IWGT Group 4

1. IWGT Group 4 Improving in vivo genotoxicity testing- the link to standard toxicity testing Summary of discussion items, conclusions and recommendations Hans-Joerg Martus Novartis Institutes for BioMedical Research Basel, Switzerland

2. Andreas Rothfuss (Chair) Masa Honma (Co-chair) Hans-Joerg Martus (Rapporteur) Marilyn Aardema Brian Burlinson Andreas Czich Sheila Galloway Shuichi Hamada Bob Heflich Jon Howe Peter Kasper David Kirkland Makoka Nakajima Mike O‘Donovan Ulla Plappert-Helbig Les Recio Maik Schuler Yoshifumi Uno IWGT Working Group 4 Group 4: Integration of genotoxicity into routine studies

3. Topics to be addressed • Combination of MN assay and Comet Assay into acute studies • Integration of the MNT into repeated-dose toxicity (RDT) studies • Integration of the Comet Assay into RDT studies • Requirements on top dose for „integrated“ RDT studies • Further tests suitable for integration (not covered due to time constraints) Group 4: Integration of genotoxicity into routine studies

4. Improving in vivo genotoxicity testing • Animal welfare recommendations to reduce animal usage • Reduce “false positive” rate in vitro • Apply smarter testing strategies in vivo • Options for improvement in vivo: Combination and Integration • Combination of acute assays into one stand-alone study. • Integration of genotoxicity endpoints into repeat dose toxicity (RDT) studies • Chances for Improvement beyond 3R’s • Improved genotoxicity risk assessment: Interpretation of genotoxicity results in conjunction with toxicity data and toxicokinetics • More efficient testing using new technologies • Potential for resource savings (compound, manpower) Group 4: Integration of genotoxicity into routine studies

5. Current Recommendations • Linking genotoxicity tests to General Toxicity is not a new concept: Integration of MN assays has been discussed since a while now… • EMS workshop, MacGregor et al., EMM 25:328-337, 1995 • IWGT, Hayashi et al, EMM 35:234-252, 2000 • More recent guidances: • REACh ITS (Integrated Testing Strategy) • Integration of genotoxicity tests (e.g. MN assay, Comet Assay) into repeat-dose tox studies, if scientifically justified • ICH S2(R) draft guidance • Integration of in vivo genotoxicity endpoints into RDT studies preferable, if scientifically justified • If more than one genotoxicity endpoint, incorporation into a single study is preferred. • ECVAM workshopon Animal reduction (Pfuhler et al., submitted) • Integration of MN assay into RDT studies “should be standard if RDT studies are foreseen for the test compound”. • Combination of acute MN and Comet Assay studies into one study. Group 4: Integration of genotoxicity into routine studies

6. Topic 1: Combination of MN assay and Comet Assay into acute studiesExample of study design (Covance) Bone Marrow & Blood Micronucleus + Comet in 3 tissues Dose 1 at 0 hrs Bone Marrow MicronucleusSampled 24 hrsafter 2nd dose Dose 2 at 24 hrs Blood Dose 3 at 45 hrs CometSampled 3hrs after 3rd dose Stomach Liver Sample 48 hrs Bleed:3 hrs Group 4: Integration of genotoxicity into routine studies

7. Topic 1: Combination of MN assay and Comet Assay into acute studies Consensus statements: • Combination of MNT and comet technically feasible and scientifically acceptable as an alternative to the separate assays • Promising results obtained with (mostly) model compounds • 3d or 4d protocols equally acceptable Group 4: Integration of genotoxicity into routine studies

8. Topic 2: Integration of MNT into repeat-dose toxicology (RDT) studies (I) Consensus statements: • Integration of MNT into RDT is scientifically acceptable • There may be situations (e.g. severe bone marrow toxicity) where an acute study is preferable Group 4: Integration of genotoxicity into routine studies

9. Topic 2: Integration of MNT into repeated-dose toxicology (RDT) studies (II) Consensus statements on „Additional early peripheral blood sampling time point on day 4“ • Early sampling not routinely required but can help in study evaluation if data show unsuitability of late sampling time point • If result from terminal sampling is negative and marked myelotoxicity is evident then the additional early sampling timepoint may provide useful data • Early sampling can be advisable to investigate erythropoiesis-related effects Group 4: Integration of genotoxicity into routine studies

10. Topic 2: Integration of MNT into repeated-dose toxicology (RDT) studies (III) Consensus statements on „Effect of Bleeding“ • Animals bled for TK or other purposes can be used for MN analysis • For rats above 9 weeks the current data suggest that bleeding might not affect response to genotoxins as long as MN frequencies in control animals are unaffected • One example exists to suggest that for rats aged 5 weeks bleeding might affect the MN response • It it advisable to use minimal volumes and low frequencies when withdrawing blood to minimize disturbance of erythropoiesis • No data to indicate generation of false-positive results in rats Group 4: Integration of genotoxicity into routine studies

11. Topic 3: integration of comet into RDT studies Consensus statements • Integration of comet into RDT studies • Integration into RDT is considered scientifically acceptable • Liver comet assay complements MNT in blood or bone marrow in detecting in vivo genotoxins • Practical issues need to be considered • Cytotoxicity • Data available so far indicate that cytotoxicity does not generate increases or decreases in DNA migration Group 4: Integration of genotoxicity into routine studies

12. Topic 4: Top dose in RDT studyBackground information: Arguments discussed within ICHS2 revision • Criteria to qualify a repeated dose test (≥ 2 weeks) as acceptable for option 2 (no or positive in vitro mammalian test): • Maximum feasible dose (formulation) • Exposure plateau • Limit dose: 1000 mg/kg • Accumulation with repeated dosing • At least 50% of acute MTD • For aneugens and certain hematotoxic clastogens: • 2 -4 day blood sampling from the multiweek study before substantial hematotoxicity develops • Acute in vivo test • Criteria to disqualify a repeated dose test for option 2: • MTD alone • Reduced exposure to parent drug with time ( 50%) (in that sex) • Typically seen in rats, especially males, and attributable to enzyme induction; not necessarily relevant to human; prevents maximal exposure to parent, although it does provide exposure to metabolite Group 4: Integration of genotoxicity into routine studies

13. Topic 4: Top dose in RDT study Consensus statements • MTD considered acceptable for in vitro negatives • For in vitro positives (or no in vitro data) MTD is acceptable in many cases such as: • RDT MTD (or exposure) close (up to two-fold) to acute MTD (or exposure) • Estimated human exposure is lower by a large margin • If deviating therefrom use proper justification Group 4: Integration of genotoxicity into routine studies

14. Group 4: Overall conclusions • Combination of MNT and comet is scientifically justified for both acute and RDT studies • Most recommendations are based on a limited data set and need to be refined in the future • Future prospects: • Evaluate genotoxins with diverse modes-of-action • Evaluate compounds with extrahepatic target tissues (comet) • Consider involving NTP for future experiments Group 4: Integration of genotoxicity into routine studies