1. Basic Aspects and Most Commonly Worldwide Employed and Validated In Vitro Assays Leon F. Stankowski, Jr., PhD Consultant, Genetic Toxicology Genotoxicity

2. OECD Guidelines In Vitro Assays Mutation Chromosome Aberration Micronucleus Agenda

3. Mutagen agent that induces a change in the DNA Clastogen agent that induces chromosome breaks (structural aberration) Aneugen agent that induces a change in chromosome number (numerical aberration) Some Terminology

4. Organisation for Economic Cooperation and Development 471 Bacterial Reverse Mutation Test 473 In Vitro Mammalian Chromosome Aberration Test 474 Mammalian Erythrocyte Micronucleus Test 475 Mammalian Bone Marrow Chromosomal Aberration Test 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes 487 In Vitro Mammalian Cell Micronucleus Test 488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays 489 In Vivo Mammalian Alkaline Comet Assay 490 In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene www.oecd.org OECD Guidelines

5. Organisation for Economic Cooperation and Development 471 Bacterial Reverse Mutation Test 473 In Vitro Mammalian Chromosome Aberration Test 474 Mammalian Erythrocyte Micronucleus Test 475 Mammalian Bone Marrow Chromosomal Aberration Test 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes 487 In Vitro Mammalian Cell Micronucleus Test 488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays 489 In Vivo Mammalian Alkaline Comet Assay 490 In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene www.oecd.org OECD Guidelines

6. Apply to all testing situations Some special modifications for human pharmaceuticals ICH - International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ICH S2(R1) www.ich.org OECD Guidelines

7. 471 Bacterial Reverse Mutation Test S. typhimurium and E. coli tester strains Reverse mutation to his or trp independence Basepair (bp) substitution – change in one base for another Frameshift (fs) mutation – insertion or deletion of one or a few a bases Mutation Assays

8. 471 Bacterial Reverse Mutation Test Requires very specific change at G:C or A:T sites to revert to his or trp independence Five tester strains required TA1535 and TA100 (bp) and TA98 (fs) and TA1537 or TA97 or TA97a (fs) and WP2 uvrA or WP2 uvrA (pKM101) or TA102 (bp) Engineered deficient in DNA excision repair error-prone DNA repair function leaky cell walls Mutation Assays

9. 471 Bacterial Reverse Mutation Test Treat 10 8 bacteria/plate (with limited his or trp) 5+ dose levels 5000 µg/plate or solubility/toxicity limit (thinning of lawn) Positive controls (diagnostic) Vehicle control ±S9 All in triplicate Incubate 48 hours Count 2- or 3-fold dose-dependent increase? Mutation Assays

10. CHO/HPRT Hemizygous + Gene Mutation Chromosome Damage -- MLA (TK +/- ) Heterozygous + - Gene Mutation Chromosome Damage - - - - From DeMarini et al. (1989)

11. 490 Mouse lymphoma (MLA) and TK6 Autosomal TK +/- TK +/- (TFT r ) 476 CHO/HPRT assay X-linked Hprt + Hprt - (TG r ) Mutation Assays

12. HPRT and TK Treat 2 - 3 x 10 6 cells 4+ dose levels 2000 µg/mL or 10 mM or solubility/toxicity limit (10 – 20% RS or RTG) Positive and vehicle controls ±S9 All in duplicate Mutation Assays

13. Subculture 2 – 7 days Select mutants with TFT or TG Concurrent cloning efficiency Incubate 7 – 14 days Count Significant, dose-dependent increase >95% control limit or GEF? Mutation Assays

14. 473 In Vitro Mammalian Chromosomal Aberration Test HPBL, RPBL, CHO, CHL cells Structural aberrations breaks/rearrangements (clastogens) Numerical aberrations polyploidy/endoreduplication (aneugens) Cytogenetics Assays

15. 473 In Vitro Mammalian Chromosomal Aberration Test Treat exponentially growing cells stimulate primary cultures with PHA 3+ dose levels 2000 µg/mL or 10 mM or solubility/toxicity limit (40 – 50% RPD, RICC, MI) Positive and vehicle controls Short treatment ±S9 Extended treatment –S9 only Single or duplicate Cytogenetics Assays

16. 473 In Vitro Mammalian Chromosomal Aberration Test Harvest all at ~1.5 cell cycles after start of treatment Arrest cultures in metaphase (Colcemid or colchicine) Score 300 cells per dose level Significant, dose-dependent increase >95% control limit? Cytogenetics Assays Break Exchange CHO Polyploidy

17. 473 In Vitro Mammalian Chromosomal Aberration Test Cytogenetics Assays dicentric HPBL break HPBL

18. 487 In Vitro Mammalian Cell Micronucleus Test HPBL, RPBL, CHO, CHL, TK6 cells Structural aberrations breaks/rearrangements (clastogens) Numerical aberrations polyploidy/endoreduplication (aneugens) Cytogenetics Assays

19. 487 In Vitro Mammalian Cell Micronucleus Test Cytogenetics Assays Double Strand Breaks Spindle Fiber Dysfunction Mitotic Cells Daughter Cells i.e. Clastogensi.e. Aneugens –CytoB

20. 487 In Vitro Mammalian Cell Micronucleus Test Cytogenetics Assays Double Strand Breaks Spindle Fiber Dysfunction Mitotic Cells Daughter Cells i.e. Clastogensi.e. Aneugens +CytoB

21. 487 In Vitro Mammalian Cell Micronucleus Test Treat exponentially growing cells stimulate primary cultures with PHA 3+ dose levels 2000 µg/mL or 10 mM or solubility/toxicity limit (50 – 60% RPD or RICC for cell lines, or CPBI or RI in primary cells and when using CytoB) Positive and vehicle controls Short treatment ±S9 Extended treatment –S9 only Single or duplicate Cytogenetics Assays

22. 487 In Vitro Mammalian Cell Micronucleus Test Harvest all at ~1.5 – 2 cell cycles after start of treatment Arrest cultures in metaphase (Colcemid or colchicine) Score 2000 cells per dose level Significant, dose-dependent increase >95% control limit? Cytogenetics Assays

23. 487 In Vitro Mammalian Cell Micronucleus Test Mechanism of Action Fluorescent In Situ Hybridization (FISH) Antikinetochore staining (CREST) Cytogenetics Assays

24. MN Negative Positive Cytogenetics Assays 487 In Vitro Mammalian Cell Micronucleus Test Mechanism of Action

25. 487 In Vitro Mammalian Cell Micronucleus Test Cytogenetics Assays Metaphase CellC+ MNC– MN Mice and human Any species

26. Thank You! Questions?

27. Thank You! Questions? leon.stankowski@wilresearch.com (31 August 2015)