1 mutagenic moa carcinogens: how high is the burden of proof ? rass telecom 09/10/08 rita schoeny,...
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Mutagenic MOA Carcinogens: Mutagenic MOA Carcinogens: How High is the Burden of How High is the Burden of
Proof ?Proof ?RASS TelecomRASS Telecom
09/10/0809/10/08
Rita Schoeny, Ph.D.Senior Science Advisor Office of Water, U.S. EPA
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DisclaimerDisclaimer
The views expressed in this The views expressed in this presentation are those of the author presentation are those of the author and do not represent the policy of and do not represent the policy of the U.S. EPA.the U.S. EPA.
Some of this Some of this isis EPA policy EPA policy
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Risk Assessment is constantly Risk Assessment is constantly evolvingevolving
Science and Judgment Science and Judgment – Describe all defaultsDescribe all defaults– Ensure they are health protective, Ensure they are health protective,
documented, departures are warranteddocumented, departures are warranted Cancer Guidelines 2005Cancer Guidelines 2005
– Use data before defaultsUse data before defaultsRather than determine how much data Rather than determine how much data
needed to depart from default needed to depart from default Including default procedures such as linear Including default procedures such as linear
low dose risklow dose risk
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Mode of Action and Cancer Mode of Action and Cancer AssessmentAssessment
MOA is the keystone to all aspects of MOA is the keystone to all aspects of the assessment process the assessment process
True for other endpointsand is the major factor in harmonization among riskassessments
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Why Do You Care about MOA ?Why Do You Care about MOA ?
MOA is key in Hazard IdentificationMOA is key in Hazard Identification– Helps describe circumstances under Helps describe circumstances under
which agent is carcinogenic (High which agent is carcinogenic (High dose? Route?)dose? Route?)
– Relevance of data for humans Relevance of data for humans MOA determines choice of Low MOA determines choice of Low
Dose ExtrapolationDose Extrapolation Life stage riskLife stage risk
MOE
Dose
Resp
on
se
(T
um
or
or
No
ntu
mo
r D
ata
)
0%
10%
EnvironmentalExposure Levels
of Interest
LED10 ED10
Nonlinear Default
EmpiricalRange of Observation
Range ofExtrapolation
x
x
xx
x
x NOAEL
LOAEL
x
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Exposure
Toxicity
Key event
Key event
Key event
““. . . a sequence of . . . a sequence of key key eventsevents and processes, and processes, starting with interaction starting with interaction of an agent with a cell, of an agent with a cell, proceeding through proceeding through operational and operational and anatomical changes, and anatomical changes, and resulting in cancer resulting in cancer formation. .formation. . . Mode of action . Mode of action is contrasted with “is contrasted with “mechanism mechanism of actionof action,” which implies a ,” which implies a more detailed understanding more detailed understanding and description of events, and description of events, often at the molecular level, often at the molecular level, than is meant by mode of than is meant by mode of actionaction””
Mode of ActionMode of Action
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Mode of Action FrameworksMode of Action Frameworks
Hypothesized MOA: Hypothesized MOA: summary description and summary description and identification of identification of key eventskey events
Experimental support:Experimental support:– Strength, consistency, Strength, consistency,
specificity of specificity of associationassociation
– Dose-response Dose-response concordanceconcordance
– Temporal relationshipTemporal relationship– Biological plausibility Biological plausibility
and coherenceand coherence Consideration of the Consideration of the
possibility of other MOAspossibility of other MOAs Relevance to humansRelevance to humans
Postulated mode of action Postulated mode of action (theory of the case)(theory of the case)
Key eventsKey events Concordance of dose-Concordance of dose-
response relationshipsresponse relationships Temporal associationTemporal association Strength, consistency and Strength, consistency and
specificity of association ofspecificity of association of tumour response with key tumour response with key
eventsevents Biological plausibility and Biological plausibility and
coherencecoherence Other modes of actionOther modes of action Uncertainties, Uncertainties,
Inconsistencies, and Data Inconsistencies, and Data GapsGaps
Assessment of postulated Assessment of postulated mode of actionmode of action
U.S. EPAU.S. EPA IPCSIPCS
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MOA/Human RelevancyMOA/Human Relevancy ILSI/IPCS ILSI/IPCS
YES
NO
NO
YESMOA notRelevant
YESMOA notRelevant
NOProceed withRisk assessment
Proceed withrisk assessment
Is the weight of evidence sufficient to establish a mode of action (MOA) in animals?
