Genotoxicity Basic Aspects and Most Commonly Worldwide Employed

Genotoxicity Basic Aspects and Most Commonly Worldwide Employed and Validated In Vitro Assays Leon F. Stankowski, Jr., PhD Consultant, Genetic Toxicology Agenda OECD Guidelines In Vitro Assays Mutation Chromosome Aberration Micronucleus

Some Terminology 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) OECD Guidelines

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 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 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 Mutation Assays 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

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 471 Bacterial Reverse Mutation Test Treat 108 bacteria/plate (with limited his or trp) 5+ dose levels 5000 g/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 CHO/HPRT Hemizygous

MLA (TK+/-) Heterozygous + + - Gene Mutation Chromosome Damage

Gene Mutation Chromosome Damage - - -

- - - From DeMarini et al. (1989) Mutation Assays 490 Mouse lymphoma (MLA) and TK6 Autosomal TK+/- TK+/- (TFTr)

476 CHO/HPRT assay X-linked Hprt+ Hprt- (TGr) Mutation Assays HPRT and TK Treat 2 - 3 x 106 cells 4+ dose levels 2000 g/mL or 10 mM or solubility/toxicity limit (10 20% RS or RTG) Positive and vehicle controls S9

All in duplicate Mutation Assays 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? Cytogenetics Assays 473 In Vitro Mammalian Chromosomal Aberration Test HPBL, RPBL, CHO, CHL cells Structural aberrations breaks/rearrangements (clastogens) Numerical aberrations polyploidy/endoreduplication (aneugens)

Cytogenetics Assays 473 In Vitro Mammalian Chromosomal Aberration Test Treat exponentially growing cells stimulate primary cultures with PHA 3+ dose levels 2000 g/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 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? CHO CHO Break Exchange Polyploidy Cytogenetics Assays 473 In Vitro Mammalian Chromosomal Aberration

Test HPBL dicentric HPBL break Cytogenetics Assays 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 487 In Vitro Mammalian Cell Micronucleus Test Double Strand Breaks i.e. Clastogens Mitotic Cells

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

Cells +CytoB Daughter Cells Spindle Fiber Dysfunction i.e. Aneugens Cytogenetics Assays 487 In Vitro Mammalian Cell Micronucleus Test Treat exponentially growing cells stimulate primary cultures with PHA

3+ dose levels 2000 g/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 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 487 In Vitro Mammalian Cell Micronucleus Test

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

Positive Cytogenetics Assays 487 In Vitro Mammalian Cell Micronucleus Test Mice and human Metaphase Cell C+ MN C MN Any species

Thank You! Questions? Thank You! Questions? [email protected] (31 August 2015)

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