11

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

22

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

33

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

44

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

55

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

66

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

77

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

88

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?

99

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

1010

ChloroformCYP2E1

Phosgene

Regenerative Cell Proliferation

Postulated Mode Of ActionPostulated Mode Of ActionMetabolism

Oxidative

Sustained Toxicity

Tumor DevelopmentKey EventsKey Events

1111

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

1212

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

1313

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

1414

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

1515

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

1616

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

1717

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

1818

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

1919

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

2020

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

2121

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

2222

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.

2323

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

2424

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

2525

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

2626

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”

2727

CyclophosphamideCyclophosphamide

Alkylating

Cytotoxic

Cytotoxic, alkylating

2828

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

2929

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)

3030

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

3131

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))

3232

So CP Is MutagenicSo CP Is Mutagenic

And it’s carcinogenicAnd it’s carcinogenic

Apply MOA Framework Apply MOA Framework

3333

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)

3434

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

3535

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

3636

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

3737

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

3838

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

3939

ChloroformCYP2E1

Phosgene

Regenerative Cell Proliferation

Postulated Mode Of ActionPostulated Mode Of ActionMetabolism

Oxidative

Sustained Toxicity

Tumor DevelopmentKey Events

4040

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)

4141

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

4242

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

4343

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

4444

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

4545

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.

4646