Can human relevancy of the MOA be reasonably excluded on the basis of fundamental, qualitative differences in key events between animals and humans?
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?
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Key EventKey Event A A “key event” “key event” is an empirically is an empirically
observable precursor step that is observable precursor step that is itself a itself a necessary elementnecessary element of the of the mode of action or is a biologically mode of action or is a biologically based marker for such an element.based marker for such an element.
Key event is necessary, but not sufficientKey event is necessary, but not sufficient
If a key event doesn’t occur, there is no If a key event doesn’t occur, there is no cancercancer
If one key event occurs, there may or may If one key event occurs, there may or may not be cancernot be cancer
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ChloroformCYP2E1
Phosgene
Regenerative Cell Proliferation
Postulated Mode Of ActionPostulated Mode Of ActionMetabolism
Oxidative
Sustained Toxicity
Tumor DevelopmentKey EventsKey Events
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MOA and KidsMOA and Kids
Supplemental Guidance for Assessing Supplemental Guidance for Assessing Susceptibility from Early-Life Susceptibility from Early-Life Exposure to CarcinogensExposure to Carcinogens– Effects observed in childhoodEffects observed in childhood– Early life exposures that contribute to Early life exposures that contribute to
later life effectslater life effects– MOA determines whetherMOA determines whether quantitativequantitative
adjustment is made adjustment is made
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Supplemental GuidanceSupplemental Guidance Use age-specific values for Use age-specific values for exposure and potencyexposure and potency When data permit, develop When data permit, develop separate potency estimatesseparate potency estimates for childhood exposurefor childhood exposure In risk characterization,In risk characterization, mutagenic MOAmutagenic MOA risk is risk is
increased by age-dependent adjustment factor increased by age-dependent adjustment factor (used with exposure info for age group)(used with exposure info for age group)
<2 yrs old, 10 fold<2 yrs old, 10 fold 2 to < 16yrs, 3 fold2 to < 16yrs, 3 fold
No MOA, linear extrapolation without ADAF; non-No MOA, linear extrapolation without ADAF; non-linear MOA, no ADAFlinear MOA, no ADAF
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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
www. epa.gov/
osa/mmoaframework/
pdfs/MMOA-ERD-FINAL
-83007.pdf
•External peer review External peer review
completed 05/08completed 05/08
•Public Comment Public Comment completed 12/07completed 12/07
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Framework on Default MOAFramework on Default MOA
““ It should also be noted that there is no It should also be noted that there is no ‘default MOA.’‘default MOA.’ The The Cancer Guidelines Cancer Guidelines offer offer some default procedures to use when no some default procedures to use when no MOA can be determined.”MOA can be determined.”
•MOA determinations follow Cancer GuidelinesFramework
•If insufficient data to support MOA, use low doselinear extrapolation and no ADAF
Determination of mutagenic MOA is as
scientifically rigorous as any other MOA
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What is a mutagen? A chemical that induces A chemical that induces
biologically relevant biologically relevant mutations in any one of a mutations in any one of a number of validated number of validated mutation assaysmutation assays
Mutation assays detect the Mutation assays detect the induction of mutantsinduction of mutants
Mutants are cells with Mutants are cells with genetic alterations that can genetic alterations that can be passed to viable be passed to viable daughter and daughter and granddaughter cells -- granddaughter cells -- heritableheritable
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What is “Mutagenic”?What is “Mutagenic”? EPA does not have a standard definition of EPA does not have a standard definition of
“mutagenic”“mutagenic” Operationally for use in “mutagenic MOA for Operationally for use in “mutagenic MOA for
cancer”cancer”– ““. . . capacity of either the . . . capacity of either the carcinogen or its carcinogen or its
metabolite to react with or bind to DNA in a manner metabolite to react with or bind to DNA in a manner that causes mutations.that causes mutations. In this context, mutagens usually In this context, mutagens usually (though not always) produce positive effects in multiple test (though not always) produce positive effects in multiple test systems for different genetic endpoints, particularly gene systems for different genetic endpoints, particularly gene mutations and structural chromosome aberrations, both mutations and structural chromosome aberrations, both in in vitrovitro and and inin
vivovivo.”.”
