent that carries over to cancer assessment. Toxicity studies

4 in animals by different routes of administration support comparison of absorption and metabolism

5 by those routes. Data on human variability in standard clinical tests may provide insight into the

6 range of human sensitivity and common mechanisms to agents that affect the tested parameters.

7

8 2.3.5. Events Relevant to Mode of Carcinogenic Action

9 Information on the biochemical and biological changes that precede tumor development

10 (which includes but is not limited to mutagenesis, increased cell proliferation, inhibition of

11 programmed cell death, and receptor activation) may provide important information in

12 determining whether a cancer hazard exists and may help inform the dose-response relationship

13 below the range of observable tumor response. Because cancer is the result of a series of genetic

14 defects in genes controlling cell growth, division, and differentiation (Vogelstein et al., 1988), the

15 ability of an agent to affect genes or gene expression is of obvious importance in evaluating its

16 influence on the carcinogenic process. Initial and key questions to examine are: Does the agent (or

17 its metabolite) interact directly with and mutate DNA to bring about changes in gene expression?

18 Does the agent bring about effects on gene expression via other processes? Furthermore,

19 carcinogenesis involves a complex series and interplay of events that alter the signals a cell

20 receives from its extracellular environment to promote growth. Many, but not all, mutagens are

21 carcinogens, and some, but not all, agents that induce cell proliferation lead to tumor

22 development. Thus, understanding the range of key influences that the chemical may have on the

23 carcinogenic process is essential for evaluating mode of action. Endpoints that provide insight

24 into an agent’s ability to alter genes and gene expression and other features of an agent’s potential

25 mode of carcinogenic action are discussed below. 

26

27 2.3.5.1. Direct DNA Reactive Effects

28 It is well known that many carcinogens are electrophiles that interact with DNA, resulting

29 in DNA adducts and breakage (referred to in these guidelines as direct DNA effects). Following

30 DNA replication, these DNA lesions can be converted into mutations and stable cytogenetic

31 alterations, which then may initiate and contribute to the carcinogenic process (Shelby and Zeiger,

32 1990; Tinwell and Ashby, 1991). Thus, studies of mutations and other genetic lesions continue to

33 be important in the assessment of potential human cancer hazard and in the understanding of an

34 agent’s mode of carcinogenic action. EPA has published testing guidelines for detecting the
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1 ability of an agent to damage DNA and produce mutations and chromosomal aberrations. Briefly,

2 standard tests for gene mutations in bacteria and mammalian cells in vitro and in vivo, and for

3 structural chromosomal aberrations in vitro and in vivo are important examples of relevant

4 methods. New molecular approaches such as mouse mutations and cancer transgenic models are

5 providing a means to examine mutation at tissue sites where the tumor response is observed

6 (Heddle and Swiger, 1996). Additionally, continued improvements in fluorescent-based

7 chromosome staining methods (FISH, fluorescent in situ hybridization) will allow the detection of

8 specific chromosomal abnormalities in relevant target tissues (Tucker and Preston, 1998). 

9 Endpoints indicative of DNA damage but not measures of mutation per se, such as DNA

10 adducts or strand breakage, can be detected in relevant target tissues and thus contribute to

11 evaluating an agent’s mutagenic potential. Evidence of chemical-specific DNA adducts (e.g.,

12 reactions at oxygen sites in DNA bases or with ring nitrogens of guanine and adenine) provides

13 information on a mutagen’s ability to directly interact with DNA (La and Swenberg, 1996). It

14 should be noted that an increase in DNA binding shown with a radioactive label incorporated in

the chemical (e.g., C14 15 ) may reflect a direct DNA reactive mechanism, but needs to be examined 

because the label may reflect reuse of C14 16 in the synthesis of DNA rather than binding. Some

17 planar molecules (e.g., 9-aminoacridine) intercalate between base pairs of DNA, which results in a

18 physical distortion in DNA that may lead to mutations when DNA replicates. As discussed

19 below, some carcinogens do not interact directly with DNA, but can produce increases in

20 endogenous levels of DNA adducts (e.g., 8-hydroxyguanine) by indirect mechanisms. 

21

22 2.3.5.2. Indirect DNA Effects or Other Effects on Genes/Gene Expression

23 Although some carcinogens may result in an elevation of mutations or cytogenetic

24 anomalies as detected in standard assays, they may do so by indirect mechanisms. These effects

25 may be brought about by chemical-cell interactions rather than the chemical (or its metabolite)

26 directly interacting with DNA. An increase in mutations might be due to cytotoxic exposures

27 causing regenerative proliferation or to mitogenic influences (Cohen and Ellwein, 1990). 

28 Increased cell division may elevate mutation by clonal expansion of initiated cells or by increasing

29 the number of genetic errors by rapid cell division and reduced time for DNA repair. Some agents

30 might result in an elevation of mutations by interfering with the enzymes involved in DNA repair

31 and recombination (Barrett and Lee, 1992). Damage to certain critical DNA repair genes or other

32 genes (e.g., the p53 gene) may result in genomic instability, which predisposes cells to further

33 genetic alterations and increases the probability of neoplastic progression (Harris and Hollstein,

34 1993; Levine, 1994). Likewise, DNA repair processes may be saturated at certain doses of a
7/02/99 2-23 DRAFT

1 chemical, and thus result in an elevation of genetic alterations. Programmed cell death (apoptosis)

2 can potentially be blocked by an agent, thereby permitting replication of cells carrying genetic

3 errors. For example, peroxisome proliferators may act by suppressing apoptotic pathways

4 (Shulte-Hermann et al., 1993; Bayly et al., 1994). At certain doses an agent may also generate

5 reactive oxygen species that produce oxidative damage to DNA and other important

6 macromolecules (Kehrer, 1993; Clayson et al., 1994; Chang et al., 1988). The role of these

7 adducts, attributable to oxidative damage (e.g., 8-hydroxyguanine), in tumorigenesis is currently

8 unclear. 

9 Several carcinogens have been shown to induce aneuploidy (Gibson et al., 1995; Barrett,

10 1992). The loss or gain of chromosomes (i.e., aneuploidy) can result in the loss of heterozygosity

11 or genomic instability (Fearon and Vogelstein, 1990; Cavenee et al., 1986). Agents that cause

12 aneuploidy typically interfere with the normal process of chromosome segregation by interacting

13 with non-DNA targets such as the proteins needed for chromosome movement. All tumors

14 (except leukemias and lymphomas) are aneuploid, but whether this is the cause or the effect of

15 tumorigenesis is not clear. Thus, it is important to understand whether the agent induces

16 aneuploidy as a key early event in the carcinogenic process or is necessary for tumor progression. 

17 It is possible for an agent to alter gene expression by transcriptional, translational, or post18 translational modifications (Barrett, 1995). For example, perturbation of DNA methylation

19 patterns may cause effects that contribute to carcinogenesis (Jones, 1986; Goodman and Counts,

20 1993; Holliday, 1987; Chuang et al., 1996). Overexpression of genes by DNA amplification has

21 been observed in certain tumors (Vainio et al., 1992). Gene amplification may result from

22 disproportionate DNA replication. Other mechanisms of altering gene expression may involve

23 cellular reprogramming through hormonal or receptor-mediated mechanisms (Ashby et al., 1994;

24 Barrett, 1992).

25 Both cell proliferation and programmed cell death are mandatory for the maintenance of

26 homeostasis in normal tissue, and when altered become important elements of the carcinogenic

27 process. The balance between the two directly affects the survival and growth of initiated cells, as

28 well as preneoplastic and tumor cell populations (i.e., increase in cell proliferation or decrease in

29 cell death) (Bellamy et al., 1995; Cohen and Ellwein, 1990, 1991; Cohen et al., 1991). Thus,

30 measures of these events contribute to the weight of the evidence for cancer hazard and to mode31 of-action understanding. In studies of proliferative effects, distinctions should be made between

32 mitogenesis and regenerative proliferation (Cohen and Ellwein, 1990, 1991; Cohen et al., 1991). 

33 In applying information from studies on cell proliferation and apoptosis to risk assessment, it is

34 important to identify the tissues and target cells involved, to measure effects in both normal and
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1 neoplastic tissue, to distinguish between apoptosis and necrosis, and to determine the dose that

2 affects these processes. Gap-junctional intercellular communication is believed to play a role in

3 tissue and organ development and in the maintenance of a normal cellular phenotype within

4 tissues. A growing body of evidence suggests that chemical interference with gap-junctional

5 intercellular communication is a contributing factor in tumor development (Swierenga and

6 Yamasaki, 1992; Yamasaki, 1995). 

7

8 2.3.5.3. Experimental Considerations in Evaluating Data on Precursor Events

9 Most testing schemes for mutagenicity and other short-term assays were designed for

10 hazard identification purposes; thus, these assays are generally conducted using acute exposures. 

11 For data on “precursor steps” to be useful in informing the dose-response curve for tumor

12 induction below the level of observation, it is important that data come from in vivo studies where

13 exposure is repeated or given over an extended period of time. Although consistency of results

14 across different assays and animal models provides a stronger basis for drawing conclusions, it is

15 desirable to have data on the precursor event in the same target organ, sex, animal strain, and

16 species as the tumor data. In evaluating an agent’s mode of action, it is usually not sufficient to

17 determine that some event commences upon dosing. It is important to understand whether it is a

18 causal event that plays a key role in the process that leads to tumor development, versus an effect

19 of the cancer process itself or simply an associated event. 

20

21 2.3.5.4. Judging Data

22 Criteria that are applicable for judging the adequacy of mechanistically based data include

23 the following:

24

25 C mechanistic relevance of the data to carcinogenicity,

26 C number of studies of each endpoint,

27 C consistency of results in different test systems and different species,

28 C similar dose-response relationships for tumor and mode of action-related effects,

29 C tests conducted in accordance with generally accepted protocols, and

30 C degree of consensus and general acceptance among scientists regarding interpretation

31 of the significance and specificity of the tests.

32

33 Although important information can be gained from in vitro test systems, a higher level of

34 confidence is generally given to data that are derived from in vivo systems, particularly those
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1 results that show a site concordance with the tumor data.

2

3 2.4. BIOMARKER INFORMATION

4 Various endpoints can serve as biological markers of events in biological systems or

5 samples. In some cases, these molecular or cellular effects (e.g., DNA or protein adducts,

6 mutation, chromosomal aberrations, levels of thyroid stimulating hormone) can be measured in

7 blood, body fluids, cells, and tissues to serve as biomarkers of exposure in both animals and

8 humans (Callemen et al., 1978; Birner et al., 1990). As such, they can do the following:

9

10 C act as an internal surrogate measure of chemical dose, representing as appropriate,

11 either recent (e.g., serum concentration) or accumulated (e.g., hemoglobin adducts)

12 exposure;

13 C help identify doses at which elements of the carcinogenic process are operating;

14 C aid in interspecies extrapolations when data are available from both experimental

15 animal and human cells; and

16 C under certain circumstances, provide insights into the possible shape of the dose17 response curve below levels where tumor incidences are observed (e.g., Choy, 1993).

18

19 Genetic and other findings (like changes in proto-oncogenes and tumor suppressor genes

20 in preneoplastic and neoplastic tissue or, possibly, measures of endocrine disruption) can indicate

21 the potential for disease and as such serve as biomarkers of effect. They, too, can be used in

22 different ways:

23

24 C The spectrum of genetic changes in proliferative lesions and tumors following chemical

25 administration to experimental animals can be determined and compared with those in

26 spontaneous tumors in control animals, in animals exposed to other agents of varying

27 structural and functional activities, and in persons exposed to the agent under study.

28 C They may provide a linkage to tumor response.

29 C They may help to identify subpopulations of individuals who may be at an elevated risk

30 for cancer, e.g., cytochrome P450 2D6/debrisoquine sensitivity for lung cancer

31 (Caporaso et al., 1989) or inherited colon cancer syndromes (Kinzler et al., 1991;

32 Peltomäki et al., 1993).

33 C As with biomarkers of exposure, it may be justified in some cases to use these

34 endpoints for dose-response assessment or to provide insight into the potential shape
2

A “key event” is an empirically observable, precursor step that is itself a necessary element of the

mode of action, or is a marker for such an element.

7/02/99 2-26 DRAFT

1 of the dose-response curve at doses below those at which tumors are induced

2 experimentally.

3

4 In applying biomarker data to cancer assessment (particularly assessments based on

5 epidemiologic data), one should consider the following:

6

7 C routes of exposure,

8 C exposure to mixtures,

9 C time after exposure,

10 C sensitivity and specificity of biomarkers, and

11 C dose-response relationships.

12

13 2.5. MODE OF ACTION-GENERAL CONSIDERATIONS AND FRAMEWORK FOR

14 ANALYSIS

15 2.5.1. General Considerations

16 The interaction of the biology of the organism and the chemical properties of the agent

17 determine whether there is an adverse effect. Thus, mode-of-action analysis is based on physical,

chemical, and biological information that helps to explain key events5 18 in an agent’s influence on

19 development of tumors. The entire range of information developed in the assessment is reviewed

20 to arrive at a reasoned judgment. An agent may work by more than one mode of action both at

21 different sites and at the same tumor site. It is felt that at least some information bearing on mode

22 of action (e.g., SAR, screening tests for mutagenicity) is present for most agents undergoing

23 assessment of carcinogenicity, even though certainty about exact molecular mechanisms may be

24 rare.

25 Inputs to mode-of-action analysis include tumor data in humans, animals, and among

26 structural analogues as well as the other key data. The more complete the data package and

27 generic knowledge about a given mode of action, the more confidence one has and the more one

28 can replace or refine default science policy positions with relevant information. Making reasoned

29 judgments is generally based on a data-rich source of chemical, chemical class, and tumor type30 specific information. Many times there will be conflicting data and gaps in the information base;

31 one must carefully evaluate these uncertainties before reaching any conclusion.
7/02/99 2-27 DRAFT

1 In making decisions about potential modes of action and the relevance of animal tumor

2 findings to humans (Ashby et al., 1990), very often the results of chronic animal studies may give

3 important clues. Some of the important factors to review include the following:

4

5 C tumor types, e.g., those responsive to endocrine influence or those produced by

6 reactive carcinogens (Ashby and Tennant, 1991);

7 C number of tumor sites, sexes, studies, and species affected or unaffected (Tennant,

8 1993);

9 C influence of route of exposure, spectrum of tumors, and local or systemic sites;

10 C target organ or system toxicity, e.g., urinary chemical changes associated with stone

11 formation, effects on immune surveillance;

12 C presence of proliferative lesions, e.g., hepatic foci, hyperplasias;

13 C progression of lesions from preneoplastic to benign to malignant with dose and time;

14 C ratio of malignant to benign tumors as a function of dose and time;

15 C time of appearance of tumors after commencing exposure;

16 C tumors invading locally, metastasizing, producing death;

17 C tumors at sites in laboratory animals with high or low spontaneous historical incidence;

18 C biomarkers in tumor cells, both induced and spontaneous, e.g., DNA or protein

19 adducts, mutation spectra, chromosome changes, oncogene activation; and

20 C shape of the dose response in the range of tumor observation, e.g., linear vs. profound

21 change in slope.

22

23 Some of the myriad of ways that information from chronic animal studies influences mode24 of-action judgments include the following. Multisite and multispecies tumor effects are often

25 associated with mutagenic agents. Tumors restricted to one sex/species may suggest an influence

26 restricted to gender, strain, or species. Late onset of tumors that are primarily benign or are at

27 sites with a high historical background incidence or show reversal of lesions on cessation of

28 exposure may point to a growth-promoting mode of action. The possibility that an agent may act

29 differently in different tissues or have more than one mode of action in a single tissue must also be

30 kept in mind.

31 Simple knowledge of sites of tumor increase in rodent studies can give preliminary clues

32 as to mode of action. Experience at the National Toxicology Program (NTP) indicates that

33 substances that are DNA reactive and produce gene mutations may be unique in producing

34 tumors in certain anatomical sites, while tumors at other sites may arise from both mutagenic or
7/02/99 2-28 DRAFT

1 nonmutagenic influences (Ashby and Tennant, 1991; Huff et al., 1991).

2 Effects on tumor sites in rodents and other mode-of-action information has been explored

3 for certain agents (Alison et al., 1994; Clayson, 1989; ECETOC, 1991; MacDonald et al., 1994;

4 McClain, 1994; Tischler et al., 1991; ILSI, 1995; Cohen and Ellwein, 1991; FASEB, 1994; Havu

5 et al., 1990; U.S. EPA, 1991c; Li et al., 1987; Grasso and Hinton, 1991; Larson et al., 1994;

6 IARC, 1990; Jack et al., 1983; Stitzel et al., 1989; Ingram and Grasso, 1991; Bus and Popp,

7 1987; Prahalada et al., 1994; Yamada et al., 1994; Hill et al., 1989; Burek et al., 1988).

8

9 2.5.2. Evaluating a Postulated Mode of Action

10

11 Peer Review

12 This section contains a framework for evaluating a postulated mode of action. In reaching

13 conclusions, the question of “general acceptance” of a mode of action will be tested as part of the

14 independent peer review that EPA obtains for its assessment and conclusions. In some cases the

15 mode of action may have already been established by development of a large body of research

16 information and characterization of the phenomenon over time. In some cases there will have

17 been development of an Agency policy, e.g., male rat thyroid disruption, or a series of previous

18 assessments in which both the mode of action and its applicability to particular cases has been

19 explored, e.g., urinary bladder stones. If so, the assessment and its peer review can be focused on

20 the evidence that a particular agent acts in this mode.

21 In other cases, the mode of action previously may not have been the subject of an Agency

22 document. If so, the data to support both the mode of action and the activity of the agent with

23 respect to it will be the subjects of EPA assessment and subsequent peer review. 

24

25 Use of the Framework

26 The framework supports a full analysis of mode-of-action information, but can also be

27 used as a screen to decide whether sufficient information is available to evaluate or the data gaps

28 are too substantial to justify further analysis. Mode-of-action conclusions are used to address the

29 question of human relevance of animal tumor responses, to address differences in anticipated

30 response among humans such as between children and adults or men and women, and as the basis

31 of decisions about the anticipated shape of the dose-response relationship. Guidance on the latter

32 appears in Section 3.

33

34
7/02/99 2-29 DRAFT

1 2.5.3. Framework for Evaluating a Postulated Carcinogenic Mode(s) of Action

2 This framework is intended to be an analytic tool for judging whether available data

3 support a mode of carcinogenic action postulated for an agent. It is based upon considerations

4 for causality in epidemiologic investigations originally articulated by Hill, but later modified by

5 others and extended to experimental studies. The original Hill criteria were applied to

6 epidemiologic data, while this framework is applied to a much wider assortment of experimental

7 data, so it retains the basic principles of Hill but is much modified in content.

8 A mode of action is composed of key events and processes starting with the interaction of

9 an agent with a cell, through operational and anatomical changes, resulting in cancer formation. 

10 “Mode” of action is contrasted with “mechanism” of action, which implies a more detailed,

11 molecular description of events than is meant by mode of action. There are many examples of

12 possible modes of carcinogenic action, such as mutagenicity, mitogenesis, inhibition of cell death,

13 cytotoxicity with reparative cell proliferation, and immune suppression. All pertinent studies are

14 reviewed in analyzing a mode of action, and an overall weighing of evidence is performed, laying

15 out the strengths, weaknesses, and uncertainties of the case as well as potential alternative

16 positions and rationales. Identifying data gaps and research needs is also part of the assessment.

17 To show that a postulated mode of action is operative, it is generally necessary to outline

18 the sequence of events leading to cancer, to identify key events that can be measured, and to

19 weigh information to determine whether there is a causal relationship between events and cancer

20 formation. In no case will it be expected that the complete sequence is known at the molecular

21 level. Instead, empirical observations made at different levels of biological organization are

22 analyzed: biochemical, cellular, physiological, tissue, organ, and system levels.

23 Several important points should be kept in mind when working with the framework:

24

25 C The topics listed for analysis should not be regarded as a checklist of necessary

26 “proofs.” The judgment whether a postulated mode of action is supported by available

27 data takes account of the analysis as a whole.

28 C The framework provides a structure for organizing the facts upon which conclusions

29 as to mode of action rest. The purpose of using the framework is to make analysis

30 transparent and allow the reader to understand the facts and reasoning behind a

31 conclusion. 

32 C The framework does not dictate an answer. The weight of evidence that is sufficient to

33 support a decision about a mode of action may be less or more depending on the

34 purpose of the analysis, e.g., screening, research needs identification, or full risk
7/02/99 2-30 DRAFT

1 assessment. To make the reasoning transparent, the purpose of the analysis ought to

2 be made apparent to the reader.

3 C Toxicokinetic studies may contribute to mode-of-action analysis by identifying the

4 active form of an agent that is central to the mode of action. Apart from contributing

5 in this way, toxicokinetics studies may reveal effects of saturation of metabolic

6 processes. These are not considered key events in a mode of action, but are given

7 separate consideration in assessing dose metrics and potential nonlinearity of the dose8 response relationship.

9 C Generally, “sufficient” support is a matter of scientific judgment in the context of the

10 requirements of the decision maker or in the context of science policy guidance

11 regarding a certain mode of action. 

12 C While a postulated mode of action may be supported for a described response in a

13 specific tissue, it may not explain other tumor responses observed. The latter will need

14 separate consideration in hazard and dose-response assessment.

15

16 It is anticipated that in a risk assessment document, the analysis of a postulated mode of

17 action will be presented before or with the characterization of an agent’s potential hazard to

18 humans.

19

20 2.5.3.1. Content of the Framework

21 The framework analysis begins with a summary description of the postulated mode of

22 action for a tumor type. (Each postulated mode of action requires separate analysis.) This is

23 followed by topics for analysis and presentation in a convenient order. For illustration, the

24 explanation of each topic includes typical questions to be addressed to the available empirical data

25 and experimental observations anticipated to be pertinent. The latter will vary from case to case. 

26 For a particular mode of action, certain observations may be established as essential in practice or

27 policy, e.g., measures of thyroid hormone levels in supporting thyroid hormone elevation as a key

28 event in carcinogenesis. A conclusion and an analysis of human relevance including

29 subpopulations are the the final parts of the analysis.

30

31 1. Summary Description of Postulated Mode of Action

32 This description briefly explains the sequence of events and processes that are considered

33 to lead to cancer formation. For example, for thyroid disruption and thyroid follicular cell tumors:

34 Thyroid hormone production is regulated by actions of the hypothalamus,
7/02/99 2-31 DRAFT

1 pituitary, and thyroid gland. Homeostasis of thyroid hormone is maintained by

2 a feedback loop between the hypothalamus and pituitary and the thyroid gland. 

3 The hypothalamus produces thyrotrophin reducing hormone (TRH), which

4 stimulates the pituitary to produce thyroid stimulating hormone (TSH) which,

5 in turn, stimulates the thyroid to produce thyroid hormone. The hypothalamus

6 and pituitary respond to high levels of circulating thyroid hormone by

7 suppressing TRH and TSH production, and to a low level by increasing them.

8 The mode of action considered is continuous elevation of TSH levels that

9 stimulates the thyroid gland to deplete its stores of thyroid hormone and

10 continues to push production resulting in hypertrophy of the production cells

11 (follicular cells) leading to hyperplasia, nodular hyperplasia, and, eventually,

12 tumors of these cells. In rats, the chain of events may be induced by direct

13 effects on hormone synthesis or by metabolic removal of circulating hormone. 

14 2. “Identification of key events” is a consideration devised for this framework. A “key

15 event” is an empirically observed precursor step consistent with a mode of action. In order to

16 judge how well data support involvement of an event in carcinogenic processes, the experimental

17 definition of the event or events must be clear and repeatable. To support an association,

18 experiments need to define and measure an event consistently.

19

20 C Can a list of events be identified that are key to the carcinogenic process?

21 C Are the events well defined? 

22

23 Pertinent observations: e.g., increased cell growth, organ weight, histology, proliferation assays,

24 hormone or other protein perturbations, receptor-ligand changes, DNA or chromosome effects,

25 cell cycle effects.

26

27 3. “Strength, consistency, specificity of association”: A statistically significant

28 association between events and a tumor response observed in well-conducted studies is

29 supportive of causation. Consistent observations in a number of such studies with differing

30 experimental designs increases that support, since different designs may reduce unknown biases. 

31 Studies showing “recovery,” i.e., absence or reduction of carcinogenicity when the event is

32 blocked or diminished, are particularly important tests of the association. Specificity of the

33 association, without evidence of other modes of action, strengthens a causal conclusion. 

34
7/02/99 2-32 DRAFT

1 C What is the level of statistical and biological significance for each event and for

2 cancer?

3 C Do independent studies and different experimental hypothesis-testing approaches

4 produce the same associations?

5 C Does the agent produce effects other than postulated?

6 C Is the key event associated with precursor lesions? 

7

8 Pertinent observations: e.g., tumor response associated with events (site of action logically relates

9 to event[s]), precursor lesions associated with events, initiation-promotion studies, stop/recovery

10 studies.

11

12 4. “Dose-response relationship”: If a key event and tumor endpoints increase with dose,

13 a causal association can be strengthened. Dose-response associations of the key event with other

14 precursor events can add further strength. Difficulty arises when an event is not causal, but

15 accompanies the process generally. Dose-response studies coupled with mechanistic studies can

16 assist in clarifying these relationships. 

17

18 C What are the correlations among doses producing events and cancer?

19

20 Pertinent observations: e.g., 2-year bioassay observation of lesions correlated with observations of

21 hormone changes and the same lesions in shorter term studies or in interim sacrifice. 

22

23 5. “Temporal relationship”: If an event is a cause of tumorigenesis, it must precede

24 tumor appearance. An event may also be observed contemporaneously or after tumor

25 appearance; these observations may add to the strength of association, but not to the temporal

26 association.

27

28 C What is the ordering of events that underlie the carcinogenic process?

29 C Is this ordering consistent among independent studies?

30

31 Pertinent observations: Studies of varying duration observing the temporal sequence of

32 events and tumorigenicity.

33

34 6. “Biological plausibility and coherence”: The postulated mode of action and the
7/02/99 2-33 DRAFT

1 events that are part of it need to be based on current understanding of the biology of cancer to be

2 accepted. If the body of information under scrutiny is consistent with other examples (including

3 structurally related agents) for which the postulated mode of action is accepted, the case is

4 strengthened. Since some modes of action can be anticipated to evoke effects other than cancer,

5 the available toxicity database on noncancer effects can contribute to this evaluation, e.g.,

6 reproductive effects of certain hormonal disturbances.

7

8 C Is the mode of action consistent with what is known about carcinogenesis in general

9 and for the case specifically?

10 C Are carcinogenic effects and events consistent across structural analogues?

11 C Is the database on the agent internally consistent in supporting the purported mode of

12 action, including relevant noncancer toxicities?

13

14 Pertinent observations: Scientific basis for considering a postulated mode of action generally,

15 given current state of knowledge of carcinogenic processes; previous examples of data sets

16 showing the mode of action; data sets on analogues; coherence of data in this case from cancer

17 and noncancer toxicity studies.

18

19 7. “Other modes of action”: This discussion covers alternative modes of action for the

20 tumor response considered and whether they are supported by the data. In addition, it provides a

21 place to discuss other tumor observations that may be arising from a different mode of action than

22 postulated.

23

24 8. “Conclusion”: This is a brief conclusion and rationale as to whether the postulated

25 mode of action is supported, also reflecting the purpose of the evaluation. The conclusion that a

26 mode of action is supported is stronger as more of the above topic analyses point in the same

27 direction, and weaker as fewer do so. The testing of the mode of action hypothesis by various

28 experimental approaches with the same result creates a stronger basis for conclusions. 

29 Characteristics of strength of support include data showing that all key events are in sequence

30 prior to tumor formation, dose and timing are consistent with the sequence, and reversal or

31 reduction of key events and effects occurs upon cessation of dosing. The conclusion should

32 address whether key event or associated metabolic information allows identification of rate33 limiting measures of either the mode of action or of toxicokinetics.

34
7/02/99 2-34 DRAFT

1 9. “Human relevance, including subpopulations” : This is an analysis of data on the

2 question whether a mode of action found to be operative in animals is also operative in humans

3 and whether any human subpopulation is apt to qualitatively respond to the mode of action

4 differently than the general population. Relevance to humans of animal responses is the default

5 assumption since metazoans appear to share the basic modes of carcinogenic action.

6 When sufficient information is developed in mature animals to show a mode of action for a

7 specific tumor type, an evaluation will be made of whether the mode of action is qualitatively

8 applicable to children (including infants and fetuses), i.e., same sequence of key events is

9 anticipated to be involved. Ideally we would have data pertinent to the question with respect to

10 the agent under assessment. In the absence of such data, a cogent biological rationale needs to

11 be developed regarding whether the mode of action is applicable to children. For the latter, the

12 evaluation would cover the scientific information at large, including such considerations as

13 age-related similarities and differences in the occurrence of the specific tumor type in the U.S.

14 population, in occurrence of identified key events of the mode of action, in pertinent biochemical,

15 physiological and toxicological processes, and in metabolism and excretion of the agent. 

16 Examples are given in case examples for chemicals T and Z in Appendix D. Based on the

17 similarities of tumors following exposure to radiation, pharmaceuticals and viruses, from a

18 qualitative standpoint, it may be anticipated that the same kind of tumors may develop following

19 childhood or adult exposure to environmental chemicals. However. when there are no

20 agent-specific data or there is not a cogent rationale supporting the comparability between

21 responses in children and adults, the mode of action will not be considered to be applicable for

22 children. It should also be noted that from a quantitative perspective, the same key events may

23 lead to greater or lesser occurrence at different agents due to toxicokinetic and exposure

24 considerations. These considerations need separate evaluation and may result in separate risk

25 estimates for the young or for that portion of a lifetime. 

26

27

28

29 2.6. WEIGHT-OF-EVIDENCE EVALUATION FOR POTENTIAL HUMAN

30 CARCINOGENICITY

31 A weight-of-evidence evaluation is a collective evaluation of all pertinent information so

32 that the full impact of biological plausibility and coherence is adequately considered. 

33 Identification and characterization of human carcinogenicity is based on human and experimental

34 data, the nature, advantages, and limitations of which have been discussed in the preceding
7/02/99 2-35 DRAFT

1 sections.

2 The subsequent sections outline: (1) the basics of weighing individual lines of evidence

3 and combining the entire body of evidence to make an informed judgment, and (2) classification

4 descriptors of cancer hazard.

5

6 2.6.1. Weight-of-Evidence Analysis

7 Judgment about the weight of evidence involves considerations of the quality and

8 adequacy of data and consistency of responses induced by the agent in question. The weight-of9 evidence judgment requires combined input of relevant disciplines. Initial views of one kind of

10 evidence may change significantly when other information is brought to the interpretation. For

11 example, a positive animal carcinogenicity finding may be diminished by other key data; a weak

12 association in epidemiologic studies may be bolstered by consideration of other key data and

13 animal findings. Factors typically considered are illustrated in figures below. Generally, no single

14 weighing factor on either side determines the overall weight. The factors are not scored

15 mechanically by adding pluses and minuses; they are judged in combination.

16

17 Human Evidence

18 Analyzing the contribution of evidence from a body of human data requires examining

19 available studies and weighing them in the context of well-accepted criteria for causation (see

20 Section 2.2.1). A judgment is made about how closely the studies satisfy these criteria,

21 individually and jointly, and how far they deviate from them. Existence of temporal relationships,

22 consistent results in independent studies, strong association, reliable exposure data, presence of

23 dose-related responses, freedom from biases and confounding factors, and high level of statistical

24 significance are among the factors leading to increased confidence in a conclusion of causality.

