Evaluation of a potential mutagenic MOA based on analysis

of the weight of evidence and using the modified Hill criteria

Martha M. Moore, Ph.D.Director, Division of Genetic and Reproductive ToxicologyNational Center for Toxicological ResearchFood and Drug AdministrationJefferson, Arkansas

Disclaimer: Not FDA Policy

TopicsTopics

Mutagenic mode of action (How high a Mutagenic mode of action (How high a burden of proof??)burden of proof??)

Overview of genetic toxicology assays—Overview of genetic toxicology assays—how to weigh the evidencehow to weigh the evidence

Strategy for using in vivo mutation for Strategy for using in vivo mutation for MOA assessment (modified Hill Criteria)MOA assessment (modified Hill Criteria)

Hazard identification vs. mode of actionHazard identification vs. mode of action

Three Steps (Questions)Three Steps (Questions) To Determine if Mutagenic MOA To Determine if Mutagenic MOA

for Tumor Inductionfor Tumor Induction

Potential mutagen? Hazard IdentificationPotential mutagen? Hazard Identification (Evaluated using standard mutation assays)(Evaluated using standard mutation assays)

In vivo rodent/human mutagen?In vivo rodent/human mutagen? (Mutagenic in the target tissue?)(Mutagenic in the target tissue?)

Is mutation a “key” event in the Is mutation a “key” event in the development of the tumor? development of the tumor?

Weigh the Evidence (MOA)Weigh the Evidence (MOA)How High a Burden of Proof?How High a Burden of Proof?

Nonmutagenic MOA

Mutagenic MOA

Mixed MOA

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.

Types of Mutagenic DamageTypes of Mutagenic Damage

Point mutationsPoint mutations

DeletionsDeletions

TranslocationsTranslocations

RecombinationRecombination

Gene ConversionGene Conversion

AmplificationAmplification

AneuploidyAneuploidy

Definitions:

Genotoxicity: Damage to the DNA or chromosome. Interactions with DNA (DNA adducts).

Mutagenicity: Damage to the DNA or chromosome structure (or number) that is passed to daughter cells.

Generally, the key question is whether the test agent is a mutagen

Assays that detect genotoxicity but Assays that detect genotoxicity but not mutagenicity (primary DNA not mutagenicity (primary DNA

damage)damage)

DNA adduct analysisDNA adduct analysisDNA strand breakageDNA strand breakageUnscheduled DNA synthesis Unscheduled DNA synthesis (UDS)(UDS)Sister chromatid exchangeSister chromatid exchange

These assays indicate whether the test agent has the capability to reach and interact with DNA or chromosomes

Positive in vivo data more significant than in vitro.

Assays that detect genotoxicityAssays that detect genotoxicity

but not mutagenicity but not mutagenicity

Chromosome aberration analysisChromosome aberration analysis Micronucleus analysisMicronucleus analysis

Positive in vivo data more significant than in vitro--however a negative in vivo does not automatically out weigh a positive in vitro

Some general considerations about Some general considerations about chromosomal aberration assayschromosomal aberration assays

Damaged chromosomes must replicate and cell Damaged chromosomes must replicate and cell division must progress todivision must progress to metaphase to generate a visible chromosomal metaphase to generate a visible chromosomal

aberration (CA)aberration (CA) cell division to generate a micronucleated cell division to generate a micronucleated

erythrocyteerythrocyte

Breakage events leading to CAs and MN are Breakage events leading to CAs and MN are generally lethal, and cells that contain them are generally lethal, and cells that contain them are rapidly eliminated from dividing cell populationsrapidly eliminated from dividing cell populations

Most visible CAs and MN are not mutations, but the Most visible CAs and MN are not mutations, but the events that generate them also generate mutationsevents that generate them also generate mutations

Assays that detect Assays that detect mutagenicitymutagenicity

Ames TestAmes Test

Mouse Lymphoma Assay (MLA)Mouse Lymphoma Assay (MLA)

HPRT mutation in various cell lines or in HPRT mutation in various cell lines or in vivo lymphocytesvivo lymphocytes