– The peer reviewers hated itThe peer reviewers hated it
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Framework: Multi-step ProcessFramework: Multi-step Process
Risk assessmentRisk assessmentis an iterative is an iterative processprocess
Visualize the Framework as series of Visualize the Framework as series of linear stepslinear steps
Step 1 is assemble relevant dataStep 1 is assemble relevant data– Genetic toxicity testing, tumor data, pk, Genetic toxicity testing, tumor data, pk,
SAR, etc.SAR, etc.– Framework describes test batteriesFramework describes test batteries
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Step 2: Evaluate Data Step 2: Evaluate Data QualityQuality
Look at primary papersLook at primary papers Judge against currentJudge against current acceptability criteria acceptability criteria
– e.g. were tests done ate.g. were tests done at cytotoxic levelscytotoxic levels
Cites publications for evaluating Cites publications for evaluating quality (e.g. Cimino 2006, OECD, ICH, quality (e.g. Cimino 2006, OECD, ICH, IWGT, DHHS 2006)IWGT, DHHS 2006)
Keep, but weighKeep, but weigh
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Gene- tox Tests Measure Gene- tox Tests Measure Different EventsDifferent Events
Gene- tox Tests Measure Gene- tox Tests Measure Different EventsDifferent Events
Genotoxicity AssaysGenotoxicity Assays
Type of Type of DamageDamage
Mouse Mouse LymphomLymphomaa
ChromosomeChromosome
Aberrations CHO Aberrations CHO cellscells
Ames Bacterial Ames Bacterial MutagenicityMutagenicity
Point mutationPoint mutation YesYes NoNo YesYes
Oligonucleotide Oligonucleotide insertion or insertion or deletiondeletion
YesYes NoNo YesYes
Allele LossAllele Loss YesYes NoNo NoNo
Small Small Chromosome Chromosome alterationalteration
YesYes ?? NoNo
Large Large Chromosome Chromosome alterationalteration
YesYes YesYes NoNo
AneuploidyAneuploidy ?? YesYes NoNoAdapted from M. Moore (2004)TERA’s Dose-Response Assessment Boot Camp
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Step 3: WOE for Mutagenic Activity Step 3: WOE for Mutagenic Activity -- 1-- 1
Evaluation requires someone expert in Evaluation requires someone expert in gene-tox (all tests don’t measure same gene-tox (all tests don’t measure same things)things)
Categorize data – suggest use of our table Categorize data – suggest use of our table in Appendix A.in Appendix A.– Put in Put in all dataall data with notes on quality with notes on quality– Use consistent terms for assay types or Use consistent terms for assay types or
endpoints: endpoints: positive, negative, inconclusive, positive, negative, inconclusive, contradictorycontradictory
– Present summary of Present summary of databasedatabase
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WOE for Mutagenic Activity -- 2WOE for Mutagenic Activity -- 2
– Conclusions across endpoints: Conclusions across endpoints: some endpoints some endpoints carry more weight than otherscarry more weight than others
e.g. Sperm head morphology may be caused by e.g. Sperm head morphology may be caused by modification of protein structuremodification of protein structure
Morphologic cell transformation does not measure Morphologic cell transformation does not measure mutationmutation
– Hierarchy of data utility Hierarchy of data utility DNA interaction ≠DNA damage ≠mutationDNA interaction ≠DNA damage ≠mutation e.g. most useful are mutations in relevant genes in e.g. most useful are mutations in relevant genes in
humanshumans
– WOE for mutagenic activity: negative, data are WOE for mutagenic activity: negative, data are inadequate, data are of questionable quality, inadequate, data are of questionable quality, data are equivocal, data are positivedata are equivocal, data are positive
No Checklist
No Minimum Data Set
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How to Weigh the Evidence as to Whether a Chemical How to Weigh the Evidence as to Whether a Chemical Causes Specific Tumors by a Mutagenic Mode of Action Causes Specific Tumors by a Mutagenic Mode of Action (Mutation is THE Key Event) (Mutation is THE Key Event)
(Listed in decreasing order of relevance/importance)(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.
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Not Finished yetNot Finished yet
Mutagenicity + carcinogenicity Mutagenicity + carcinogenicity ≠≠Mutagenic MOAMutagenic MOA
Apply MOA FrameworkHypothesized MOAExperimental support:
–Dose-response concordance–Strength, consistency, specificity of association–Temporal relationship–Biological plausibility and coherence
Consideration of the possibility of other MOAsRelevance to humans
Step 4
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Key EventsKey Events DNA changes resulting in mutationDNA changes resulting in mutation “ “ For a chemical to act by a mutagenic MOA, For a chemical to act by a mutagenic MOA,
either the chemical or its direct metabolite is either the chemical or its direct metabolite is the agent inducing the mutations that initiate the agent inducing the mutations that initiate cancer.”cancer.”