25 Generally, the weight of human evidence increases with the number of adequate studies

26 that show comparable results on populations exposed to the same agent under different

27 conditions. The analysis takes into account all studies of high quality, whether showing positive

28 associations or null results, or even protective effects. In weighing positive studies against null

29 studies, possible reasons for inconsistent results should be sought, and results of studies that are

30 judged to be of high quality are given more weight than those from studies judged to be

31 methodologically less sound. See Figure 2-1.
7/02/99 2-36 DRAFT

Increase weight Decrease weight 

Number of independent studies with

consistent results

Few studies

Equally well-designed and conducted studies

with null results

Most causal criteria satisfied: Few causal criteria satisfied

Temporal relationship

Strong association

Reliable exposure data

Dose-response relationship

Freedom from bias and confounding 

Biological plausibility

High statistical significance

a

Figure 2-1. Factors for weighing human evidence.
7/02/99 2-37 DRAFT

1 Generally, no single factor is determinative. For example, strength of association is one of

2 the causal criteria. A strong association (i.e., a relatively large risk) is more likely to indicate

3 causality than a weak association. However, finding of a large excess risk in a single study must

4 be balanced against the lack of consistency as reflected by null results from other equally well5 designed and well-conducted studies. In this situation, the positive association of a single study

6 may either suggest the presence of chance, bias, or confounding, or reflect different exposure

7 conditions. On the other hand, evidence of weak but consistent associations across several

8 studies suggests either causality or that the same confounder may be operating in all of these

9 studies.

10

11 Animal Evidence

12 Evidence from long-term or other carcinogenicity studies in laboratory animals constitutes

13 the second major class of information bearing on carcinogenicity. See Figure 2-2. As discussed

14 in Section 2.2.2, each relevant study must be reviewed and evaluated as to its adequacy of design

15 and conduct as well as the statistical significance and biological relevance of its findings. Factors

16 that usually increase confidence in the predictivity of animal findings are those of (1) multiplicity

17 of observations in independent studies; (2) severity of lesions, latency, and lesion progression; and

18 (3) consistency in observations.
7/02/99 2-38 DRAFT

Increase weight Decrease weight

Number of independent studies with 

consistent results

Single study

Inconsistent results

Same site across species, structural

analogues

Multiple observations

 Species

 Sites

 Sexes

Single site/species/sex

Severity and progression of lesions

 Early-in-life tumors/malignancy

 Dose-response relationships

 Lesion progression

 Uncommon tumor

Benign tumors only

High background of incidence tumors

Route of administration like human 

exposure

Route of administration unlike human

exposure

a

Figure 2-2. Factors for weighing animal evidence.
7/02/99 2-39 DRAFT

1 Other Key Data

2 Additional information bearing on the qualitative assessment of carcinogenic potential may

3 be gained from comparative pharmacokinetic and metabolism studies, genetic toxicity studies,

4 SAR analysis, and other studies of an agent’s properties. See Figure 2-3. Information from these

5 studies helps to elucidate potential modes of action and biological fate and disposition. The

6 knowledge gained supports interpretation of cancer studies in humans and animals and provides a

7 separate source of information about carcinogenic potential.
7/02/99 2-40 DRAFT

Increase weight Decrease weight

A rich set of other key data are available Few or poor data

Physicochemical data or

Data indicate reactivity with macromolecules Inadequate data necessitate use of default

assumptions

Structure-activity relationships support

hazard potential

 or

Comparable metabolism and toxicokinetics

between species

Data show that animal findings are not

relevant to humans

Toxicological and human clinical data support

tumor findings

Biomarker data support attribution of 

effects to agent

Mode-of-action data support causal 

interpretation of human evidence or 

relevance of animal evidence

a

Figure 2-3. Factors for weighing other data.
7/02/99 2-41 DRAFT

1 Totality of Evidence

2 In reaching a view of the entire weight of evidence, all data and inferences are merged. 

3 Figure 2-4 indicates the generalities. In fact, possible weights of evidence span a broad

4 continuum that cannot be capsulized. Most of the time the data in various lines of evidence fall in

5 the middle of the weights represented in the four figures in this section.
7/02/99 2-42 DRAFT

Increase Weight Decrease Weight 

Evidence of human causality Data not available or do not show causality

Evidence of animal effects relevant 

to humans

Data not available or not relevant

Coherent inferences Conflicting data

Comparable metabolism and toxicokinetics 

between species

Metabolism and toxicokinetics not

comparable

Mode of action comparable across species Mode of action not comparable across species

a

Figure 2-4. Factors for weighing totality of evidence.
7/02/99 2-43 DRAFT

1 The following section and the weight-of-evidence narrative discussed in Section 2.8

2 provide a way to state a conclusion and capture this complexity in a consistent way.

3

4 2.6.2. Descriptors for Summarizing Weight of Evidence

5

6 To express conclusions about the weight of evidence for human carcinogenic potential,

7 standard descriptors are utilized as part of the narrative (see Section 2.7.2.). The descriptors are

8 not meant to replace an explanation of the nuances of the biological evidence, but rather to

9 summarize it. Applying a descriptor is a matter of judgment and cannot be reduced to a formula.

10 Each standard descriptor may be applicable to a wide variety of potential data sets and weights of

11 evidence. There will always be gray areas, gradations, and borderline cases. That is why the

12 descriptors are presented only in the context of a weight of evidence narrative. Using them within

13 a narrative preserves and presents the complexity that is an essential part of the hazard

14 characterization. Risk managers should consider the entire range of information included in the

15 narrative rather than focusing simply on the descriptor.

16 Different conclusions may be reached for a single agent when carcinogenicity is dose or

17 route dependent. For instance, the agent is likely to be carcinogenic by one route of exposure but

18 not by others (Section 2.3.3). In this instance, more than one descriptor is used, one for each

19 route of exposure. Another example would be that an agent is likely carcinogenic above a certain

20 dose range but not likely to be carcinogenic below that range. 

21 The descriptors are standardized and are to be used consistently from case to case. They

22 are part of the first sentence of the narrative. The discussions below explain descriptors which

23 appear in italics, and along with Appendices A and C, illustrate their use, including by route of

24 exposure.

25 "Carcinogenic To Humans”

26

27 This descriptor is appropriate when there is convincing epidemiologic evidence

28 demonstrating causality between human exposure and cancer. 

29

30 This descriptor is also appropriate when there is an absence of conclusive epidemiologic

31 evidence to clearly establish a cause and effect relationship between human exposure and cancer,

32 but there is compelling evidence of carcinogenicity in animals and mechanistic information in

33 animals and humans demonstrating similar mode(s) of carcinogenic action. It is used when all of 

34 the following conditions are met:
7/02/99 2-44 DRAFT

1 • There is evidence in a human population(s) of association of exposure to the

2 agent with cancer, but not enough to show a causal association, and

3 C There is extensive evidence of carcinogenicity, and 

4 C The mode(s) of carcinogenic action and associated key events have been identified in

5 animals, and

6 C The keys events that precede the cancer response in animals have been observed in the

7 human population(s) that also shows evidence of an association of exposure to the

8 agent with cancer.

9

10 “Likely To be Carcinogenic To Humans”

11

12 This descriptor is appropriate when the available tumor effects and other key data are

13 adequate to demonstrate carcinogenic potential to humans. Adequate data are within a spectrum. 

14 At one end is evidence for an association between human exposure to the agent and cancer and

15 strong experimental evidence of carcinogenicity in animals; at the other, with no human data, the

16 weight of experimental evidence shows animal carcinogenicity by a mode or modes of action that

17 are relevant or assumed to be relevant to humans.

18

19 “Suggestive Evidence of Carcinogenicity, 

20 but Not Sufficient to Assess Human Carcinogenic Potential”

21

22 This descriptor is appropriate when the evidence from human or animal data is suggestive

23 of carcinogenicity, which raises a concern for carcinogenic effects but is judged not sufficient for a

24 conclusion as to human carcinogenic potential. Examples of such evidence may include: a

25 marginal increase in tumors that may be exposure-related, or evidence is observed only in a single

26 study, or the only evidence is limited to certain high background tumors in one sex of one species.

27 Dose-response assessment is not indicated for these agents. Further studies would be needed to

28 determine human carcinogenic potential. 

29

30 "Data Are Inadequate for An Assessment of Human Carcinogenic Potential”

31

32 This descriptor is used when available data are judged inadequate to perform an

33 assessment. This includes a case when there is a lack of pertinent or useful data or when existing

34 evidence is conflicting, e.g., some evidence is suggestive of carcinogenic effects, but other equally
7/02/99 2-45 DRAFT

1 pertinent evidence does not confirm a concern. 

2 

3 "Not likely To Be Carcinogenic To Humans”

4

5 This descriptor is used when the available data are considered robust for deciding that

6 there is no basis for human hazard concern. The judgment may be based on— 

7

8 C Extensive human experience that demonstrates lack of carcinogenic effect (e.g., 

9 phenobarbital).

10

11 C Animal evidence that demonstrates lack of carcinogenic effect in at least two well12 designed and well-conducted studies in two appropriate animal species (in the absence

13 of human data suggesting a potential for cancer effects).

14

15 C Extensive experimental evidence showing that the only carcinogenic effects observed

16 in animals are not considered relevant to humans (e.g., showing only effects in the

17 male rat kidney due to accumulation of "2u-globulin). 

18

19 C Evidence that carcinogenic effects are not likely by a particular route of exposure

20 (Section 2.3.3.)

21 C Evidence that carcinogenic effects are not anticipated below a defined dose range.

22

23

24 2.7. TECHNICAL HAZARD CHARACTERIZATION

25 The hazard characterization has two functions. First, it presents results of the hazard

26 assessment and an explanation of how the weight-of-evidence conclusion was reached. It explains

27 the potential for human hazard, anticipated attributes of its expression, and mode-of-action

28 considerations for dose response. Second, it contains the information needed for eventual

29 incorporation into a risk characterization consistent with EPA guidance on risk characterization

30 (U.S. EPA, 1995).

31 The characterization summarizes the conclusions reached concerning the mode of action

32 of the agent and devotes particular attention to a clear statement of the strengths and weaknesses

33 of the inferences made and their relation to the framework for analyzing described in Chapter 2. 

34 The implications of the mode of action for the dose-response assessment are clearly stated, along
7/02/99 2-46 DRAFT

1 with the degree of confidence in those conclusions.

2 The characterization qualitatively describes the conditions under which the agent’s effects

3 may be expressed in human beings. These qualitative hazard conditions are ones that are

4 observable in the tumor and other key data without having done either quantitative dose-response

5 or exposure assessment. The description includes how expression is affected by route of

6 exposure and dose levels and durations of exposure. Implications for disproportionate risks in

7 particular subpopulations, including fetuses and children, are identified when such information

8 exists. 

9 The discussion of limitations of dose as a qualitative aspect of hazard addresses the

10 question of whether reaching a certain dose range appears to be a precondition for a hazard to be

11 expressed; for example, when carcinogenic effects are secondary to another toxic effect that

12 appears only when a certain dose level is reached. The assumption is made that an agent that

13 causes internal tumors by one route of exposure will be carcinogenic by another route, if it is

14 absorbed by the second route to give an internal dose. Conversely, if there is a route of exposure

15 by which the agent is not absorbed (does not cross an absorption barrier; e.g., the exchange

16 boundaries of skin, lung, and digestive tract through uptake processes) to any significant degree,

17 hazard is not anticipated by that route. An exception to the latter statement would be when the

18 site of contact is also the target tissue of carcinogenicity. Duration of exposure may be a

19 precondition for hazard if, for example, the mode of action requires cytotoxicity or a physiologic

20 change, or is mitogenicity, for which exposure must be sustained for a period of time before

21 effects occur. The characterization could note that one would not anticipate a hazard from

22 isolated, acute exposures. The above conditions are qualitative ones regarding preconditions for

23 effects, not issues of relative absorption or potency at different dose levels. The latter are dealt

24 with under dose-response assessment (Section 3), and their implications can only be assessed after

25 human exposure data are applied in the characterization of risk.

26 The characterization describes conclusions about mode-of-action information and its

27 support for recommending dose-response approaches.

28 The hazard characterization routinely includes the following in support of risk

29 characterization:

30

31 C a summary of results of the assessment;

32 C identification of the kinds of data available to support conclusions and explanation of

33 how the data fit together, highlighting the quality of the data in each line of evidence,

34 e.g., tumor effects, short-term studies, structure-activity relationships), and
7/02/99 2-47 DRAFT

1 highlighting the coherence of inferences from the different kinds of data;

2 C strengths and limitations (uncertainties) of the data and assessment, including

3 identification of default assumptions invoked in the face of missing or inadequate data;

4 C identification of alternative interpretations of data that are considered equally

5 plausible;

6 C identification of any subpopulations believed to be more susceptible to the hazard than

7 the general population, especially attending to fetuses, infants, and children;

8 C conclusions about the agent’s mode of action and recommended dose-response

9 approaches; and

10 C significant issues regarding interpretation of data that arose in the assessment. Typical

11 ones may include:

12 -- determining causality in human studies,

13 -- dosing (MTD), background tumor rates, relevance of animal tumors to

14 humans;

15 -- weighing studies with positive and null results, considering the influence of

16 other available kinds of evidence; and

17 -- drawing conclusions based on mode-of-action data versus using a default

18 assumption about the mode of action.

19

20 2.8. WEIGHT-OF-EVIDENCE NARRATIVE

21 The weight-of-evidence narrative summarizes the results of hazard assessment employing

22 the descriptors defined in Section 2.6.1. The narrative (about two pages in length) explains an

23 agent’s human carcinogenic potential and the conditions of its expression. If data do not allow a

24 conclusion as to carcinogenicity, the narrative explains the basis of this determination. An

25 example narrative appears below. More examples appear in Appendix A.

26 The items regularly included in a narrative are:

27

28 C name of agent and Chemical Abstracts Services number, if available;

29 C conclusions (by route of exposure) about human carcinogenicity, using a standard

30 descriptor from Section 2.6.1;

31 C summary of human and animal tumor data on the agent or its structural analogues,

32 their relevance, and biological plausibility;

33 C other key data (e.g., structure-activity data, toxicokinetics and metabolism, short-term

34 studies, other relevant toxicity or clinical data);
7/02/99 2-48 DRAFT

1 C discussion of possible mode(s) of action and appropriate dose-response approach(es);

2 and

3 C conditions of expression of carcinogenicity, including route, duration, and magnitude

4 of exposure.

5

6 Example Narrative

7 Aromatic Compound

8 CAS# XXX

9 CANCER HAZARD SUMMARY

10 Aromatic compound (AR) is carcinogenic to humans by all routes of exposure.

11 The weight of evidence of human carcinogenicity is based on (a) consistent evidence of

12 elevated leukemia incidence in studies of exposed workers and significant increases of genetic

13 damage in bone marrow cells and blood lymphocytes of exposed workers; (b) significantly

14 increased incidence of cancer in both sexes of several strains of rats and mice; (c) genetic damage

15 in bone marrow cells of exposed rodents and effects on intracellular signals that control cell

16 growth.

17 AR is readily absorbed by all routes of exposure and rapidly distributed throughout the

18 body. The mode of action of AR is not understood. A dose-response assessment that assumes

19 linearity of the relationship is recommended as a default.

20

21 SUPPORTING INFORMATION

22 Data include numerous human epidemiologic and biomonitoring studies, long-term

23 bioassays, and other data on effects of AR on genetic material and cell growth processes. The

24 key epidemiologic studies and animal studies are well conducted and reliable. The other data are

25 generally of good quality also.

26

27 Human Effects

28 Numerous epidemiologic and case studies have reported an increased incidence or a causal

29 relationship associating exposure to AR and leukemia. Among the studies are five for which the

30 design and performance as well as follow-up are considered adequate to demonstrate the causal

31 relationship. Biomonitoring studies of exposed workers have found dose-related increases in

32 chromosomal aberrations in bone marrow cells and blood lymphocytes.

33
7/02/99 2-49 DRAFT

1 Animal Effects

2 AR caused increased incidence of tumors in various tissues in both sexes of several rat and

3 mouse strains. AR also caused chromosomal aberrations in rabbits, mice, and rats--as it does in

4 humans.

5

6 Other Key Data

7 AR itself is not DNA reactive and is not mutagenic in an array of test systems both in vitro

8 and in vivo. Metabolism of AR yields several metabolites that have been separately studied for

9 effects on carcinogenic processes. Some have mutagenic activity in test systems and some have

10 other effects on growth controls inside cells.

11

12 MODE OF ACTION

13 No rodent tumor precisely matches human leukemia in pathology. The closest parallel is a

14 mouse cancer of blood-forming tissue. Studies of the effects of AR at the cell level in this model

15 system are ongoing. As yet, the mode of action of AR is unclear, but most likely the carcinogenic

16 activity is associated with one or a combination of its metabolites. It is appropriate to apply a

17 linear approach to the dose-response assessment pending a better understanding because: (a)

18 genetic damage is a typical effect of AR exposure in mammals, and (b) metabolites of AR produce

19 mutagenic effects in addition to their other effects on cell growth controls; AR is a multitissue

20 carcinogen in mammals, suggesting that it is affecting a common controlling mechanism of cell

21 growth.
1. For this discussion, “exposure” means contact of an agent with the outer boundary of an

organism. “Applied dose” means the amount of an agent presented to an absorption barrier and

available for absorption. “Internal dose” means the amount crossing an absorption barrier (e.g.,

the exchange boundaries of skin, lung, and digestive tract) through uptake processes. “Delivered

dose” for an organ or cell means the amount available for interaction with that organ or cell (U.S.

EPA, 1992a).

2. A “key event” is an empirically observed precursor consistent with a mode of action.

7/02/99 3-1 DRAFT

1 3. DOSE-RESPONSE ASSESSMENT

2

3 Dose-response assessment evaluates potential risks to humans at exposure levels of

4 interest. The approach to dose-response assessment for a particular agent is based on the

5 conclusion reached as to its mode of action (Sections. 2.4 -2.5). The evaluation first covers the

relationship of the dose6 6 to the degree of response in the dose range of observation in experiments

7 or human studies. This evaluation is then followed by extrapolation to estimate response at lower

8 environmental exposure levels (ILSI, 1995). In general, three extrapolations may be made: from

9 high to low doses, from animal to human responses, and from one route of exposure to another. 

10 Cancer is a disease that develops through many cell and tissue changes over time. 

11 Traditional dose-response assessment procedures using tumor incidence as the response have

12 seldom taken into account the effects of key events within the whole biological process, even

13 though these events are the determinants of the overall dose-response. This has been due to lack

14 of empirical data and understanding about these events. As more data become available and our

15 understanding about how agents cause cancer improves, they can be used in dose-response

16 assessment along with the traditional procedures. These guidelines encourage use of these new

17 data as they become available to improve dose-response assessment.

In this discussion, “response” data include measures of key events7 18 considered integral to

19 the carcinogenic process, in addition to tumor incidence. These responses may include changes in

20 DNA, chromosomes, or other key macromolecules; effects on growth signal transduction,

21 including induction of hormonal changes; or physiological or toxic effects that affect cell

22 proliferation. Key events are precursors to cancer pathology; they may include proliferative

23 events diagnosed as precancerous, but not pathology that is judged to be cancer. Analysis of such

24 responses may be done along with those of tumor incidence to enhance the tumor dose-response

25 analysis. If dose-response analysis of non tumor key events is more informative about the

26 carcinogenic process for an agent, it is used in lieu of, or in conjunction with, tumor incidence
7/02/99 3-2 DRAFT

1 analysis for the overall dose-response assessment.

2 “Dose” means the “human equivalent dose” as discussed in Section 3.3, unless otherwise

3 noted. When animal responses are used in the assessment, the animal dose is adjusted to human

4 equivalence. The preferred approach for this is to use toxicokinetic modeling to compare species. 

5 If this is not possible given the data available, a default factor for allometric scaling of oral dose is

6 provided. For adjustment of inhalation dose, the EPA’s Reference Concentration (RfC)

7 methodology is used. 

8

9

10 Coverage of the Chapter

11 This chapter covers: 1) consideration of mode of action in selecting dose-response

12 assessment approaches, 2) assessment of observed data and extrapolation procedures, 3) 

13 analyses of response data and 4) analyses of dose data. The final section discusses dose-response

14 characterization.

15

16 3.1 HUMAN STUDIES

17 Analysis of human studies in the observed range is determined according to the type of

18 study and how dose and response are measured in the study. In some cases the agent may have

19 discernible interactive effects with another agent (e.g., asbestos and smoking), making possible

20 estimation of contribution of the agent and others as risk factors. Also, in some cases, estimation

21 of population risk in addition to, or in lieu of, individual risk may be appropriate. The following

22 discussions are addressed mainly to animal data. Nevertheless, if human data permit, the

23 principles or approaches below apply for performing dose-response assessment in two parts--

24 range of observation and range of extrapolation, for deriving a point of departure, and for linear

25 or margin of exposure analysis according to mode of action (NRC, 1999; Teta, 1999). The

26 approach is tailored to the nature of the human data and the mode of action data available, if any.

27

28

29 3.2. MODE OF ACTION AND DOSE-RESPONSE APPROACH

30

31 The cancer dose-response relationship(s) for a chemical is considered in a two step

32 process. First is the determination of the mode of action and dose response for each tumor type

33 that results in a significant increase in tumor incidence. Second is an analysis of the information

34 bearing on all tumor types that are increased in incidence by the chemical. The overall synthesis
7/02/99 3-3 DRAFT

1 includes consideration of the number of sites, their consistency across sexes, strains and species,

2 the strength of the mode of action information for each tumor type, the anticipated relevance of

3 each tumor type to humans, and the consistency of the means of estimating risks across tumor

4 types.

5 For each tumor the mode of action and other information may support one of the

6 following dose response extrapolations: 1) linear, 2) nonlinear using a margin of exposure

7 (MOE) analysis, or 3) both linear and nonlinear (MOE) analyses. In rare cases, detailed mode of

8 action information may be available which allow the formulation of a biologically based model. 

9 Examples include the following:

10

11 Factors Supporting a Linear Approach

12 Any of the following conclusions leads to selection of a linear dose-response assessment

13 approach:

14 C There is an absence of sufficient tumor mode of action information. 

15 C The chemical has direct DNA mutagenic activity or other indications of DNA effects

16 that are consistent with linearity. 

17 C Human exposure or body burden is high and near doses associated with key events in

18 the carcinogenic process (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin)

19 C Mode of action analysis does not support direct DNA effects, but the dose-response

20 relationship is expected to be linear (e.g., certain receptor-mediated effects) 

21

22 Factors Supporting a Nonlinear Approach

23 Any of the following conclusions leads to selection of a nonlinear (margin of exposure)

24 approach to dose-response assessment:

25 C A tumor mode of action supporting nonlinearity applies (e.g., some cytotoxic and

26 hormonal agents such as disruptors of hormone homeostasis), and the chemical does

27 not demonstrate mutagenic effects consistent with linearity.

28 C A mode of action supporting nonlinearity has been demonstrated, and the chemical has

29 some indication of mutagenic activity, but it is judged not to play a significant role in

30 tumor causation.

31

32 Factors Supporting Both Linear And Nonlinear Approaches

33 Any of the following conclusions leads to selection of both a linear and nonlinear approach

34 to dose-response assessment. Relative support for each dose response method and advice on the
7/02/99 3-4 DRAFT

1 use of that information needs to be presented. In some cases, evidence for one mode of action is

2 stronger that for the other, allowing emphasis to be placed on that dose-response approach. In

3 other cases, both modes of action are equally possible, and both dose-response approaches should

4 be emphasized.

5

6 C Modes of action for a single tumor type support both linear and nonlinear dose

7 response in different parts of the dose-response (e.g., 4,4' methylene chloride).

8 C A tumor mode of action supports different approaches at high and low dose; e.g., at

9 high dose, nonlinearity, but, at low dose, linearity (e.g., formaldehyde).

10 C The agent is not DNA-reactive and all plausible modes of action are consistent with

11 nonlinearity, but not fully established (arsenic).

12 C Modes of action for different tumor types support differing approaches, e.g., nonlinear

13 for one and linear due to lack of mode of action for the other (e.g., trichloroethylene).

14

15 The use of biologically based models is covered below.

16

17 3.3. DOSE-RESPONSE ANALYSIS

18 3.3.1. Modeling the Overall Process--Biologically-based Models

19 Generally applicable biologically-based models may be applied such as the two-stage

20 models of initiation plus clonal expansion and progression developed by Moolgavkar and

21 Knudson (1981), Chen and Farland (1991) and others. These models of the carcinogenic process

22 continue to be improved, but are not yet standard methods. No model of this kind is available for

23 standard application. 

24 If data are extensive and sufficient to quantitatively relate specific key events in the cancer

25 process to neoplasia, and the purpose of the assessment is such as to justify investing the

26 necessary resources, a biologically-based model may be developed on an agent-specific basis.

27 Before developing such a model, extensive data are needed to build its form as well as to estimate

28 how well it conforms with the observed data to support confidence in results. Theoretical

29 estimates of critical parameters, such as cell proliferation rates, are not used to enable application

30 of such a model in the absence of data (Portier, 1987). It is possible that different models will

31 provide equivalent fits to the observed data but differ substantially in their projections below the

32 observed range. This is often the case when a model is over-parameterized (that is, there are

33 more parameters to be estimated than data points to be fitted), so that different combinations of

34 parameter estimates can yield similar results in the observed range. For this reason, critical
7/02/99 3-5 DRAFT

1 parameters of a biologically based model, such as mutation and proliferation rates, are measured

2 in the laboratory and not estimated by curve-fitting to tumor incidence data. This approach helps

3 reduce model uncertainty (i.e., uncertainty due to choice of models or model structure) and

4 ensures that the models do not give answers that are biologically unrealistic. This approach also

5 provides a robustness of results (i.e., results are not likely to change substantially when fitted to a

6 slightly different data set), if the mode of action is sufficiently understood so that model

7 parameters represent rates and other quantities associated with known key events in tumor

8 development. 

9 Such models are to be distinguished from toxicokinetic models (i.e., physiologically based

10 pharmacokinetic” models) which address dose issues, as discussed in Section 3.3.2. Effects on

11 dose such as saturation of metabolic pathways may introduce nonlinearities in the dose-response

12 relationship, but are not modes of action, and are dealt within arriving at an appropriate dose

13 metric.

14

15 3.3.2. Analysis in the Range of Observation

16 This section covers use of information about key events which may be in the context of

17 either human or animal data. It then discusses curve-fitting and selecting a point of departure

18 with regard to animal data. Last, it discusses human data.

19

20 3.3.2.1. Applying Information About Key Events

21 Even though a biologically-based model may not be feasible, information about key events

22 in the process can be used in the assessment. The principle underlying these Guidelines is to use

23 approaches that include as much information about these events as possible. When such

24 information is available, it may be used in a variety of ways: 

25 1) If an event(s) is quantitatively described and considered key to cancer development, its

26 dose-response assessment in the range of observation can be used in conjunction with, or in lieu

27 of, the dose-response for tumor incidence to establish the point of departure for extrapolation. 

28 [Caution must be used in using rates of molecular events such as mutation or cell proliferation or

29 of signal transduction. Such rates may be difficult to relate to cell or tissue changes overall. The

30 timing of observations of these phenomena, as well as the cell type involved, need to be linked to

31 other precursor events to ensure the measurement is truly a “key”event (see Section 2.5). In

32 many cases such rates are more appropriately used as in "2)" or "3)" below.]

33 2) Quantitative description of a key event(s) can be used to test whether the dose34 response for tumor incidence can be confidently extended to support a lower point of departure
7/02/99 3-6 DRAFT

for linear extrapolation than the tumor data alone would support (e.g., to an LED01 1 from an

LED10 2 ). 

3 3) Quantitative information on a key event(s) can be used to address the question of how

4 quickly risk decreases as dose decreases in a margin of exposure analysis.

5

6 3.3.2.2. Procedures for Analysis in the Range of Observation of Animal Studies

7

8 Curve-fitting

9 A curve-fitting procedure is used that is appropriate to the kind of response data in the

10 range of observation. This may be tumor incidence or data on a key event(s). For incidence

11 information, the Agency applies a standard curve-fitting procedure to provide consistency among

12 assessments. This procedure models incidence, adjusted for background, as an increasing function

13 of dose; it is available to the public on the Agency’s World Wide Web site for immediate use or

14 for downloading (reference to be provided). The procedure identifies situations in which the

15 standard algorithm fails to yield a reliable point of departure, signaling the need for additional

16 judgment and an alternative analysis.

17 For tumor incidence studies that provide time-to-tumor information, more elaborate

18 models would be appropriate. The Agency intends to provide a time-to-tumor version of its

19 standard procedure in the future.

20 For non tumor data, curve-fitting procedures are used that are appropriate to the kind of

21 response data in the observed range, and are explained in each case (reference to benchmark

22 models to be provided). 

23 NOAEL/LOAEL

24 As discussed below, the observed range of data may be represented by a

25 NOAEL/LOAEL procedure when a margin of exposure analysis is chosen as the default

26 procedure for nonlinear dose-response extrapolation.

27

28 3.3.2.3. Point of Departure for Extrapolation from Observed Animal Data

29 A point of departure from observed data--for tumor incidence, or for key event(s)--is

30 estimated to mark the beginning of extrapolation. This is a point that is either a data point or an

31 estimated point that can be considered to be in the range of observation, without significant

32 extrapolation. Depending on the kind of data available and the purpose of the analysis, there are

33 differing procedures for estimating the point of departure. The point of departure employs the

34 human equivalent dose.
7/02/99 3-7 DRAFT

1 Incidence data are most amenable to curve-fitting procedures. For example, tumor data

2 from a rodent bioassay are traditionally modeled with curve-fitting procedures. Some key event

3 data may also be in the form of incidence data (e.g., hyperplasia), but more likely will be

4 continuous data for which currently there are not standard and consistent modeling procedures.

5 Continuous data include, for instance, tissue weight changes or blood levels of a hormone.

6 NOAEL/LOAEL procedures are available for continuous and other data as needed.

7

8 Point of Departure Using Data Suitable for Curve-fitting

9 When a curve-fitting procedure is applied to tumor data (see Figure 3-1) or to incidence

data on a key event, the point of departure used in most cases is the LED10 10 --the 95% lower

11 confidence limit on a dose associated with 10% extra risk adjusted for background. For tumor

12 data, it is used as a matter of science policy to provide consistency among assessments. It is also

13 useful in comparing results with assessment of noncancer endpoints (U.S. EPA, 1991d). The 10%

14 level is selected because a 10% response is at or just below the limit of sensitivity for discerning a

15 statistically significant tumor increase in most long-term rodent studies (Haseman, 1983), and is

16 within the observed range for many other kinds of toxicity studies. Use of the lower limit takes

17 experimental variability and sample size into account. If a tumor incidence study has greater than

usual sensitivity and an observed response is below LED10 18 , then a lower point for linear

extrapolation can be used to improve the assessment. [The ED10 19 (central estimate) is appropriate

20 for use in relative hazard/potency ranking among agents for priority setting because it is a more

21 confident comparison point among many assessments than an extrapolated point. Because of its

convenience for comparison uses, the ED10 22 is always presented for reference with its upper and

23 lower 95% confidence limits.] 

The LED10 24 is adopted as the standard point of departure for non tumor key event or

25 toxicity incidence data in order to harmonize curve-fitting procedures between cancer and

26 noncancer toxicity assessments. Because the NOAEL in study protocols for non tumor toxicity

27 can range from about a 5% to a 30% effect level (Faustman et al., 1994), adopting the 10%

28 effect level as the standard point of departure will accommodate most of these data sets without

departing the range of observation. The LED10 29 can be regarded as an improved and harmonized

30 estimate of the NOAEL.
3-8

Figure 3-1. Graphical presentation of data and extrapolation.

6/17/99 3-8 DRAFT--DO NOT CITE OR QUOTE
7/02/99 3-9 DRAFT

1 Point of Departure Using Data Suitable for a NOAEL/LOAEL Procedure

2 The point of departure may be a NOAEL when a margin of exposure analysis is the

3 nonlinear dose-response approach. The kinds of data available and the circumstances of the

4 assessment both contribute to deciding to estimate a NOAEL or LOAEL which is not as rigorous

5 or as ideal as curve-fitting, but can be appropriate. The NOAEL/LOAEL procedure is used to

6 maintain consistency among assessments while still encouraging quantitative analyses of the data

7 by modeling to explore underlying phenomena.

