Basic Aspects and Most Commonly Worldwide Employed and Validated In Vitro Assays
Leon F. Stankowski, Jr., PhDConsultant, Genetic Toxicology
Genotoxicity
• OECD Guidelines• In Vitro Assays
• Mutation• Chromosome Aberration• Micronucleus
Agenda
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
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
OECD Guidelines
• 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 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
Mutation Assays
CHO/HPRTHemizygous
+
GeneMutation
ChromosomeDamage
- -
MLA (TK+/-)Heterozygous
+
-
GeneMutation
ChromosomeDamage
-
-
-
-
From DeMarini et al. (1989)
• 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 µg/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?
Mutation 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 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
• 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 CHO
Polyploidy
• 473 In Vitro Mammalian Chromosomal Aberration Test
Cytogenetics Assays
dicentric
HPBLbreak
HPBL
• 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
Cytogenetics Assays
Double Strand Breaks Spindle Fiber Dysfunction
MitoticCells
DaughterCells
i.e. Clastogens i.e. Aneugens
–CytoB
• 487 In Vitro Mammalian Cell Micronucleus Test
Cytogenetics Assays
Double Strand Breaks Spindle Fiber Dysfunction
Mitotic Cells
DaughterCells
i.e. Clastogens i.e. Aneugens
+CytoB
• 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
• 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
MN MN
Negative Positive
Cytogenetics Assays
• 487 In Vitro Mammalian Cell Micronucleus Test• Mechanism of Action
• 487 In Vitro Mammalian Cell Micronucleus Test
Cytogenetics Assays
Metaphase Cell C+ MN C– MN
Mice and human
Any species
Thank You!
Questions?