““This is contrasted with a MOA wherein mutagenicity This is contrasted with a MOA wherein mutagenicity occurs as an indirect effect of another key event in occurs as an indirect effect of another key event in carcinogenesis.”carcinogenesis.”
Properties for mutagenicity as the key event Properties for mutagenicity as the key event Long list in Long list in GuidelinesGuidelines: early tumor response, initiator, : early tumor response, initiator,
target tissue is exposed to DNA-reactive chemical, target tissue is exposed to DNA-reactive chemical, mutation is early event, mutation in oncogenes, etcmutation is early event, mutation in oncogenes, etc
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InitiatingInitiating
MutationMutationTumorTumor
Multiple events
Toxicity
Altered Gene Expression
Cell Proliferation
InitiatingInitiatingMutationMutation
Multiple events
TumorTumor
Mutagenic CarcinogenMutagenic Carcinogen
Nonmutagenic CarcinogenNonmutagenic Carcinogen
Tumor Induction: Time-related Tumor Induction: Time-related Accumulation of EventsAccumulation of Events
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Applying the MOA FrameworkApplying the MOA Framework
Types of data supporting WOETypes of data supporting WOE– Consistency across assaysConsistency across assays– Induction of Induction of ≥ 1 type of ≥ 1 type of
effecteffect– Effects Effects in vivoin vivo– Mutation in absence of cytotoxicityMutation in absence of cytotoxicity– Belongs to a class of compounds with Belongs to a class of compounds with
established mutagenic MOA established mutagenic MOA Including the “Supplemental Guidance 12”Including the “Supplemental Guidance 12”
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CyclophosphamideCyclophosphamide
Alkylating
Cytotoxic
Cytotoxic, alkylating
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Postulated Mode Of ActionPostulated Mode Of ActionCPCPMetabolismMetabolism
Cyt p 450sCyt p 450s
Phosphoramide mustard, PAM
Phosphoramide mustard, PAM
Acrolein Acrolein
DNA damageDNA damage
MutationsMutations
Tumor Tumor DevelopmentDevelopment
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Cyclophosphamide GAPCyclophosphamide GAP
-6
-4
-2
0
2
4
6
8
LOG DOSE UNITS
GENETIC ACTIVITY PROFILE
PROKARY LOW EUK PLNT INS MAMM VITRO HUMAN F MAMM VIVO HU
50-18-0
IARCS6
1987
CYCLOPHOSPHAMIDE
BRP
ECD
ECL
BSD
FAS
SA0
SA5
7AS
8AS9AS
SASE
CK
ECW
EC2
ECR S
SD
SCG
SCH
SCF
SCR
SZFA
NF
SCN
DMM
DMX
CMD
DMH
LMD
DMN
URP
GCOG9H
G5T
SIC
MIS
SIR
SIS
SITSIA
CIC
CIS
CIT
MBT
TCMTCS
TRR
DIH
UHT
SHL
SHT
SIH
CHL
CHT
BFA
BFH
HMAHMHHMM
DVA
UVM
UVR
UVAGVA
MST
SVA
MVM
MVRMVCMVA
CBA
CLA
CCC
CGC
CGG
COE
DLM
DLRMHT
SLH
CLHCVH
IARC human carcinogen (group 1: human - sufficient, animal - sufficient)
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CP In Vivo Tests: AnimalsCP In Vivo Tests: Animals
Gene Mutation AssaysGene Mutation Assays– Positive Mouse Spot Test (2.5-10 Positive Mouse Spot Test (2.5-10
mg/kg)mg/kg)– Positive Muta Mouse (lacZ) 100 mg/kg Positive Muta Mouse (lacZ) 100 mg/kg
x 5 days in bone marrowx 5 days in bone marrow
– BB66CC33FF11 mouse (lacI) 100 mg/kg MF mouse (lacI) 100 mg/kg MF increased in lungs and urinary bladder increased in lungs and urinary bladder
– No transgenic studies in ratsNo transgenic studies in rats
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CP In Vivo Tests: HumansCP In Vivo Tests: Humans
– Micronuclei peripheral blood lymphocytes Micronuclei peripheral blood lymphocytes (PBL) & buccal epithelials 26/26 nurses (PBL) & buccal epithelials 26/26 nurses handling CPhandling CP
– Structural chromosome aberrations & SCE, Structural chromosome aberrations & SCE, gene mutations or DNA damage (Comet gene mutations or DNA damage (Comet assay) in PBL or bone marrow, patientsassay) in PBL or bone marrow, patients
– Structural chromosome aberrations in Structural chromosome aberrations in children children
– Mutation of p53 in bladder tumors Mutation of p53 in bladder tumors (cumulative doses of 6-125 mg/kg)(cumulative doses of 6-125 mg/kg)
– 6-Thioguanine-resistant T lymphocytes from 6-Thioguanine-resistant T lymphocytes from multiple sclerosis patients (750 mg/mmultiple sclerosis patients (750 mg/m22))
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So CP Is MutagenicSo CP Is Mutagenic