8 The circumstances of an assessment can also lead to choosing a NOAEL/LOAEL

9 approach. If several data sets for key events and tumor response are available for an agent, and

10 they are a mixture of continuous and incidence data, the most practicable way to assess them

11 together is through a NOAEL/LOAEL approach. The purpose of the assessment also may lead to

12 a decision to use the NOAEL/LOAEL approach. A preliminary or screening assessment to decide

13 whether risk concern is high or low or to decide on additional data requirements is one example. 

14 Similarly, the nature of the regulatory decision may be served well by this approach to assessment.

15

16 3.3.3. Analysis in the Range of Extrapolation--Default Procedures

17 Extrapolation from the point of departure to lower doses is usually necessary, and in the

18 absence of a data set rich enough to support a biologically based model, is conducted using one of

19 the two default procedures described below. The Agency has adopted these procedures as a

20 matter of science policy based on current hypotheses of the potential shapes of dose-response

21 curves for differing modes of action at low doses. The choice of the procedure to be used in an

22 individual case is a judgment based on the agent's mode of action (See Section 3.2). 

23

24 3.3.3.1. Linear Procedure

25 For linear extrapolation, a straight line is drawn from the point of departure expressed as a

26 human equivalent dose (Section 3.3.2) to the origin--zero incremental dose, zero incremental

27 response to give a probability of extra risk. The slope of the line expresses extra risk per dose

28 unit (Flamm and Winbush, 1984; Gaylor and Kodell, 1980; Krewski et al., 1984). Risk is the

29 product of the slope and anticipated exposure. This approach to assessing risk is considered

30 generally conservative of public health, including sensitive subpopulations, in the absence of

31 specific information about the extent of human variability in sensitivity to effects. When a linear

32 extrapolation procedure is used, the risk characterization summary also displays the degree of

33 extrapolation from empirical data by showing the margin of exposure associated with exposure

34 scenarios of interest as below. 
3. A reference dose (RfD) or reference concentration (RfC) for noncancer toxicity is an estimate

with uncertainty spanning perhaps an order of magnitude of daily exposure to the human

population (including sensitive subgroups) that is anticipated to be without appreciable deleterious

effects during a lifetime. It is arrived at by dividing empirical data on effects by uncertainty

factors that consider inter- and intraspecies variability, extent of data on all important chronic

exposure toxicity endpoints, and availability of chronic as opposed to subchronic data.

7/02/99 3-10 DRAFT

1 3.3.3.2. Nonlinear Extrapolation

2 A default assumption of nonlinearity is appropriate when there is no evidence for linearity

3 and sufficient evidence to support an assumption of nonlinearity. The mode of action may lead to

4 a dose-response relationship that is nonlinear, with response falling much more quickly than

5 linearly with dose, or being most influenced by individual differences in sensitivity. Alternatively,

6 the mode of action may theoretically have a threshold, e.g., the carcinogenicity may be a

7 secondary effect of toxicity or of an induced physiological change that is itself a threshold

8 phenomenon (see Appendix C, example 5, or Appendix D, example 2 ). The EPA does not

9 generally try to distinguish between modes of action that might imply a "true threshold" from

10 others with a nonlinear dose-response relationship. Except in unusual cases where extensive

11 information is available, it is not possible to distinguish between these empirically.

12 As a matter of science policy under this analysis, nonlinear probability functions are not

13 fitted to the response data to extrapolate quantitative low-dose risk estimates because different

14 models can lead to a very wide range of results, and there is currently no basis, generally, to

15 choose among them. Thus, the default procedure for nonlinear extrapolation is to conduct a

16 margin of exposure analysis, as described below, to evaluate concern for levels of exposure. 

17

18 3.3.3.2.1. Margin of Exposure Analysis

19 A margin of exposure is defined as the point of departure divided by the environmental

20 exposure of interest. The environmental exposures of interest, for which margins of exposure are

21 estimated, may be actual or projected exposure levels. A risk manager decides whether a given

22 margin of exposure is acceptable under applicable management policy criteria. The risk

23 assessment provides supporting information to assist the decisionmaker in this determination.

24 A margin of exposure analysis is applicable if data are sufficient to presume a non-linear

25 dose-response function containing a significant change in slope. If, in a particular case, the

26 evidence indicates a biological threshold, as in the case of carcinogenicity being secondary to

another toxicity that has a threshold, an RfD8 27 or RfC like approach may be estimated and

28 considered in cancer assessment. In this case, the RfD or RfC is an estimate with uncertainty
7/02/99 3-11 DRAFT

1 spanning perhaps an order of magnitude of daily exposure to the human population (including

2 sensitive subgroups) that is anticipated to be without a cancer hazard despite a lifetime of

3 exposure. In many cases, data may be insufficient to determine an RfD and/or an RfC for the

4 cancer endpoint. In that case, a margin of exposure analysis provides useful input to the decision5 maker regarding the distance between an exposure of interest and the range of observation where

6 cancer risk is inferred to be sub-linear.

7

8 To support a risk manager's consideration of the margin of exposure, all of the pertinent

9 hazard, dose-response, and human exposure information is characterized so as to provide insights

10 about the scientific community’s current understanding of the phenomena that may be occurring

11 as dose (exposure) decreases substantially below the observed data. The goal is to provide as

12 much information as possible about the risk reduction that accompanies lowering of exposure and

13 the adequacy of a margin of exposure based on scientific input, recognizing that, in some cases,

14 legislative, sociological, and/or technological issues may also impact on the decision regarding the

15 acceptability of a given margin of exposure. The discussion below describes the general principles

16 and major elements to be considered in a margin of exposure analysis. The Agency will develop

17 more specific guidance on the margin of exposure approach, as recommended (SAB, 1999). The

18 guidance will be peer reviewed and published separately as part of the Agency’s implementation

19 activity of these guidelines.

20

21 For a margin of exposure analysis, the point of departure would ideally be the dose where

22 the key events in tumor development would not occur in a heterogenous human population, thus

23 representing an actual “no effect level.” Therefore, it is recommended that margin of exposure

24 analyses be based on precursor responses rather than tumor incidences, since precursor events

25 can often be detected with greater sensitivity( i.e. both earlier and at lower doses), providing

26 further input to the decision regarding acceptability of the margin of exposure. An analysis of an

27 actual point of departure derived from available data, however, would often contain residual

28 uncertainty regarding its designation as an actual no effect level for cancer in the population. The

29 earlier the precursor event in the carcinogenic process and the larger the margin of exposure the

30 more likely the exposure of interest will be without appreciable risk of cancer. To this end, some

31 important points to address in the analysis of the point of departure and the margin of exposure

32 include the following:

33

34 • Nature of the response. Is the point of departure based on tumors or on a key event
7/02/99 3-12 DRAFT

1 that is a precursor to tumors? A mode of action can be represented by a sequence of

2 dose-response curves, where an early key event arises at a low dose, subsequent key

3 events at higher doses, and tumors at a still higher dose. For example, a mode of

4 action that begins with bladder stones and progresses through epithelial irritation and

5 hyperplasia before producing tumors can be represented by a sequence of dose6 response curves for stones, irritation, hyperplasia, and tumors, each curve higher on

7 the dose scale than its immediate precursor. A nonlinear dose-response assessment

8 considers more than tumors as it identifies a dose where events that can lead to tumor

9 development would not occur. Identification of a key event does not imply that it is

10 adverse in itself, only that it is an observable step preceding tumor development. 

11 Basing a dose-response assessment on key events is intended to protect against not

12 only the observation of adverse effects, but also earlier damage that can lead to later

13 tumor development. 

14

15 Thus, it is most desirable to estimate a dose-response curve for the key event precipitating

16 tumor development, and use this curve to estimate the point of departure. However, lack

17 of quantitative information on the key event may make it necessary to use tumor data

18 instead of key event data. In this case, the analysis of the margin of exposure must

19 contain an estimate of the dose-response curve for tumors plus have sufficient discussion

20 of the difference (on the dose scale) between no effect levels and effect levels for key

21 events and for tumors. A larger margin of exposure may be needed to account for

22 possible differences between the dose-response curves for the key events and for tumors,

23 and to assure decision-makers that cancer risk for the heterogeneous population

24 (including sensitive subgroups) is not appreciable. 

25

26 • Slope of the observed dose-response curve. Have we reached a dose where tumors

27 or (preferably) the key precursor events would not occur? A 10-percent incidence is

28 typically used as a point of departure because it reflects the lowest incidence that

29 experimental studies can typically detect. This does not, however, mean that a 10-

30 percent incidence represents a level where tumors or the key precursor events would

31 not occur. To account for this limitation, one needs to consider the slope of the dose32 response curve, which describes how sharply the incidence declines below the point of

33 departure. If the dose-response curve at the point of departure is relatively steep, the

34 point of departure represents a point on the dose-response curve where occurrence of
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1 the key event(s) declines rapidly with decreasing dose. On the other hand, if the dose2 response curve is relatively shallow, then the point where the effect virtually

3 disappears may lie far below the point of departure. In short, the margin of exposure

4 needs to be larger if the analysis is based on a response(s) that has a shallow dose5 response curve compared to an analysis based on a response with a steep dose6 response curve. More guidance needs to be developed to define quantitatively what

7 constitutes a steep versus a shallow dose-response curve.

8

9 • Human sensitivity compared with experimental animals. How sensitive is the human

10 population compared with the tested animals? For this comparison, all doses should

11 have already been converted to equivalent human doses, using either a physiologically

12 based toxicokinetic model, a cross-species dosimetry model, or the default cross13 species scaling factor. These dose conversions reflect interspecies differences in

14 toxicokinetics, not toxicodynamics. When information is not sufficient to quantify

15 human sensitivity with regard to the toxicodynamics compared with the tested animals,

16 this uncertainty needs to be taken into account in the discussion of an adequate margin

17 of exposure. As with noncancer assessment, the default assumption is that the most

18 sensitive humans are more sensitive than the test animals. Depending on the data

19 available on the sensitivity of the test species to the agent and the endpoint of concern

20 as compared to humans, the margin of exposure decision may need to incorporate

21 more or less conservatism. 

22

23 • Nature and extent of human variability in sensitivity. Is there information on sensitive

24 individuals that would be part of a heterogeneous human population? Pertinent

25 information would come from human studies, since animal studies, particularly those

26 using homogeneous animal strains, do not provide information about human

27 variability. When information is not sufficient to quantify the extent of human

28 variability in sensitivity, this uncertainty should be reflected in the discussion of an

29 adequate margin of exposure (also see discussion below on human exposure). 

30

31 • Human Exposure. The evaluation of margin of exposure also takes into account the

32 expected pattern of human exposure to an agent including the magnitude, frequency,

33 and duration of exposure. Some modes of action involve significant duration of

34 exposure before tumorigenicity results. For example, stimulus of cell growth through
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1 hormonal or other signal disruption or as a result of damage from toxicity is reversible

2 if the exposure is for a short time, since homeostasis brings a return to normal levels

3 after cessation of exposure. Thus, for a specialized population that is occasionally and

4 briefly exposed to an agent with such a mode of action, an adequate of margin of

5 exposure would be smaller than for chronic exposure. As the duration of exposure or

6 frequency of exposure increases, an adequate margin of exposure would increase

7 accordingly.

8

9 Furthermore, if the population exposed in a particular scenario is wholly or largely

10 composed of a subpopulation of special concern (e.g. children) for whom evidence

11 indicates a special sensitivity to the agent’s mode of action, an adequate margin of

12 exposure would be larger than for general population exposure.

13

14 To provide input regarding scientific considerations regarding the acceptability of a margin of

15 exposure by the risk manager, the risk assessment along with risk characterization explicitly

16 considers all of the hazard and dose-response and human exposure factors together. This input on

17 the margin of exposure is not solely a composite of individual adjustment factors to account for

18 missing data or knowledge gaps as discussed above. Rather, each case calls for individual 

19 judgment, taking all of these points as a whole. It is appropriate to provide a graphical

20 representation of the data and dose-response modeling in the observed range, also showing

21 exposure levels of interest to the decision-maker (See figure 3-1.). In order to provide a frame of

22 reference, by way of comparison, a straight line extrapolation may be displayed to show what risk

23 levels would be associated with decreasing dose, if the dose-response were linear. 

24

25 3.3.3.3. Linear and Nonlinear Extrapolations

26 Both linear and nonlinear procedures may be used in particular cases. If a mode of action

27 analysis finds substantial support for differing modes of action for different tumor sites, an

28 appropriate procedure is used for each. Both procedures may also be appropriate to discuss

29 implications of complex dose-response relationships. For example, if it is apparent that an agent

30 is both DNA reactive and is highly active as a promotor at high doses, and there are insufficient

31 data for modeling, both linear and nonlinear default procedures may be needed to decouple and

32 consider the contribution of both phenomena.

33

34 3.3.3.4. Use of Toxicity Equivalence Factors and Relative Potency Estimates
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1 A toxicity equivalence factor (TEF) procedure is one used to derive quantitative dose2 response estimates for agents that are members of a category or class of agents. TEFs are based

3 on shared characteristics that can be used to order the class members by carcinogenic potency

4 when cancer bioassay data are inadequate for this purpose (U.S. EPA, 1991c). The ordering is by

5 reference to the characteristics and potency of a well-studied member or members of the class. 

6 Other class members are indexed to the reference agent(s) by one or more shared characteristics

7 to generate their TEFs. The TEFs are usually indexed at increments of a factor of 10. Very good

8 data may permit a smaller increment to be used. Shared characteristics that may be used are, for

9 example, receptor-binding characteristics, results of assays of biological activity related to

10 carcinogenicity, or structure-activity relationships.

11 TEFs are generated and used for the limited purpose of assessment of agents or mixtures

12 of agents in environmental media when better data are not available. When better data become

13 available for an agent, its TEF should be replaced or revised. Criteria for constructing TEFs are

14 given in U.S. EPA (1991b). The criteria call for data that are adequate to support summing doses

15 of the agents in mixtures. To date, adequate data to support use of TEF's has been found in only

16 one class of compounds (dioxins) (U.S. EPA, 1989a).

17 Relative potencies can be similarly derived and used for agents with carcinogenicity or

18 other supporting data. These are conceptually similar to TEFs, but they are less firmly based in

19 science and do not have the same level of data to support them. They are used only when there is

20 no better alternative.

21 The uncertainties associated with both TEFs and relative potencies are explained

22 whenever they are used.

23

24 3.4. RESPONSE DATA

25 Response data for analysis include tumor incidence data from human or animal studies as

26 well as data on other responses as they relate to an agent's carcinogenicity, such as effects on

27 growth control processes or cell macromolecules or other toxic effects. Tumor incidence data are

28 ordinarily the basis of dose-response assessment, but other response data can augment such

29 assessment or provide separate assessments of carcinogenicity or other important effects.

30 Data on carcinogenic processes underlying tumor effects may be used to support

31 biologically based or case-specific models. Other options for such data exist. If confidence is

32 high in the linkage of a precursor effect and the tumor effect, the assessment of tumor incidence

33 may be extended to lower dose levels by linking it to the assessment of the precursor effect

34 (Swenberg et al., 1987). Even if a quantitative link is not appropriate, the assessment for a
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1 precursor effect may provide a view of the likely shape of the dose-response curve for tumor

2 incidence below the range of tumor observation (Cohen and Ellwein, 1990; Choy, 1993). If

3 responses other than tumor incidence are regarded as better representations of the carcinogenicity

4 of the agent, they may be used in lieu of tumor responses. For example, if it is concluded that the

5 carcinogenic effect is secondary to another toxic effect, the dose-response for the other effect will

6 likely be more pertinent for risk assessment. As another example, if disruption of hormone

7 activity is the key mode of action of an agent, data on hormone activity may be used in lieu of

8 tumor incidence data.

9 If adequate positive human epidemiologic response data are available, they provide an

10 advantageous basis for analysis since concerns about interspecies extrapolation do not arise. 

11 Adequacy of human exposure data for quantification is an important consideration in deciding

12 whether epidemiologic data are the best basis for analysis in a particular case. If adequate

13 exposure data exist in a well-designed and well-conducted epidemiologic study that detects no

14 effects, it may be possible to obtain an upper-bound estimate of the potential human risk to

15 provide a check on plausibility of available estimates based on animal tumor or other responses,

16 e.g., do confidence limits on one overlap the point estimate of the other?

17 When animal studies are used, response data from a species that responds most like

18 humans should be used if information to this effect exists. If this is unknown and an agent has

19 been tested in several experiments involving different animal species, strains, and sexes at several

20 doses and different routes of exposure, all of the data sets are considered and compared, and a

21 judgment is made as to the data to be used to best represent the observed data and important

22 biological features such as mode of action. Appropriate options for presenting results include:

23 ! use of a single data set,

24 ! combining data from different experiments (Stiteler et al., 1993; Vater et al., 1993),

25 ! showing a range of results from more than one data set,

26 ! showing results from analysis of more than one statistically significant tumor response

27 based on differing modes of action,

28 ! representing total response in a single experiment by combining animals with

29 statistically significant tumors at more than one site, or

30 ! a combination of these options.

31 The approach judged to best represent the data is presented with the rationale for the judgment,

32 including the biological and statistical considerations involved. The following are some points to

33 consider:

34 ! quality of study protocol and execution,
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1 ! proportion of malignant neoplasms,

2 ! latency of onset of neoplasia,

3 ! number of data points to define the relationship of dose and response,

4 ! background incidence in test animal,

5 ! differences in range of response among species, sexes, strains,

6 ! most sensitive responding species, and

7 ! availability of data on related precursor events to tumor development.

8 Analyses of carcinogenic effects other than tumor incidence are similarly presented and evaluated

9 for their contribution to a best judgment on how to represent the biological data for dose10 response assessment.

11

12 3.5. DOSE DATA

13 Whether animal experiments or epidemiologic studies are the sources of data, questions

14 need to be addressed in arriving at an appropriate measure of dose for the anticipated

15 environmental exposure. Among these are:

16 ! whether the dose is expressed as an environmental concentration, applied dose, or

17 delivered dose to the target organ,

18 ! whether the dose is expressed in terms of a parent compound, one or more

19 metabolites, or both,

20 ! the impact of dose patterns and timing where significant,

21 ! conversion from animal to human doses, where animal data are used, and

22 ! the conversion metric between routes of exposure where necessary and appropriate.

23 In practice, there may be little or no information on the concentration or identity of the active

24 form at a target; being able to compare the applied and delivered doses between routes and

25 species is the ideal, but is rarely attained. Even so, the objective is to use available data to obtain

26 as close to a measure of internal or delivered dose as possible.

27 The following discussion assumes that the analyst will have data of varying detail in

28 different cases about toxicokinetics and metabolism. Discussed below are approaches to basic

29 data that are most frequently available, as well as approaches and judgments for improving the

30 analysis based on additional data. The estimation of dose in human studies is tailored to the form

31 of dose data available.

32
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1 3.5.1. Interspecies Adjustment of Dose--Adult Human

2 When adequate data are available, the doses used in animal studies can be adjusted to

3 equivalent human doses using toxicokinetic information on the particular agent. The methods

4 used should be tailored to the nature of the data on a case-by-case basis. In rare cases, it may also

5 be possible to make adjustments based on toxicodynamic considerations. In most cases, however,

6 there are insufficient data available to compare dose between species. In these cases, the estimate

7 of human equivalent dose is based on science policy default assumptions. The defaults described

8 below are modified or replaced whenever better comparative data on toxicokinetic or metabolic

9 relationships are available. The availability and discussion of the latter also may permit reduction

10 or discussion of uncertainty in the analysis.

11 For oral exposure, the default assumption is that delivered doses are related to applied

12 dose by a power of body weight. This assumption rests on the similarities of mammalian

13 anatomy, physiology, and biochemistry generally observed across species. This assumption is

14 more appropriate at low applied dose concentrations where sources of nonlinearity, such as

15 saturation or induction of enzyme activity, are less likely to occur. To derive an equivalent human

16 oral dose from animal data, the default procedure is to scale daily applied doses experienced for a

lifetime in proportion to body weight raised to the 0.75 power (W0.75 17 ). Equating exposure

18 concentrations in parts per million units for food or water is an alternative version of the same

default procedure because daily intakes of these are in proportion to W0.75 19 . The rationale for this

20 factor rests on the empirical observation that rates of physiological processes consistently tend to

maintain proportionality with W0.75 21 . A more extensive discussion of the rationale and data

22 supporting the Agency's adoption of this scaling factor is in U.S. EPA, 1992b. Information such

23 as blood levels or exposure biomarkers or other data that are available for interspecies comparison

24 are used to improve the analysis when possible.

25 The default procedure to derive an human equivalent concentration of inhaled particles

26 and gases is described in U.S. EPA (1994) and Jarabek (1995a,b). The methodology estimates

27 respiratory deposition of inhaled particles and gases and provides methods for estimating internal

28 doses of gases with different absorption characteristics. The method is able to incorporate

29 additional toxicokinetics and metabolism to improve the analysis if such data are available.

30

31 3.5.2. Adjustment of Dose from Adults to Children

32 Slope factors and unit risk estimates for lifetime exposure incorporate exposure factors

33 that are based on adults (specifically, body weight, breathing rate, and drinking water ingestion

34 rate). When these unit risk estimates are used to assess risks from less-than-lifetime exposure that
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1 occurs during childhood, adjustments for differences between adults and children may be

2 appropriate.

3 Inhalation unit risk estimates: Section 3.5.1 specifies that the inhalation methodology

4 (U.S. EPA, 1994) be used for inhaled concentrations when agent-specific data are insufficient to

5 develop a case-specific dosimetry model. The methodology incorporates exposure factors based

on a 70-kg adult who breathes at a plausibly high rate of 20 m3 6 /d. Because children breathe more

7 air per unit of body weight (U.S. EPA, 1998), use of adult exposure factors may not be

8 appropriate. Consequently, inhalation unit risk estimates are adjusted to reflect a child's body

9 weight and breathing rate. For example, the following calculation adjusts an (adult) unit risk

estimate of 1x10-4 per ug/m3 so that it applies to a 9-kg infant who breathes 4.5 m3 10 /d:

(1x10-4 per ug/m3) x (4.5 m3/d / 20 m3/d) / (9 kg / 70 kg) = 1.75x10-4 per ug/m3 11 .

12 For inhaled gases and aerosols, this adjustment is intended to provide the same degree of

13 health-conservatism for children and adults. For inhaled particles, the adjustment does not take

14 into account the different size and spacing of airways of children and adults; this difference could

15 result in children and adults retaining particles with a different size distribution and different

16 toxicologic properties. To reduce this uncertainty, EPA is developing a default dosimetry model

17 for children that is based on children's inhalation parameters.

18 Drinking water unit risk estimates: Similarly, drinking water unit risk estimates

19 incorporate exposure factors based on a 70-kg adult who drinks water at a plausibly high rate of

20 2 L/d. Because children drink more water per unit of body weight (U.S. EPA, 1997c), use of

21 adult exposure factors may not be appropriate. Consequently, drinking water unit risk estimates

22 will be adjusted to reflect a child's body weight and drinking water ingestion rate.

23 Oral slope factors: Oral slope factors incorporate a cross-species scaling factor based on

equivalence of mg/kg3/4 24 -d (U.S. EPA, 1992b). This cross-species factor is intended to achieve

25 equivalence in lifetime cancer risk in different mammalian species. When risks from childhood

26 exposure are being assessed, the child's weight is not substituted for an adult weight in the cross27 species scaling factor. There are several reasons why using the child's weight in the cross-species

28 factor may not be appropriate:

29 C Using the child's weight instead of an adult weight assumes that children have faster

30 metabolism, leading to faster clearance, smaller body burdens, and smaller risks. 

31 Although children generally metabolize and eliminate many chemicals faster than

32 adults, this is not true in all cases (Renwick, 1998).

33 C The data supporting the 3/4-power factor pertain to cross-species equivalence, a

34 fundamentally different question from determining equivalence across different life
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1 stages of a single species.

2 C Although exposure may begin during childhood, subsequent events that complete the

3 carcinogenesis process may continue into adulthood.

4 Using an adult body weight is also a science policy choice that provides some degree of

5 health-conservatism for children in view of the uncertainties in extrapolating risks to children. 

6 Quantitatively, the effect of this choice is rather modest; for example, basing the scaling factor on

7 a 70-kg adult instead of a 10-kg child results in risk estimates that are 1.6 times higher

([70/10][1-3/4] 8 = 1.6).

9 Dermal exposure: The risk of distal-site cancers from the fraction of a dermal exposure

10 that is systemically absorbed is sometimes assessed by reducing the oral slope factor by a dermal

11 absorption factor that reflects the ratio of absorption by the dermal route to absorption by the oral

12 route. Use of a dermal absorption factor based on adults could increase the uncertainty in a risk

13 assessment of childhood exposure. Neonates, especially premature infants, have much greater

14 skin absorption than older children or adults (Schilter et al., 1996).

15 The risk of skin cancer from dermal exposure, in particular, from the fraction that remains

16 on the skin and is not systemically absorbed, has generally not been addressed because methods to

17 do so have not been developed. In order to assess children’s risks from this important pathway,

18 methodological research is needed in this area.

19

20 3.5.3. Toxicokinetic Analyses

21 Physiologically based mathematical models are potentially the most comprehensive way to

22 account for toxicokinetic processes affecting dose. Models build on physiological compartmental

23 modeling and attempt to incorporate the dynamics of tissue perfusion and the kinetics of enzymes

24 involved in metabolism of an administered compound.

25 A comprehensive model requires the availability of empirical data on the carcinogenic

26 activity contributed by parent compound and metabolite or metabolites and data by which to

27 compare kinetics of metabolism and elimination between species. A discussion of issues of

28 confidence accompanies presentation of model results (Monro, 1992). This includes

29 considerations of model validation and sensitivity analysis that stress the predictive performance

30 of the model. When a delivered dose measure is used in animal to human extrapolation of dose31 response data, the assessment should discuss the confidence in the assumption that the

32 toxicodynamics of the target tissue(s) will be the same in both species. Toxicokinetic data can

33 improve dose-response assessment by accounting for sources of change in proportionality of

34 applied to internal or delivered dose at various levels of applied dose. Many of the sources of
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1 potential nonlinearity involve saturation or induction of enzymatic processes at high doses. An

2 analysis that accounts for nonlinearity (for instance, due to enzyme saturation kinetics) can assist

3 in avoiding overestimation or underestimation of low dose-response otherwise resulting from

4 extrapolation from a sublinear or supralinear part of the experimental dose-response curve

5 (Gillette, 1983). Toxicokinetic processes tend to become linear at low doses, an expectation that

6 is more robust than low-dose linearity of response (Hattis, 1990). Accounting for toxicokinetic

7 nonlinearities allows better description of the shape of the curve at relatively high levels of dose in

8 the range of observation, but cannot determine linearity or nonlinearity of response at low dose

9 levels (Lutz, 1990a; Swenberg et al., 1987).

10 Toxicokinetic modeling results may be presented as the preferred method of estimating

11 human equivalent dose or in parallel discussion with default assumptions depending on relative

12 confidence in the modeling.

13

14 3.5.4. Route-to-Route Extrapolation

15 Judgments frequently need to be made about the carcinogenicity of an agent through a

16 route of exposure different than the one in the underlying studies. For example, exposures of

17 interest may be through inhalation of an agent tested primarily through animal feeding studies or

18 through ingestion of an agent that showed positive results in human occupational studies from

19 inhalation exposure.

20 Route-to-route extrapolation has both qualitative and quantitative aspects. For the

21 qualitative aspect, the assessor weighs the degree to which positive results through one route of

22 exposure in human or animal studies support a judgment that similar results would have been

23 observed in appropriate studies using the route of exposure of interest. In general, confidence in

24 making such a judgment is strengthened when the tumor effects are observed at a site distant from

25 the portal of entry and when absorption through the route of exposure of interest is similar to

26 absorption via the tested routes. In the absence of contrary data, the qualitative default

27 assumption is that, if the agent is absorbed by a route to give an internal dose, it may be

28 carcinogenic by that route. (See section 2.7.1.)

29 When a qualitative extrapolation can be supported, quantitative extrapolation may still be

30 problematic in the absence of adequate data. The differences in biological processes among

31 routes of exposure (oral, inhalation, dermal) can be great because of, for example, first-pass

32 effects and differing results from different exposure patterns. There is no generally applicable

33 method for accounting for these differences in uptake processes in quantitative route-to-route

34 extrapolation of dose-response data in the absence of good data on the agent of interest. 
7/02/99 3-22 DRAFT

1 Therefore, route-to-route extrapolation of dose data relies on a case-by-case analysis of available

2 data. When good data on the agent itself are limited, an extrapolation analysis can be based on

3 expectations from physical and chemical properties of the agent, properties and route-specific

4 data on structurally analogous compounds, or in vitro or in vivo uptake data on the agent. Route5 to-route uptake models may be applied if model parameters are suitable for the compound of

6 interest. Such models are currently considered interim methods; further model development and

7 validation is awaiting the development of more extensive data (see generally, Gerrity and Henry,

8 1990). For screening or hazard ranking, route-to-route extrapolation may be based on assumed

9 quantitative comparability as a default, as long as it is reasonable to assume absorption by

10 compared routes. When route-to-route extrapolation is used, the assessor's degree of confidence

11 in both the qualitative and quantitative extrapolation should be discussed in the assessment and

12 highlighted in the dose-response characterization.

13

14 3.5.5. Dose Averaging

15 The cumulative dose received over a lifetime, expressed as lifetime average daily dose, is

16 generally considered an appropriate default measure of exposure to a carcinogen (Monro, 1992). 

17 The assumption is made that a high dose of a carcinogen received over a short period of time is

18 equivalent to a corresponding low dose spread over a lifetime. While this is a reasonable default

19 assumption based on theoretical considerations, departures from it are expected. Another

20 approach is needed in some cases, such as when dose-rate effects are noted (e.g., formaldehyde). 

21 Cumulative dose may be replaced, as appropriate and justified by the data, with other dose

22 measures. In such cases, modifications to the default assumption are made to take account of

23 these effects; the rationale for the selected approach is explained.

24 In cases where a mode of action or other feature of the biology has been identified that has

25 special dose implications for sensitive subpopulations (e.g., differential effects by sex or

26 disproportionate impacts of early-life exposure), these are explained and are recorded to guide

27 exposure assessment and risk characterization. Special problems arise when the human exposure

28 situation of concern suggests exposure regimens (e.g., route and dosing schedule) that are

29 substantially different from those used in the relevant animal studies. These issues are explored

30 and pointed out for attention in the exposure assessment and risk characterization.

31

32 3.6. DISCUSSION OF UNCERTAINTIES

33 The exploration of significant uncertainties in data for dose and response and in

34 extrapolation procedures is part of the assessment. The presentation distinguishes between model
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1 uncertainty and parameter uncertainty. Model uncertainty is an uncertainty about a basic

2 biological question. For example, a default, linear dose-response extrapolation may have been

3 made based on tumor and other key evidence supporting the view that the model for an agent's

4 mode of action is a DNA-reactive process. Discussion of the confidence in the extrapolation is

5 appropriately done qualitatively or by showing results for alternatives that are equally plausible. It

6 is not useful, for example, to conduct quantitative uncertainty analysis running multiple forms of

7 linear models. This would obviate the function of the policy default.

8 Parameter uncertainties deal with numbers representing statistical or analytical measures

9 of variance or error in data or estimates. Uncertainties in parameters are described quantitatively,

10 if practicable, through sensitivity analysis and statistical uncertainty analysis. With the recent

11 expansion of readily available computing capacity, computer methods are being adapted to create

12 simulated biological data that are comparable with observed information. These simulations can

13 be used for sensitivity analysis, for example, to analyze how small, plausible variations in the

14 observed data could affect dose-response estimates. These simulations can also provide

15 information about experimental uncertainty in dose-response estimates, including a distribution of

16 estimates that are compatible with the observed data. Because these simulations are based on the

17 observed data, they cannot assist in evaluating the extent to which the observed data as a whole

18 are idiosyncratic rather than typical of the true situation. If quantitative analysis is not possible,

19 significant parameter uncertainties are described qualitatively. In either case, the discussion

20 highlights uncertainties that are specific to the agent being assessed, as distinct from those that are

21 generic to most assessments.