Transgenic rodents (Big Blue, GPT/Delta)Transgenic rodents (Big Blue, GPT/Delta)

Positive in vivo data more significant than in vitro--however a negative in vivo does not automatically out weigh a positive in vitro

Standard GeneticStandard Genetic Toxicology Battery Toxicology Battery

Ames Test or other bacterial assay

Mouse Lymphoma Tk assay or in vitro cytogenetics

In vivo cytogenetics

Genotoxicity Assays MeasureGenotoxicity Assays MeasureDifferent Genetic EventsDifferent Genetic Events

(One should not expect to obtain the same results with all assays)(One should not expect to obtain the same results with all assays)

Mutation Ames MN Chrom Abs MLA

Point Mutation yes no no yes

Insertion/Deletion yes no no yes

Allele Loss no no no yes

Multi-locus Mutation no no no yes

Small Chrom. Damage no yes no yes

Large Chrom. Damage no yes yes yes

Aneuploidy no yes yes yes

Weight-of-evidence assessmentWeight-of-evidence assessment

Need to assess each genotoxicity Need to assess each genotoxicity experiment for quality/consistency of data experiment for quality/consistency of data (just because it is published does not mean (just because it is published does not mean that the data is valid).that the data is valid).

Need to integrate the data from the various Need to integrate the data from the various assays based upon an understanding of the assays based upon an understanding of the assays (what types of events they do and assays (what types of events they do and do not detect).do not detect).

Mutagen = Mutagenic CarcinogenMutagen = Mutagenic Carcinogen

Cancer is a multi-stage, multiple event Cancer is a multi-stage, multiple event diseasedisease

Mutation is involved in the etiology of Mutation is involved in the etiology of cancercancer

Cancer is a disease of mutation and cell Cancer is a disease of mutation and cell divisiondivision

Increased mutations can occur by the Increased mutations can occur by the induction of new mutations or by increasing induction of new mutations or by increasing the “spontaneous” mutation ratethe “spontaneous” mutation rate

Genotoxicity data informs MOA for cancer risk assessment

Key Question: Does the agent mediate the induction of tumors via a mutational mode of action?

MOA Evaluation Should involveMOA Evaluation Should involve

Assessment of mutation in the target Assessment of mutation in the target tissuetissue

Time to mutation (temporality)Time to mutation (temporality)

Dose response concordance (Mutation Dose response concordance (Mutation and tumor induction)and tumor induction)

Initiating

Mutation Tumor

Multiple events

Multi-Stage Tumor Induction Requires the Accumulation of Events over Time

(Many/most rodent carcinogens require long chronic exposure)

Toxicity/

Cell Proliferation

InitiatingMutation

Multiple events

Tumor

Mutagenic Carcinogen

Nonmutagenic Carcinogen

Mutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periods

Nonmutagenic carcinogens would be expected to be negative after long chronic treatment, or show a positive response only after long chronic treatment

Temporality—time-to-mutation vs time-to-tumor

Time in Weeks

Mu

tan

t F

req

uen

cy

Predictions

If mutation is THE key event:

The mutation dose response will lead the tumor dose response

If the tumor dose response leads the mutation dose response or the mutation response is negative after long chronic exposures

Consistent with mutation is not the key event

Dose Response Concordance:

Predictions

How do you evaluate doseHow do you evaluate dose response concordance? response concordance?

(A work in progress)(A work in progress)

Visual inspection of the curvesVisual inspection of the curves

Quantitative modeling to compare mutation Quantitative modeling to compare mutation and tumor responseand tumor response

Benchmark dose (BMD) (single dose Benchmark dose (BMD) (single dose assessment)assessment)

Compare the probability of an “adverse” Compare the probability of an “adverse” MF response with the probability of tumor MF response with the probability of tumor response (dose response curve response (dose response curve assessment)assessment)

Unfortunately most of the available in Unfortunately most of the available in vivo mutation studies were conducted vivo mutation studies were conducted for hazard identification—not optimally for hazard identification—not optimally

designed for MOA evaluation designed for MOA evaluation

We present 2 case studies that can be used to We present 2 case studies that can be used to demonstrate the general approachdemonstrate the general approach

