4.2.3.3 — Genotoxicity

Genotoxicity studies: in vitro (Ames, chromosomal aberration) and in vivo (micronucleus)

Requirements by Phase

IND Phase 1: required IND Phase 2: required IND Phase 3: required NDA: required

Regulatory Requirements (ICH S2R1)

• [Preclinical (before marketing authorization)] Completion of either standard test battery option (negative results, appropriate protocols, adequate evaluation) provides sufficient assurance of absence of genotoxic activity, and no additional tests are warranted.
• [Preclinical] In vivo genotoxicity endpoints should be incorporated into toxicity studies for repeated administrations, if scientifically justified.
• [Preclinical] A single bacterial mutation (Ames) test is considered sufficient if it is clearly negative or positive, and carried out with a fully adequate protocol.
• [Preclinical] For in vitro cytogenetic assays (metaphase chromosome aberrations or micronuclei), cytotoxicity should not exceed a reduction of about 50% in cell growth.
• [Preclinical] For the Mouse Lymphoma L5178Y Cell Tk Gene Mutation Assay (MLA), at the top dose there should be 80-90% cytotoxicity as measured by an RTG between 20-10%.
• [Preclinical] Small increases in apparent genotoxicity (statistically significant but within historical control confidence intervals, or weak/equivocal and not reproducible) are not considered biologically meaningful, and no further testing is called for.
• [Preclinical] Positive results in the Ames test are thought to indicate DNA reactivity, and extensive follow-up testing to assess in vivo mutagenic and carcinogenic potential would be warranted.
• [Preclinical] If in vitro mammalian cell positive results occur only under conditions not occurring in vivo (pH, osmolality, precipitates) or only at the most toxic concentrations (≥80% RTG for MLA, ≥50% growth suppression for cytogenetics), a single in vivo test is considered sufficient.
• [Preclinical] Negative results in appropriate in vivo assays (two, with adequate justification for the endpoints measured and demonstration of exposure) are considered sufficient to demonstrate absence of significant genotoxic risk.
• [Preclinical] For in vivo studies, it is considered sufficient to treat animals with a positive control only periodically, and not concurrently with every assay, after a laboratory has established competence in the use of the assay.

Source: ICH S2(R1)

Cross-References: Positive results may require follow-up with Carcinogenicity studies. Summarized in the Toxicology Written Summary.