Mutagenic MOA Carcinogens: How High is the Burden of Proof ?

1. Mutagenic MOA Carcinogens: How High is the Burden of Proof ? RASS Telecom 09/10/08 Rita Schoeny, Ph.D. Senior Science Advisor Office of Water, U.S. EPA

2. Disclaimer • The views expressed in this presentation are those of the author and do not represent the policy of the U.S. EPA. Some of this is EPA policy

3. Risk Assessment is constantly evolving • Science and Judgment • Describe all defaults • Ensure they are health protective, documented, departures are warranted • Cancer Guidelines 2005 • Use data before defaults • Rather than determine how much data needed to depart from default • Including default procedures such as linear low dose risk

4. Mode of Action and Cancer Assessment • MOA is the keystone to all aspects of the assessment process True for other endpoints and is the major factor in harmonization among risk assessments

5. Why Do You Care about MOA ? • MOA is key in Hazard Identification • Helps describe circumstances under which agent is carcinogenic (High dose? Route?) • Relevance of data for humans • MOA determines choice of Low Dose Extrapolation • Life stage risk

6. “. . . a sequence of key events and processes, starting with interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation. . . Mode of action is contrasted with “mechanism of action,” which implies a more detailed understanding and description of events, often at the molecular level, than is meant by mode of action” Toxicity Mode of Action Exposure Key event Key event Key event

7. Hypothesized MOA: summary description and identification of key events Experimental support: Strength, consistency, specificity of association Dose-response concordance Temporal relationship Biological plausibility and coherence Consideration of the possibility of other MOAs Relevance to humans Postulated mode of action (theory of the case) Key events Concordance of dose-response relationships Temporal association Strength, consistency and specificity of association of tumour response with key events Biological plausibility and coherence Other modes of action Uncertainties, Inconsistencies, and Data Gaps Assessment of postulated mode of action Mode of Action Frameworks IPCS U.S. EPA

8. MOA/Human RelevancyILSI/IPCS NO Is the weight of evidence sufficient to establish a mode of action (MOA) in animals? Proceed with risk assessment YES Can human relevancy of the MOA be reasonably excluded on the basis of fundamental, qualitative differences in key events between animals and humans? YES MOA not Relevant NO Can human relevancy of the MOA be reasonably excluded on the basis of quantitative differences in either kinetic or dynamic factors between animals and humans? NO YES Proceed with Risk assessment MOAnot Relevant

9. Key Event • A “key event” is an empirically observable precursor step that is itself a necessary element of the mode of action or is a biologically based marker for such an element. Key event is necessary, but not sufficient If a key event doesn’t occur, there is no cancer If one key event occurs, there may or may not be cancer

10. Postulated Mode Of Action Chloroform CYP2E1 Metabolism Oxidative Phosgene Sustained Toxicity Regenerative Cell Proliferation Key Events Tumor Development

11. MOA and Kids • Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens • Effects observed in childhood • Early life exposures that contribute to later life effects • MOA determines whetherquantitative adjustment is made

12. Supplemental Guidance • Use age-specific values for exposure and potency • When data permit, develop separate potency estimates for childhood exposure • In risk characterization,mutagenic MOArisk is increased by age-dependent adjustment factor (used with exposure info for age group) • <2 yrs old, 10 fold • 2 to < 16yrs, 3 fold • No MOA, linear extrapolation without ADAF; non-linear MOA, no ADAF

13. Public Comment • completed 12/07 Framework for Determining a Mutagenic Mode of Action for Carcinogenicity Using EPA’s 2005 Cancer Guidelines and Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens • External peer review • completed 05/08 www. epa.gov/ osa/mmoaframework/ pdfs/MMOA-ERD-FINAL -83007.pdf

14. Framework on Default MOA • “It should also be noted that there is no ‘default MOA.’ The Cancer Guidelines offer some default procedures to use when no MOA can be determined.” • MOA determinations follow Cancer Guidelines • Framework • If insufficient data to support MOA, use low dose • linear extrapolation and no ADAF Determination of mutagenic MOA is as scientifically rigorous as any other MOA

15. What is a mutagen? • A chemical that induces biologically relevant mutations in any one of a number of validated mutation assays • Mutation assays detect the induction of mutants • Mutants are cells with genetic alterations that can be passed to viable daughter and granddaughter cells -- heritable