Riddelliine (consistent with mutagenic MOA)Riddelliine (consistent with mutagenic MOA)

Dichloroacetic Acid (DCA) (consistent with Dichloroacetic Acid (DCA) (consistent with nonmutagenic MOA)nonmutagenic MOA)

Case study 1: Visual inspection and BMD analysis for riddelline: (Consistent with mutagenic MOA)

Riddelliine

Dose (mg/kg/day)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Can

cer

inci

denc

e (%

) at

104

wee

ks

0

20

40

60

80

100

0

20

40

60

80

100Observed mutation frequencyPredicted mutation frequencyObserved cancer incidencePredicted cancer incidence

Mut

ant F

requ

ency

( x

10-

6 ) a

t 12

wee

ks

BMDU10(M)=0.28BMDL10(C)=0.30

BMD10(M)=0.16 BMD10(C)=0.39

Case study 2: Visual inspection and BMD analysis for DCA (60-week exposure for MF)(Consistent with nonmutagenic MOA)

Dichloroacetic Acid

Dose (g/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0M

uta

nt

Fre

qu

en

cy (

x 1

0-6)

at

60

we

eks

0

20

40

60

80

100

0

20

40

60

80

100

Observed mutation frequencyPredicted mutation frequencyObserved cancer incidencePredicted cancer incidence

Can

cer

Inci

de

nce

(%

) at

10

0 w

eeks

BMDU10(C)=0.26BMDL10(M)=0.54

BMD10(C)=0.22 BMD10(M)=0.97

General Features of the Hazard General Features of the Hazard Identification DesignIdentification Design

Acute exposure (single or small # of Acute exposure (single or small # of doses)doses)

IWGT recommended design is 28 days IWGT recommended design is 28 days treatment and 3 days for mutant treatment and 3 days for mutant manifestationmanifestation

Generally a negative control and 2 dosesGenerally a negative control and 2 doses

Generally the high dose is the MTD and Generally the high dose is the MTD and the low dose is approximately ½ the top the low dose is approximately ½ the top dosedose

MOA Basic DesignMOA Basic Design Same species/strain as cancer studySame species/strain as cancer study

Same exposure route as cancer studySame exposure route as cancer study

Multiple doses (6 or 7 or more) based on tumor data--Multiple doses (6 or 7 or more) based on tumor data--Enough doses to adequately “define” the dose response Enough doses to adequately “define” the dose response curvecurve

Chronic treatment ( up to 12 months) modeled on the Chronic treatment ( up to 12 months) modeled on the tumor bioassaytumor bioassay

Interim analysis of MF (to define time-to-mutation)Interim analysis of MF (to define time-to-mutation)

Detection of mutation in the target tissue(s)Detection of mutation in the target tissue(s)

Evaluate the dose response concordanceEvaluate the dose response concordance

Bottom-line Questions for MOA Bottom-line Questions for MOA AssessmentAssessment

What happens?What happens? When does it happen?When does it happen? At what dose does it happen?At what dose does it happen?

This information can then be used to This information can then be used to develop a timeline for the various develop a timeline for the various events and also to understand the events and also to understand the dose response curves for the various dose response curves for the various eventsevents

ConclusionsConclusions A weight of the evidence assessment of all of A weight of the evidence assessment of all of

the gene-tox data is useful for MOA the gene-tox data is useful for MOA

The most important question is whether the The most important question is whether the chemical induces mutation in the target chemical induces mutation in the target tissuetissue

Future research to assess mutation as a key Future research to assess mutation as a key event should use a MOA design rather than event should use a MOA design rather than hazard ID design hazard ID design

Further work needed to assess dose response Further work needed to assess dose response concordanceconcordance

AcknowledgementsAcknowledgements

Robert Heflich, NCTRRobert Heflich, NCTR Mugamine Manjanatha, NCTRMugamine Manjanatha, NCTR Lynne Haber, Lynne Haber, TERATERA