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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
slide6
“. . . 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
slide13

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

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

slide22

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
  • 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
  • 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

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
  • 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

Cytotoxic, alkylating

Alkylating

Cytotoxic

postulated mode of action1
Postulated Mode Of Action

CP

MetabolismCyt p 450s

Phosphoramide mustard, PAM Acrolein

DNA damage

Tumor

Development

Mutations

cp in vivo tests animals
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
cp in vivo tests humans
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)
so cp is mutagenic
So CP Is Mutagenic
  • And it’s carcinogenic
  • Apply MOA Framework
dose resp concordance
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)

cp dose resp concordance
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
slide35

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

cp temporal associations
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
cp database plausibility coherence
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
moa relevance
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

postulated mode of action2
Postulated Mode Of Action

Chloroform

CYP2E1

Metabolism

Oxidative

Phosgene

Sustained Toxicity

Regenerative Cell Proliferation

Key Events

Tumor Development

mutagenicity lines of evidence
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

mutagenicity ccl 3 conclusions
Mutagenicity CCl3: Conclusions
  • Weight of Evidence
    • Mutagenicity is not a component of chloroform induced neoplasia
metabolism conclusions
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
moa conclusions for chloroform
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

consider
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.
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