16. What is “Mutagenic”? • EPA does not have a standard definition of “mutagenic” • Operationally for use in “mutagenic MOA for cancer” • “. . . capacity of either the carcinogen or its metabolite to react with or bind to DNA in a manner that causes mutations.In this context, mutagens usually (though not always) produce positive effects in multiple test systems for different genetic endpoints, particularly gene mutations and structural chromosome aberrations, both in vitro and in vivo.” • The peer reviewers hated it

17. Framework: Multi-step Process • Risk assessment is an iterative process • Visualize the Framework as series of linear steps • Step 1 is assemble relevant data • Genetic toxicity testing, tumor data, pk, SAR, etc. • Framework describes test batteries

18. Step 2: Evaluate Data Quality • Look at primary papers • Judge against current acceptability criteria • e.g. were tests done at cytotoxic levels • Cites publications for evaluating quality (e.g. Cimino 2006, OECD, ICH, IWGT, DHHS 2006) • Keep, but weigh

19. Gene- tox Tests Measure Different Events TERA’s Dose-Response Assessment Boot Camp Adapted from M. Moore (2004)

20. Step 3: WOE for Mutagenic Activity -- 1 • Evaluation requires someone expert in gene-tox (all tests don’t measure same things) • Categorize data – suggest use of our table in Appendix A. • Put in all data with notes on quality • Use consistent terms for assay types or endpoints: positive, negative, inconclusive, contradictory • Present summary of database

21. WOE for Mutagenic Activity -- 2 • Conclusions across endpoints: some endpoints carry more weight than others • e.g. Sperm head morphology may be caused by modification of protein structure • Morphologic cell transformation does not measure mutation • Hierarchy of data utility • DNA interaction ≠DNA damage ≠mutation • e.g. most useful are mutations in relevant genes in humans • WOE for mutagenic activity: negative, data are inadequate, data are of questionable quality, data are equivocal, data are positive No Checklist No Minimum Data Set

22. How to Weigh the Evidence as to Whether a Chemical Causes Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event) (Listed in decreasing order of relevance/importance) Cancer relevant oncogene/tumor suppressor gene mutations can be detected in the target tissue following chemical exposure Surrogate gene mutations can be detected in the target tissue following chemical exposure 3. DNA adducts (known to be mutagenic adducts) can be detected in the target tissue following chemical exposure Primary DNA damage can be detected in the target tissue following chemical exposure Gene mutations and/or DNA adducts or other measures of primary DNA damage can be detected in vivo. 6. Evidence that the chemical can induce mutations, cytogenetic damage, DNA adducts and/or primary DNA damage in vitro.

23. Not Finished yet • Mutagenicity + carcinogenicity ≠ Mutagenic MOA Apply MOA Framework • Hypothesized MOA • Experimental support: • Dose-response concordance • Strength, consistency, specificity of association • Temporal relationship • Biological plausibility and coherence • Consideration of the possibility of other MOAs • Relevance to humans Step 4

24. Key Events • DNA changes resulting in mutation • “ For a chemical to act by a mutagenic MOA, either the chemical or its direct metabolite is the agent inducing the mutations that initiate cancer.” • “This is contrasted with a MOA wherein mutagenicity occurs as an indirect effect of another key event in carcinogenesis.” • Properties for mutagenicity as the key event • Long list in Guidelines: early tumor response, initiator, target tissue is exposed to DNA-reactive chemical, mutation is early event, mutation in oncogenes, etc

25. Tumor Induction: Time-related Accumulation of Events Mutagenic Carcinogen Multiple events Initiating Mutation Tumor Nonmutagenic Carcinogen Toxicity Altered Gene Expression Cell Proliferation Initiating Mutation Multiple events Tumor

26. Applying the MOA Framework • Types of data supporting WOE • Consistency across assays • Induction of ≥ 1 type of effect • Effects in vivo • Mutation in absence of cytotoxicity • Belongs to a class of compounds with established mutagenic MOA • Including the “Supplemental Guidance 12”

27. Cyclophosphamide Cytotoxic, alkylating Alkylating Cytotoxic

28. Postulated Mode Of Action CP MetabolismCyt p 450s Phosphoramide mustard, PAM Acrolein DNA damage Tumor Development Mutations

29. Cyclophosphamide GAP

30. CP In Vivo Tests: Animals • Gene Mutation Assays • Positive Mouse Spot Test (2.5-10 mg/kg) • Positive Muta Mouse (lacZ) 100 mg/kg x 5 days in bone marrow • B6C3F1 mouse (lacI) 100 mg/kg MF increased in lungs and urinary bladder • No transgenic studies in rats