22 Estimation of the applied dose in a human study has numerous uncertainties such as the

23 exposure fluctuations that humans experience compared with the controlled exposures received

24 by animals on test. In a prospective cohort study, there is opportunity to monitor exposure and

25 human activity patterns for a period of time that supports estimation of applied dose (U.S. EPA,

26 1992a). In a retrospective study, exposure may be based on monitoring data but is often based on

27 human activity patterns and levels reconstructed from historical data, contemporary data, or a

28 combination of the two. Such reconstruction is accompanied by analysis of uncertainties

29 considered with sensitivity analysis in the estimation of dose (Wyzga, 1988; U.S. EPA, 1986a). 

30 These uncertainties can also be assessed for any confounding factor for which a quantitative

31 adjustment of dose-response data is made (U.S. EPA, 1984).

32
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1 3.7. TECHNICAL Dose-response CHARACTERIZATION

2 As with hazard characterization, the dose-response characterization serves the dual

3 purposes of presenting a technical characterization of the assessment results and supporting the

4 risk characterization.

5 The characterization presents the results of analyses of dose data, of response data, and of

6 dose-response. When alternative approaches are plausible and persuasive in selecting dose data,

7 response data, or extrapolation procedures, the characterization follows the alternative paths of

8 analysis and presents the results. The discussion covers the question of whether any should be

9 preferred over others because it (or they) better represents the available data or corresponds to

10 the view of the mechanism of action developed in the hazard assessment. The results for different

11 tumor types by sex and species are provided along with the one(s) preferred. Similarly, results for

12 responses other than tumor incidence are shown if appropriate.

13 Numerical dose-response estimates are presented to one significant figure to prevent an

14 inappropriate sense of high precision. However, since rounding can introduce significant errors in

15 a calculation, the rounding should be performed explicitly in the presentation of results; the actual

16 calculations are not done with intermediate rounding. Numbers are qualified as to whether they

17 represent central tendency or upper bounds and whether the method used is inherently more likely

18 to overestimate or underestimate (Krewski et al., 1984). 

19 In cases where a mode of action or other feature of the biology has been identified that has

20 special implications for early-life exposure, differential effects by sex, or other concerns for

21 sensitive subpopulations, these are explained. Similarly, any expectations that high dose-rate

22 exposures may alter the risk picture for some portion of the population are described. These and

23 other perspectives are recorded to guide exposure assessment and risk characterization. Whether

24 the lifetime average daily dose or another measure of dose should be considered for differing

25 exposure scenarios is discussed.

26 Uncertainty analyses, qualitative or quantitative if possible, are highlighted in the

27 characterization.

28 The dose-response characterization routinely includes the following, as appropriate for the

29 data available:

30 ! identification of the kinds of data available for analysis of dose and response and for

31 dose-response assessment,

32 ! results of assessment as above,

33 ! explanation of analyses in terms of quality of data available,

34 ! selection of study/response and dose metric for assessment,
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1 ! discussion of implications of variability in human susceptibility, including for

2 susceptible subpopulation,

3 ! applicability of results to varying exposure scenarios--issues of route of exposure, dose

4 rate, frequency, and duration,

5 ! discussion of strengths and limitations (uncertainties) of the data and analyses that are

6 quantitative as well as qualitative, and

7 ! special issues of interpretation of data, such as:

8 -- selecting dose data, response data, and dose-response approach(es),

9 -- use of meta-analysis,

10 -- uncertainty and quantitative uncertainty analysis.
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4. TECHNICAL EXPOSURE CHARACTERIZATION

1 Exposure assessment is the determination (qualitative and quantitative) of the magnitude,

2 frequency, and duration of exposure (EPA, 1992). The following section provides a brief

3 overview of exposure assessment principles with an emphasis on issues related to carcinogenic

4 risk assessment. The information presented here should be used in conjunction with other

5 guidances including: the 1992 Guidelines for Exposure Assessment, the 1995 Policy and

6 Guidance for Risk Characterization, the 1997 Exposure Factors Handbook, the 1997 Policy for

7 Use of Probabilistic Analysis in Risk Assessments, and the 1997 Guiding Principles for Monte

8 Carlo Analysis. In addition, program specific guidelines for exposure assessment should be

9 consulted.

10 Exposure assessment generally consists of four major steps: defining the assessment

11 questions, selecting or developing the conceptual and mathematical models, collecting data or

12 selecting and evaluating available data, and exposure characterization. Each of these steps is

13 briefly described below.

14 Defining the Assessment Questions

15 In providing a clear and unambiguous statement of the purpose and scope of the exposure

16 assessment (EPA, 1997a), consider the following.

17 < The management objectives of the assessment will determine whether deterministic

18 screening level analyses are adequate or whether full probabilistic exposure

19 characterization is needed.

20 < Identify and include all important sources (e.g., pesticide applications), pathways (e.g.,

21 food or water), and routes (e.g., ingestion, inhalation, and dermal) of exposure in the

22 assessment. If a particular source, pathway, or route is omitted, a clear and

23 transparent explanation should be provided.

24 < Separate analyses should be conducted for each definable subgroup within the

25 population of interest. In particular, subgroups that are believed to be highly exposed

26 or susceptible to a particular health effect should be studied. This includes people with
7/02/99 4-2 DRAFT

1 certain diseases or genetic susceptibilities, and others whose behavior or physiology

2 may lead to higher exposure or susceptibility. Consider the following examples.

3 < Physiological differences between men and women (e.g., body weight and

4 inhalation rate) may lead to important differences in exposures. See, for example,

5 the discussion in the Exposure Factors Handbook, Appendix 1A (EPA, 1997c).

6 < Pregnant and lactating women may have exposures that differ from the general

7 population (e.g., slightly higher water consumption) (EPA, 1997c). Further,

8 exposure to pregnant women may result in exposure to the developing fetus.

9 (NAS, 1993).

10 < Children consume more food per body weight than adults while consuming fewer

11 types of foods (ILSI, 1992, NAS, 1993 and EPA, 1997c). In addition, children

12 engage in crawling and mouthing (i.e., putting hands and objects in the mouth) 

13 behaviors which can increase their exposures.

14 < The elderly and disabled may have important differences in their exposures due to

15 a more sedentary lifestyle (EPA, 1997c). In addition, the health status of this

16 group may affect their susceptibility to the detrimental effects of exposure.

17 For further guidance, see the Guidelines for Exposure Assessment, § 3 (EPA, 1992).

18 Selecting or Developing the Conceptual and Mathematical Models

19 Carcinogen risk assessment models are generally based on the premise that risk is

20 proportional to total lifetime dose. Therefore, the exposure metric used for carcinogenic risk

21 assessment is the Lifetime Average Daily Dose (LADD). The LADD is typically used in

22 conjunction with the Cancer Slope Factor (CSF) to calculate individual excess cancer risk. It is

23 an estimate of the daily intake of a carcinogenic agent throughout the entire life of an individual. 

24 Depending on the objectives of the assessment, the LADD may be calculated deterministically

25 (using point estimates for each factor to derive a point estimate of the exposure) or stochastically

26 (using probability distributions to represent each factor and such techniques as Monte Carlo

27 analysis to derive a distribution of the LADD) (EPA, 1997b). Stochastic analyses may help to
7/02/99 4-3 DRAFT

1 identify certain population segments that are highly exposed and may need to be assessed as a

2 special subgroup. For further guidance, see the Guidelines for Exposure Assessment, § 5.3.5.2

3 (EPA, 1992).

4 When the route of exposure is inhalation or dermal contact, derivation of the LADD will

5 often require an approach to “route-to-route extrapolation.” The CSF and other measures of

6 toxicity are typically derived from oral administered doses in animal studies. Therefore, for

7 ingestion exposures in a human population it is not usually necessary to make adjustments to

8 account for route specific differences in absorption and uptake. However, for inhalation and

9 dermal exposures, such adjustments may be necessary. For further guidance, see the Guidelines

10 for Exposure Assessment, § 2.1.4 (EPA, 1992).

11 As discussed elsewhere in these guidelines, there may be cases where the mode of action

12 indicates that dose rates are important in the carcinogenic process. In these cases, short term,

13 less-than-lifetime exposure estimates may be more appropriate for risk assessment than the

14 LADD. Such estimates could be used to calculate the margin (MOE) that exists between

15 exposure and the point of departure derived in the dose-response assessment.

16 Collecting Data or Selecting and Evaluating Available Data

17 After the assessment questions have been defined and the conceptual and mathematical

18 models have been developed, it is necessary to compile and evaluate existing data or, if necessary,

19 to collect new data. Depending on the exposure scenario under consideration, data on a wide

20 variety of exposure factors may be needed. The U.S. EPA Exposure Factors Handbook (EPA,

21 1997c) contains a large compilation of exposure data with some analysis and recommendations.

22 Some of these data are organized by age groups to assist with assessing such subgroups as

23 children. See, for example, the Exposure Factors Handbook, Volume 1, Chapter 3 (EPA, 1997c). 

24 When using these existing data, it is important to evaluate the quality of the data and the extent to

25 which the data are representative of the population under consideration. The U.S. EPA Guidance

26 for Data Quality Assessment (EPA, 1996) and program specific guidances can provide further

27 assistance for evaluating existing data. 

28 When existing data fail to provide an adequate surrogate for the needs of a particular
7/02/99 4-4 DRAFT

1 assessment, it will be necessary to collect new data. Such data collection efforts should be guided

2 by the references listed above (e.g., the Guidance for Data Quality Assessment and program

3 specific guidance). Once again, subgroups of concern are an important consideration in any data

4 collection effort.

5 Exposure Characterization

6 The exposure characterization is a technical characterization that presents the assessment

7 results and supports the risk characterization. It provides a statement of the purpose, scope, and

8 approach used in the assessment, identifying the exposure scenarios and population subgroups

9 covered. It provides estimates of the magnitude, frequency, duration, and distribution of

10 exposures among members of the exposed population as the data permit. It identifies and

11 compares the contribution of different sources, pathways, and routes of exposure. In particular, a

12 qualitative discussion of the strengths and limitations (uncertainties) of the data and models are

13 presented.

14 The discussion of uncertainties is a critical component of the exposure characterization. 

15 Uncertainties can arise out of problems with the conceptual and mathematical models.

16 Uncertainties can also arise from poor data quality and data that are not quite representative of

17 the population or scenario of interest. Consider the following examples of uncertainties. 

18 < National data (i.e., data collected to represent the entire U.S. population) may not be

19 representative of exposures occurring within a regional or local population.

20 < Use of short term data to infer chronic, lifetime exposures must be done with caution. 

21 Using short term data to estimate long term exposures has the tendency to

22 underestimate the number of people exposed, while overestimating the exposure levels

experienced by those in the upper end (i.e., above the 90th 23 percentile) of the exposure

24 distribution. For further guidance, refer to the Guidelines for Exposure Assessment, §

25 5.3.1 (EPA, 1992). 

26 < Children’s behavior may lead to relatively high but intermittent exposures (EPA,

27 1998). This pattern of exposure, “one that gradually declines over the developmental

28 period and which remains relatively constant thereafter” is not accounted for in the

29 LADD model (ILSI, 1992). Further the physiological characteristics of children may
7/02/99 4-5 DRAFT

1 lead to important differences in exposure. Some of these differences can be accounted

2 for in the LADD model. For further guidance, see the Guidelines for Exposure

3 Assessment, § 5.3.5.2 (EPA, 1992).

4 Overall, the exposure characterization should provide a full description of the sources,

5 pathways, and routes of exposure. The characterization also should include a full description of

6 the populations assessed. In particular highly exposed or susceptible subgroups should be

7 discussed. For further guidance on the exposure characterization, consult the 1992 Guidelines for

8 Exposure Assessment (EPA, 1992), the 1995 Policy and Guidance for Risk Characterization

9 (EPA, 1995b and a) and EPA's Rule Writer's Guide to Executive Order 13045 (especially

10 Attachment C: Technical Support for Risk Assessors--Suggestions for Characterizing Risks to

11 Children) (EPA, 1999).
7/02/99 5-1 DRAFT

5. RISK CHARACTERIZATION

1 5.1. PURPOSE

2 EPA has developed general guidance on risk characterization for use in all of its risk

3 assessment activities. Administrator Carol Browner has issued a policy statement on risk

4 characterization, the core of which is the following mandate:

5

6 Each risk assessment prepared in support of decision making at EPA

7 should include a risk characterization that follows the principles and reflects the

8 values outlined in this policy. A risk characterization should be prepared in a

9 manner that is clear, transparent, reasonable, and consistent with other risk

10 characterizations of similar scope prepared across programs in the Agency. 

11 Further, discussion of risk in all EPA reports, presentations, decision packages,

12 and other documents should be substantively consistent with the risk

13 characterization. The nature of the risk characterization will depend upon the

14 information available, the regulatory application of the risk information, and the

15 resources (including time) available. In all cases, however, the assessment

16 should identify and discuss all the major issues associated with determining the

17 nature and extent of the risk and provide commentary on any constraints

18 limiting fuller exposition. (U.S. EPA, 1995)

19

20 EPA is also developing a Risk Characterization Handbook (draft available as publication number

21 EPA/600/R-99/025, dated March 1999), which provides detailed guidance to Agency staff. The

22 discussion below does not attempt to duplicate this material but summarizes its applicability to

23 carcinogen risk assessment. 

24 The risk characterization process includes an integrative analysis of the major results of

25 the risk assessment which is summarized for the risk manager in a nontechnical discussion that

26 minimizes the use of technical terms. It is an appraisal of the science that informs the risk

27 manager in his/her public health decisions, as do other decision-making analyses of economic,

28 social, or technology issues. It also serves the needs of other interested readers. The summary is

29 an information resource for preparation of risk communication information, but being somewhat

30 technical, is not itself the usual vehicle for communication with every audience.

31 The integrative analysis brings together the assessments of hazard, dose response, and

32 exposure to make risk estimates for the exposure scenarios of interest. This analysis is generally

33 much more extensive than the Risk Characterization Summary. It may be peer-reviewed or

34 subject to public comment along with the summary in preparation for an Agency decision. The

35 integrative analysis may be titled differently by different EPA programs (e.g., “Staff Paper” for
7/02/99 5-2 DRAFT

1 criteria air pollutants), but it typically will identify exposure scenarios of interest in decision

2 making and present risk analyses associated with them. Some of the analyses may concern

3 scenarios in several media; others may examine, for example, only drinking water risks. The

4 integrative analysis also may be the document that contains quantitative analyses of uncertainty.

5 The values supported by a risk characterization throughout the process are transparency

6 in environmental decision making, clarity in communication, consistency in core assumptions and

7 science policies from case to case, and reasonableness. While it is appropriate to err on the side

8 of protection of health and the environment in the face of scientific uncertainty, common sense

9 and reasonable application of assumptions and policies are essential to avoid unrealistic estimates

10 of risk (U.S. EPA, 1995). Both integrative analyses and the Risk Characterization Summary

11 present an integrated and balanced picture of the analysis of the hazard, dose response, and

12 exposure. The risk analyst should provide summaries of the evidence and results and describe the

13 quality of available data and the degree of confidence to be placed in the risk estimates. 

14 Important features include the constraints of available data and the state of knowledge, significant

15 scientific issues, and significant science and science policy choices that were made when

16 alternative interpretations of data existed (U.S. EPA, 1995). Choices made about using default

17 assumptions or data in the assessment are explicitly discussed in the course of analysis, and if a

18 choice is a significant issue, it is highlighted in the summary.

19

20 5.2. APPLICATION

21 Risk characterization is a necessary part of generating any Agency report on risk, whether

22 the report is preliminary, to support allocation of resources toward further study, or

23 comprehensive, to support regulatory decisions. In the former case, the detail and sophistication

24 of the characterization are appropriately small in scale; in the latter case, appropriately extensive. 

25 Even if a document covers only parts of a risk assessment (hazard and dose-response analyses, for

26 instance), the results of these are characterized.

27 Risk assessment is an iterative process that grows in depth and scope in stages from

28 screening for priority making, to preliminary estimation, to fuller examination in support of

29 complex regulatory decision making. Default assumptions are used at every stage because no

30 database is ever complete, but they are predominant at screening stages and are used less as more

31 data are gathered and incorporated at later stages. Various provisions in EPA-administered

32 statutes require decisions based on findings that represent all stages of iteration. There are close

33 to 30 provisions within the major statutes that require decisions based on risk, hazard, or

34 exposure assessment. For example, Agency review of pre-manufacture notices under Section 5 of
7/02/99 5-3 DRAFT

1 the Toxic Substances Control Act relies on screening analyses, while requirements for industry

2 testing under Section 4 of that act rely on preliminary analyses of risk or simply of exposure. At

3 the other extreme, air quality criteria under the Clean Air Act rest on a rich data collection

4 required by statute to undergo reassessment every few years. There are provisions that require

5 ranking of hazards of numerous pollutants--by its nature a screening level of analysis--and other

6 provisions that require a full assessment of risk. Given this range in the scope and depth of

7 analyses, not all risk characterizations can or should be equal in coverage or depth. The risk

8 assessor must carefully decide which issues in a particular assessment are important to present,

9 choosing those that are noteworthy in their impact on results. For example, health effect

10 assessments typically rely on animal data since human data are rarely available. The objective of

11 characterization of the use of animal data is not to recount generic issues about interpreting and

12 using animal data. Agency guidance documents cover these. Instead, the objective is to call out

13 any significant issues that arose within the particular assessment being characterized and inform

14 the reader about significant uncertainties that affect conclusions.

15

16 5.3. PRESENTATION OF RISK CHARACTERIZATION SUMMARY

17 The presentation is a nontechnical discussion of important conclusions, issues, and

18 uncertainties that uses the hazard, dose-response, exposure, and integrative analyses for technical

19 support. The primary technical supports within the risk assessment are the hazard

20 characterization, dose-response characterization, and exposure characterization described in this

21 guideline. The risk characterization is derived from these. The presentation should fulfill the aims

22 outlined in the purpose section above.

23

24 5.4. CONTENT OF RISK CHARACTERIZATION SUMMARY

25 Specific guidance on hazard, dose response, and exposure characterization appears in

26 previous sections. Overall, the risk characterization routinely includes the following, capturing

27 the important items covered in hazard, dose response, and exposure characterization:

28

29 C primary conclusions about hazard, dose response, and exposure, including equally

30 plausible alternatives;

31 C nature of key supporting information and analytic methods;

32 C risk estimates and their attendant uncertainties, including key uses of default

33 assumptions when data are missing or uncertain;

34 C statement of the extent of extrapolation of risk estimates from observed data to
7/02/99 5-4 DRAFT

1 exposure levels of interest (i.e., margin of exposure) and its implications for certainty

2 or uncertainty in quantifying risk;

3 C significant strengths and limitations of the data and analyses, including any major peer

4 reviewers’ issues;

5 C appropriate comparison with similar EPA risk analyses or common risks with which

6 people may be familiar; and

7 C comparison with assessment of the same problem by another organization.
7/02/99 6-1 DRAFT

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APPENDIX A. WEIGHT-OF-EVIDENCE NARRATIVES

1 This appendix contains several general illustrations of weight-of-evidence narratives.

2

3 NARRATIVE #1

4 Substance #1

5 CAS# XXX

6 CANCER HAZARD SUMMARY

7 Substance 1 is likely to be carcinogenic to humans by all routes of exposure. The weight

8 of evidence of human carcinogenicity of Substance 1 is based on (a) findings of carcinogenicity in

9 rats and mice of both sexes by oral and inhalation exposures; (b) its similarity in structure to other

10 chlorinated organics that are known to cause liver and kidney damage, and liver and kidney

11 tumors in rats and mice; (c) suggestive evidence of a possible association between Substance 1

12 exposure of workers in the laundry and dry cleaning industries and increased cancer risk in a

13 number of organ systems; and (d) human and animal data indicating that Substance 1 is absorbed

14 by all routes of exposure.

15 In comparison with other agents designated as likely carcinogens, the overall weight of

16 evidence for Substance 1 places it at the low end of the grouping. This is because one cannot

17 attribute observed excess cancer risk in exposed workers solely to Substance 1. Moreover, there

18 is considerable scientific uncertainty about the human significance and relevance of certain rodent

19 tumors associated with exposure to Substance 1 and other chlorinated organics, but insufficient

20 evidence about mode of action. Hence, the human relevance of the animal evidence of

21 carcinogenicity relies on a default assumption.

22 There is no clear evidence about the mode of action for each tumor type induced in rats

23 and mice. Available evidence suggests that Substance 1 induces cancer mainly by promoting cell

24 growth rather than via direct mutagenic action, although a mutagenic mode of action for rat

25 kidney tumors cannot be ruled out. The dos-response assessment should, therefore, adopt both

26 default approaches, nonlinear and linear. It is recognized that the latter approach likely

27 overestimates risk at low doses if the mode of action is primarily growth promoting. This

28 approach, however, may be useful for screening analyses.

29
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1 SUPPORTING INFORMATION

2 Human Data

3 A number of epidemiologic studies of dry cleaning and laundry workers have reported

4 elevated incidences of lung, cervix, esophagus, kidney, blood, and lymphoid cancers. Many of

5 these studies are confounded by coexposure to other petroleum solvents, making them limited for

6 determining whether the observed increased cancer risks are causally related to Substance 1. The

7 only investigation of dry cleaning workers with no known exposure to other chemicals did not

8 evaluate other confounding factors such as smoking, alcohol consumption, and low

9 socioeconomic status to exclude the possible contribution of these factors to cancer risks.

10

11 Animal Data

12 The carcinogenic potential of Substance 1 has been adequately investigated in two chronic

13 studies in two rodent species, the first study by gavage and the second study by inhalation.

14 Substance 1 is carcinogenic in the liver in both sexes of mice when tested by either route of

15 exposure. It causes marginally increased incidences of mononuclear cell leukemia (MCL) in both

16 sexes of rats and low incidences of a rare kidney tumor in male rats by inhalation. No increases in

17 tumor incidence were found in rats treated with Substance 1 by gavage. This rat study was

18 considered limited because of high mortality of the animals.

19 Although Substance 1 causes increased incidences of tumors at multiple sites in two

20 rodent species, controversy surrounds each of the tumor endpoints concerning their relevance

21 and/or significance to humans (see discussion under Mode of Action).

22

23 Other Key Data

24 Substance 1 is a member of a class of chlorinated organics that often cause liver and

25 kidney toxicity and carcinogenesis in rodents. Like many chlorinated hydrocarbons, Substance 1

26 itself has tested negative in a battery of standard genotoxicity tests using bacterial and mammalian

27 cell systems, including human lymphocytes and fibroblast cells. There is evidence, however, that a

28 minor metabolite generated by an enzyme found in rat kidney tissue is mutagenic. This kidney

29 metabolite has been hypothesized to be related to the development of kidney tumors in the male

30 rat. This metabolic pathway appears to be operative in the human kidney.

31 Human data indicate that Substance 1 is readily absorbed via inhalation, but to a much

32 lesser extent by skin contact. Animal data show that Substance 1 is absorbed well by the oral

33 route.

34
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1 MODE OF ACTION

2 The mechanisms of Substance 1-induced mouse liver tumors are not completely

3 understood. One mechanism has been hypothesized to be mediated by a genotoxic epoxide

4 metabolite generated by enzymes found in the mouse liver, but there is a lack of direct evidence in

5 support of this mechanism. A more plausible mechanism that still needs to be further defined is

6 related to liver peroxisomal proliferation and toxicity by TCA (trichloroacetic acid), a major

7 metabolite of Substance 1. However, there are no definitive data indicating that TCA induces

8 peroxisomal proliferation in humans.

9 The mechanisms by which Substance 1 induces kidney tumors in male rats are even less

10 well understood. The rat kidney response may be related to either kidney toxicity or the activity

11 of a mutagenic metabolite of the parent compound.

12 The human relevance of Substance 1-induced MCL in rats is unclear. The biological

13 significance of marginally increased incidences of MCL has been questioned by some, since this

14 tumor occurs spontaneously in the tested rat strain at very high background rates. On the other

15 hand, it has been considered by others to be a true finding because there was a decreased time to

16 onset of the disease and the disease was more severe in treated as compared with untreated

17 control animals. The exact mechanism by which Substance 1 increases incidence of MCL in rats

18 is not known.

19 Overall, there is not enough evidence to justify high confidence in a conclusion about any

20 single mode of action; it would appear that more than a single mode operates in different rodent

21 tissues. The apparent lack of mutagenicity of Substance 1 itself and its general growth-promoting

22 effect on high-background tumors, as well as its toxicity toward mouse liver and rat kidney tissue,

23 support the view that its predominant mode of action is cell growth promoting rather than

24 mutagenic. A mutagenic contribution to the renal carcinogenicity due to a metabolite cannot be

25 entirely ruled out.

26

27 NARRATIVE #2

28 Substance #2

29 CAS# XXX

30 CANCER HAZARD SUMMARY

31 There is suggestive evidence for carcinogenicity of Substance 2, but it is not sufficient for

32 assessment of human carcinogenic potential.

33 The evidence on carcinogenicity consists of (a) data from an oral animal study showing a

34 response only at the highest dose in female rats, with no response in males, and (b) the fact that
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1 other low-molecular-weight chemicals in this class have shown tumorigenicity in the respiratory

2 tract after inhalation. The one study of Substance 2 effects by the inhalation route was not

3 adequately performed. The available evidence is too limited to describe human carcinogenicity

4 potential or support dose-response assessment.

5

6 SUPPORTING INFORMATION

7 Human Data

8 An elevated incidence of cancer was reported in a cohort of workers in a chemical plant

9 who were exposed to a mixture of chemicals, including Substance 2 as a minor component. The

10 study is considered inadequate because of the small size of the cohort studied and the lack of

11 adequate exposure data.

12

13 Animal Data

14 In a long-term drinking water study in rats, an increased incidence of adrenal cortical

15 adenomas was found in the highest dosed females. No other significant finding was made. The

16 oral rat study was well conducted by a standard protocol. In a 1-year study in hamsters at one

17 inhalation dose, no tumors were seen. This study was inadequate because of high mortality and

18 consequent short duration. The chemical is very irritating and is a respiratory toxicant in

19 mammals. The animal data are too limited for conclusions to be drawn.

20

21 Structural Analogue Data

22 Substance 2’s structural analogues, formaldehyde and acetaldehyde, both have

23 carcinogenic effects on the rat respiratory tract.

24

25 Other Key Data

26 The weight of results of mutagenicity tests in bacteria, fungi, fruit flies, and mice leads to

27 an overall conclusion of not mutagenic; Substance 2 is lethal to bacteria to a degree that makes

28 testing difficult and test results difficult to interpret. The chemical is readily absorbed by all

29 routes.

30

31 MODE OF ACTION

32 Data are not sufficient to judge whether there is a carcinogenic mode of action.

33

34
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1 NARRATIVE #3

2 Substance #3

3 CAS# XXX

4 CANCER HAZARD SUMMARY

5 Substance 3 is carcinogenic to humans by all routes of exposure. Although several

6 studies in workers fall short of establishing causality, when considered together, suggest an

7 elevated risk of lung cancer after long-term exposure to Substance 3. More importantly, animal

8 cancer bioassay studies and mechanistic studies in both animals and exposed humans have

9 provided strong consistent results that support a level of concern equal to having conclusive

10 epidemiologic evidence. The weight of evidence of human carcinogenicity of Substance 3 is based

11 on (a) consistent evidence of carcinogenicity at multiple sites in both sexes of rats and mice by

12 oral and inhalation exposure; (b) epidemiologic evidence suggestive of a possible association

13 between exposure of industrial workers to Substance 3 and elevated risk of lung cancer, which is

14 the tumor type consistently found in different test species and with different routes of

15 administration; (c) mutagenic effects in numerous in vivo and in vitro test systems, which are

16 similar to those found in humans; (d) a similar profile of p53 mutations in transgenic rodent and

17 human lung tumor tissue; (e) membership in a class of DNA-reactive compounds that are

18 regularly observed to cause carcinogenic and mutagenic effects in animals. Due to its ready

19 absorption by all routes of exposure and rapid distribution throughout the body, Substance 3 is

20 expected to pose a risk by all routes of exposure. The strong evidence of a mutagenic mode of

21 action supports dose-response assessment that assumes linearity of the relationship.

22

23 SUPPORTING INFORMATION

24 Human Data

25 Elevated risks of lung cancer different than that associated with smoking have been

26 reported in exposed workers in several studies. The interpretation of the studies separately is

27 complicated by the lack of consistency in dose-response, latency period, and average age of

28 appearance exposures, as well as by exposure to other agents. So, there is no single study that

29 demonstrates that Substance 3 caused the effects. Nevertheless, several of the studies together

30 are considered suggestive of Substance 3 carcinogenicity because they consistently show cancer

31 elevation in the same tissue. Biomonitoring studies of exposed workers find DNA damage in

32 blood lymphocytes and the degree of DNA damage correlates with the level and duration of

33 Substance 3 exposure. More importantly, a mechanistic linkage is found for humans by

34 observation of a similar profile of mutation in the p53 gene from the lung tumor tissue of the p53
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1 transgenic mouse and exposed workers. This mutation spectra is consistent with the type of

2 predominant DNA adducts induced by Substance 3. This evidence provides strong support to the

3 positive suggestion from the worker cancer studies. 

4 Animal Data

5 Substance 3 causes cancer in multiple tissue sites in rats and mice of both sexes by oral

6 and inhalation exposure. In particular, there is a consistent trend of a similar tumor site found in

7 the human studies, namely, an elevated incidence of lung tumor in different species/sexes and by

8 different routes of exposure. The database is more extensive than usual and the studies are of

9 good quality. The observation of multisite, multispecies carcinogenic activity by an agent is

10 considered to be very strong evidence and is often the case with highly mutagenic agents. There

11 are also strong evidence in many studies showing that Substance 3 is mutagenic across different

12 phylogenetic levels including rodents, as well as in peripheral cells of exposed humans--a property

13 that is very highly correlated with carcinogenicity. Further strengthening the concern for human

14 cancer risk is a similar p53 mutation spectra observed in lung tumor tissue from the p53

15 transgenic mouse and human cancer biopsies. In humans, a large number of the cases had a

16 mutation in p53 with a predominance of GC to AT transitions. The mutation spectra of

17 Substance 3 associated lung tumors differed from patterns reported for sporadic and smoking

18 related tumors.

19

20 Structural Analogue Data

21 SAR analysis indicates that Substance 3 is a highly DNA-reactive agent. Structurally

22 related chemicals, also exhibit mutagenic and carcinogenic effects in laboratory animals.

23

24 Other Key Data

25 The structure and DNA reactivity of Substance 3 support potential carcinogenicity. Both

26 properties are highly correlated with carcinogenicity. Numerous positive mutagenicity tests in

27 vitro and in vivo add to this support and are reinforced by observation of similar genetic damage

28 in exposed workers.

29 Substance 3 is experimentally observed to be readily absorbed by all routes and rapidly

30 distributed through the body.

31

32 MODE OF ACTION

33 All of the available data in both humans and animals, strongly indicate a mutagenic mode

34 of action, with a particular human target in lung tissue. A mechanistic linkage is found for rodents
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1 and humans by observations of a similar profile of mutations in the p53 gene from the lung tumor

2 tissue of the p53 transgenic mouse and exposed workers. This mutation spectra is consistent with

3 the type of predominant DNA adducts induced by Substance 3. The tumor suppressor gene, p53,

4 is a frequently mutated gene in human tumors, including lung. The consistent finding of

5 mutagencity in experimental assays and human biomonitoring studies, the finding of p53

6 mutations in transgenic animal and human lung tumor tissue, all points to a mutagenic mode of

7 action and supports assuming linearity of the dose-response relationship.

8

9 NARRATIVE #4

10 Substance #4

11 CAS# XXX

12 CANCER HAZARD SUMMARY

13 This chemical is likely to be carcinogenic to humans by all routes of exposure. Its

14 carcinogenic potential is indicated by (a) tumor and toxicity studies on structural analogues, which

15 demonstrate the ability of the chemical to produce thyroid follicular cell tumors in rats and

16 hepatocellular tumors in mice following ingestion, and (b) metabolism and hormonal information

17 on the chemical and its analogues, which contributes to a working mode of action and associates

18 findings in animals with those in exposed humans. In comparison with other agents designated as

19 likely carcinogens, the overall weight of evidence for this chemical places it at the lower end of

20 the grouping. This is because there is a lack of tumor response data on this agent itself.

