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

Workgroup members Martha Moore Chair Masa Honma Co-chair Julie Clements Rapporteur Takumi Awogi George Douglas Aoi Kimura Wolfgang Muster Mike ODonovan 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

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

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

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

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

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

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

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

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

Preliminary Data (unaudited) POSITIVEEQUIVO.NEGATIVEINCONCL. POSITIVE NEGATIVE 001 (water!) 37 NTP CALLNTP CALL REANALYSIS CALL

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.

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

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

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

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

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

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