1. Group 6 Follow-up of in vivo positive results Follow-up of 2005 IWGT and subsequent work done by HESI IVGT on follow-up of in vitro positive results. Takes into consideration the new data and information available on dose-response curves and mode of action. 5 TH INTERNATIONAL WORKSHOP ON GENOTOXICITY TESTING Basel, August 17-19, 2009

2. NameRegione-mail 1Jan Van BentemEurope, NetherlandsRegulatoryJan.van.Benthem@rivm.nl 2Ricardo CrebelliEurope, ItalyRegulatorycrebelli@iss.it 3Kerry DearfieldUSARegulatoryKerry.Dearfield@fsis.usda.gov 4George DouglasCanadaRegulatoryGeorge_Douglas@hc-sc.gc.ca 5Peter FarmerEurope, UKAcademicpbf1@leicester.ac.uk 6Elmar GockeEurope, SwitzerlandIndustryElmar.gocke@roche.com 7Baskhar GollapudiUSAIndustryBBGollapudi@dow.com 8Makoto HayashiJapanRegulatoryhayashi@anpyo.or.jp 9David LovellEurope, UKAcademicD.Lovell@surrey.ac.uk 10Werner LutzEurope, GermanyAcademiclutz@toxi.uni-wuerzburg.de 11Takehiko NohmiJapanAcademicnohmi@nihs.go.jp 12Jim MacGregor, Rapporteur USAConsultantjtmacgregor@earthlink.net 13Daniel MarzinEurope, FranceRegulatorydaniel.marzin@pasteur-lille.fr 14Martha MooreUSARegulatoryMMMoore@nctr.fda.gov 15David PhillipsEurope, UKAcademicDavid.Phillips@icr.ac.uk 16Lutz Műller, Co-Chair Europe, SwitzerlandIndustryLutz.mueller@roche.com 17V é ronique Thybaud, Chair Europe, FranceIndustryveronique.thybaud@sanofi- aventis.com Tripartite group: three main regions, Scientists from academic laboratories, regulatory authorities and industry Participants of In vivo follow-up workgroup Not able to attend

3. Topics: Topic 1: Use of in vitro results in the design and interpretation of in vivo assays Topic 2: Quantitative aspects of the dose response curve Topic 3: Evaluation and impact of Mode of Action Topic 4: Update on in vivo models C*: agreement both on principle and wording No C*: agreement on principle, wording to be refined (lack of time). 5 TH IWGT in Basel, 2009 Group 6: Follow-up of positives results

4. Topic 1 Use of in vitro results in the design and interpretation of in vivo assays 2005 IWGT and IVGT flow charts Kerry Dearfield 5 TH IWGT in Basel, 2009 Group 6: Follow-up of positives results

5. IVGT Review group: Flow chart for follow-up actions

6. Topic 1: Statements The endpoint studied in vivo needs to be appropriate, e.g., the same as that found increased in vitro or a surrogate shown to be appropriate for predicting that same endpoint. It is recognized that some systems are not specific to a single endpoint, and this must be taken into account. C* The selection of tissue(s) should consider tissue exposure, ADME information compound metabolism, and other toxicological considerations. C* If in vitro test(s) are positive, and in vivo tests are conducted, the risk can be considered to be negligible if follow-up test(s) in appropriate tissues and endpoints in vivo at appropriate doses show that the in vitro results are probably not of concern in vivo. C* 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

7. Topic 2: Quantitative aspects of the dose response curve Viracept case study: Genotoxicity data in animals, dose-response curves and statistical assessment, and risk assessment for the human exposure. Elmar Gocke and Lutz Müller Quantitative aspects of in vivo risk and the potential role of new flow cytometric genetic toxicity assays Jim MacGregor Analysis of dose-response curves (threshold, NOEL, etc) Werner Lutz Potential modifiers of dose-response curves. Could additional effects (e.g. induction of cell proliferation) lower the apparent threshold? Véronique Thybaud 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

8. Topic 2: Statements For risk assessment – in vivo follow up of positives (after review of Viracept data): Transgenic animals are acceptable surrogates to internal genes for risk assessment. C* For risk assessment, internal dose is a key element, and may include: –Cmax/AUC –Surrogate endpoints, DNA/protein adducts. Nonlinear response curves can be shown in vivo, even with DNA-reactive agents, but must be demonstrated with appropriate data. –Statistical scrutiny must be applied. 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

