1. 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 Toxicology
National Center for Toxicological Research
Food and Drug Administration
Jefferson, Arkansas
Disclaimer: Not FDA Policy

2. Topics Mutagenic mode of action (How high a burden of proof??)
Overview of genetic toxicology assays—how to weigh the evidence
Strategy for using in vivo mutation for MOA assessment (modified Hill Criteria)
Hazard identification vs. mode of action

3. Three 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?

4. Weigh the Evidence (MOA) How High a Burden of Proof?
Nonmutagenic MOA
Mutagenic MOA
Mixed MOA

5. 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)
Cancer relevant oncogene/tumor suppressor gene mutations can be detected in the target tissue following chemical exposure
Surrogate gene mutations can be detected in the target tissue following chemical exposure
DNA adducts (known to be mutagenic adducts) can be detected in the target tissue following chemical exposure
Primary DNA damage can be detected in the target tissue following chemical exposure
Gene 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.

6. Types of Mutagenic Damage
Point mutations
Deletions
Translocations
Recombination
Gene Conversion
Amplification
Aneuploidy

7. 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

8. Assays that detect genotoxicity but not mutagenicity (primary DNA damage)
DNA adduct analysis
DNA strand breakage
Unscheduled DNA synthesis (UDS)
Sister 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.

9. Assays that detect genotoxicity but not mutagenicity
Chromosome aberration analysis
Micronucleus analysis
Positive in vivo data more significant than in vitro--however a negative in vivo does not automatically out weigh a positive in vitro

10. Some general considerations about chromosomal aberration assays
Damaged chromosomes must replicate and cell division must progress to
metaphase to generate a visible chromosomal aberration (CA)
cell division to generate a micronucleated erythrocyte
Breakage events leading to CAs and MN are generally lethal, and cells that contain them are rapidly eliminated from dividing cell populations
Most visible CAs and MN are not mutations, but the events that generate them also generate mutations

11. Assays that detect mutagenicity
Ames Test
Mouse Lymphoma Assay (MLA)
HPRT mutation in various cell lines or in vivo lymphocytes
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

12. Standard Genetic Toxicology Battery
Ames Test or other bacterial assay
Mouse Lymphoma Tk assay or in vitro cytogenetics
In vivo cytogenetics

13. Genotoxicity Assays Measure Different Genetic Events (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

14. Weight-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).

15. Mutagen = Mutagenic Carcinogen
Cancer is a multi-stage, multiple event disease
Mutation is involved in the etiology of cancer
Cancer is a disease of mutation and cell division
Increased mutations can occur by the induction of new mutations or by increasing the “spontaneous” mutation rate

16. Genotoxicity data informs MOA for cancer risk assessment
Key Question: Does the agent mediate the induction of tumors via a mutational mode of action?

17. MOA Evaluation Should involve
Assessment of mutation in the target tissue
Time 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 Carcinogen
Multiple events
Initiating
Mutation
Tumor
Nonmutagenic Carcinogen
Initiating
Mutation
Multiple events
Toxicity/
Cell Proliferation
Tumor

19. Temporality—time-to-mutation vs time-to-tumor
Predictions
Mutagenic carcinogens would be expected to show a positive mutation response after relatively short treatment periods
Mutant Frequency
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

20. Dose Response Concordance: 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

21. How do you evaluate dose response concordance? (A work in progress)
Visual inspection of the curves
Quantitative modeling to compare mutation and tumor response
Benchmark dose (BMD) (single dose assessment)
Compare the probability of an “adverse” MF response with the probability of tumor response (dose response curve assessment)

22. Unfortunately most of the available in vivo mutation studies were conducted for hazard identification—not optimally designed for MOA evaluation
We present 2 case studies that can be used to demonstrate the general approach
Riddelliine (consistent with mutagenic MOA)
Dichloroacetic Acid (DCA) (consistent with nonmutagenic MOA)

23. Case study 1: Visual inspection and BMD analysis for riddelline: (Consistent with mutagenic MOA)
BMD10(M)=0.16
BMD10(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)=0.22
BMD10(M)=0.97

26. General 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 manifestation
Generally a negative control and 2 doses
Generally the high dose is the MTD and the low dose is approximately ½ the top dose

27. MOA Basic Design Same species/strain as cancer study
Same exposure route as cancer study
Multiple doses (6 or 7 or more) based on tumor data--Enough doses to adequately “define” the dose response curve
Chronic treatment ( up to 12 months) modeled on the tumor bioassay
Interim analysis of MF (to define time-to-mutation)
Detection of mutation in the target tissue(s)
Evaluate the dose response concordance

28. Bottom-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

29. Conclusions
A weight of the evidence assessment of all of the gene-tox data is useful for MOA
The most important question is whether the chemical induces mutation in the target tissue
Future research to assess mutation as a key event should use a MOA design rather than hazard ID design
Further work needed to assess dose response concordance

30. Acknowledgements Robert Heflich, NCTR Mugamine Manjanatha, NCTR
Lynne Haber, TERA