21 Biological information on the compound is contradictory in terms of how to quantitate

22 potential cancer risks. The information on disruption on thyroid-pituitary status argues for using

23 a margin of exposure evaluation. However, the chemical is an aromatic amine, a class of agents

24 that are DNA-reactive and induce gene mutation and chromosome aberrations, which argues for

25 low-dose linearity. Additionally, there is a lack of mode-of-action information on the mouse liver

26 tumors produced by the structural analogues, also pointing toward a low-dose linear default

27 approach. In recognition of these uncertainties, it is recommended to quantitate tumors using

28 both nonlinear (to place a lower bound on the risks) and linear (to place an upper bound on the

29 risks) default approaches. Given the absence of tumor response data on the chemical per se, it is

30 recommended that tumor data on close analogues be used to possibly develop toxicity equivalent

31 factors or relative potencies.

32 Overall, this chemical is an inferential case for potential human carcinogenicity. The

33 uncertainties associated with this assessment include (1) the lack of carcinogenicity studies on the

34 chemical, (2) the use of tumor data on structural analogues, (3) the lack of definitive information
7/02/99 A-8 DRAFT

1 on the relevance of thyroid-pituitary imbalance for human carcinogenicity, and (4) the different

2 potential mechanisms that may influence tumor development and potential risks.

3

4 SUPPORTING INFORMATION

5 Human Data

6 Worker exposure has not been well characterized or quantified, but recent medical

7 monitoring of workers exposed over a period of several years has uncovered alterations in

8 thyroid-pituitary hormones (a decrease in T3 and T4 and an increase in TSH) and symptoms of

9 hypothyroidism. A urinary metabolite of the chemical has been monitored in workers, with

10 changes in thyroid and pituitary hormones noted, and the changes were similar to those seen in an

11 animal study.

12

13 Animal Data

14 The concentration of the urinary metabolite in rats receiving the chemical for 28 days was

15 within twofold of that in exposed workers, a finding associated with comparable changes in

16 thyroid hormones and TSH levels. In addition, the dose of the chemical given to rats in this study

17 was essentially the same as that of an analogue that had produced thyroid and pituitary tumors in

18 rats. The human thyroid responds in the same way as the rodent thyroid following short-term,

19 limited exposure. Although it is not well established that thyroid-pituitary imbalance leads to

20 cancer in humans as it does in rodents, information in animals and in exposed humans suggests

21 similar mechanisms of disrupting thyroid-pituitary function and the potential role of altered TSH

22 levels in leading to thyroid carcinogenesis.

23

24 Structural Analogue Data

25 This chemical is an aromatic amine, a member of a class of chemicals that has regularly

26 produced carcinogenic effects in rodents and gene and structural chromosome aberrations in

27 short-term tests. Some aromatic amines have produced cancer in humans.

28 Close structural analogues produce thyroid follicular cell tumors in rats and hepatocellular

29 tumors in mice following ingestion. The thyroid tumors are associated with known perturbations

30 in thyroid-pituitary functioning. These compounds inhibit the use of iodide by the thyroid gland,

31 apparently due to inhibition of the enzyme that synthesizes the thyroid hormones (T3, T4). 

32 Accordingly, blood levels of thyroid hormones decrease, which induces the pituitary gland to

33 produce more TSH, a hormone that stimulates the thyroid to produce more of its hormones. The

34 thyroid gland becomes larger because of increases in the size of individual cells and their
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1 proliferation, and upon chronic administration of the chemical, tumors develop. Thus, thyroid

2 tumor development is significantly influenced by disruption in the thyroid-pituitary axis.

3

4 Other Key Data

5 The chemical can be absorbed by the oral, inhalation, and dermal routes of exposure.

6

7 MODE OF ACTION

8 Data on the chemical and on structural analogues indicate the potential association of

9 carcinogenesis with perturbation of thyroid-pituitary homeostasis. Structural analogues are

10 genotoxic, thus raising the possibility of different mechanisms by which this chemical may

11 influence tumor development.

12

13 NARRATIVE #5

14 Substance #5

15 CAS# XXX

16 CANCER HAZARD SUMMARY

17 Substance 5 is likely to be a human carcinogen by all routes of exposure. Findings are

18 based on very extensive and significant experimental findings that include (a) tumors at multiple

19 sites in both sexes of two rodent species via three routes of administration relevant to human

20 exposure; (b) close structural analogues that produce a spectrum of tumors like those from

21 Substance 5; (c) significant evidence for the production of reactive Substance 5 metabolites that

22 readily bind to DNA and produce gene mutations in many systems, including cultured mammalian

23 and human cells; and (d) two null studies and one positive epidemiologic study; in the positive

24 study, there may have been exposure to Substance 5. These findings support a decision that

25 Substance 5 might produce cancer in exposed humans. In comparison to other agents considered

26 likely human carcinogens, the overall weight of evidence for Substance 5 puts it near the top of

27 the grouping. Given the agent’s mutagenicity, which can influence the carcinogenic process, a

28 linear dose-response extrapolation is recommended.

29 Uncertainties include the lack of adequate information on the mutagenicity of Substance 5

30 in mammals or humans in vivo, although such effects would be expected.

31
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1 SUPPORTING INFORMATION

2 Human Data

3 The information on the carcinogenicity of Substance 5 from human studies is inadequate. 

4 Two studies of production workers have not shown significant increases in cancer from exposure

5 to Substance 5 and other chemicals. An increase in lymphatic cancer was reported in a mortality

6 study of grain elevator workers who may have been exposed to Substance 5 (and other

7 chemicals).

8

9 Animal Data

10 Substance 5 produced tumors in four chronic rodents studies. Tumor increases were

11 noted in males and females of rats and mice following oral dermal and inhalation exposure (rat--

12 oral and two inhalation, mouse--oral and dermal). It produces tumors both at the site of

13 application (e.g., skin with dermal exposure) and at sites distal to the portal of entry into the body

14 (e.g., mammary gland) following exposure from each route. Tumors at the same site were noted

15 in both sexes of a species (blood vessel), both species (forestomach) and via different routes of

16 administration (lung). Some tumors developed after very short latency, metastasized extensively,

17 and produced death, an uncommon findings in rodents. The rodent studies were well designed

18 and conducted except for the oral studies, in which the doses employed caused excessive toxicity

19 and mortality. However, given the other rodent findings, lower doses would also be anticipated

20 to be carcinogenic.

21

22 Structural Analogue Data

23 Several chemicals structurally related to Substance 5 are also carcinogenic in rodents. 

24 Among four that are closest in structure, tumors like those seen for Substance 5 were often noted

25 (e.g., forestomach, mammary, lung), which helps to confirm the findings for Substance 5 itself. In

26 sum, all of the tumor findings help to establish animal carcinogenicity and support potential human

27 carcinogenicity for Substance 5.

28

29 Other Key Data

30 Substance 5 itself is not reactive, but from its structure it was expected to be metabolized

31 to reactive forms. Extensive metabolism studies have confirmed this presumption and have

32 demonstrated metabolites that bind to DNA and cause breaks in the DNA chain. These lesions

33 are readily converted to gene mutations in bacteria, fungi, higher plants, insects, and mammalian

34 and human cells in culture. There are only a limited number of reports on the induction of
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1 chromosome aberrations in mammals and humans; thus far they are negative.

2

3 MODE OF ACTION

4 Human carcinogens often produce cancer in multiple sites of multiple animal species and

5 both sexes and are mutagenic in multiple test systems. Substance 5 satisfies these findings. It

6 produces cancer in males and females of rats and mice. It produces gene mutations in cells across

7 all life forms--plants, bacteria, and animals--including mammals and humans. Given the

8 mutagenicity of Substance 5 exposure and the multiplicity and short latency of Substance 5 tumor

9 induction, it is reasonable to use a linear approach for cancer dose-response extrapolation.
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APPENDIX B. RESPONSES TO THE NATIONAL ACADEMY OF SCIENCES

NATIONAL RESEARCH COUNCIL REPORT SCIENCE AND JUDGMENT IN RISK

ASSESSMENT (NRC, 1994)

1 Recommendations of the National Academy of Sciences National Research Council

2 In 1994, the National Academy of Sciences published a report, Science and Judgment in

3 Risk Assessment. The report was written by a Committee on Risk Assessment of Hazardous Air

4 Pollutants formed under the Academy's Board on Environmental Studies and Toxicology,

5 Commission on Life Sciences, National Research Council. The report was called for under

6 Section 112(o)(1)(A,B) of the Clean Air Act Amendments of 1990, which provided for the EPA

7 to arrange for the Academy to review:

8 C risk assessment methodology used by EPA to determine the carcinogenic risk

9 associated with exposure to hazardous air pollutants from source categories and

10 subcategories subject to the requirements of this section, and

11 C improvements in such methodology.

12 Under Section 112(o)(2)(A,B), the Academy was to consider the following in its review:

13 C the techniques used for estimating and describing the carcinogenic potency to humans

14 of hazardous air pollutants, and

15 C the techniques used for estimating exposure to hazardous air pollutants (for

16 hypothetical and actual maximally exposed individuals as well as other exposed

17 individuals).

18 To the extent practicable, the Academy was also to review methods of assessing adverse human

19 health effects other than cancer for which safe thresholds of exposure may not exist [Section

20 112(o)(3)]. The Congress further provided that the EPA Administrator should consider, but need

21 not adopt, the recommendations in the report and the views of the EPA Science Advisory Board

22 with respect to the report. Prior to the promulgation of any standards under Section 112(f), the

23 Administrator is to publish revised guidelines for carcinogenic risk assessment or a detailed

24 explanation of the reasons that any recommendations contained in the report will not be

25 implemented [Section 112(o)(6)].

26 The following discussion addresses the recommendations of the 1994 report that are

27 pertinent to the EPA cancer risk assessment guidelines. Guidelines for assessment of exposure, of

28 mixtures, and of other health effects are separate EPA publications. Many of the

29 recommendations were related to practices specific to the exposure assessment of hazardous air
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1 pollutants, which are not covered in cancer assessment guidelines. Recommendations about these

2 other guidelines or practices are not addressed here.

3

4 Hazard Classification

5 The 1994 report contains the following recommendation about classifying cancer hazard:

6 C The EPA should develop a two-part scheme for classifying evidence on

7 carcinogenicity that would incorporate both a simple classification and a narrative

8 evaluation. At a minimum, both parts should include the strength (quality) of the

9 evidence, the relevance of the animal model and results to humans, and the relevance

10 of the experimental exposures (route, dose, timing, and duration) to those likely to be

11 encountered by humans.

12 The report also presented a possible matrix of 24 boxes that would array weights of evidence

13 against low, medium, or high relevance, resulting in 24 codes for expressing the weight and

14 relevance.

15 These guidelines adopt five standard hazard descriptors and a narrative for presentation of

16 the weight-of-evidence findings. The descriptors are used within the narrative. There is no

17 matrix of alphanumerical weight-of-evidence boxes.

18 The issue of an animal model that is not relevant to humans has been dealt with by not

19 including an irrelevant response in the weighing of evidence, rather than by creating a weight of

20 evidence and then appending a discounting factor as the NRC scheme would do. The issue of

21 relevance is more complex than the NRC matrix makes apparent. Often the question of relevance

22 of the animal model applies to a single tumor response, but one encounters situations in which

23 there are more tumor responses in animals than the questioned one. Dealing with this complexity

24 is more straightforward if it is done during the weighing of evidence rather than after as in the

25 NRC scheme. Moreover, the same experimental data are involved in deciding on the weight of

26 evidence and the relevance of a response. It would be awkward to go over the same data twice.

27 In recommending that the relevance of circumstances of human exposure be taken into

28 account, the NRC appears to assume that all of the actual conditions of human exposure will be

29 known when the classification is done. This is not the case. More often than not, the hazard

30 assessment is applied to risks associated with exposure to different media or environments at

31 different times. In some cases, there is no priority to obtaining exposure data until the hazard

32 assessment has been done. The approach of these guidelines is to characterize hazards as to

33 whether their expression is intrinsically limited by route of exposure or by reaching a particular

34 dose range based strictly on toxicological and other biological features of the agent. Both the use
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1 of descriptors and the narrative specifically capture this information. Other aspects of appropriate

2 application of the hazard and dose-response assessment to particular human exposure scenarios

3 are dealt with in the characterization of the dose-response assessment, e.g., the applicability of the

4 dose-response assessment to scenarios with differing frequencies and durations.

5 The NRC scheme apparently intended that the evidence would be weighed, then given a

6 low, medium, or high code for some combination of relevance of the animal response, route of

7 exposure, timing, duration, or frequency. The 24 codes contain none of this specific information,

8 and, in fact, do not communicate what the conclusion is about. To make the codes communicate

9 the information apparently intended would require some multiple of the 24 in the NRC scheme. 

10 As the number of codes increases, their utility for communication decreases.

11 Another reason for declining to use codes is that they tend to become outdated as research

12 reveals new information that was not contemplated when they were adopted. This has been the

13 case with the classification system under the 1986 EPA guidelines.

14 Even though these guidelines do not adopt a matrix of codes, their method of using

15 descriptors and narratives captures the information the NRC recommended as the most important,

16 and in the EPA’s view, in a more transparent manner.

17

18 Dose-Response

19 The 1994 report contains the following recommendations about dose-response issues:

20 C EPA should continue to explore, and when scientifically appropriate, incorporate

21 toxicokinetic models of the link between exposure and biologically effective dose (i.e.,

22 dose reaching the target tissue).

23 C Despite the advantages of developing consistent risk assessments between agencies by

24 using common assumptions (e.g., replacing surface area with body weight to the 0.75

25 power), EPA should indicate other methods, if any, that would be more accurate.

26 C EPA should continue to use the linearized multistage model as a default option but

27 should develop criteria for determining when information is sufficient to use an

28 alternative extrapolation model.

29 C EPA should continue to use as one of its risk characterization metrics upper-bound

30 potency estimates of the probability of developing cancer due to lifetime exposure. 

31 Whenever possible, this metric should be supplemented with other descriptions of

32 cancer potency that might more adequately reflect the uncertainty associated with the

33 estimates.
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1 C EPA should adopt a default assumption for differences in susceptibility among humans

2 in estimating individual risks.

3 C In the analysis of animal bioassay data on the occurrence of multiple tumor types, the

4 cancer potencies should be estimated for each relevant tumor type that is related to

5 exposure, and the individual potencies should be summed for those tumors.

6 Toxicokinetic models are encouraged in these guidelines, with discussion of appropriate

7 considerations for their use. When there are questions as to whether such a model is more

8 accurate in a particular case than the default method for estimating the human equivalent dose,

9 both alternatives may be used. It should be noted that the default method for inhalation exposure

10 is a toxicokinetic model.

11 The rationale for adopting the oral scaling factor of body weight to the 0.75 power has

12 been discussed above in the explanation of major defaults. The empirical basis is further explored

13 in U.S. EPA (1992b). The more accurate approach is to use a toxicokinetic model when data

14 become available, or to modify the default when data are available, as encouraged under these

15 guidelines. As the U.S. EPA (1992b) discussion explores in depth, data on the differences among

16 animals in response to toxic agents are basically consistent with using a power of 1.0, 0.75, or

17 0.66. The Federal agencies chose the power of 0.75 for the scientific reasons given in the

18 previous discussion of major defaults; these were not addressed specifically in the NRC report. It

19 was also considered appropriate, as a matter of policy, for the agencies to agree on one factor. 

20 Again, the default for inhalation exposure is a model that is constructed to become better as more

21 agent-specific data become available.

22 EPA proposes not to use a computer model such as the linearized multistage model as a

23 default for extrapolation below the observed range. The reason is that the basis for default

24 extrapolation is a theoretical projection of the likely shape of the curve, considering mode of

25 action. For this purpose, a computer model looks more sophisticated than a straight-line

26 extrapolation, but is not. The extrapolation will be by straight line as explained in the explanation

27 of major defaults. This was also recommended by workshop reviewers of a previous draft of

28 these guidelines (U.S. EPA, 1994b). In addition, a margin-of-exposure analysis is proposed in

29 cases in which the curve is thought to be nonlinear, based on mode of action. In both cases, the

30 observed range of data will be modeled by curve fitting in the absence of supporting data for a

31 biologically based or case-specific model.

32 The result of using straight-line extrapolation is thought to be an upper bound on low33 dose potency to the human population in most cases, but as discussed in the major defaults

34 section, it may not always be. Exploration and discussion of uncertainty of parameters in curve-
7/02/99 B-5 DRAFT

1 fitting a model of the observed data or in using a biologically based or case-specific model is

2 called for in the dose-response assessment and characterization sections of these guidelines.

3 The issue of a default assumption for human differences in susceptibility has been

4 addressed under the major defaults discussion in Section 1.3 with respect to margin-of-exposure

5 analysis. EPA has considered but decided not to adopt a quantitative default factor for human

6 differences in susceptibility when a linear extrapolation is used. In general, EPA believes that

7 linear extrapolation is sufficiently conservative to protect public health. Linear approaches (both

8 LMS and straight-line extrapolation) from animal data are consistent with linear extrapolation on

9 the same agents from human data (Goodman and Wilson, 1991; Hoel and Portier, 1994). If

10 actual data on human variability in sensitivity are available they will, of course, be used.

11 In analyzing animal bioassay data on the occurrence of multiple tumor types, these

12 guidelines outline a number of biological and other factors to consider. The objective is to use

13 these factors to select response data (including nontumor data as appropriate) that best represent

14 the biology observed. As stated in Section 3 of the guidelines, appropriate options include use of

15 a single data set, combining data from different experiments, showing a range of results from

16 more 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 animals

18 with tumors, or a combination of these options. The approach judged to best represent the data is

19 presented with the rationale for the judgment, including the biological and statistical

20 considerations involved. EPA has considered the approach of summing tumor incidences and

21 decided not to adopt it. While multiple tumors may be independent, in the sense of not arising

22 from metastases of a single malignancy, it is not clear that they can be assumed to represent

23 different effects of the agent on cancer processes. In this connection, it is not clear that summing

24 incidences provides a better representation of the underlying mode(s) of action of the agent than

25 combining animals with tumors or using another of the several options noted above. Summing

26 incidences would result in a higher risk estimate, a step that appears unnecessary without more

27 reason.

28

29 Risk Characterization

30 C When EPA reports estimates of risk to decisionmakers and the public, it should

31 present not only point estimates of risk, but also the sources and magnitudes of

32 uncertainty associated with these estimates.

33 C Risk managers should be given characterizations of risk that are both qualitative and

34 quantitative, i.e., both descriptive and mathematical.
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1 C EPA should consider in its risk assessments the limits of scientific knowledge, the

2 remaining uncertainties, and the desire to identify errors of either overestimation or

3 underestimation.

4 In part as a response to these recommendations, the Administrator of EPA issued

5 guidelines for risk characterization and required implementation plans from all programs in EPA

6 (U.S. EPA, 1995). The Administrator's guidance is followed in these cancer guidelines. The

7 assessments of hazard, dose-response, and exposure will all have accompanying technical

8 characterizations covering issues of strengths and limitations of data and current scientific

9 understanding, identification of defaults utilized in the face of gaps in the former, discussions of

10 controversial issues, and discussions of uncertainties in both their qualitative and, as practicable,

11 their quantitative aspects.
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APPENDIX C. CASE STUDY EXAMPLES FOR HAZARD EVALUATION

1 This section provides examples of substances that fit the descriptors above. These

2 examples are based on available information about real substances and are selected to illustrate the

3 principles for weight-of-evidence evaluation and the application of the classification scheme.

4 These case studies show the interplay of differing lines of evidence in making a conclusion. 

5 Some particularly illustrate the role that “other key data” can play in conclusions.

6

7 Example 1: “Carcinogenic to Humans”--Route-Dependent/Linear Extrapolation

8 Human Data

9 Substance 1 is an aluminosilicate mineral that exists in nature with a fibrous habit. Several

10 descriptive epidemiologic studies have demonstrated very high mortality from malignant

11 mesothelioma, mainly of the pleura, in three Turkish villages where there was a contamination of

12 this mineral and where exposure had occurred from birth. Both sexes were equally affected and

13 at an unusually young age.

14

15 Animal Data

16 Substance 1 has been studied in a single long-term inhalation study in rats at one exposure

17 concentration that showed an extremely high incidence of pleural mesothelioma (98% in treated

18 animals versus 0% in concurrent controls). This is a rare malignant tumor in the rat and the onset

19 of tumors occurred at a very early age (as early as 1 year). Several studies involving injection into

20 the body cavities of rats or mice (i.e., pleural or peritoneal cavities) also produced high incidences

21 of pleural or peritoneal mesotheliomas. No information is available on the carcinogenic potential

22 of substance 1 in laboratory animals via oral and dermal exposures.

23

24 Other Key Data

25 Information on the physical and chemical properties of substance 1 indicates that it is

26 highly respirable to humans and laboratory rodents. It is highly insoluble and is not likely to be

27 readily degraded in biological fluid.

28 No information is available on the deposition, translocation, retention, lung clearance, and

29 excretion of the substance after inhalation exposure or ingestion. Lung burden studies have

30 shown the presence of elevated levels of the substance in lung tissue samples of human cases of

31 pleural mesotheliomas from contaminated villages compared with control villages.
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1 No data are available on genetic or related effects in humans. The substance has been

2 shown to induce unscheduled DNA synthesis in human cells in vitro and transformation and

3 unscheduled DNA synthesis in mouse cells.

4 The mechanisms by which this substance causes cancer in humans and animals are not

5 understood, but appear to be related to its unique physical, chemical, and surface properties. Its

6 fiber morphology is similar to a known group of naturally occurring silicate minerals that have

7 been known to cause respiratory cancers in humans(including pleural mesothelioma) from

8 inhalation exposure and genetic changes.

9

10 Evaluation

11 Human evidence is judged to establish a causal link between exposure to substance 1 and

12 human cancer. Even though the human evidence does not satisfy all criteria for causality, this

13 judgment is based on a number of unusual observations: large magnitude of the association,

14 specificity of the association, demonstration of environmental exposure, biological plausibility,

15 and coherence based on the entire body of knowledge of the etiology of mesothelioma.

16 Animal evidence demonstrates a causal relationship between exposure and cancer in

17 laboratory animals. Although available data are not optimal in terms of design (e.g., the use of

18 single dose, one sex only), the judgment is based on the unusual findings from the only inhalation

19 experiment in rats (i.e., induction of an uncommon tumor, an extremely high incidence of

20 malignant neoplasms, and onset of tumors at an early age). Additional evidence is provided by

21 consistent results from several injection studies showing an induction of the same tumors by

22 different modes of administration in more than one species.

23 Other key data, while limited, support the human and animal evidence of carcinogenicity. 

24 It can be inferred from human and animal data that this substance is readily deposited in the

25 respiratory airways and deep lung and is retained for extended periods of time after first exposure. 

26 Information on related fibrous substances indicates that the modes of action are likely mediated by

27 the physical and chemical characteristics of the substance (e.g., fiber shape, high aspect ratio, a

28 high degree of insolubility in lung tissues).

29 Insufficient data are available to evaluate the human carcinogenic potential of substance 1

30 by oral exposure. Even though there is no information on its carcinogenic potential via dermal

31 uptake, it is not expected to pose a carcinogenic hazard to humans by that route because it is very

32 insoluble and is not likely to penetrate the skin.

33
7/02/99 C-3 DRAFT

1 Conclusion

2 It is concluded that substance 1 is carcinogenic to humans by inhalation exposure. The

3 weight of evidence of human carcinogenicity is based on (a) exceptionally increased incidence of

4 malignant mesothelioma in epidemiologic studies of environmentally exposed human populations;

5 (b) significantly increased incidence of malignant mesothelioma in a single inhalation study in rats

6 and in several injection studies in rats and mice; and (c) supporting information on related fibrous

7 substances that are known to cause cancer via inhalation and genetic damage in exposed

8 mammalian and human mesothelial cells. The human carcinogenic potential of substance 1 via

9 oral exposure cannot be determined on the basis of insufficient data. It is not likely to pose a

10 carcinogenic hazard to humans via dermal uptake because it is not anticipated to penetrate the

11 skin.

12 The mode of action of this substance is not understood. In addition to this uncertainty,

13 dose-response information is lacking for both human and animal data. Epidemiologic studies

14 contain observations of significant excess cancer risks at relatively low levels of environmental

15 exposure. The use of linear extrapolation in a dose-response relationship assessment is

16 appropriate as a default since mode-of-action data are not available.

17

18 Example 2: “Carcinogenic to Humans”-- Any Exposure Conditions/Linear Extrapolation 

19 Human Data

20 Substance 2 is an alkylating agent that is used extensively as a chemical intermediate in

21 organic synthesis, particularly in the synthesis of plastics and resins. Several cohort studies of

22 workers using substance 2 have been conducted. Four studies of chemical workers exposed to

23 substance 2 (as well as other agents) found an increased mortality rate from lung cancer. The

24 excess was primarily found in small subgroups with high-level exposure. Although smoking was a

25 confounding factor, the predominant lung tumor found was small-cell carcinoma, which is distinct

26 from the squamous cell carcinomas usually found in smokers. Although the type of lung cancer

27 was consistent among the four studies, the dose-response, latency period, and average age of

28 appearance was not consistent. Furthermore, there are confounding exposures to other

29 chemicals. No increase in mortality rate was observed in two studies, one of which had exposures

30 higher than the studies reporting an increased incidence of lung cancer.

31
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1 Animal Data

2 A multisite tumor response in rats and mice of both sexes is found in 2-year rodent

3 bioassay studies when substance 2 is administered by various routes. In particular, the induction

4 of lung tumors is consistently found across different studies, species, and routes of administration. 

5 For example, when administered by inhalation, substance 2 induced a dose-related increase in the

6 incidences of lung tumors in female and male mice (B6C3F1); and squamous cell carcinomas of

7 the lung and nasal tumors in male rats (F344). When administered by subcutaneous injection,

8 substance 2 induced a statistically significant response for pulmonary tumors and local

9 fibrosarcomas in mice of both sexes. An oral gavage 2-year study resulted in an elevated

10 incidence of lung tumors in male rats and both sexes of mice, forestomach tumors in both sexes of

11 rats and mice, liver tumors in both sexes of rats, and urinary bladder tumors in both sexes of mice.

12 Substance 2 produced lung and forestomach tumors in the p53 mouse cancer transgenic assay

13 when administered via gavage. It is an initiator of skin tumors in mice.

14

15 Other Key Data

16 Substance 2 is a liquid but can exist as a vapor at room temperature given its high vapor

17 pressure. It is readily absorbed dermally. Studies in rats indicate that, once absorbed, substance

18 2 is uniformly distributed throughout the body. It is metabolized by hydrolysis and by conjugation

19 with glutathione. The ability to form glutathione conjugate varies across animal species, with the

20 rat being most active, followed by mice.

21

22 Substance 2 induces cell transformation in the Syrian Hamster Embryo assay. It is a

23 direct-acting alkylating agent and is consistently mutagenic when tested in a variety of

24 nonmammalian and mammalian assays, including in vivo rodent tests. It has been shown to form

25 DNA adducts and to produce predominantly GC to AT transitions. Substance 2 produces similar

26 genetic lesions in rodents and humans. It was found to cause dose-related increases, HPRT

27 mutations, and chromosome aberrations in peripheral blood lymphocytes of exposed workers. A 

28 similar p53 mutation spectra has been found in lung tumor tissue from the p53 transgenic mouse

29 and human cancer biopsies. In humans, a large number of the cases had a mutation in p53, with a

30 predominance of GC to AT transitions. The mutation spectra of substance-2-associated lung

31 tumors differed from patterns reported for sporadic and smoking-related tumors.

32 SAR analysis indicates that substance 2 is a highly DNA-reactive agent. Structurally

33 related chemicals also exhibit mutagenic and carcinogenic effects in laboratory animals.

34
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1 Evaluation

2 Available epidemiologic studies, taken together, suggest that a causal association between

3 exposure to substance 2 and elevated risk of cancer is plausible. This judgment is based on small

4 but consistent excesses of lung tumors that are distinct from smoking-related lung cancer in the

5 studies of highly exposed workers. The evidence is close and indicates that causal interpretation

6 is credible, but not conclusively demonstrated because of certain inconsistencies in the available

7 studies, possible bias, and confounding factors that could not be adequately excluded.

8 Extensive evidence indicates that substance 2 is carcinogenic to laboratory animals in

9 multiple species and at multiple tissue sites with multiple routes of exposure. There is an

10 induction of malignant tumors to an unusual degree with regard to incidence. In particular, there

11 is a consistent dose-related induction of lung tumors across different species and routes of

12 administration in well-designed and conducted studies. This tumor response is similar to that

13 reported in exposed humans. 

14 The potential human carcinogenicity of substance 2 is reinforced by observations of similar

15 genetic damage (DNA adducts, HPRT mutations, chromosomal aberrations) in experimental tests

16 and exposed workers. The genetic effects induced in experimental animals are dose related and

17 observed at exposures lower than those that produce lung tumors in rodent bioassays. A

18 mechanistic linkage is found for rodents and humans by observations of a similar profile of

19 mutations in the p53 gene from the lung tumor tissue of the p53 transgenic mouse and exposed

20 workers. This mutation spectra is consistent with the type of predominant DNA adducts induced

21 by substance 2. 

22 Substance 2 belongs to a well-defined, structurally related class of substances whose

23 members are carcinogenic in rodents and are likely to be human carcinogens. 

24

25 Conclusion

26 It is concluded that substance 2 is “carcinogenic to humans” by all routes of exposure.

27 The weight of evidence of human carcinogenicity is based on (a) consistent evidence of

28 carcinogenicity in rats and mice by oral and inhalation exposure; (b) epidemiologic evidence

29 suggestive of a causal association between exposure and elevated risk of lung cancer, which is the

30 tumor type consistently induced in different test species and with different routes of

31 administration; (c) evidence of genetic damage in blood lymphocytes of exposed workers; (d)

32 mutagenic effects in numerous in vivo and in vitro test systems, which are similar to those found

33 in humans; (e) similar profile of p53 mutations in rodent and human lung tumor tissue; (f) 

34 membership in a class of DNA-reactive compounds that have been shown to cause carcinogenic
7/02/99 C-6 DRAFT

1 and mutagenic effects in animals; and (g) ability to be absorbed by all routes of exposure,

2 followed by rapid distribution throughout the body.

3 The evidence is compelling that the mutagenic properties of substance 2 in experimental

4 animals and humans are an important influence on the carcinogenic process. Thus, substance 2

5 acts through a mode of action that is operative in humans and would therefore reasonably be

6 anticipated to cause cancer in humans. A linear extrapolation should be assumed in dose-response

7 assessment.

8

9 Example 3: “Likely Human Carcinogen”--Any Exposure Conditions/Linear Extrapolation

10 Human Data

11 Substance 3 is a brominated alkane. Three studies have investigated the cancer mortality

12 of workers exposed to this substance. No statistically significant increase in cancer at any site was

13 found in a study of production workers exposed to substance 3 and several other chemicals. 

14 Elevated cancer mortality was reported in a much smaller study of production workers. An

15 excess of lymphoma was reported in grain workers who may have had exposure to substance 3

16 and other chemical compounds. These studies are considered inadequate due to their small

17 cohort size; lack of or poorly characterized exposure concentrations; or concurrent exposure of

18 the cohort to other potential or known carcinogens.

19

20 Animal Data

21 The potential carcinogenicity of substance 3 has been extensively studied in an oral gavage

22 study in rats and mice of both sexes, two inhalation studies of rats of different strains of both

23 sexes, an inhalation study in mice of both sexes, and a skin painting study in female mice.

24 In the oral study, increased incidences of squamous-cell carcinoma of the forestomach

25 were found in rats and mice of both sexes. Additionally, there were increased incidences of liver

26 carcinomas in female rats, hemangiosarcomas in male rats, and alveolar/bronchiolar adenoma of

27 the lung of male and female mice. Excessive toxicity and mortality were observed in the rat study,

28 especially in the high-dose groups, which resulted in early termination of the study, and similar

29 time-weighted average doses for the high- and low-treatment groups.