31. CP In Vivo Tests: Humans • Micronuclei peripheral blood lymphocytes (PBL) & buccal epithelials 26/26 nurses handling CP • Structural chromosome aberrations & SCE, gene mutations or DNA damage (Comet assay) in PBL or bone marrow, patients • Structural chromosome aberrations in children • Mutation of p53 in bladder tumors (cumulative doses of 6-125 mg/kg) • 6-Thioguanine-resistant T lymphocytes from multiple sclerosis patients (750 mg/m2)

32. So CP Is Mutagenic • And it’s carcinogenic • Apply MOA Framework

33. Dose Resp Concordance • Mutation is key event • Expect mutations and / or DNA interaction at lower dose than tumors • Mutation is not the key event • Expect increased mutation at doses higher than those required for tumor induction (the increase in mutations likely results as a secondary effect of cytotoxicity or cell proliferation)

34. Rodents Lowest effective dose [induction of SCE in rat bone marrow (0.62 mg/kg)] Consistent with data showing significant tumor formation in the urinary bladder of male rats at 1.25 and 2.5 mg/kg/day (488 mg total) Humans Chromosome aberrations & SCEs 2 hrs after dosing 33-40 mg/kg p53 mutations at a cumulative dose of 6 g Cohort of 6171survivors of non-Hodgkin's lymphoma; 48 developed cancer of the urinary tract – those receiving a total dose of 20g had a 2.4-fold  risk of bladder cancer;20-49g, a 6-fold  risk CP Dose / Resp Concordance

35. Temporality: Evaluate time-to-mutation Mutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periods Mutant Frequency Time in Weeks Nonmutagenic carcinogens would be expected to be negative after long chronic treatment, or show a positive response only after long chronic treatment

36. CP TEMPORAL ASSOCIATIONS • SCE bone marrow of Fischer 344 rats dosed with 20 mg/kg (ip) CP after 30 min. (1 hr after 5 or 10 mg/kg) • Chromosomal aberration & micronuclei in human bone marrow 24 hrs post therapeutic dose of 40 mg/kg (iv) • Cytotoxicity & regenerative proliferation in the rat also occur early: • Bladder damage (ulceration of mucosa, necrosis of bladder epithelium)—1 day • Regeneration of bladder epithelia – 36 hrs • Hyperplasia of bladder epithelia – 48 hrs • Malignant bladder tumors — 40-60 weeks

37. CP Database Plausibility & Coherence • Qualitative & quantitative data for key events leading to tumors • Concordance of most key events in animal models & humans • No stop/recovery studies found, but there is evidence suggesting that CP-associated cancers may occur up to several years after drug treatment has ceased. • Gaps in human data (e.g., DNA adducts & cell proliferation) do not compromise the analysis

38. MOA Relevance Rats Humans PAM generation Yes Yes DNA adducts Yes Plausible Mutagenicity Yes Yes Bladder cytotoxicity Yes Yes Epithelial regeneration Yes Plausible Hyperplasia Yes Yes Bladder tumors Yes Yes

39. Postulated Mode Of Action Chloroform CYP2E1 Metabolism Oxidative Phosgene Sustained Toxicity Regenerative Cell Proliferation Key Events Tumor Development

40. CCl3 Genetic Activity Profile

41. Mutagenicity: Lines of Evidence Negative in vitro Conflicting evidence in vivo Initiation-Promotion Studies CCl3 is not an initiator Molecular Based Approaches Negative for tumors in p53 +/- transgenic mouse cancer bioassay Negative for mutations in LacI transgenic B6C3F1 mice Negative for mutation in rat GST transfected bacteria

42. Mutagenicity CCl3: Conclusions • Weight of Evidence • Mutagenicity is not a component of chloroform induced neoplasia

43. Metabolism: Conclusions • Predominate pathway • P450 (CYP2E1)-mediated oxidative pathway • Phosgenekey reactive metabolite • The following play little, if any role in chloroform induced tumors-- • Reductive P450 metabolism & free radical production • GST catalyzed conjugation

44. MOA Conclusions for Chloroform Hypothesized MOA Well Supported Other MOAs NOT Well Supported Human Relevance Presumed (also epidemiological data on chlorinated water) Applies to Children (but not more susceptible) Consistent with Nonlinear Dose Response Risk Approach Based on Protection Against Sustained Toxicity/Proliferation

45. Consider • What data are available? • Screening genetox data, batteries of test designed for hazard identification • What data are optimal? • Real, live MOA data (e.g. time course studies in relevant human genes) • What data are practical? • Something less than what was available for cyclophosphamide • Requires some strategic thought in test design.