And it’s carcinogenicAnd it’s carcinogenic
Apply MOA Framework Apply MOA Framework
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Dose Resp ConcordanceDose Resp Concordance
Mutation is key eventMutation is key event– Expect mutations and / or DNA Expect mutations and / or DNA
interaction at lower dose than tumors interaction at lower dose than tumors Mutation is not the key eventMutation is not the key event
– Expect increased mutation at doses Expect increased mutation at doses higher than those required for tumor higher than those required for tumor inductioninduction (the increase in mutations likely results as a
secondary effect of cytotoxicity or cell proliferation)
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CP Dose / Resp ConcordanceCP Dose / Resp Concordance RodentsRodents
– Lowest effective dose Lowest effective dose [induction of SCE in rat [induction of SCE in rat bone marrow (0.62 bone marrow (0.62 mg/kg)]mg/kg)]
– Consistent with data Consistent with data showing significant showing significant tumor formation in the tumor formation in the urinary bladder of male urinary bladder of male rats at 1.25 and 2.5 rats at 1.25 and 2.5 mg/kg/day (488 mg mg/kg/day (488 mg total)total)
HumansHumans– Chromosome Chromosome
aberrations & SCEs 2 aberrations & SCEs 2 hrs after dosing hrs after dosing 33-40 33-40 mg/kgmg/kg
– p53 mutations at a p53 mutations at a cumulative dose of cumulative dose of 6 g6 g
– Cohort of 6171survivors Cohort of 6171survivors of non-Hodgkin's of non-Hodgkin's lymphoma; 48 lymphoma; 48 developed cancer of the developed cancer of the urinary tract – those urinary tract – those receiving a total dose of receiving a total dose of 20g20g had a 2.4-fold had a 2.4-fold risk risk of bladder cancer;of bladder cancer; 20-20-49g49g, a 6, a 6-fold -fold risk risk
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Mutagenic carcinogens would be expected to show a positive Mutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periodsmutation response after relatively short treatment periods
Nonmutagenic carcinogens would be expected to be Nonmutagenic carcinogens would be expected to be negative after long chronic treatment, or show a positive negative after long chronic treatment, or show a positive response only after long chronic treatmentresponse only after long chronic treatment
Temporality: Evaluate time-to-mutationTemporality: Evaluate time-to-mutation
Time in Weeks
Mu
tan
t F
req
uen
cy
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CP TEMPORAL ASSOCIATIONSCP TEMPORAL ASSOCIATIONS
SCE bone marrow of Fischer 344 rats dosed with SCE bone marrow of Fischer 344 rats dosed with 20 mg/kg (ip) CP after 20 mg/kg (ip) CP after 30 min30 min. (. (1 hr1 hr after 5 or 10 after 5 or 10 mg/kg)mg/kg)
Chromosomal aberration & micronuclei in human Chromosomal aberration & micronuclei in human bone marrow bone marrow 24 hrs24 hrs post therapeutic dose of 40 post therapeutic dose of 40 mg/kg (iv)mg/kg (iv)
Cytotoxicity & regenerative proliferation in the rat Cytotoxicity & regenerative proliferation in the rat also occur early:also occur early:– Bladder damage (ulceration of mucosa, necrosis of Bladder damage (ulceration of mucosa, necrosis of
bladder epithelium)—1 daybladder epithelium)—1 day– Regeneration of bladder epithelia – 36 hrsRegeneration of bladder epithelia – 36 hrs– Hyperplasia of bladder epithelia – 48 hrsHyperplasia of bladder epithelia – 48 hrs– Malignant bladder tumors — 40-60 weeks Malignant bladder tumors — 40-60 weeks
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CP Database Plausibility & CP Database Plausibility & CoherenceCoherence
Qualitative & Qualitative & quantitative quantitative data for key events data for key events leading to tumorsleading to tumors
Concordance of most key events in animal Concordance of most key events in animal models & humansmodels & humans
No stop/recovery studies found, but there is No stop/recovery studies found, but there is evidence suggesting that CP-associated evidence suggesting that CP-associated cancers may occur up to several years after cancers may occur up to several years after drug treatment has ceased. drug treatment has ceased.