30 In the first inhalation study in rats and mice, increased incidences of carcinomas and

31 adenocarcinomas of the nasal cavity and hemangiosarcoma of the spleen were found in exposed

32 animals of each species of both sexes. Treated female rats also showed increased incidences of

33 alveolar/bronchiolar carcinoma of the lung and mammary gland fibroadenomas. Treated male rats

34 showed an increased incidence of peritoneal mesothelioma. In the second inhalation study in rats
7/02/99 C-7 DRAFT

1 (single exposure only), significantly increased incidences of hemangiosarcoma of the spleen and

2 adrenal gland tumors were seen in exposed animals of both sexes. Additionally, increased

3 incidences of subcutaneous mesenchymal tumors and mammary gland tumors were induced in

4 exposed male and female rats, respectively.

5 Lifetime dermal application of substance 3 to female mice resulted in significantly

6 increased incidences of skin papillomas and lung tumors.

7 Several chemicals structurally related to substance 3 are also carcinogenic in rodents. The

8 spectrum of tumor responses induced by related substances was similar to those seen with

9 substance 3 (e.g., forestomach, mammary gland, and lung tumors).

10

11 Other Key Data

12 Substance 3 exists as a liquid at room temperature and is readily absorbed by ingestion,

13 inhalation, and dermal contact. It is widely distributed in the body and is eliminated in the urine

14 mainly as metabolites (e.g., glutathione conjugate).

15 Substance 3 is not itself DNA-reactive, but is biotransformed to reactive metabolites, as

16 inferred by findings of its covalent binding to DNA and induction of DNA strand breaks, both in

17 vivo and in vitro. Substance 3 has been shown to induce sister chromatid exchanges, mutations,

18 and unscheduled DNA synthesis in human and rodent cells in vitro. Reverse and forward

19 mutations have been consistently produced in bacterial assays and in vitro assays using eukaryotic

20 cells. Substance 3, however, did not induce dominant lethal mutations in mice or rats, or

21 chromosomal aberrations or micronuclei in bone marrow cells of mice treated in vivo.

22

23 Evaluation

24 Available epidemiologic data are considered inadequate for an evaluation of a causal

25 association of exposure to the substance and excess of cancer mortality due to major study

26 limitations.

27 There is extensive evidence that substance 3 is carcinogenic in laboratory animals. 

28 Increased incidences of tumors at multiple sites have been observed in multiple studies in two

29 species of both sexes with different routes of exposure. It induces tumors both at the site of entry

30 (e.g., nasal tumors via inhalation, forestomach tumors by ingestion, skin tumors with dermal

31 exposure) and at distal sites (e.g., mammary gland tumors). Additionally, it induced tumors at the

32 same sites in both species and sexes via different routes of exposure (e.g., lung tumors). With the

33 exception of the oral study in which the employed doses caused excessive toxicity and mortality,

34 the other studies are considered adequately designed and well conducted. Overall, given the
7/02/99 C-8 DRAFT

1 magnitude and extent of animal carcinogenic responses to substance 3, coupled with similar

2 responses to structurally related substances, these animal findings are judged to be highly relevant

3 and predictive of human responses.

4 Other key data, while not very extensive, are judged to be supportive of carcinogenic

5 potential. Substance 3 has consistently been shown to be mutagenic in mammalian cells, including

6 human cells, and in nonmammalian cells; thus, mutation is likely a mode of action for its

7 carcinogenic activity. However, the possible involvement of other modes of action has not been

8 fully investigated. Furthermore, induction of genetic changes from in vivo exposure to substance

9 3 has not been demonstrated.

10

11 Conclusion

12 Substance 3 is likely to be carcinogenic to humans. In comparison with other agents

13 designated as likely human carcinogens, the overall weight of evidence for substance 3 puts it at

14 the high end of the grouping.

15 The weight of evidence of human carcinogenicity is based on animal evidence and other

16 key evidence. Human data are inadequate for an evaluation of human carcinogenicity. The

17 overall weight of evidence is based on (a) extensive animal evidence showing induction of

18 increases of tumors at multiple sites in both sexes of two rodent species via three routes of

19 administration relevant to human exposure; (b) tumor data of structural analogues exhibiting

20 similar patterns of tumors in treated rodents; (c) in vitro evidence for mutagenic effects in

21 mammalian cells and nonmammalian systems; and (d) its ability to be absorbed by all routes of

22 exposure followed by rapid distribution throughout the body.

23 Some uncertainties are associated with the mechanisms of carcinogenicity of substance 3. 

24 Although there is considerable evidence indicating that mutagenic events could account for

25 carcinogenic effects, there is still a lack of adequate information on the mutagenicity of substance

26 3 in vivo in animals or humans. Moreover, alternative modes of action have not been explored. 

27 Nonetheless, available data indicate a likely mutagenic mode of action. Linear extrapolation

28 should be assumed in dose-response assessment.

29

30 Example 4: “Likely Human Carcinogen”--All Routes/Linear and Nonlinear Extrapolation

31 Human Data

32 Substance 4 is a chlorinated alkene solvent. Several cohort studies of dry cleaning and

33 laundry workers exposed to substance 4 and other solvents reported significant excesses of

34 mortality due to cancers of the lung, cervix, esophagus, kidney, bladder, lymphatic and
7/02/99 C-9 DRAFT

1 hematopoietic system, colon, or skin. No significant cancer risks were observed in a subcohort of

2 one of these investigations of dry cleaning workers exposed mainly to substance 4. Possible

3 confounding factors such as smoking, alcohol consumption, or low socioeconomic status were

4 not considered in the analyses of these studies.

5 A large case-control study of bladder cancer did not show any clear association with dry

6 cleaning. Several case-control studies of liver cancer identified an increased risk of liver cancer

7 with occupational exposure to organic solvents. The specific solvents to which workers were

8 exposed and exposure levels were not identified.

9

10 Animal Data

11 The potential carcinogenicity of substance 4 has been investigated in two long-term

12 studies in rats and mice of both sexes by oral administration and inhalation.

13 Significant increases in hepatocellular carcinomas were induced in mice of both sexes

14 treated with substance 4 by oral gavage. No increases in tumor incidence were observed in

15 treated rats. Limitations in both experiments included control groups smaller than treated groups,

16 numerous dose adjustments during the study, and early mortality due to treatment-related

17 nephropathy.

18 In the inhalation study, there were significantly increased incidences of hepatocellular

19 adenoma and carcinoma in exposed mice of both sexes. In rats of both sexes, there were

20 marginally significant increased incidences of mononuclear cell leukemia (MCL) when compared

21 with concurrent controls. The incidences of MCL in control animals, however, were higher than

22 historical controls from the conducting laboratory. The tumor finding was also judged to be

23 biologically significant because the time to onset of tumor was decreased and the disease was

24 more severe in treated than in control animals. Low incidences of renal tubular cell adenomas or

25 adenocarcinomas were also observed in exposed male rats. The tumor incidences were not

26 statistically significant, but there was a significant trend.

27

28 Other Key Data

29 Substance 4 has been shown to be readily and rapidly absorbed by inhalation and ingestion

30 in humans and laboratory animals. Absorption by dermal exposure is slow and limited. Once

31 absorbed, substance 4 is primarily distributed to and accumulated in adipose tissue and the brain,

32 kidney, and liver. A large percentage of substance 4 is eliminated unchanged in exhaled air, with

33 urinary excretion of metabolites comprising a much smaller percentage. The absorption and

34 distribution profiles of substance 4 are similar across species including humans.
7/02/99 C-10 DRAFT

1 Two major metabolites (trichloroacetic acid [TCA] and trichloroethanol), which are

2 formed by a P-450-dependent mixed-function oxidase enzyme system, have been identified in all

3 studied species, including humans. There is suggestive evidence for the formation of an epoxide

4 intermediate based on the detection of two other metabolites (oxalic acid and trichloroacetyl

5 amide). In addition to oxidative metabolism, substance 4 also undergoes conjugation with

6 glutathione. Further metabolism by renal beta-lyases could lead to two minor active metabolites

7 (trichlorovinyl thiol and dichlorothiokente).

8 Toxicokinetic studies have shown that the enzymes responsible for the metabolism of

9 substance 4 can be saturated at high exposures. The glutathione pathway was found to be a

10 minor pathway at low doses, but more prevalent following saturation of the cytochrome P-450

11 pathway. Comparative in vitro studies indicate that mice have a greater capacity to metabolize to

12 TCA than rats and humans. Inhalation studies also indicate saturation of oxidative metabolism of

13 substance 4, which occurs at higher dose levels in mice than in rats and humans. Based on these

14 findings, it has been postulated that the species differences in the carcinogenicity of substance 4

15 between rats and mice may be related to the differences in the metabolism to TCA and glutathione

16 conjugates.

17 Substance 4 is a member of the class of chlorinated organics that often cause liver and

18 kidney toxicity and carcinogenesis in rodents. Like many chlorinated organics, substance 4 itself

19 does not appear to be mutagenic. Substance 4 was generally negative in in vitro bacterial systems

20 and in vivo mammalian systems. However, a minor metabolite formed in the kidney by the

21 glutathione conjugation pathway has been found to be a strong mutagen.

22 The mechanisms of induced carcinogenic effects of substance 4 in rats and mice are not

23 completely understood. It has been postulated that mouse liver carcinogenesis is related to liver

24 peroxisomal proliferation and toxicity of the metabolite TCA. Information on whether or not

25 TCA induces peroxisomal proliferation in humans is not definitive. The induced renal tumors in

26 male rats may be related either to kidney toxicity or the activity of a mutagenic metabolite. The

27 mechanisms of increases in MCL in rats are not known.

28

29 Evaluation

30 Available epidemiologic studies, taken together, provide suggestive evidence of a possible

31 causal association between exposure to substance 4 and cancer incidence in the laundry and dry

32 cleaning industries. This is based on consistent findings of elevated cancer risks in several studies

33 of different populations of dry cleaning and laundry workers. However, each individual study is

34 compromised by a number of study deficiencies including small numbers of cancers, confounding
7/02/99 C-11 DRAFT

1 exposure to other solvents, and poor exposure characterization. Others may interpret these

2 findings collectively as inconclusive.

3 There is considerable evidence that substance 4 is carcinogenic to laboratory animals. It

4 induces tumors in mice of both sexes by oral and inhalation exposure and in rats of both sexes via

5 inhalation. However, owing to incomplete understanding of the mode of action, the predictivity

6 of animal responses to humans is uncertain.

7 Animal data of structurally related compounds showing common target organs of toxicity

8 and carcinogenic effects (but lack of mutagenic effects) provide additional support for the

9 carcinogenicity of substance 4. Comparative toxicokinetic and metabolism information indicates

10 that the mouse may be more susceptible to liver carcinogenesis than rats and humans. This may

11 indicate differences of the degree and extent of carcinogenic responses, but does not detract from

12 the qualitative weight of evidence of human carcinogenicity. The toxicokinetic information also

13 indicates that oral and inhalation are the major routes of human exposure.

14

15 Conclusion

16 Substance 4 is likely to be carcinogenic to humans by all routes of exposure. The weight

17 of evidence of human carcinogenicity is based on: (a) demonstrated evidence of carcinogenicity in

18 two rodent species of both sexes via two relevant routes of human exposure; (b) the substance’s

19 similarity in structure to other chlorinated organics that are known to cause liver and kidney

20 toxicity and carcinogenesis in rodents; (c) suggestive evidence of a possible association between

21 exposure to the substance in the laundry and dry cleaning industries and increased cancer

22 incidence; and (d) human and animal data indicating that the substance is absorbed by all routes of

23 exposure.

24 There is considerable scientific uncertainty about the human significance of certain rodent

25 tumors associated with substance 4 and related compounds. In this case, the human relevance of

26 the animal evidence of carcinogenicity relies on the default assumption.

27 Overall, there is not enough evidence to give high confidence in a conclusion about any

28 single mode of action; it appears that more than one is plausible in different rodent tissues. 

29 Nevertheless, the lack of mutagenicity of substance 4 and its general growth-promoting effect on

30 high background tumors, as well as its toxicity toward mouse liver and rat kidney tissue, support

31 the view that the predominant mode is growth-promoting rather than mutagenic. A mutagenic

32 contribution to carcinogenicity due to a metabolite cannot be ruled out. The dose-response

33 assessment should, therefore, adopt both default approaches, nonlinear and linear extrapolations. 
7/02/99 C-12 DRAFT

1 The latter approach is very conservative since it likely overestimates risk at low doses in this case,

2 and is primarily useful for screening analyses.

3

4 Example 5: “Likely/Not Likely Human Carcinogen”--Range of Dose Limited, Margin-of5 Exposure Extrapolation

6 Human Data

7 Substance 5 is a metal-conjugated phosphonate. No human tumor or toxicity data exist

8 on this chemical.

9

10 Animal Data

11 Substance 5 caused a statistically significant increase in the incidence of urinary bladder

12 tumors in male, but not female, rats at 30,000 ppm (3%) in the diet in a long-term study. Some of

13 these animals had accompanying urinary tract stones and toxicity. No bladder tumors or adverse

14 urinary tract effects were seen in two lower dose groups (2,000 and 8,000 ppm) in the same

15 study. A chronic dietary study in mice at doses comparable to those in the rat study showed no

16 tumor response or urinary tract effects. A 2-year study in dogs at doses up to 40,000 ppm

17 showed no adverse urinary tract effects.

18

19 Other Key Data

20 Subchronic dosing of rats confirmed that there was profound development of stones in the

21 male bladder at doses comparable to those causing cancer in the chronic study, but not at lower

22 doses. Sloughing of the epithelium of the urinary tract accompanied the stones.

23 There was a lack of mutagenicity relevant to carcinogenicity. In addition, there is nothing

24 about the chemical structure of substance 5 to indicate DNA reactivity or carcinogenicity.

25 Substance 5 is composed of a metal, an ethanol, and a simple phosphorus-oxygen26 containing component. The metal is not absorbed from the gut, whereas the other two

27 components are absorbed. At high doses, ethanol is metabolized to carbon dioxide, which makes

28 the urine more acidic; the phosphorus level in the blood and calcium in the urine are increased. 

29 Chronic testing of the phosphorus-oxygen-containing component alone in rats did not show any

30 tumors or adverse effects on the urinary tract.

31 Because substance 5 is a metal complex, it is not likely to be readily absorbed from the

32 skin.

33

34 Evaluation
7/02/99 C-13 DRAFT

1 Substance 5 produced cancer of the bladder and urinary tract toxicity in male, but not

2 female rats and mice, and dogs failed to show the toxicity noted in male rats. The mode of action

3 developed from the other key data to account for the toxicity and tumors in the male rats is the

4 production of bladder stones. At high but not lower subchronic doses in the male rat, substance 5

5 leads to elevated blood phosphorus levels; the body responds by releasing excess calcium into the

6 urine. The calcium and phosphorus combine in the urine and precipitate into multiple stones in

7 the bladder. The stones are very irritating to the bladder; the bladder lining is eroded and cell

8 proliferation occurs to compensate for the loss of the lining. Cell layers pile up, and finally,

9 tumors develop. Stone formation does not involve the chemical per se but is secondary to the

10 effects of its constituents on the blood and, ultimately, the urine. Bladder stones, regardless of

11 their cause, commonly produce bladder tumors in rodents, especially the male rat.

12

13 Conclusion

14 Substance 5, a metal aliphatic phosphonate, is likely to be carcinogenic to humans only

15 under high-exposure conditions following oral and inhalation exposure that lead to bladder stone

16 formation, but is not likely to be carcinogenic under low-exposure conditions. It is not likely to

17 be a human carcinogen via the dermal route, given that the compound is a metal conjugate that is

18 readily ionized and its dermal absorption is not anticipated. The weight of evidence is based on

19 (a) bladder tumors only in male rats; (b) the absence of tumors at any other site in rats or mice; (c)

20 the formation of calcium-phosphorus-containing bladder stones in male rats at high, but not low,

21 exposures that erode bladder epithelium and result in profound increases in cell proliferation and

22 cancer; and (d) the absence of structural alerts or mutagenic activity.

23 There is a strong mode-of-action basis for the requirements of (a) high doses of substance

24 5, (b) which lead to excess calcium and increased acidity in the urine, (c) which result in the

25 precipitation of stones, and (d) the necessity of stones for toxic effects and tumor hazard

26 potential. Lower doses fail to perturb urinary constituents, lead to stones, produce toxicity, or

27 give rise to tumors. Therefore, dose-response assessment should assume nonlinearity.

28 A major uncertainty is whether the profound effects of substance 5 may be unique to the

29 rat. Even if substance 5 produced stones in humans, there is only limited evidence that humans

30 with bladder stones develop cancer. Most often human bladder stones are either passed in the

31 urine or lead to symptoms resulting in their removal. However, since one cannot totally dismiss

32 the male rat findings, some hazard potential may exist in humans following intense exposures. 

33 Only fundamental research could illuminate this uncertainty.

34
7/02/99 C-14 DRAFT

1 Example 6: “Suggestive” Evidence

2 Human Data

3 Substance 6 is an unsaturated aldehyde. In a cohort study of workers in a chemical plant

4 exposed to a mixture of chemicals with substance 6 as a minor component, a greater risk of

5 cancer was reported than was expected. This study is considered inadequate because of multiple

6 exposures, small cohort, and poor exposure characterization.

7

8 Animal Data

9 Substance 6 was tested for potential carcinogenicity in a drinking water study in rats, an

10 inhalation study in hamsters, and a skin painting study in mice. No significant increases in tumors

11 were observed in male rats treated with substance 6 at three dose levels in drinking water. 

12 However, a significant increase of adrenal cortical adenomas was found in the only treated female

13 dose group administered a dose equivalent to the high dose of males. This study used a small

14 number of animals (20 per dose group).

15 No significant finding was detected in the inhalation study in hamsters. This study is

16 inadequate due to the use of too few animals, short duration of exposure, and inappropriate dose

17 selection (use of a single exposure that was excessively toxic as reflected by high mortality).

18 No increase in tumors was induced in the skin painting study in mice. This study is of

19 inadequate design for carcinogenicity evaluation because of several deficiencies: small number of

20 animals, short duration of exposure, lack of reporting about the sex and age of animals, and purity

21 of test material.

22 Substance 6 is structurally related to low-molecular-weight aldehydes that generally

23 exhibit carcinogenic effects in the respiratory tracts of laboratory animals via inhalation exposure. 

24 Three skin painting studies in mice and two subcutaneous injection studies of rats and mice were

25 conducted to evaluate the carcinogenic potential of a possible metabolite of substance 6

26 (identified in vitro). Increased incidences of either benign or combined benign and malignant skin

27 tumors were found in the dermal studies. In the injection studies of rats and mice, increased

28 incidences of local sarcomas or squamous cell carcinoma were found at the sites of injection. All

29 of these studies are limited by the small number of test animals, the lack of characterization of test

30 material, and the use of single doses.

31

32 Other Key Data

33 Substance 6 is a flammable liquid at room temperature. Limited information on its

34 toxicokinetics indicates that it can be absorbed by all routes of exposure. It is eliminated in the
7/02/99 C-15 DRAFT

1 urine mainly as glutathione conjugates. Substance 6 is metabolized in vitro by rat liver and lung

2 microsomal preparations to a dihydroxylated aldehyde.

3 No data were available on the genetic and related effects of substance 6 in humans. It did

4 not induce dominant lethal mutations in mice. It induced sister chromatid exchanges in rodent

5 cells in vitro. The mutagenicity of substance 6 is equivocal in bacteria. It did not induce DNA

6 damage or mutations in fungi.

7

8 Evaluation

9 Available human data are judged suggestive, but not sufficient for an evaluation of any

10 causal relationship between exposure to substance 6 and human cancer.

11 The carcinogenic potential of substance 6 has not been adequately studied in laboratory

12 animals due to serious deficiencies in study design, especially the inhalation and dermal studies. 

13 There is suggestive evidence of carcinogenicity in the drinking water study in female rats. 

14 However, the significance of that study to a potential for human response is uncertain since the

15 finding is limited to occurrence of benign tumors in one sex, and at the high dose only. Additional

16 suggestion for animal carcinogenicity comes from observation that a possible metabolite is

17 carcinogenic at the site of administration. This metabolite, however, has not been studied in vivo. 

18 Overall, the animal evidence is judged to be suggestive for human carcinogenicity.

19 Other key data, taken together, do not add significantly to the overall weight of evidence

20 of carcinogenicity. SAR analysis indicates that substance 6 would be DNA-reactive. However,

21 mutagenicity data are inconclusive. Limited in vivo data do not support a mutagenic effect. 

22 While there is some evidence of DNA damage in rodent cells in vitro, there is either equivocal or

23 no evidence of mutagenicity in nonmammalian systems.

24

25 Conclusion

26 While there is a suggestion of animal carcinogenicity, the data are inadequate for a

27 judgment about the human carcinogenicity potential of substance 6. Both human and animal data

28 are judged inadequate for an evaluation. There is evidence suggestive of potential carcinogenicity

29 on the basis of limited animal findings and SAR considerations. Data are not sufficient to judge

30 whether there is a mode of carcinogenic action. Additional studies are needed for a full evaluation

31 of the potential carcinogenicity of substance 6. Hence, dose-response assessment is not

32 appropriate.

33
7/02/99 C-16 DRAFT

1 Example 7: “Not Likely to be a Human Carcinogen”--Appropriately Studied Chemical in

2 Animals Without Tumor Effects

3 Human Data

4 Substance 7, a plant extract, has not been studied for its toxic or carcinogenic potential in

5 humans.

6

7 Animal Data

8 Substance 7 has been studied in four chronic studies in three rodent species. In a feeding

9 study in rats, males showed a nonsignificant increase in benign tumors of the parathyroid gland in

10 the high-dose group, where the incidence in concurrent controls greatly exceeded the historical

11 control range. Females demonstrated a significant increase in various subcutaneous tumors in the

12 low-dose group, but findings were not confirmed in the high-dose group, and there was no dose13 response relationship. These effects were considered as not adding to the evidence of

14 carcinogenicity. No tumor increases were noted in a second adequate feeding study in male and

15 female rats. In a mouse feeding study, no tumor increases were noted in dosed animals. There

16 was some question as to the adequacy of the dosing; however, it was noted that in the mouse 90-

17 day subchronic study that a dose of twice the high dose in the chronic study led to significant

18 decrements in body weight. In a hamster study there were no significant increases in tumors at

19 any site. No structural analogues of substance 7 have been tested for cancer.

20

21 Other Key Data

22 There are no structural alerts that would suggest that substance 7 is a DNA-reactive

23 compound. It is negative for gene mutations in bacteria and yeast, but positive in cultured mouse

24 cells. Tests for structural chromosome aberrations in cultured mammalian cells and in rats are

25 negative; however, the animals were not tested at sufficiently high doses. Substance 7 binds to

26 proteins of the cell division spindle; therefore, there is some likelihood for producing numerical

27 chromosome aberrations, an endpoint that is sometimes noted in cancers. In sum, there is limited

28 and conflicting information concerning the mutagenic potential of the agent.

29 The compound is absorbed via oral and inhalation exposure but only poorly via the skin.

30

31 Evaluation

32 The only indication of a carcinogenic effect comes from the finding of benign tumors in

33 male rats in a single study. There is no confirmation of a carcinogenic potential from dosed
7/02/99 C-17 DRAFT

1 females in that study, in males and females in a second rat study, or from mouse and hamster

2 studies.

3 There is no structural indication that substance 7 is DNA-reactive, there is inconsistent

4 evidence of gene mutations, and chromosome aberration testing is negative. The agent binds to

5 cell division spindle proteins and may have the capacity to induce numerical chromosome

6 anomalies. Further information on gene mutations and in vivo structural and numerical

7 chromosome aberrations may be warranted.

8

9 Conclusion

10 Substance 7 is not likely to be carcinogenic to humans via all relevant routes of exposure. 

11 This weight-of-evidence judgment is largely based on the absence of significant tumor increases in

12 chronic rodent studies. Adequate cancer studies in rats, mice, and hamsters fail to show any

13 carcinogenic effect; a second rat study showed an increase in benign tumors at a site in dosed

14 males, but not females.
7/02/99 D-1 DRAFT

APPENDIX D. CASE STUDY EXAMPLES FOR MODE-OF-ACTION EVALUATION

1 This appendix contains case examples to illustrate the application of the framework for

2 mode-of-action analysis. Evaluations of mode-of-action information will ordinarily appear

3 before or within the hazard characterization section of a risk assessment. Since these examples

4 are given outside of a risk assessment, the basic data that underlie the evaluation are

5 summarized first for reference, followed by the mode-of-action analysis.

6

7 D.1.0. EXAMPLE 1: CHEMICAL T (THYROID DISRUPTION)

8

9 D.1.1. HAZARD DATA SUMMARY

10 D.1.1.1. Data Availability

11 Data include a rat chronic/carcinogenicity feeding study, an 18-month CD-1 mouse

12 carcinogenicity study, a 1-year dog feeding study, a subchronic feeding study in the rat, a 4-week

13 and 1-year subchronic feeding study in the dog, a 21-day dermal study in the rat, developmental

14 toxicity studies in the rat and rabbit, a two-generation reproduction study in the rat, mutagenicity

15 studies, metabolism studies, and special subchronic mechanistic studies. 

16

17 D.1.1.1.1. Rat

18 D.1.1.1.1.1. 24-month toxicity. Male and female Sprague-Dawley rats received chemical T in the

19 diet for 24 months. Thyroid follicular cell tumor incidence was increased in male but not female

20 animals (see Table D-1). Tumor incidence in the two high-dose male groups was higher than in

21 historical control studies. Thyroid and liver weights were increased in the two high-dose groups. A

22 few renal tubular adenomas occurred in dosed male and female animals, but there was no statistical

23 significance. SGPT was increased in high-dose animals; some other liver enzymes were increased at

24 various times.

25
7/02/99 D-2 DRAFT

Table D-1. Thyroid follicular cell tumor incidence in male rats

Tumor Dose (ppm in diet)

0 1 10 100 1000 3000 a

 Benign 1/50 b 2/47 0/49 2/47 8/49 12/48 b

Malignant 1/50 b 1/47 0/49 0/47 1/49 4/48

Combined 2/50 b 3/47 0/49 2/47 9/49 14/48 b

 aTwo animals had both benign and malignant tumors. 

 bStatistically significant for trend noted at control; pairwise comparison noted at dose level.
7/02/99 D-3 DRAFT

1 D.1.1.1.1.2. Special subchronic studies. Groups of male Sprague-Dawley rats were fed

2 chemical T at 3000 ppm in the diet for 7, 14, 28, 56, or 90 days. Starting at 7 days, TSH levels

were significantly increased and T4 3 values were significantly decreased. There were also

4 significant increases in thyroid and liver weights and for follicular cell hypertrophy and

hyperplasia. Hepatic UDPGT activity for T4 5 was increased, while hepatic 5’-monodeiodinase

6 activity was either unaffected or decreased. Radioiodine uptake into the thyroid gland was

7 measured. The percent of the dose per gram of thyroid tissue was equivalent in 3000 ppm and

8 control groups, as was protein-bound iodide per mg of thyroid protein. Activities of hepatic aryl

9 hydrocarbon hydroxylase, ethoxycoumarin O-dehydrase, and cytochrome P-450 were significantly

10 increased in chemical T dosed animals.

11 Groups of male Sprague-Dawley rats were fed chemical T (30, 100, 300, 1000, 3000

12 ppm) for 56 days; some animals were taken off chemical T for another 56 or 112 days to evaluate

13 reversibility of effects. Thyroid weights were significantly increased in the top two doses, while

14 liver weights were increased in the top three doses. T4 UDPGT activity was increased in the top

15 two doses. T4 was decreased and TSH increased at the top dose, along with increases in the

16 incidence of follicular cell hypertrophy and hyperplasia. Upon stopping chemical T dosing, all

17 parameters returned to normal except for thyroid weight. Elimination of radioiodine-labeled T4

18 from the blood and into the bile was measured after 56 days of chemical T dosing. Blood

19 clearance was twice as fast in dosed animals as in controls, while there was a 40% increase in the

20 rate of excretion of the hormone into the bile of treated animals. 

21

22 D.1.1.1.2. Dog

23 D.1.1.1.2.1. Subchronic toxicity. Subchronic feeding of chemical T (0, 10, 100, 1000, 5000

24 ppm) produced an increase in thyroid weight and hyperplasia of the gland at 5000 ppm. There

25 was hepatocellular hypertrophy at 1000 ppm and above.

26

27 D.1.1.1.2.2. 12-month toxicity. One-year feeding of chemical T (1, 20, 200, 2000 ppm) led to

28 hepatocellular hypertrophy/hyperplasia at 200 and 2000 ppm but not at 0 or 20 ppm. At 2000

29 ppm, absolute and relative liver weights were increased. At 2000 ppm, there were increases in

30 SGOT, SGPT, GGT, and ALK, and decreases in cholesterol, albumin, and total protein.

31

32 D.1.1.1.3. Mouse

33 D.1.1.1.3.1. 18-month toxicity. In an 18-month chemical T feeding study (0, 1, 10, 100, 400,

34 800 ppm), there were no increases in tumor incidence at any site. Absolute and relative liver
7/02/99 D-4 DRAFT

1 weights were statistically significantly increased over controls at the highest dose level, as were

2 kidney weights in the female. Increases in liver enzymes were noted at various intervals, including

3 SGPT, SGOT, and ALK. Dose levels in the study were considered adequate.

4

5 D.1.1.2. Mutagenicity

6 Negative results were seen in four strains of Salmonella with or without metabolic

7 activation; negative results in assay of forward mutation of HGPT locus of Chinese hamster ovary

8 cells (dosing probably not sufficient); negative results in mouse bone marrow micronucleus assay;

9 negative results in assay for unscheduled DNA synthesis in rat hepatocytes pretreated with

10 chemical T. The compound does not have a structure that suggests electrophilicity.

11

12 D.1.2. SUMMARY DESCRIPTION OF POSTULATED MODE OF ACTION 

13 Thyroid hormone production is regulated by actions of the hypothalamus, pituitary, and

14 thyroid glands. Homeostasis of thyroid hormone is maintained by a feedback loop among the

15 hypothalamus and pituitary and the thyroid gland. The hypothalamus produces thyrotrophin

16 reducing hormone (TRH), which stimulates the pituitary to produce thyroid stimulating hormone

17 (TSH) which, in turn, stimulates the thyroid to produce thyroid hormone. The hypothalamus and

18 pituitary respond to a high level of circulating thyroid hormone by suppressing TRH and TSH

19 production, and to a low level by increasing them. The mode of action considered is continuous

20 elevation of TSH levels that stimulates the thyroid gland to deplete its stores of thyroid hormone

21 and continues to push production, resulting in hypertrophy of the production cells (follicular cells)

22 leading to hyperplasia, nodular hyperplasia and, eventually, tumors of these cells. In rats, the

23 chain of events may be induced by direct effects on hormone synthesis or by metabolic removal of

24 circulating hormone.

25

26 D.1.3. KEY EVENTS

27 The key events considered with respect to chemical T-induced tumorigenesis in male rats

include hormone changes in TSH, T4, and T3, and changes in hepatic T4 28 -UDPGT, indicators of

liver microsomal enzyme induction, enhanced liver metabolism, increased biliary excretion of T4 29 ,

30 increase in thyroid weight and liver weight, and thyroid follicular cell hypertrophy/hyperplasia. 

31 These events have been well defined and measured in male rats in subchronic studies, augmenting

32 observations at interim and terminal sacrifice in a chronic study.
7/02/99 D-5 DRAFT

1 D.1.4. STRENGTH, CONSISTENCY, SPECIFICITY OF ASSOCIATION OF TUMOR

2 RESPONSE WITH KEY EVENTS

3 The thyroid tumor response in the chronic study at the highest dose was associated with

4 hypertrophy/hyperplasia in the thyroid and increase in weight of the thyroid. In subchronic

5 studies, the organ weight and hypertrophy/hyperplasia were shown to appear and reverse in

6 statistically significant degrees under the same conditions of dose and time as the appearance and

7 reversal of changes in thyroid hormone levels and thyroid hormone metabolism. Stop/recovery

8 studies showed that cessation of dosing was followed in turn by return of hormone levels to

9 control levels, reduction in liver and thyroid weights, and reversal of hyperplasia in thyroid

10 follicular cells. The only sign slow to reverse was thyroid weight after the longest dosing period. 

