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
Mode of Action
Exposure
Key event

• . . . 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

Key event
Key event
7
Mode of Action Frameworks
IPCS
U.S. EPA

• 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

• 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

8
MOA/Human Relevancy ILSI/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
MOA not 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 doesnt 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 whether quantitative 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 MOA risk is
  increased by age-dependent adjustment factor
  (used with exposure info for age group)
• lt2 yrs old, 10 fold
• 2 to lt 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 EPAs 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-FIN
AL -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
Genotoxicity Assays Genotoxicity Assays Genotoxicity Assays
Type of Damage Mouse Lymphoma Chromosome Aberrations CHO cells Ames Bacterial Mutagenicity
Point mutation Yes No Yes
Oligonucleotide insertion or deletion Yes No Yes
Allele Loss Yes No No
Small Chromosome alteration Yes ? No
Large Chromosome alteration Yes Yes No
Aneuploidy ? Yes No
Adapted from M. Moore (2004)
TERAs Dose-Response Assessment Boot Camp
20
Step 3 WOE for Mutagenic Activity -- 1

• Evaluation requires someone expert in gene-tox
  (all tests dont 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
Metabolism Cyt 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 its 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
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

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
• Phosgene key 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.

46
(No Transcript)