Gaps in human data (e.g., DNA adducts & cell Gaps in human data (e.g., DNA adducts & cell proliferation) do not compromise the analysis proliferation) do not compromise the analysis
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MOA RelevanceMOA Relevance
RatsRats HumansHumans
PAM generationPAM generation Yes Yes YesYesDNA adductsDNA adducts YesYes PlausiblePlausibleMutagenicityMutagenicity YesYes YesYesBladderBladdercytotoxicitycytotoxicity Yes Yes YesYesEpithelial regenerationEpithelial regeneration Yes Yes
PlausiblePlausibleHyperplasiaHyperplasia Yes Yes YesYesBladder tumorsBladder tumors Yes Yes YesYes
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ChloroformCYP2E1
Phosgene
Regenerative Cell Proliferation
Postulated Mode Of ActionPostulated Mode Of ActionMetabolism
Oxidative
Sustained Toxicity
Tumor DevelopmentKey Events
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CClCCl33 Genetic Activity Profile Genetic Activity Profile
-4
-2
0
2
4
6
8
LOG DOSE UNITS
GENETIC ACTIVITY PROFILE
PROKARY LOW EUK PLNT INS MAMM VITRO HUMAN F MAMM VIVO HU
67-66-3
IARC_V73
1999
CHLOROFORM
BRP
ECL
DSB F
AS
0AS
5AS
7AS
8AS
9AS
SAS
WCE
2CE
GCS
SCH
GNA
SCR
FNA
NCS
ANN
XMD A
ID
PRU
AI
U H9G
CIS
SI
R
T7S
LHU
HI
U
SHL
LHC
BFA
MMH
AVD
RPU
MVU
AVG
SVA
MVM
MVR
CBA
IARC possible human carcinogen (group 2B: human - inadequate, animal - suff icient)
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Mutagenicity: Mutagenicity: Lines of Evidence
Negative Negative in vitroin vitro Conflicting evidence Conflicting evidence in vivoin vivo Initiation-Promotion StudiesInitiation-Promotion Studies
– CCl3 is not an initiatorCCl3 is not an initiator Molecular Based ApproachesMolecular Based Approaches
– NegativeNegative for tumors in p53 +/- transgenic mouse for tumors in p53 +/- transgenic mouse cancer bioassaycancer bioassay
– NegativeNegative for mutations in LacI transgenic B6C3F1 for mutations in LacI transgenic B6C3F1 micemice
– NegativeNegative for mutation in rat GST transfected for mutation in rat GST transfected bacteria bacteria
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Mutagenicity CClMutagenicity CCl33: Conclusions: Conclusions
Weight of EvidenceWeight of EvidenceMutagenicity is not a component Mutagenicity is not a component
of chloroform induced neoplasiaof chloroform induced neoplasia
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Predominate pathwayPredominate pathway– P450 (CYP2E1)-mediated oxidative P450 (CYP2E1)-mediated oxidative
pathway pathway PhosgenePhosgene key reactive metabolitekey reactive metabolite
Metabolism: Metabolism: ConclusionsConclusions
The following play little, if any role in chloroform induced tumors--
–Reductive P450 metabolism & free radical production –GST catalyzed conjugation
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MOA Conclusions for ChloroformMOA Conclusions for Chloroform
Hypothesized MOA Well SupportedOther 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
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ConsiderConsider
What data are available?What data are available?– Screening genetox data, batteries of test Screening genetox data, batteries of test
designed for hazard identificationdesigned for hazard identification What data are optimal?What data are optimal?
– Real, live MOA data (e.g. time course studies in Real, live MOA data (e.g. time course studies in relevant human genes)relevant human genes)
What data are practical?What data are practical?– Something less than what was available for Something less than what was available for
cyclophosphamidecyclophosphamide– Requires some strategic thought in test design. Requires some strategic thought in test design.
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