11 Strength, consistency, and specificity of association were well established in the studies.

12

13 D.1.5. DOSE-RESPONSE RELATIONSHIP

14 Dose correlations exist for parameters in the chronic and subchronic studies for all of the

15 relevant parameters. Thyroid follicular cell tumors, thyroid hypertrophy/hyperplasia, and

16 increased thyroid and liver weight are noted at similar doses, usually at dietary levels of 1000 and

3000 ppm chemical T. Correspondingly in the subchronic study, at 3000 ppm T4 17 is depressed

while TSH is elevated. At 1000 and 3000 ppm, hepatic T4 18 -UDPGT activity is statistically

significantly elevated, and there is an increase in biliary excretion of T4 19 at 3000 ppm. The only

20 parameter showing significant effect at a dose below 100 ppm chemical T was liver weight

21 increase in a subchronic study at 300 ppm.

22

23 D.1.6. TEMPORAL ASSOCIATION

24 The chronic study, together with the three subchronic studies of key events observing

25 effects after different durations at one dose, at multiple doses, and after recovery, shows events

26 occurring in the following sequence: (1) increase in hepatic glucuronidation, de-iodination and

excretion of T4 27 , as well as its elimination from the blood; (2) a rise in circulating TSH; (3) an

28 increase in thyroid weight and thyroid follicular cell hypertrophy; (4) thyroid follicular cell

29 hyperplasia; and (5) thyroid follicular cell tumors. The stop experiments indicate reversal of the

30 thyroid and liver weight increases as well as reversal of hormone and other protein measures. 

31 While reversal of thyroid weight increase in the recovery study was less after a longer duration of

32 treatment, hypertrophy/hyperplasia did reverse after the longer duration. 

33

34 D.1.7. BIOLOGICAL PLAUSIBILITY AND COHERENCE OF THE DATABASE
7/02/99 D-6 DRAFT

1 Under EPA science policy (U.S. EPA, 1998a), determination of the antithyroid activity of

2 a chemical requires empirical demonstration of five items: (1) increases in thyroid growth, (2) 

3 changes in thyroid and pituitary hormones, (3) location of the site(s) of antithyroid action, (4) 

4 dose-response correlations among various key precursor events and tumor incidence, and (5) 

5 reversibility of effects following treatment cessation. The database on chemical T documents all

6 such information.

7 Thyroid tumorigenesis, particularly in the male rat, has been observed to be associated

8 with exposure to a number of industrial chemicals, pesticides, and pharmaceuticals. A significant

9 number of these appear to work in a manner similar to chemical T, by enhancing thyroid hormone

10 metabolism and excretion by the liver.

11 Thyroid tumors did not appear in the female rats in the 2-year study. Thyroid hypertrophy

12 and hyperplasia were observed in the females 6 months after their appearance in males. As is

13 noted with other chemicals, the female rat is less sensitive to the effect of antithyroid chemicals

14 regarding key events and tumor development. Hepatic enlargement and effects are noted in the

15 mouse and dog studies, as they are in the rat. In addition, dogs receiving high doses of chemical

16 T show enlargement of the thyroid gland.

17

18 D.1.8. OTHER MODES OF ACTION

19 Chemical T does not belong to a class of chemicals that is expected to generate reactive

20 metabolites, and no related chemicals have been tested for carcinogenicity. Short-term studies

21 demonstrate that the chemical does not increase gene mutations in Salmonella (Ames test) or

22 cultured mammalian cells (maximal dosage may not have been reached), micronuclei in bone

23 marrow cells, and unscheduled DNA synthesis in cultured cells. No other modes of action, apart

24 from thyroid disruption, are described to account for the thyroid tumors.

25 Several sites of action were investigated as being the source of the antithyroid effects of

26 chemical T. The chemical does not inhibit the entry of inorganic iodide into the thyroid (iodide

27 pump) or block the organification and incorporation of iodide into thyroid hormone (thyroid

peroxidase); likewise, it does not inhibit monodeiodinase, which blocks the conversion of T4 28 to

T3 29 .

30 Chemical T administration leads to renal adenomas in male and female rats; the response

31 lacked statistical significance. The mode of action for the thyroid tumors does not account for the

32 renal tumors. Assessment of the significance and mode of action of the renal tumors requires

33 separate analysis.

34
7/02/99 D-7 DRAFT

1 D.1.9. CONCLUSION

2 The weight of evidence supports a conclusion that chemical T acts by inducing hepatic

3 metabolism and biliary elimination of thyroid hormone, prompting increased production of TSH,

4 which ultimately results in thyroid follicular cell neoplasia as postulated.

5

6 D.1.10. RELEVANCE OF THE MODE OF ACTION TO HUMANS

7

8 Relevance to humans

9 Chemical T affects the liver of rats, mice, and dogs, and the thyroid of rats and dogs.

10 Given the breadth of responses, it is possible that humans may respond similarly. The subject of

11 the relevance of an antithyroid mode of action for thyroid tumors is extensively covered in the

12 Agency’s policy for the assessment of this mode of action (U.S. EPA, 1998a). In summary the

13 policy states: 

14

15 The role of thyroid-pituitary disruption in cancer development in humans is much

16 less convincing than in animals. Iodide deficiency is associated with increases in

17 thyroid cancer in some studies but not others. Similarly, an association between

18 either inborn errors of metabolism affecting thyroid hormone output or

19 autoimmune-related Graves’ disease and cancer is suggested but not proved. It

20 seems that TSH may at least play some permissive role in carcinogenesis in

21 humans. Accordingly, one cannot qualitatively reject the animal model; it seems

22 reasonable that it may serve as an indicator of a potential human thyroid cancer

23 hazard. However, to the extent that humans are susceptible to the tumor-inducing

24 effects of thyroid-pituitary disruption, and given that definitive human data are not

25 available, it would appear that quantitatively humans are less sensitive than rodents

26 in regard to developing cancer from perturbations in thyroid-pituitary status.

27

28 The measured key events and their effects, as well as effects of reversal of the events, are

29 consistent with what is known about the regulation of thyroid hormone balance, and the

30 postulated carcinogenic mode of action as summarized above.

31 Thyroid tumorigenesis, particularly in the male rat, has been observed to be associated

32 with exposure to a number of pesticides and pharmaceuticals. A pattern of thyroid organ growth,

33 frequently liver growth, thyroid hormone changes, or changes in hormone metabolism has been
7/02/99 D-8 DRAFT

1 seen with a large proportion of these compounds. Chemical T effects are parallel to these other

2 cases.

3 Thyroid tumors did not appear in the female rats in the 2-year study. Thyrotrophy and

4 hyperplasia were observed in the females with a 6-month lag after their appearance in the male. 

5 The female is apparently more tolerant of thyroid disruption; whether tumors would have been

6 seen in the females if the 2-year study had been extended is uncertain.

7 Relevance to subpopulations

8 Thyroid hormones are regulated within rather narrow ranges, with normal adult human

9 serum values often being given as T4--4 to 11 ug/dL and T3--80 to 180 ng/dL. TSH levels

10 extend over a broader range--0.4 to 8 ug/ml, due to the incorporation in recent years of more

11 sensitive laboratory methods that have extended the normal range to lower values (Ingbar &

12 Woeber, 1981; Surks et al., 1990). The upper bound on normal TSH has not changed, and it is

13 the one of import to considerations of antithyroid effects of chemicals. During development

14 somewhat higher levels for each of the hormones are noted, with adult hormone values being

15 reached beyond about 10 years of age (Nicholson and Pesce, 1992). Growth of the thyroid gland

16 continues for the first 15 years of life, going from about 1 gram at birth to an adult size of about

17 17 grams (Fisher and Klein, 1981; Larsen, 1982). Early developmental inability to synthesize

18 adequate thyroid hormone leads to altered physical and mental development (cretinism)

19 (DiGeorge, 1992; Goldey et al., 1995) and is treatable. The control of normal thyroid growth

20 during development is not totally known, although the increase in gland size may be independent

21 of TSH stimulation (Logothetopoulus, 1963). Extended deviations in human thyroid hormone

22 levels either above or below the normal range are associated with hyperthyroidism and

23 hypothyroidism, respectively and are treated in the U.S. to restore balance. 

24 Thyroid cancer is a rare condition in the U.S., occurring with an incidence of about

25 0.004% per year (Greenspan & Strewler, 1997). The incidence is predominantly in persons over

26 30, and increases in older persons; in children the incidence is at the 1 per million rate. Mortality

27 rates per 100,000 are above zero only for those older than 35 (Ries et al., 1999). 

28 It is recognized that the human thyroid is susceptible to ionizing radiation, the only

29 verified human thyroid carcinogen. Children are known to be more sensitive than adults to the

30 carcinogenic effects of radiation (NRC, 1990; IAEA, 1996). The nature and consequences of

31 radiation have differences from thyroid disruption by inborn deficits or possible chemical influence

32 that is not mutagenic. The major effect of ionizing radiation on the thyroid is thought to be due to

33 mutation. Antithyroid effects can also be induced at elevated radiation doses due to cytotoxicity

34 of follicular cells with resulting reduction in thyroid hormone and elevation of TSH. Mutagenic
7/02/99 D-9 DRAFT

1 chemicals, however, do not act totally like radiation: (a) X rays penetrate the body and target

2 organs without having to be absorbed. Chemicals must be absorbed and distributed to target

3 organs. (b) Unlike most organic chemicals, radioiodine is actively transported and concentrated in

4 the thyroid gland, and it becomes incorporated into nascent thyroglobulin. (c) Given that the size

5 of the thyroid gland is smaller in children than in adults, for a given blood level of radioiodine, the

6 internal dose to the thyroid of a child is greater than that for an adult. (d) Radioiodine in the

7 Chernobyl accident was picked up by cattle and incorporated into milk. Due to differences in

8 milk consumption, the external dose presented to children was greater than to adults.(e) Single

9 quanta of radiation result in a series of ionizations within biological material, each of which can

10 react with DNA to induce mutations and affect the carcinogenic process. Chemicals are much less

11 efficient: they frequently need to be metabolized to active intermediates, with each molecule

12 interacting singly with DNA, usually by forming adducts which can be converted to mutations. (f)

13 The spectrum of mutagenic effects vary with the source. Ionizing radiation often results in

14 deletions and other structural chromosomal aberrations, while chemicals not uncommonly

15 produce more gene mutations. (g) The thyroid of children is more sensitive to carcinogenic effects

16 of external radiation on a per unit dose basis than in adults, especially for children less than 5

17 years of age. Sensitivity decreases with advancing age and seems to disappear in adulthood. It is

18 estimated that, overall, children may be two or more times more sensitive to carcinogenic effects

19 of external emitters than are adults (NRC, 1990).

20 The evidence supports the view that Chemical T’s mode of action will not be different for

21 children. Thyroid cancer is very rare in younger age groups and lower in incidence and mortality

22 than for older adults. It does not appear that the young have any propensity for thyroid cancer

23 from which one could infer some underlying cancer process that differs from adults (absent

24 ionizing radiation treatment or incidents, discussed above). The basic elements of thyroid

25 function and hormone homeostasis are the same in children and adults with a period of growth

26 during which children reach lower adult balances. The chemical disruption mode of action of

27 Chemical T in animals, to the extent that it is applicable to humans, appears equally applicable to

28 human subpopulations. It is not expected to share the features of radiation.

29

30
7/02/99 D-10 DRAFT

1

2 D.2.0. EXAMPLE 2: CHEMICAL Z (BLADDER TUMOR)

3

4 D.2.1. HAZARD DATA SUMMARY

5 D.2.1.1. Data Availability

6 Data include a rat chronic/carcinogenicity feeding study, an 18 month CD-1 mouse

7 carcinogenicity study, a three-generation reproduction study in the rat, and a 2-year feeding study

8 in dogs. There are no data on the effects in humans of exposure to chemical Z. 

9 A 13-week feeding study in rats included interim sacrifices at 2, 4, and 8 weeks and

10 establishment of 16-week recovery groups at 8 weeks and a 21-week recovery group at 13

11 weeks. 

12 D.2.1.2. Tumor Observations

13 D.2.1.2.1. Tumor Response

14 D.2.1.2.1.1. Rats. Administration of chemical Z in the diet to male Sprague-Dawley rats at dose

15 levels of 30,000 ppm or more for 2 years resulted in an increase in bladder urothelial tumors in

16 male rats. Statistically significant increases (p<0.05) were noted at the high dose only

17 (40,000/30,000 ppm) in the incidences of transitional cell papillomas, carcinomas, combined

18 papillomas and carcinomas, and hyperplasia in the 2-year SD rat bioassay (Table D-2). Bladder

19 calculi were observed in some animals but correlation between stones and tumors was not evident

20 at final sacrifice.
7/02/99 D-11 DRAFT

 Table D-2. Incidence of transitional cell lesions and stones in the bladder 

 of males from a 2-year SD rat study

Parameter Dose (ppm)

0 2000 8000 40,000/30,000

N 73 75 78 78

Lesion

 Papilloma 1 1 1 5

 Carcinoma 2 2 1 16

Combined 3 3 2 21

 Hyperplasia 5 7 5 29

Stones 0 0 0 5
7/02/99 D-12 DRAFT

1 D.2.1.2.1.2. Mice. No increase in tumor incidences was observed in an 18-month bioassay with

2 mice.

3

4 D.2.1.3. Mutagenicity

5 Chemical Z has not shown mutagenic activity based on results of Salmonella sp. or

6 micronucleus assays. No evidence exists that the chemical produces effects on DNA synthesis nor

7 does it appear to be clastogenic. There are no structural alerts suggesting mutagenic potential for

8 the chemical.

9

10 D.2.1.4. Toxicity, Uroliths, and Hyperplasia

11 There was a strong association among disruptions in urinary physiology, toxicity, uroliths,

12 and hyperplasia in the 13-week study in mid-dose and high-dose animals (30,000 and 50,000 ppm

13 respectively, [p<.05]). In the control and 8,000 ppm group, no animals had stones and no animals

14 had hyperplasia (see Table D-3).

15
7/02/99 D-13 DRAFT

 Table D-3. Incidence of bladder hyperplasia and stones in male SD rats 

 treated up to 13 weeks 

Parameter 2 weeks 8 weeks 13 weeks

Dosea 123412341234

N 10 10 10 10 10 10 10 9 10 10 10 6

Papillary

hyperplasia

007800970056

Simple 

hyperplasia

0020

Stones 0 0 3 4 0 0 9 8 0 0 7 6

a

Dose (ppm): 1 = control, 2 = 8000, 3 = 30,000, 4 = 50,000.
7/02/99 D-14 DRAFT

1 D.2.1.4.1. Thirteen-Week Study

2 Urothelial toxicity and disruptions in urinary physiology and urothelial toxicity appeared

3 early in the study. Early changes in urinary physiology (decreased pH and increased cation

4 concentration) were observed following 2 weeks of treatment and persisted throughout the

5 duration of the study. Urothelial toxicity was expressed as edema, cystitis, and hyperplasia; 

6 hyperplasia (simple and papillary transitional cell combined) increased in overall incidence with

7 continued treatment. It was present in 70% of mid-dose (30,000 ppm) animals and 80% of high8 dose (50,000 ppm) animals following 2 weeks of exposure, and in 70% of the mid-dose group

9 and 100% of the high-dose group at 13 weeks. There was some indication of a decrease in

10 severity of hyperplasia at 13 weeks when compared to earlier time periods, as there was an

11 apparent shift from the incidence of papillary hyperplasia to simple hyperplasia and a decrease in

12 the combined incidence of hyperplasia in the 30,000 ppm group of animals.

13 Uroliths were found to be present as early as 2 weeks (0%, 0%, 30%, and 40%) and the

14 incidence increased over the period of the study. The incidence of uroliths at termination of the

15 13-week study was 0%, 0%, 70%, and 100%, but there was a decrease in size and number of

16 stones per animal at 13 weeks.

17

18 D.2.1.4.2. Three-Generation Reproduction Study in Rats 

19 High dose levels (>20,000 ppm in the diet) led to formation of lesions in the urinary tract of

20 males and females of the F1, F2, and F3 generations. The lesions included hemorrhage of the

21 bladder wall, increased pelvic dilation, and papillary necrosis. In the F3 generation, additional

22 effects noted in renal tissue were hyperplasia of the transitional epithelium and desquamation of

23 cells in the lumen of the urinary tract. The changes were associated with crystalline or calcareous

24 deposits.

25

26 D.2.1.5. Reversibility of Effects

27 There was strong evidence of reversibility of bladder stones and bladder hyperplasia. When

28 animals that had been treated for 8 weeks were returned to basal diet for 16 weeks, uroliths were

29 found in 30% of 30,000 ppm animals and 25% of high-dose animals. Bladder hyperplasia

30 (papillary and transitional cell combined) was reduced to 25% and 30% in each of these two dose

31 groups (Table D-4). An analysis of individual animal data revealed a strong correlation between

32 the incidence of uroliths and hyperplasia at the termination of the recovery period.

33
7/02/99 D-15 DRAFT

 Table D-4. Reversal of incidence of bladder hyperplasia 

 and stones following 8 weeks treatment and 16 weeks recovery

Parameter Dose (ppm)

0 8000 30,000 50,000

N 10 10 10 8

Papillary

hyperplasia

00 2 1

Simple

hyperplasia

00 1 1

Stones 0 0 3 2
7/02/99 D-16 DRAFT

1 D.2.1.6. Blood and Urine Chemistry

2 Chemical Z administration resulted in increases in blood phosphorus and carbon dioxide

3 (data not shown). Urinalyses (Table D-5) showed elevated calcium levels, reduced urinary

4 phosphorus, and a profound lowering of urinary pH (5.0), which began at 2 weeks and persisted

5 throughout the 13-week study in the 30,000 and 50,000 ppm group of rats. These changes

6 occurred in the presence of bladder stones, which were reported to consist of 33% calcium and

7 23% phosphorus. 

8
7/02/99 D-17 DRAFT

Table D-5. Clinical chemistry values (urine) in male SD rats treated up to 13 weeks 

 

Parameter 2 weeks 8 weeks 13 weeks

Dose 1 2 3 4 1 2 3 4 1 2 3 4

N 10 10 10 10 10 10 10 9 10 10 10 6

Calcium -

mg/dL

6 11 56b 36c 11 11 18 65b 5 7 14b 58b

Phosphorus

- mg/dL

90 62 2b 13c 109 90 19 1b 57 67 26 1b

pH 7 6.5 5b 5b 7.4 6.9 5.8b 5.0b 7.

2

6.

7

6.0b 5.0b

Stones 0 0 3 4 0 0 9 8 0 0 7 6

a

Dose (ppm): 1 = control, 2 = 8000, 3 = 30,000, 4 = 50,000.

b

p<.01. c

p<.05
7/02/99 D-18 DRAFT

1 D.2.1.7. Metabolism

2 Upon ingestion by rats, the ethyl moiety of chemical Z is rapidly absorbed, hydrolyzed to a

3 phosphite, and oxidized via acetaldehyde and acetate to carbon dioxide and water. Absorption of

4 the phosphite moiety leads to increased blood phosphorus levels. There is also an increase in

5 blood calcium load, which leads to increased excretion of calcium via the urine. Ethyl phosphite

6 moieties and carbon dioxide are also eliminated via the urine. A marked depression of urinary pH

7 (5.0) results from acidification of the urine by carbon dioxide. An aluminum moiety of the parent

8 chemical is poorly absorbed, and most is eliminated in the feces. The phosphite metabolite, the

9 major urinary metabolite, was not shown to express carcinogenic potential when administered to

10 Sprague-Dawley rats at dose levels up to 32,000 ppm. It also does not express any mutagenic

11 potential and does not have any structural alerts.

12

13 D.2.1.8. Structure-Activity Relationships

14 There are no data on structurally related chemicals.

15

16 D.2.2. MODE-OF-ACTION ANALYSIS 

17 D.2.2.1. Summary Description of Postulated Mode of Action

18 Chemical Z produces transitional cell tumors in male Sprague-Dawley rats. The mode of

19 action includes disruption in urinary physiology, including precipitation of calcium and

20 phosphorus and formation of bladder calculi. The stones irritate the urothelium of the bladder,

21 followed by transitional cell hyperplasia and bladder tumor formation. Disruption of urinary

22 physiology is a consequence of a metabolic sequence involving (1) absorption and metabolism of

23 the ethyl moiety to carbon dioxide, resulting in a reduction in urinary pH; and (2) absorbtion of

24 the phosphite moiety, which leads to increased blood phosphorus levels and increased release of

25 calcium into the urine. Increases in water consumption followed by increased urinary volume may

26 contribute to bladder toxicity, but a precise role of increased urinary volume has not been

27 established.

28 The mode of action for chemical Z is consistent with other data that demonstrate that solid

29 masses in the rodent bladder, regardless of their origin--insertion of solid materials, including inert

30 pellets, precipitation of administered chemicals (e.g., melamine) or disruption of urinary

31 physiology (e.g., diethylene glycol)--lead to urothelial toxicity and the formation of tumors.

32

33 D.2.2.2. Key Events
7/02/99 D-19 DRAFT

1 The key precursor events associated with bladder tumor formation following administration

2 of chemical Z to rats include increased blood phosphorus and carbon dioxide, elevated urinary

3 calcium and volume, decreased urinary pH and phosphorus, formation of bladder stones, and

4 irritation and hyperplasia of the urothelium.

5

6 D.2.2.3. Strength, Consistency, and Specificity of Association of Tumor Response with Key

7 Events

8 The only tumor response seen in animal studies is bladder tumors in male Sprague-Dawley

9 rats. Studies in dogs and mice showed no effect on the bladder. The rat tumor response was seen

10 only at high doses that lead to key precursor effects: altered urinary physiology (volume, calcium,

11 pH) results in stones and produces toxicity and hyperplasia of the urothelium. The high-dose

12 changes were noted in a rat chronic, a rat subchronic, and a three-generation reproduction study

13 in rats. The key events, including hyperplasia, were observed to be reversible in subchronic

14 stop/recovery studies. Administration of the major metabolite of chemical Z, monosodium

15 phosphite, fails to reduce urinary pH, increase urinary volume, or produce nonneoplastic or

16 neoplastic lesions of the bladder. The database on chemical Z is sufficient to evaluate the

17 proposed mode of action despite the absence of more complete information on the composition of

18 the stones and questions regarding the absence of toxicity following the administration of

19 monosodium phosphite. There is a high degree of confidence that the findings accurately reflect

20 the effects associated with administration of the chemical. No data gaps were identified that

21 would substantially alter the evaluation of the proposed mode of action.

22

23 D.2.2.4. Dose-Response (D/R) Relationships

24 The 2-year bioassay showed urothelial hyperplasia, transitional cell papillomas, and

25 transitional cell carcinomas and a few bladder stones at 40,000/30,000 ppm. Of 78 high-dose

26 animals, 37% showed bladder tumors. Tumors, hyperplasia, and stones were not increased at

27 8000 ppm. A special 13-week feeding study demonstrated that key events--increased urinary

28 calcium levels, decreased urinary phosphorus levels, decreased pH, bladder stones, irritation,

29 edema, and hyperplasia--occurred consistently only at dose levels of 30,000 ppm or greater. A

30 strong dose-response correlation was shown between calculus formation and hypercalciuria,

31 acidic urine, and bladder hyperplasia. In a rat reproduction study, bladder effects were noted at

32 24,000 ppm but not at 12,000 ppm.

33

34 D.2.2.5. Temporal Association
7/02/99 D-20 DRAFT

1 A subchronic rat study with serial sacrifices at 2, 4, 8, and 13 weeks, including evaluation

2 of 16-week recovery groups after 8 weeks and a 21-week recovery group after 13 weeks, was

3 performed. By 2 weeks of administration, chemical Z produced stones that filled the bladder and

4 resulted in advanced papillary hyperplasia. The number and size of stones was greatest at two

5 weeks and there was a progressive decrease over the 13 week period. Early changes in urinary

6 physiology (decreased urinary pH, increased calcium concentration, and decreased phosphorus

7 concentration) were observed following 2 weeks of treatment and persisted throughout the

8 duration of the study. Observation of the 8-week treatment/16-week recovery groups showed

9 that incidence of both stones and hyperplasia significantly decreased as compared with incidence

10 in animals sacrificed at 8 weeks. Also, upon cessation of dosing at 13 weeks, the incidence of

11 animals with stones, the incidence of papillary hyperplasia, and the severity of hyperplasia

12 decreased significantly by the end of a 21-week recovery period (data not shown). The changes

13 noted within 2 weeks of dosing appear to have set in motion a series of events beginning with

14 increased urinary calcium concentrations, followed or accompanied by stone formation, irritation

15 of the bladder urothelium, hyperplasia and, eventually, neoplasia.

16

17 D.2.2.6. Biological Plausibility and Coherence of the Database

18 Long-term and subchronic studies with chemical Z have demonstrated a dose correlation

19 between development of stones and bladder tumor formation in male rats. Data from the 13-

20 week study indicate a rapid onset of effects (changes in urinary parameters, formation of stones,

21 and hyperplasia within 2 weeks of dosing) and adaptation of treated animals to chemical Z

22 exposure by 13 weeks (decreased numbers and size of stones per animal, decreased severity of

23 hyperplasia). Tumors were observed only at doses at which key events were observed. 

24 Additional bioassay data provide support for the association of tumors in rats with the key

25 events in rats and the absence of both tumors and similar key events in other species treated with

26 chemical Z. Treatment of rats in a three-generation reproduction study at high dose levels

27 (>20,000 ppm in the diet) led to formation of lesions in the urinary tract of males and females.

28 When administered to dogs at dose levels up to 40,000 ppm in the diet for up to 2 years, the

29 chemical produced minimal toxic effects overall, no effects on the urinary tract, and no tumors. 

30 Chemical Z produced no effects in mice when administered up to a dose level of 20,000/30,000

31 ppm in the diet for 2 years.

32 Observations with chemical Z are in keeping with those observed in many other

33 experimental settings. Stones, regardless of their chemical makeup, are irritating to the rodent

34 bladder, causing irritation, hyperplasia, and eventually neoplasia.
7/02/99 D-21 DRAFT

1 There are some uncertainties regarding the role of certain findings following chemical Z

2 administration. Generally, an increase in urinary pH is associated with the precipitation of calcium

3 and phosphorus-containing stones in rats. However, stones are formed in the presence of a low

4 urinary pH in rats administered chemical Z. It is also unclear whether or not the acidic

5 environment of the urine (most likely a consequence of the conversion of the ethyl moiety to

6 carbon dioxide in the blood) contributes to or enhances any effects noted in bladder tissue in rats.

7 There was a paucity of stones in high-dose animals at termination of the 2-year study but a higher

8 incidence of bladder tumors, which suggests that bladder stones may not be the causative factor

9 involved in bladder tumor formation. Other considerations discount this presumption. First, a

10 number of the high-dose animals showed hydronephrosis or dilation of the ureters, presumptive

11 indications of past urinary tract obstruction. Second, the 13-week study provided evidence that

12 bladder calculi develop rapidly (within 2 weeks), but then decreased in frequency and size. The

13 decrease in size and number of bladder calculi was accompanied by a decrease in severity of

14 bladder hyperplasia in animals treated with 30,000 ppm of chemical Z. Third, it is recognized that

15 a constant ppm of an agent in the diet results in a reduction in dose per unit body weight as an

16 animal grows. Finally, the increased urinary volume or decreased urinary pH may have led to a

17 dissolution of stones over time.

18 The absence of bladder stones and urothelial toxicity following administration of the major

19 metabolite, monosodium phosphite, is puzzling, as one might expect administration to rats would

20 lead to similar bladder effects as with chemical Z. However, the metabolite when administered to

21 rats, leads to an increase in blood levels of phosphorus but does not alter urinary volume or pH as

22 would be expected with an increase in sodium consumption. Considering the high dose-level of

23 metabolite administered to rats (32,000 ppm), it is unlikely an additional bioassay using higher

24 dose-levels would provide useful information.

25

26 D.2.2.7. Other Modes of Action

27 Chemical Z is not mutagenic in short-term tests and it does not have a structure

28 suggesting biological reactivity. No other modes of action, apart from that postulated, are in

29 evidence. The fact that bladder tumors were the sole tumors seen in rats and that no other species

30 showed tumors or other toxicities like those in the rat make it less likely that the agent has another

31 generalized mode of action.

32

33 D.2.2.8. Conclusion
7/02/99 D-22 DRAFT

1 The available bioassay data on chemical Z are sufficient to support the postulated mode of

2 action that the chemical, which lacks mutagenic potential, leads to bladder tumor formation in

3 male rats through a sequence of key events involving perturbations in urinary physiology,

4 especially increased calcium concentration, calculus formation, urothelial irritation, hyperplasia,

5 and neoplasia.

6

7 D.2.3. RELEVANCE OF THE MODE OF ACTION TO HUMANS

8

9 Bacterial infection, urinary stones or a combination of the two may be risk factors for

10 human urinary tract cancer (Burin et al., 1995; Davis et al., 1984; Gonzalez et al., 1991; Kawai et

11 al., 1994; Hiatt et al., 1982). Infection of the bladder with Schistosoma haematobium leads to

12 bladder tumors, and part of its action may be associated with stone formation (IARC, 1995). A

13 significant relationship has also been shown between spinal cord injury and bladder cancer;

14 chronic infection and stones are found in individuals so affected (Bickel et al., 1991; Broecker et

15 al., 1981; Dolin et al., 1994; El-Marsi and Fellows, 1981; Stonehill et al., 1996). Case control

16 epidemiologic studies (relative risks less than three) suggest associations between bladder cancer

17 and urinary tract stones (Burin et al., 1995; Gonzalez et al. 1991). A large cohort study supports

18 the association shown between bladder stones and bladder cancer (Chow et al., 1991). Taken as a

19 whole, stones may play some role (particularly, along with infection) in bladder cancer formation. 

20 Bladder cancer is a disease of advancing age, with about 2/3 of all cases occurring among persons

21 aged 65 years or older (Hankey et al., 1993). 

22

23 Stones occur much more frequently in the upper urinary tract than in the bladder of

24 humans (about 10% of urinary stones are found in the bladder), presumably because the upright

25 posture of humans predisposes them to expelling stones through the urethra once a stone passes

26 from the kidney to the bladder (Hiatt et al., 1982; Johnson et al. 1979; DeSesso, 1995). This

27 characteristic, as well as the pain which accompanies such stones and leads to their surgical

28 removal. Stones in the rodent bladder tend to be retained, because of their horizontal position. 

29 These findings suggest suggest that there may be a lower susceptibility of humans compared to

30 rodents to the development of urinary tract tumors associated with stones.

31

32 Precipitation of chemicals in the urinary tract with the formation of stones is a common

33 finding, with about 12% of males and 5% of females having a history over a lifetime of at least

34 one stone (Johnson et al., 1979). Compared to adults, urinary stone formation in children is an
7/02/99 D-23 DRAFT

1 uncommon occurrence except in individuals with a predisposing condition, such as, various inborn

2 errors of metabolism (e.g., cystinuria) and congenital malformations (Gearhart et al., 1991). The

3 prevalence of urinary stones in children is about 1 case per 20,000 per year (0.005%) (Khoory et

4 al., 1998). Only about 5% of stones are initially manifest during the first 20 years of life (Johnson

5 et al., 1979). Causes of urinary stones in children are remarkably similar to those of adults

6 (Khoory et al., 1998; Stapleton, 1996). Like with adults, the urine of children varies in pH and

7 osmolality, particularly in response to diet and physiologic stressors (e.g., exercise, heat). Urinary

8 excretion of chemicals occurs throughout life, although there may be quantitative differences

9 associated with a number of factors including disease states and nutritional status. Stones used to

10 be more common in children in developed countries than they are now, largely due to

11 malnutrition, which is still a problem in developing nations today (Trinchieri, 1996). 