9. Topic 2: Statements Hazard-screening is sufficient for public health protection if current screening tests (in vitro mutation and chromosomal aberration plus in vivo chromosomal damage) are negative, but when in vivo genotoxicity is identified then additional in vivo data are necessary to define risk in relation to exposure. Dose and exposure metrics must be justified in each situation. Cross-species extrapolation should consider the same factors used for other toxicity endpoints: e.g., relative metabolism, PK differences, surface area scaling, plus DNA repair differences and relative apoptosis efficiency. C* Secondary factors (i.e., potential modifiers of dose-response curves) such as cell proliferation may modify mutagenic responses, therefore risk assessment must take this into account. C* –The impact of the secondary mechanisms) may be tissue specific. C* 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

10. Topic 2: Statements Agents documented to induce genetic damage via interaction with non-DNA targets are expected to exhibit a threshold below which damage does not occur. –For such threshold mechanisms, the NOGEL is appropriate metric to which additional safety margins may be applied. For DNA-reactive genotoxicants: –DNA primary damage can be used for exposure, while stable mutations should be used for risk assessment. –Some agents may exhibit a [practical threshold]: i.e., a dose below which exposure does not add significantly to background rates of DNA damage. –Additional data are necessary to determine if generalizations can be made to define practical thresholds of concern for genotoxic/mutagenic damage. –Consensus is needed about appropriate analytical (statistical) methods of defining thresholds and risk levels (e.g., margin of exposure). 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

11. Topic 3 : Evaluation and impact of Mode of Action Genotoxic versus non-genotoxic mechanisms: feed-back from Genotoxic and Carcinogenic Thresholds in Tokyo. Takehiko Nohmi Use of in vivo mutation data to inform MOA for cancer. Martha Moore Examples of species, tissue and high dose specific effect, and mechanistic studies. Daniel Marzin Evaluation (and regulation) of in vivo positives with adequate negative carcinogenicity data: example a substance recently evaluated in an European regulatory body. Riccardo crebelli 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

12. Topic 3: Statements The better the information and data (e.g., MOA) the more certainty you will have in interpreting the dose-response curve. This leads to less uncertainty when determining an acceptable exposure level. C* In case of MOA analysis and extrapolation to human: –All data should be used, not only genotoxicity data. –In vitro studies may be designed to address mechanistic questions, and aid extrapolation to human. 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

13. Topic 3: Statements Mode of action of individual compounds is decisive for risk assessment. Mechanisms underlying the shape of the dose- response curve should be investigated more thoroughly in vitro and in vivo. Genotoxicity should be examined in target organs of chemical carcinogens, using the same species and strains, when possible. 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

14. Topic 3: Statements (may need more work) In vivo mutagenicity can be associated with adverse effects other than cancer, and this endpoint warrants consideration in risk assessment. –Negative carcinogenicity data may not give by default reassurance on the lack of genotoxicity in vivo for in vitro genotoxins. –For in vitro genotoxins the possibility of somatic (and eventually germ cell) effects in vivo is to be considered, even in presence of negative carcinogenicity data. 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

15. Topic 4: Update on in vivo Models Current status of F344 gpt delta rats for in vivo mutagenesis. Takehiko Nohmi Comparison between transgenes and endogenous genes. George Douglas 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

16. Topic 4: Statements TGR assays provide data of comparable quality and predictivity for carcinogenicity compared to other standard mutagenicity tests. They can be used effectively to follow up results of other in vivo tests, (as well as in vitro tests). TGR assays fill a need in current regulatory practices (e.g. in vivo follow-up). Need to continue development of in vivo assays, esp. multi-endpoint, multi-species assays (including Human). Promising assays include: –Pig-a, flow cytometric micronucleus assays –gptΔ rats and mice. 5 TH IWGT in Basel, 2009 Follow-up of in vivo positives results

17. Thank you! 5 TH INTERNATIONAL WORKSHOP ON GENOTOXICITY TESTING Basel, August 17-19, 2009