Presentation on theme: "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,"— Presentation transcript:
1Evaluation of a potential mutagenic MOA based on analysis of the weight of evidence and using the modified Hill criteriaMartha M. Moore, Ph.D.Director, Division of Geneticand Reproductive ToxicologyNational Center for Toxicological ResearchFood and Drug AdministrationJefferson, ArkansasDisclaimer: Not FDA Policy
2Topics Mutagenic mode of action (How high a burden of proof??) Overview of genetic toxicology assays—how to weigh the evidenceStrategy for using in vivo mutation for MOA assessment (modified Hill Criteria)Hazard identification vs. mode of action
3Three Steps (Questions) To Determine if Mutagenic MOA for Tumor Induction Potential mutagen? Hazard Identification(Evaluated using standard mutation assays)In vivo rodent/human mutagen?(Mutagenic in the target tissue?)Is mutation a “key” event in the development of the tumor?
4Weigh the Evidence (MOA) How High a Burden of Proof? Nonmutagenic MOAMutagenic MOAMixed MOA
5How 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 exposureSurrogate gene mutations can be detected in the target tissue following chemical exposureDNA adducts (known to be mutagenic adducts) can be detected in the target tissue following chemical exposurePrimary DNA damage can be detected in the target tissue following chemical exposureGene mutations and/or DNA adducts or other measures of primary DNA damage can be detected in vivo.Evidence that the chemical can induce mutations, cytogenetic damage, DNA adducts and/or primary DNA damage in vitro.
6Types of Mutagenic Damage Point mutationsDeletionsTranslocationsRecombinationGene ConversionAmplificationAneuploidy
7Definitions: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
8Assays that detect genotoxicity but not mutagenicity (primary DNA damage) DNA adduct analysisDNA strand breakageUnscheduled DNA synthesis (UDS)Sister chromatid exchangeThese assays indicate whether the test agent has the capability to reach and interact with DNA or chromosomesPositive in vivo data more significant than in vitro.
9Assays that detect genotoxicity but not mutagenicity Chromosome aberration analysisMicronucleus analysisPositive in vivo data more significant than in vitro--however a negative in vivo does not automatically out weigh a positive in vitro
10Some general considerations about chromosomal aberration assays Damaged chromosomes must replicate and cell division must progress tometaphase to generate a visible chromosomal aberration (CA)cell division to generate a micronucleated erythrocyteBreakage events leading to CAs and MN are generally lethal, and cells that contain them are rapidly eliminated from dividing cell populationsMost visible CAs and MN are not mutations, but the events that generate them also generate mutations
11Assays that detect mutagenicity Ames TestMouse Lymphoma Assay (MLA)HPRT mutation in various cell lines or in vivo lymphocytesTransgenic 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
12Standard Genetic Toxicology Battery Ames Test or other bacterial assayMouse Lymphoma Tk assay or in vitro cytogeneticsIn vivo cytogenetics
13Genotoxicity Assays Measure Different Genetic Events (One should not expect to obtain the same results with all assays)MutationAmes MN Chrom Abs MLAPoint Mutation yes no no yesInsertion/Deletion yes no no yesAllele Loss no no no yesMulti-locus Mutation no no no yesSmall Chrom. Damage no yes no yesLarge Chrom. Damage no yes yes yesAneuploidy no yes yes yes
14Weight-of-evidence assessment Need to assess each genotoxicity experiment for quality/consistency of data (just because it is published does not mean that the data is valid).Need to integrate the data from the various assays based upon an understanding of the assays (what types of events they do and do not detect).
15Mutagen = Mutagenic Carcinogen Cancer is a multi-stage, multiple event diseaseMutation is involved in the etiology of cancerCancer is a disease of mutation and cell divisionIncreased mutations can occur by the induction of new mutations or by increasing the “spontaneous” mutation rate
16Genotoxicity data informs MOA for cancer risk assessment Key Question: Does the agent mediate the induction of tumors via a mutational mode of action?
17MOA Evaluation Should involve Assessment of mutation in the target tissueTime to mutation (temporality)Dose response concordance (Mutation and tumor induction)
18(Many/most rodent carcinogens require long chronic exposure) Multi-Stage Tumor Induction Requires the Accumulation of Events over Time(Many/most rodent carcinogens require long chronic exposure)Mutagenic CarcinogenMultiple eventsInitiatingMutationTumorNonmutagenic CarcinogenInitiatingMutationMultiple eventsToxicity/Cell ProliferationTumor
19Temporality—time-to-mutation vs time-to-tumor PredictionsMutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periodsMutant FrequencyTime in WeeksNonmutagenic carcinogens would be expected to be negative after long chronic treatment, or show a positive response only after long chronic treatment
20Dose Response Concordance: Predictions If mutation is THE key event:The mutation dose response will lead the tumor dose responseIf the tumor dose response leads the mutation dose response or the mutation response is negative after long chronic exposuresConsistent with mutation is not the key event
21How do you evaluate dose response concordance? (A work in progress) Visual inspection of the curvesQuantitative modeling to compare mutation and tumor responseBenchmark dose (BMD) (single dose assessment)Compare the probability of an “adverse” MF response with the probability of tumor response (dose response curve assessment)
22Unfortunately most of the available in vivo mutation studies were conducted for hazard identification—not optimally designed for MOA evaluationWe present 2 case studies that can be used to demonstrate the general approachRiddelliine (consistent with mutagenic MOA)Dichloroacetic Acid (DCA) (consistent with nonmutagenic MOA)
23Case study 1: Visual inspection and BMD analysis for riddelline: (Consistent with mutagenic MOA) BMD10(M)=0.16BMD10(C)=0.39
24(Consistent with nonmutagenic MOA) Case study 2: Visual inspection and BMD analysis for DCA (60-week exposure for MF)(Consistent with nonmutagenic MOA)BMD10(C)=0.22BMD10(M)=0.97
26General Features of the Hazard Identification Design Acute exposure (single or small # of doses)IWGT recommended design is 28 days treatment and 3 days for mutant manifestationGenerally a negative control and 2 dosesGenerally the high dose is the MTD and the low dose is approximately ½ the top dose
27MOA Basic Design Same species/strain as cancer study Same exposure route as cancer studyMultiple doses (6 or 7 or more) based on tumor data--Enough doses to adequately “define” the dose response curveChronic treatment ( up to 12 months) modeled on the tumor bioassayInterim analysis of MF (to define time-to-mutation)Detection of mutation in the target tissue(s)Evaluate the dose response concordance
28Bottom-line Questions for MOA Assessment What happens?When does it happen?At what dose does it happen?This information can then be used to develop a timeline for the various events and also to understand the dose response curves for the various events
29ConclusionsA weight of the evidence assessment of all of the gene-tox data is useful for MOAThe most important question is whether the chemical induces mutation in the target tissueFuture research to assess mutation as a key event should use a MOA design rather than hazard ID designFurther work needed to assess dose response concordance
30Acknowledgements Robert Heflich, NCTR Mugamine Manjanatha, NCTR Lynne Haber, TERA