12

13 Chemical Z is converted to metabolic derivatives through simple hydrolysis, a chemical

14 conversion that does not depend on enzymatic activity. It is not plausible that differences in levels

15 of enzymatic activity, such as detoxification via hepatic metabolism or metabolism in other tissues

16 will alter, qualitatively, responses in population subgroups such as the aged, the infirm, or infants

17 and children who may be exposed to Chemical Z. 

18

19 In summary, the potential human carcinogenic hazard of the chemical cannot be dismissed

20 for Chemical Z. Chemical Z poses a carcinogenic hazard to humans only under conditions that

21 would lead to the formation of bladder stones. It is reasonable to conclude that the mode of

22 action involving stone formation for Chemical Z that has been developed for adult animals may be

23 applicable to young animals and to children. Information suggests that effects in the young may

24 not be any greater than in adults and, in fact, the young may be less susceptible unless there are

25 rare extenuating factors. 

26

27

28

29 3.0. EXAMPLE 3: CHEMICAL D

30

31 D.3.1. HAZARD DATA SUMMARY 

32 D.3.1.1. Data Availability

33 Human data are inadequate to establish a basis for carcinogenicity. Experimental data

34 include:
7/02/99 D-24 DRAFT

1 C Three chronic toxicity and carcinogenicity studies in rats and mice: an inhalation study,

2 an oral dietary study, and an oral gavage study; 

3 C Subchronic studies by the oral and inhalation routes in rats and mice; 

4 C Inhalation developmental toxicity studies in rats and rabbits; 

5 C An inhalation two-generation reproductive toxicity study in the rat; 

6 C In vitro and in vivo genotoxicity studies; 

7 C Toxicokinetic and metabolism studies; and

8 C Protein binding studies.

9

10 D.3.1.2. Carcinogenicity/Chronic Toxicity 

11 Chemical D has been shown to cause increased tumor incidences in rats and mice. The

12 tumor responses seem to be dependent on the tested animal species, sex, dose, and route of

13 administration. Results of available chronic bioassays are summarized in Table D-6. 

14
7/02/99 D-25 DRAFT

Table D-6. Summary results of chronic bioassays

Study/dose F344 rats B6C3F1 mice

Oral gavage

Rat study: 0, 25, 50 mg/kg 

(5 d/wk for 2 yr)

Mouse study: 0, 50, 100

mg/kg (5 d/wk for 2 yr)

Forestomach: 

Papillomas (males: 1/50,

2/50, 8/50; females: 0/50,

2/50, 3/50)

Carcinomas (males only0/50, 0,50, 4/50)

Basal cell and epithelial

hyperplasia (dose-related;

males and females)

Liver:

Adenomas (males:1/50, 6/50,

7/50)

Carcinomas (males: 0/50,

1/50, 3/50)

Forestomach: 

Papillomas (males: 0/50,

1/50, 5/49; females: 0/50,

2/50, 7/50) 

Carcinomas (females only:

0/50,1/50, 4/50)

Basal cell and epithelial

hyperplasia (dose-related;

males and females) 

Lung:

Adenomas (males: 2/50, 4/50,

8/49; females: 2/50, 4/50,

7/50)

Carcinomas (males only:

0/52, 2/52, 4/49) 

Oral dietary

Rat study: 0, 2.5, 12.5, 25

mg/kg/day for 2 yr

Mouse study: 0, 2.5. 25, 50

mg/kg/day for 2 yr

Forestomach:

Basal cell and epithelial

hyperplasia (dose-related;

males and females)

Liver:

Adenomas (significant in

males only: 2/50, 1/50, 6/50,

9/50)

No histopathologic changes 

Inhalation

Rat study: 0, 5, 20, 60 ppm (6

hr/d 5 d/wk for 2yr)

Mouse study: 0, 5, 20, 60

ppm (6 hr/d 5 d/wk for 2yr)

Nasal cavity:

Epithelial hyperplasia (doserelated; males and females)

Nasal cavity:

Epithelial hyperplasia (doserelated; males and females)

Lung:

Adenomas (males only: 2/50,

3/50, 6/50)
7/02/99 D-26 DRAFT

1 C In rats, chemical D caused dose-related increases in liver tumors (males only) and

2 forestomach tumors (both sexes) via oral gavage, but only liver tumors (males high

3 dose only) by ingestion. No tumors were found in an inhalation study.

4 C In mice, chemical D caused dose-related increases in forestomach and lung tumors

5 (both sexes) by oral gavage, but no tumors were observed in the oral dietary study.

6 Chemical D only induced an increased incidence of lung tumors in male mice exposed

7 to the high dose by inhalation.

8 C Nonneoplastic changes were observed in the forestomach of treated rats (gavage and

9 dietary studies) and mice (gavage only) of both sexes. Chemical D also induced

10 nonneoplastic changes in the nasal mucosa of rats and mice of both sexes via

11 inhalation.

12

13 D.3.1.3. Subchronic Toxicity

14 Subchronic toxicity studies have been conducted in rats and mice by the oral and

15 inhalation routes. The primary organs affected were the forestomach (rats) and the liver (mice) via

16 oral exposure, and the nasal cavity and respiratory tract of both rodent species via inhalation. 

17

18 D.3.1.3.1. Oral Studies

19 Groups of F344 rats (10 animals of each sex per dose group) were administered 0, 5, 15,

20 50, or 100 mg/kg/day of chemical D via their diets for 13 weeks. Dose-related decreases in body

21 weight gain were observed in treated males and females. Basal cell hyperplasia and hyperkeratosis

22 of the forestomach was found in males and females rats treated with chemical D at the three

23 highest doses. 

24 B6C3F1 mice (10 animals of each sex per dose group) were administered 0, 25, 50, 100,

25 or 175 mg/kg/day via their diets for 13 weeks. Body weight gains of treated males and females

26 were depressed in a dose-related manner compared to controls. Histologic changes were noted in

27 the liver and were characterized as decreased hepatocyte size in all treatment groups. This

28 observation was consistent with decreased hepatocellular cytoplasmic glycogen.

29

30 D.3.1.3.2. Inhalation Studies

31 F344 rats (10 animals of each sex per dose group) were exposed to 0, 10, 30, 90, or 150

32 ppm of chemical D for 6 hr/day, 5 days/week for 13 weeks. Treatment-related effects included

33 depressed body weight gain (at 30 ppm and greater), degenerative changes in nasal olfactory
7/02/99 D-27 DRAFT

1 epithelium, and hyperplasia of respiratory epithelium in both males and females (at 90 and 150

2 ppm). 

3 B6C3F1 mice (10 animals of each sex per dose group) were exposed to 0, 10, 30, 90, or

4 150 ppm of chemical D for 6 hr/day, 5 days/week for 13 weeks. Treatment-related effects

5 included depressed body weight gain (at 30 ppm and greater), and histopathologic changes in the

6 respiratory and olfactory epithelium of the nasal mucosa of both sexes exposed to 30, 90, and 150

7 ppm). 

8

9 D.3.1.4. Developmental and Reproductive Toxicity

10 Pregnant F344 rats and New Zealand White rabbits were exposed to 0, 20, 60, or 120

11 ppm of chemical D during gestation days 6-15 (rats) and 6-18 (rabbits). Maternal effects

12 (decrease body weight gain) were observed in rabbits and rats, in all treatment groups. A slight

13 but statistically significant increase in the incidence of delayed ossification of the vertebral centra

14 was observed in rats exposed to the high dose level. No developmental effects were observed in

15 the rabbit study.

16 Exposure of F344 rats to 0, 10, 30, or 90 ppm of chemical D for up to two generations

17 did not induce any effects on reproductive parameters or neonatal growth and survival in any of

18 the generations. Parental effects were limited to epithelial degeneration of the nasal mucosa of the

19 adults rats exposed to 90 ppm.

20

21 D.3.1.5. Mutagenicity

22 Chemical D was tested in many assays for gene mutation and chromosomal aberrations, as

23 well as assays indicative of DNA damage, DNA strand breaks, and DNA alkylation. A

24 heterogeneous database is found (a few in vitro positive responses and several negative results). 

25 It has been suggested that this heterogeneity is due to different studies that have used different

26 test materials containing varying levels of impurities. 

27 A few studies demonstrated that chemical D was weakly positive in the Ames bacterial

28 assays in the presence of liver microsomes. Addition of cytosolic enzymes, presumably containing

29 the detoxification enzyme glutathione transferase (GST), abolished mutagenic activity. Studies

30 for chromosomal aberrations in vitro assays using mammalian cells have tended to be negative. 

31 There are a few positive results reported, but these are inconsistent with negative studies

32 conducted in the same assay.

33 There are very few in vivo genotoxicity studies on chemical D. Chemical D has been

34 found to be negative in a mouse micronucleus assay when tested up to oral doses of 175 mg/kg. 
7/02/99 D-28 DRAFT

1 Chemical D has been reported to produce sister chromatid exchanges (SCEs) in mice. It should

2 be noted that this assay has a low specificity for predicting carcinogenesis (i.e., a high rate of false

3 positives compared to results of the rodent cancer bioassay). No dominant lethal effects (i.e.,

4 germ cell genetic damage) were found in rats exposed to chemical D by inhalation up to 150 ppm. 

5

6 In vivo DNA binding studies were conducted in rats and mice. Rats were exposed acutely

7 to chemical D at doses of 0, 10, 25, or 100 mg/kg. Mice were exposed acutely by inhalation to

chemical D at 0, 30, and 60 ppm. No significant DNA binding (as measured by 32 8 P postlabeling

9 assay) was seen in liver tissue from treated rats and lung tissue from exposed mice. In the mouse,

10 DNA strand breakage was also studied by alkaline elution. Negative results were reported.

11

12 D.3.1.6. Toxicokinetic and Metabolism Studies

13 Toxicokinetic and metabolism studies in rats and mice have demonstrated that chemical D

14 was rapidly absorbed by the oral and inhalation route. Blood half-lives were less than 10 minutes. 

15 Mercapturic acid conjugate of chemical D was the only major metabolite detected in the urine of

16 treated rats and mice (about 80-90% of administered dose). Conjugated metabolites of chemical

17 D epoxide were not detected in the urine of treated rats and mice. 

18 Significant dose-related decreases in hepatic and lung tissues of GSH were observed in

19 rats treated acutely with chemical D at oral doses of 0, 5, 20, 50, or 100 mg/kg, and in mice

20 exposed acutely by inhalation to 0, 30, 60, or 150 ppm, respectively. 

21

22 D.3.1.7. Protein Binding Studies

23 Chemical D was found to bind with tissue proteins in the forestomach and liver of rats

24 treated acutely with oral doses 10, 50, and 100 mg/kg. Chemical D binding to tissue proteins was

25 also found in the lung of mice exposed via acute inhalation at 30, 60, or 100 ppm.

26

27 D.3.2. MODE-OF-ACTION ANALYSIS

28 D.3.2.1. Summary Description of Postulated Mode of Action 

29 It is postulated that chemical D causes tumors in rats and mice only when it is

30 administered at high doses and/or by bolus administration that overwhelms the detoxifying

31 mechanisms. The tumorigenic responses also appear to be closely associated with tissue toxicity

32 (e.g., rat and mouse forestomach) and high background spontaneous tumors (e.g., mouse lung, rat

33 liver). These observations, coupled with the lack of significant in vivo mutagenic activity, lead to

34 the postulation that chemical D-induced tumorigenicity is likely to be operated by a nonmutagenic
7/02/99 D-29 DRAFT

1 mode of action, and appears to be secondary to toxicity and reparative cell proliferation. At high

2 doses, a mutagenic mode of action may also be involved.

3

4 It is postulated that once absorbed, chemical D is biotransformed spontaneously or by

5 microsomal mixed functional oxidases (MFO) to an epoxide derivative that can react directly with

6 DNA. Both parent chemical D and the epoxide derivative are rapidly conjugated with glutathione

7 (GSH), which then can be excreted in the urine, mainly as the mercapturic acid conjugate of

8 chemical D. Under normal physiologic conditions, i.e., at nonsaturating doses, chemical D is

9 effectively detoxified as glutathione conjugate, and epoxidation does not take place in any great

10 extent. At high doses, chemical D is expected to react chemically with thiols in proteins, causing

11 tissue toxicity (forestomach), depleting tissue GSH, and causing proliferation of high background

12 spontaneous foci of altered cells (rat liver and mouse lung) leading to tumorigenesis. As less GSH

13 is available for detoxifying chemical D, more chemical D is metabolized to the mutagenic epoxide

14 derivative, which may play a role in the carcinogenic process. 

15

16 D.3.2.2. Key Events

17 D.3.2.2.1. Metabolism

18 It is hypothesized that epoxidation of chemical D does not take place to any great extent

19 since conjugated metabolite(s) of chemical D epoxide have not been detected in the urine of

20 treated rats and mice. This finding was based only on acute exposure to chemical D. The

21 metabolic profile of chemical D might differ under repeated exposures, particularly because

22 chemical D has been found to deplete tissue GSH. Additional in vitro and in vivo metabolism

23 studies are needed to further elucidate the potential role of MFO and epoxidation of chemical D.

24

25 D.3.2.2.2. Tissue Toxicity

26 It is postulated that chemical D-induced tumorigenicity is secondary to toxicity. The only

27 target organ that exhibits both toxicity and tumorigenicity is the forestomach of rats and mice.

28 Liver and lung toxicities have not been observed in chronic studies, although they have been

29 reported in subchronic studies at higher doses. On the other hand, nasal toxicity was observed in

30 exposed rats and mice, but no tumors were found.

31 Furthermore, the data supporting the postulated mechanism(s) of chemical D-induced

32 toxicity are limited. It is hypothesized to be mediated by chemical D binding to tissue proteins.

33 The only available information is the finding from acute oral and inhalation studies showing dose34 related chemical D binding to proteins of the liver and forestomach of rats, and lung of mice,
7/02/99 D-30 DRAFT

1 respectively. Additional studies are needed to investigate the potential toxicity of chemical D at

2 the biochemical, molecular, cellular, and tissue levels. 

3

4 D.3.2.2.3. Depletion of GSH

5 The ability of chemical D to deplete tissue GSH has been demonstrated to take place only

6 in the liver and forestomach of rats following acute ingestion and in the lung of mice via acute

7 inhalation. Additional data are needed to examine the effects of chemical D on GSH levels in

8 target organs as well as unaffected organs after repeated exposure.

9

10 D.3.2.2.4. Proliferation Activity

11 There is no information to substantiate the postulate that chemical D promotes highly

12 spontaneous rat liver or mouse lung altered cells. Cell proliferation and mutation spectra studies

13 are needed to examine the proliferative potential of chemical D.

14

15 

16 D.3.2.3. Strength, Consistency, Specificity of Association of Tumor Response With Key

17 Events

18 As discussed above, the postulated key events have not been clearly established. Thus, it is

19 difficult to determine how well these key events relate to the observed tumorigenic responses. In

20 general, the relationship between toxic and carcinogenic effects of chemical D on the forestomach

21 of rats and mice is relatively stronger and more consistent than its effects on the rat liver and the

22 mouse lung.

23

24 D.3.2.3.1. Forestomach Tumors

25 Subchronic studies and chronic studies in rats and mice demonstrate that the forestomach

26 is the primary target by oral exposure to chemical D. The rat appears to be more susceptible to

27 chemical D-induced forestomach toxicity than the mouse. 

28 Dose-related neoplastic and nonneoplastic lesions of the forestomach were observed in

29 treated rats and mice of both sexes when chemical D was administered by gavage. In contrast,

30 only hyperplastic lesions of the forestomach were found in male and female rats following

31 subchronic and chronic dietary exposures to chemical D. No histopathologic changes were

32 observed in the forestomach of treated mice in the subchronic and chronic dietary studies.

33

34 D.3.2.3.2. Liver Tumors
7/02/99 D-31 DRAFT

1 Chronic exposure of chemical D caused increased incidences of hepatic adenomas in male

2 rats when administered in the diet and by gavage. However, nonneoplastic changes in the liver

3 were not observed in male rats after chronic or subchronic oral exposure to chemical D.

4

5 D.3.2.3.3. Lung Tumors

6 Chemical D induced increased incidences of lung adenomas in exposed mice via chronic

7 inhalation (males only) and oral gavage. Nonneoplastic changes in the lung of exposed mice were

8 not reported in the chronic study. 

9

10 D.3.2.4. Dose-Response Relationships

11 As discussed above, dose correlations were demonstrated for chemical D-induced toxicity

12 and/or carcinogenicity in the various target tissues of treated rats and mice. Dose-related

13 depletion of tissue GSH was demonstrated with chemical D. However, no dose-related data are

14 available for other toxicokinetic and metabolic parameters (absorption, uptake, distribution,

15 metabolism, clearance and excretion of chemical D and metabolites), in vivo DNA binding, and

16 other key events (e.g., cytotoxicity, cell proliferation) that are postulated to be involved in the

17 tumorigenic process.

18

19 D.3.2.5. Temporal Association

20 While there are limited data indicating an association between chemical D-induced

21 carcinogenicity and related toxicity (mostly for the forestomach), there are no data to discern the

22 temporal association of these effects. Moreover, no data are available to establish the sequence of

23 key events at the biochemical, molecular, or cellular levels that might mediate the tumorigenic

24 responses.

25

26 D.3.2.6. Biological Plausibility and Coherence of the Database

27 The postulated mode of action for chemical D-induced forestomach tumors in rats and

28 mice appears plausible and coherent with current knowledge. Many chemicals that are strong

29 irritants have been shown to cause forestomach tumors via bolus administration. Similarly, the

30 mouse lung appears to be more susceptible to the carcinogenic actions of many toxicants by

31 inhalation. On the other hand, the observation that chemical D induces liver tumors only in the rat

32 is not consistent with the general observation that the mouse is more susceptible than the rat to

33 the carcinogenic effects of many halogenated hydrocarbons. 

34
7/02/99 D-32 DRAFT

1 D.3.2.7. Other Modes of Action

2 Chemical D bears a structural resemblance to several short-chain halogenated

3 hydrocarbons that are also animal carcinogens. Chemical D is expected to generate a mutagenic

4 epoxide. Chemical D has been shown to exhibit weak mutagenic responses in a number of in vitro

5 bacterial assays in the presence of liver microsomes, although addition of cytosolic enzymes,

6 presumably containing GST, has been shown to abolish the mutagenic activity. Several

7 cytogenetic assays demonstrated that chemical D can cause chromosomal damage in mammalian

8 cells. Thus, a mutagenic mode of action cannot be entirely ruled out for chemical D.

9

10 D.3.2.8. Conclusion

11 There is little evidence to support a conclusion that chemical D-induced tumorigenicity in

12 rats and mice is mediated by a nonlinear mode of action. The key events responsible for the

13 tumorigenic responses are not well defined and a temporal association of these key events has not

14 been fully investigated. Furthermore, it is still not possible to rule out a mutagenic mode of action

15 by chemical D. Additional data on the chemical interactions of chemical D with macromolecules,

16 and the nature of cytotoxic insults in target tissues and their relationship to tumor formation are

17 needed.
7/02/99 E-1 DRAFT

APPENDIX E. NONLINEAR DOSE-RESPONSE:

MARGIN OF EXPOSURE ANALYSIS

1 [To Be Developed]
7/02/99 F-1 DRAFT

1 APPENDIX F. DOSE-RESPONSE ASSESSMENT FOR A CARCINOGEN POSING

2 HIGHER RISKS AFTER CHILDHOOD EXPOSURE

3

4

5

6 a. Introduction

7 Compound K is a carcinogenic to humans by all exposure routes. This conclusion is based

8 on: (1) consistent epidemiologic evidence of a causal association between occupational exposure

9 and the development of angiosarcoma, an extremely rare tumor; (2) suggestive epidemiological

10 evidence that cancers of the brain, lung, and lymphopoietic system are associated with exposure;

11 (3) consistent evidence of carcinogenicity in rats, mice, and hamsters via the oral and inhalation

12 routes; (4) mutagenicity and DNA adduct formation by compound K and its metabolites in

13 numerous in vivo and in vitro test systems; and (5) efficient absorption via all routes of exposure

14 tested, followed by rapid distribution throughout the body.

15 Carcinogenicity involves genetic toxicity and is understood in some detail. Compound K

16 is metabolized to a reactive metabolite, probably an epoxide, which is believed to be the ultimate

17 carcinogenic metabolite. The reactive metabolite then binds to DNA, forming DNA adducts that,

18 if not repaired, ultimately lead to mutations and tumor formation. Therefore, a linear

19 extrapolation was used in the dose-response assessment. Because of uncertainty regarding

exposure levels in the occupationally exposed cohorts, an inhalation unit risk of 2x10-6 per ug/m3 20

21 was based on chronic inhalation studies in rats (not presented here).

22 Evidence has also been reported indicating increased sensitivity to early-life exposure. 

23 This case study shows how to use such evidence in a quantitative risk assessment. To focus on

24 early-life exposures, the hazard assessment and dose-response assessment for chronic exposure

(including derivation of the inhalation unit risk of 2x10-6 per ug/m3 25 ) are not presented here.

26

27 b. Dose-response data for early-life exposure

28 A dose-rate study compared responses to different dosing regimens, in which rats inhaled

29 compound K for 100 hours, starting at 13 weeks of age or 1 day of age (see table F-1). No effect

30 was observed for 100-hr exposures starting at 13 weeks, but 100-hr exposure starting at 1 day

31 had a clear carcinogenic effect, causing both angiosarcomas and hepatomas.

32 Tumor incidences in the newborn rats were also compared with rats inhaling compound K

33 for 52 weeks starting later in life (at 13 weeks) (see table F-2). Angiosarcoma incidence was

34 comparable from 52-week exposure starting at 13 weeks and 5-week exposure starting at 1 day. 
7/02/99 F-2 DRAFT

1 Hepatoma incidence, however, was high after newborn exposure but virtually absent after chronic

2 exposure starting later in life.

3 These data illustrate two phenomena that indicate higher cancer risks from childhood

4 exposure: (1) high incidence of a tumor (angiosarcomas) also caused by adult exposure, and

5 (2) occurrence of another tumor (hepatomas) not associated with adult exposure. The data

6 suggest that risks from short-term, early-life exposure may not be reversible even in the absence

7 of further exposure. The data do not, however, help us understand why early-life exposure poses

8 greater risks. It could be that the metabolized dose is higher in newborns than in adults (either

9 through more efficient metabolism, slower elimination, or a higher saturation point), alternatively,

10 metabolized doses could be comparable in newborns and adults, but newborns could be

11 biologically more sensitive to the same dose. Without understanding the mode of action early in

12 life, we can nonetheless use these data to estimate the higher cancer risks caused by early-life

13 exposure.

14

15 c. Dose conversion

16 Extensive pharmacokinetic studies show that the carcinogenic effects are caused by a

17 metabolite and that metabolism becomes saturated below the tested doses. A PBPK model was

18 fitted and validated (using independent data) to convert the experimental inhaled concentrations

19 to equivalent human concentrations (see table F-3). This involved two steps: (1) convert

20 experimental concentrations in air (ppm) to tissue concentrations in rat liver (mg metabolite per

21 L liver), and (2) convert these tissue concentrations to equivalent human concentrations in air

(ppm or mg/m3 22 ). The inhalation unit risk for chronic adult exposure was derived using doses

23 from this model.

24 Although the PBPK model was fitted using data on mature rats and adult human males,

25 dose estimates from this model were also used for dose-response modeling of tumors from early26 life exposure. Similarly, although liver tissue concentrations were used as the dose metric in the

27 PBPK model, this model was also used for angiosarcomas and angiomas at all sites (NTP

28 guidance indicates that these tumors should be combined). Although the ideal would be to have

29 pharmacokinetic information on various tissue concentrations in children, these studies have not

30 been conducted. The lack of this information introduces some uncertainty into the results. Use of

31 the PBPK model reflects a conscious decision that a credible dose-response model would be

32 based on saturable metabolism and not on administered concentrations alone.

33 Although it is standard practice to calculate lifetime average daily doses for carcinogens

34 (U.S. EPA, 1992), a different approach may be appropriate when considering effects of childhood
7/02/99 F-3 DRAFT

1 exposures if children are more sensitive than adults. Specifically, it may not be appropriate to

2 average childhood exposures over a full lifetime, since that implies that childhood exposure is

3 equivalent to full-life exposure at a lower rate. Consequently, the dose estimates from the PBPK

4 model are not averaged over a lifetime. Instead, the average dose during the early-life period (in

5 this experiment, 5 weeks) is used. That is, the administered concentration is reduced to reflect

6 intermittent exposure of 4 hr/d, 5 d/wk, but there is no further reduction by the ratio of the early7 life period (5 wk) to a lifetime. This childhood exposure estimate is applied to the childhood8 specific unit risk estimate calculated below. (If a unit risk estimate could not be calculated from

9 the early-life experiments and the adult unit risk estimate were used instead, the adult unit risk

10 would be adjusted for children as discussed in section 3.5.2.)

11

12 d. Analysis in the range of observation

13 In the range of observation, incidences of angiosarcomas or hepatomas (from table F-1)

14 are modeled separately as functions of equivalent human concentration based on metabolized dose

15 (from table F-3) using a quantal polynomial model of the form

p(d) = 1 - exp(-q1d - . . . - qkdk

), q1 16 , . . . , qk$ 0

The resulting points of departure are LEC10 = 36 ppm for angiosarcomas and LEC10 17 = 33 ppm for

hepatomas. Converting these to units of ug/m3 (for this compound, 1 ppm = 2600 ug/m3 18 ) yields

LEC10 = 9.4x104

 ug/m3 for angiosarcomas and LEC10 = 8.6x104 ug/m3 19 for hepatomas.

20

21 e. Extrapolation to lower doses

22 The available mechanistic information, which indicates a reactive metabolite that binds to

23 DNA and forms DNA adducts that ultimately lead to mutations and tumor formation, supports

24 linear extrapolation to lower doses. Linear extrapolation follows the line from the point of

departure to the origin (zero dose, zero excess risk). The slope of this line is 0.10/LEC10 25 . 

Accordingly, the unit risk estimates are 1.1x10-6 per ug/m3

 for angiosarcomas and 1.2x10-6 26 per

ug/m3 27 for hepatomas.

28

29 f. Combining unit risk estimates for multiple tumor types

30 To obtain an estimate of overall cancer risk, the unit risks for the induced tumor types are

31 combined. In the absence of individual animal pathology data, a neutral assumption is that the

32 tumor types are independent. In this case, the induction of angiosarcomas but not hepatomas by

33 later-life exposure suggests that these tumor types are caused by different modes of action and

34 may be independent. Under an assumption of independence, the combined unit risk is
7/02/99 F-4 DRAFT

1.1x10-6 + 1.2x10-6 - (1.1x10-6 x 1.2x10-6) = 2.3x10-6 per ug/m3 1

2

3 g. Strengths and limitations of the data

4 Although the data on newborn animals come from one rat strain over a limited range of

5 inhalation concentrations and there are no epidemiologic studies of children exposed to this

6 compound, the animal results indicate a potential for an increased susceptibility to tumors if

7 children are exposed. Another limitation is that individual animal data are not available to

8 determine whether animals with angiosarcomas are more likely to have hepatomas. Without these

9 data, an assumption of independence was made when combining unit risks across multiple tumor

10 sites.

11 The conversion used in this assessment to obtain the human continuous exposure

12 concentrations in ppm from the corresponding human dose metric in mg/L was a linear one. This

13 conversion methods seems simplistic given the complexity of the human body. This conversion

14 may be not be unreasonable, however, because this compound is rapidly and efficiently absorbed,

15 converted to water-soluble metabolites, and excreted.

16

17 h. Application to less-than-lifetime exposure scenarios

18 Two observations about the early-life studies have implications for how this assessment

19 would be applied to less-than-lifetime exposure scenarios, particularly during childhood.

20 1. The exposure period in the early-life experiment (weeks 1-5) does not overlap that of

21 the chronic experiments (weeks 14-65) used to estimate the inhalation unit risk for

22 chronic adult exposure. Therefore, the full lifetime cancer risk can be approximated by

23 adding risks from these nonoverlapping exposure periods. 

24 2. Because the effects of early-life exposure are different from effects of later exposures, it

25 would not be appropriate to prorate childhood exposures as if they were received at a

26 proportionately lower rate over a full lifetime.

27 These observations imply that the potential for increased sensitivity to childhood exposure

28 is not reflected in the unit risk estimated from later-life exposures. The following examples

29 illustrate how to combine early-life and later-life unit risk estimates.

30

Example 1. Full lifetime exposure (birth through death) to 1 ug/m3 31

32 The total risk is made up of two components, an early-life risk and a later-life risk.

Risk from early-life exposure: (2.3x10-6 per ug/m3

) x (1 ug/m3) = 2.3x10-6 33

Risk from later-life exposure: (2x10-6 per ug/m3) x (1 ug/m3) = 2x10-6 34
7/02/99 F-5 DRAFT

Total risk: 4.3x10-6 1

2

Example 2. Exposure to 2 ug/m3 3 from ages 30-60

4 Because exposure begins at age 30, there is no early-life component. The later-life

5 component is prorated as a duration of 30 years over an assumed lifespan of 70 years.

6 Risk from early-life exposure: Not applicable

Risk from later-life exposure: (2x10-6 per ug/m3) x (2 ug/m3) x (30/70) = 1.7x10-6 7

Total risk: 1.7x10-6 8

9

Example 3. Exposure to 5 ug/m3 10 from ages 0-10

11 Here there is an early-life component that is not prorated. The later-life component is,

12 however, prorated as 10 out of 70 years.

Risk from early-life exposure: (2.3x10-6 per ug/m3) x (5 ug/m3) = 1.2x10-5 13

Risk from later-life exposure: (2x10-6 per ug/m3) x (5 ug/m3) x (10/70) = 1.4x10-6 14

Total risk: 1.3x10-5 15
7/02/99 F-6 DRAFT

1 Table F-1. Comparison of tumor incidences in male and female Sprague-Dawley rats from 100-hr inhalation

2 exposures to newborn and mature rats

3 Angiosarcomas and angiomas (all sites) Liver hepatomas

4 Inhaled concentration

5 (ppm) Controla 6000 ppm 10,000 ppm Controla 6000 ppm 10,000 ppm

6 4 hr/d, 5 d/wk, 5 wk,

7 starting at age 13 wk 1/277 3/120 2/118 0/277 0/120 1/118

8 1 hr/d, 4 d/wk, 25 wk,

9 starting at age 13 wk 1/277 5/118 4/119 0/277 0/118 0/119

10 4 hr/d, 1 d/wk, 25 wk,

11 starting at age 13 wk 1/277 4/120 4/119 0/277 2/120 0/119

12 4 hr/d, 5 d/wk, 5 wk,

13 starting at age 1 day 1/277 20/42 18/44 0/277 20/42 20/44 

14 a 15 One control group served for all exposure patterns

16
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1 Table F-2. Comparison of tumor incidences in male and female Sprague-Dawley rats from 5-wk newborn

2 exposure and 52-wk later-life exposure

3 Angiosarcomas and angiomas (all sites) Liver hepatomas

4 Inhaled concentration

5 (ppm) Control 6000 ppm 10,000 ppm Control 6000 ppm 10,000 ppm

6 4 hr/d, 5 d/wk, 52 wk,

7 starting at age 13 wk 2/58 22/59 13/60 0/58 1/59 1/60

8 4 hr/d, 5 d/wk, 5 wk,

starting at age 1 daya 9 1/277 20/42 18/44 0/277 20/42 20/44

10 a 11 Repeated from table F-1

12
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1 Table F-3. Results of PBPK modeling

2 Inhaled concentration (ppm) Control 6000 ppm 10,000 ppm

3 Internal dose of metabolite (mg metabolite / L liver) 0 395 404

4 Equivalent continuous human inhaled concentration (ppm) 0 251 257

5
7/02/99 G-1 DRAFT

APPENDIX G. RESPONSE TO COMMENTS ON 

OTHER SCIENCE ISSUES

1 [To Be Developed]