Group 1B: Suitable Top Concentration for Tests with Mammalian Cells -MLA Workgroup-

1. Group 1B: Suitable Top Concentration for Tests with Mammalian Cells-MLA Workgroup- 5TH INTERNATIONAL WORKSHOP ON GENOTOXICITY TESTING Basle, August 17-19, 2009

2. Martha Moore Chair Masa Honma Co-chair Julie Clements Rapporteur Takumi Awogi George Douglas Aoi Kimura Wolfgang Muster Mike O’Donovan Rita Schoeny Freddie van Goethem Bhaskar Gollapudi* Shinobu Wakuri* * Unable to attend Affiliations of panel: 4 regulatory, 4 pharma, 2 CRO One representative of the chemical industry in audience Workgroup members

3. Agenda items for discussion • Listing the various regulatory decisions/uses for MLA data • Appropriate Gold Standard to which MLA data should be compared • Re-evaluation of NTP MLA data using the current IWGT recommendations • Recommended top concentration for non pharmaceuticals • Proposals and conclusions

4. Regulatory decisions utilising MLA data • Pre clinical safety evaluation for potential carcinogens • Identification of mutagens (mutation is the endpoint of concern) • Support for hazard identification (weight of the evidence to address question - is chemical a mutagen or carcinogen?) • Use for hazard assessment/Mode of Action and Risk Assessment • Classification and Labelling

5. Discussion of “Gold standard” – to which MLA data should be compared • In vitro gene mutation assays need to be evaluated against repeat dose in vivo mutation assays • Using mutation as a surrogate for cancer does not , and would not be expected to, give a perfect correlation with carcinogenicity • Gold Standard is not readily available

6. Observations • Acceptance criteria and data interpretation (Neg/Pos) for the assay well established • In some circumstances it is important to understand the mechanism/mode of action for mutation induction within the MLA itself – follow up studies

7. Observations 2 • Pharmaceuticals often have high molecular weights • The average molecular weight of the chemicals that go through testing in the chem/agchem world is approximately 250

8. Observations 3 Straw poll • Participants, both workgroup and audience, were asked to indicate their support for the ICH proposal for pharmaceuticals to lower top concentration from 10mM to 1mM • Workgroup 7 For, 3 Against • Audience 9 For, 3 Against

9. NTP MLA Data Re-analysis Bhaskar Gollapudi, Melissa Schisler, & Martha Moore (NOTE: Not workgroup analysis)

10. Re-analysis Approach • So far, analyzed 244 chemicals using data from NTP publications • IWGT criteria, decision rules, and expert judgment used to classify chemical assay responses into • Positive • Equivocal • Inconclusive • Negative • Lack of colony sizing data precluded negative classification (with one exception – water!) • Data were examined for • Background CE (65-120) and MF (35-140) • MF of positive control (IMF >300 at RTG>10%) • Dose selection (at least one dose between 10-20% RTG) • Data consistency

11. Preliminary Data (unaudited) REANALYSIS CALL NTP CALL

12. Take Home Messages • Careful reanalysis of NTP MLA data using current standards is in progress. • A significant proportion of NTP experiments are not valid by current standards • Low background MF • Low induced MF in positive controls • NTP Positive calls were often based on MF data below 10% RTG. • Strict adherence to IWGT acceptance criteria would have resulted in substantially more tests as Inconclusive calls • Approximately 50% of the positive calls in the NTP data may need re-testing. • Use of NTP calls in deriving correlations (e.g., to carcinogenicity) may lead to inaccurate conclusions.

13. IWGT MLA Workgroup Conclusions • Consensus that top concentration for testing (10 mM) needs to be re-considered and further evaluated • Several proposals put forward • Each one discussed and voted upon • Number of workgroup members ready to support proposal based on available information • Number of workgroup members felt proposal had merit and should be further developed, investigated and evaluated

14. Proposal Number 1 • Top concentration 1000 µg/ml or 10 mM whichever is lower (BG proposal) • Number of workgroup members ready to endorse this now – 0 • Number of workgroup members who consider it is a viable proposition warranting further evaluation - 6

15. Proposal Number 2 • Lower top concentration for non pharmaceuticals to 1 mM • Number of workgroup members ready to endorse this now – 6 • Number of workgroup members who consider it is a viable proposition warranting further evaluation - 3

16. Proposal Number 3 • Lower top concentration for non pharmaceuticals to 2 mM • Number of workgroup members ready to endorse this now – 1 • Number of workgroup members who consider it is a viable proposition warranting further evaluation - 6

17. Proposal Number 4 for Complex Mixtures • For complex mixtures, top concentration proposed to be 5000 µg/ml (or as high as considered appropriate in each case) • Number of workgroup members ready to endorse this now – 4 • Number of workgroup members who consider it is a viable proposition warranting further evaluation - 3

18. Issues needing further discussion • Agree on data required and Gold Standard for evaluation • Agree what level of concordance is required • Formulate strategy to obtain data • Make data driven decision