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

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Mutagenic moa carcinogens how high is the burden of proof

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


Disclaimer
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


Risk assessment is constantly evolving
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


Mode of action and cancer assessment
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


Why do you care about moa
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


“. . . 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


Mode of action frameworks

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


Moa human relevancy ilsi ipcs
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


Key event
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


Postulated mode of action
Postulated Mode Of Action

Chloroform

CYP2E1

Metabolism

Oxidative

Phosgene

Sustained Toxicity

Regenerative Cell Proliferation

Key Events

Tumor Development


Moa and kids
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


Supplemental guidance
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


  • 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


    Framework on default moa
    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


    What is a mutagen
    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


    What is mutagenic
    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


    Framework multi step process
    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


    Step 2 evaluate data quality
    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


    Gene tox tests measure different events
    Gene- tox Tests Measure Different Events

    TERA’s Dose-Response Assessment Boot Camp

    Adapted from M. Moore (2004)


    Step 3 woe for mutagenic activity 1
    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


    Woe for mutagenic activity 2
    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


    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.


    Not finished yet
    Not Finished yet Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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


    Key events
    Key Events Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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


  • Tumor induction time related accumulation of events
    Tumor Induction: Time-related Accumulation of Events Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    Mutagenic Carcinogen

    Multiple events

    Initiating

    Mutation

    Tumor

    Nonmutagenic Carcinogen

    Toxicity

    Altered Gene Expression

    Cell Proliferation

    Initiating

    Mutation

    Multiple events

    Tumor


    Applying the moa framework
    Applying the MOA Framework Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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”


    Cyclophosphamide
    Cyclophosphamide Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    Cytotoxic, alkylating

    Alkylating

    Cytotoxic


    Postulated mode of action1
    Postulated Mode Of Action Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    CP

    MetabolismCyt p 450s

    Phosphoramide mustard, PAM Acrolein

    DNA damage

    Tumor

    Development

    Mutations


    Cyclophosphamide gap
    Cyclophosphamide GAP Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)


    Cp in vivo tests animals
    CP In Vivo Tests: Animals Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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


    Cp in vivo tests humans
    CP In Vivo Tests: Humans Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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)


    So cp is mutagenic
    So CP Is Mutagenic Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • And it’s carcinogenic

    • Apply MOA Framework


    Dose resp concordance
    Dose Resp Concordance Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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)


    Cp dose resp concordance

    Rodents Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    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


    Temporality: Evaluate time-to-mutation Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    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


    Cp temporal associations
    CP TEMPORAL ASSOCIATIONS Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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


    Cp database plausibility coherence
    CP Database Plausibility & Coherence Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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


    Moa relevance
    MOA Relevance Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    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


    Postulated mode of action2
    Postulated Mode Of Action Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    Chloroform

    CYP2E1

    Metabolism

    Oxidative

    Phosgene

    Sustained Toxicity

    Regenerative Cell Proliferation

    Key Events

    Tumor Development


    Ccl 3 genetic activity profile
    CCl Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)3 Genetic Activity Profile


    Mutagenicity lines of evidence
    Mutagenicity: Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)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


    Mutagenicity ccl 3 conclusions
    Mutagenicity CCl Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)3: Conclusions

    • Weight of Evidence

      • Mutagenicity is not a component of chloroform induced neoplasia


    Metabolism conclusions
    Metabolism: Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)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


    Moa conclusions for chloroform
    MOA Conclusions for Chloroform Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    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


    Consider
    Consider Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event)

    • 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.


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