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
1
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
2
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
3
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
4
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
determines choice of Low
Dose Extrapolation
 Life stage risk
es
ow
(L
x
ce
)
se
Do
x
Es
tim
at
e)
en
fid
5%
t9
n
Co
tra
l
Environmental
Exposure Levels
of Interest
n
o
it
m
Li
Empirical
Range of
Observation
(C
en
Response (Tumor or Nontumor Data)
 MOA
x
10%
x
Lin
au
Def
ear
lt
Range of
Extrapolation
x
0%
LED10
ED10
Nonlinear Default
5
x NOAEL
MOE
Dose
x LOAEL
Mode of Action

“. . . 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
Exposure
Key event
Key event
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
Toxicity
6
Mode of Action Frameworks
IPCS
U.S. EPA




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 doseresponse 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
7
MOA/Human Relevancy ILSI/IPCS
Is the weight of evidence
sufficient to establish a mode of
action (MOA) in animals?
NO
Proceed with
risk assessment
YES
MOA not
Relevant
YES
Can human relevancy of the MOA be
reasonably excluded on the basis of
fundamental, qualitative differences in
key events between
animals and humans?
NO
MOA not
Relevant
YES
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
Proceed with
Risk assessment
8
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
9
Postulated Mode Of Action
Chloroform
Sustained Toxicity
Regenerative Cell Proliferation
Key Events
Tumor Development
10
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
11
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)

 <2
yrs old, 10 fold
 2 to < 16yrs, 3 fold

No MOA, linear extrapolation without
ADAF; non-linear MOA, no ADAF
12
Framework for Determining
•Public Comment
completed 12/07
a Mutagenic Mode of
•External peer review
Action for Carcinogenicity
completed 05/08
Using EPA’s 2005 Cancer
Guidelines and Supplemental
Guidance for Assessing
www. epa.gov/
Susceptibility from Early-Life
osa/mmoaframework/
Exposure to Carcinogens
pdfs/MMOA-ERD-FINAL
-83007.pdf
13
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
14
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
15
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
16
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
17
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
18
Gene- tox Tests Measure Different
Events
Genotoxicity Assays
Type of Damage
Mouse
Lymphoma
Chromosome
Aberrations CHO cells
Ames Bacterial
Mutagenicity
Point mutation
Yes
No
Yes
Oligonucleotide
Yes
insertion or deletion
No
Yes
Allele Loss
Yes
No
No
Small Chromosome
alteration
Yes
?
No
Large Chromosome
alteration
Yes
Yes
No
Aneuploidy
?
Yes
No
TERA’s Dose-Response Assessment Boot Camp
Adapted from M. Moore
(2004)
19
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
20
WOE for Mutagenic Activity -- 2
– Conclusions across endpoints: some endpoints
carry more weight than others
 e.g.
No Checklist
Sperm head morphology may be caused by
modification of protein structure
 Morphologic cell transformation does not measure
mutation
– Hierarchy of data utility
interaction
≠DNA damage
≠mutation
No
Minimum
Data
Set
 DNA
 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
21
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)
1.
Cancer relevant oncogene/tumor suppressor gene
mutations can be detected in the target tissue following
chemical exposure
2.
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
4.
Primary DNA damage can be detected in the target tissue
following chemical exposure
5.
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.
22
Not Finished yet
 Mutagenicity
+ carcinogenicity ≠
Mutagenic MOA
Apply MOA Framework
Step 4
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
23
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
24
Tumor Induction: Time-related
Accumulation of Events
Mutagenic Carcinogen
Initiating
Multiple events
Tumor
Mutation
Nonmutagenic Carcinogen
Toxicity
Altered Gene
Expression
Cell Proliferation
Initiating
Mutation
Multiple events
Tumor
25
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”
26
Cyclophosphamide
Cytotoxic, alkylating
Alkylating
Cytotoxic
27
Postulated Mode Of Action
CP
Metabolism
Cyt p 450s
DNA damage
Tumor
Development
Mutations
28
Cyclophosphamide GAP
CYCLOPHOSPHAMIDE
50-18-0
IARCS6
1987
GENETIC ACTIVITY PROFILE
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MAMM VIVO
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IARC human carcinogen (group 1: human - sufficient, animal - sufficient)
29
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
30
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)
31
So CP Is Mutagenic
 And
it’s carcinogenic
 Apply
MOA Framework
32
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)
33
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; 2049g, a 6-fold  risk
34
Temporality: Evaluate time-to-mutation
Mutant Frequency
Mutagenic carcinogens would be expected to show a positive
mutation response after relatively short treatment periods
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
35
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
36
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
37

MOA Relevance
Rats
PAM generation
DNA adducts
Mutagenicity
Bladder
cytotoxicity
Epithelial regeneration
Hyperplasia
Bladder tumors
Humans
Yes
Yes
Yes
Yes
Plausible
Yes
Yes
Yes
Yes
Yes
Yes
Plausible
Yes
Yes
38
Postulated Mode Of Action
Chloroform
Sustained Toxicity
Regenerative Cell Proliferation
Key Events
Tumor Development
39
CCl3 Genetic Activity Profile
CHLOROFORM
67-66-3
IARC_V73
1999
GENETIC ACTIVITY PROFILE
8
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PROKARY
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IARC possible human carcinogen (group 2B: human - inadequate, animal - sufficient)
40
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
41
Mutagenicity CCl3: Conclusions
Weight of Evidence
Mutagenicity is not a component
of chloroform induced neoplasia
42
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

43
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
44
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.
45
46