4.2.3.3.1 — Genotoxicity In Vitro

Bacterial reverse mutation assay (Ames test) and in vitro mammalian chromosomal aberration or micronucleus test

Requirements by Phase

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

Content Requirements

• In vitro non-mammalian cell system: bacterial reverse mutation assay (Ames test)
• In vitro mammalian cell system: chromosomal aberration or micronucleus test
• Standard battery per ICH S2(R1)
• GLP compliance required

Expected Deliverables

• Ames test study report (GLP)
• In vitro chromosomal aberration or micronucleus test report (GLP)
• Genotoxicity summary table

ICH Guidelines: S2(R1)

Regulatory Requirements (FDA IND PHASE 2 3 CMC)

• [Phase 2] All components used in the manufacture of the drug product should be identified by their established names and a reference to a quality standard.

Regulatory Requirements (ICH M3R2)

• If genotoxicity endpoints are incorporated into a general toxicity study, the maximum dose should be MFD, MTD, or 1000 mg/kg/day limit dose.
• [Phase 1 (multiple dose)] For multiple-dose clinical development trials, an additional assessment for chromosomal damage in a mammalian system(s) should be completed.
• [before Phase 2] A complete battery of genotoxicity tests should be completed before initiation of Phase II trials.
• If a positive genotoxicity finding occurs, an assessment and possibly additional testing should be conducted to determine appropriateness of further human administration.
• [before Phase 1] All female reproduction toxicity studies and the standard battery of genotoxicity tests should be completed before including WOCBP not using highly effective birth control or with unknown pregnancy status in any clinical trial.
• [before Phase 1] Before including pregnant women in clinical trials, all female reproduction toxicity studies and the standard battery of genotoxicity tests should be conducted.
• [before Phase 1] Repeated-dose toxicity studies of appropriate duration in adult animals, the core safety pharmacology package, and the standard battery of genotoxicity tests should be available before initiation of trials in pediatric populations.
• Carcinogenicity studies are not recommended to support pediatric clinical trials unless there is a significant cause for concern (e.g., genotoxicity evidence, pro-carcinogenic risk).
• Combination genotoxicity, safety pharmacology, or carcinogenicity studies are generally not recommended if individual agents have been tested according to current standards.

Regulatory Requirements (ICH Q3AR2)

• [Development] If considered desirable, a minimum screen (e.g., genotoxic potential) should be conducted. A study to detect point mutations and one to detect chromosomal aberrations, both in vitro, are considered an appropriate minimum screen.

Regulatory Requirements (ICH Q5B)

• [Development, Registration] For extrachromosomal expression systems, the percent of host cells retaining the expression construct should be determined.
• [Development, Registration] For extrachromosomal expression systems, the expression construct should be isolated and the nucleotide sequence encoding the product should be verified without further cloning.
• [Development, Registration] For cells with chromosomal copies of the expression construct, the nucleotide sequence encoding the product could be verified by recloning and sequencing of chromosomal copies.

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.

Regulatory Requirements (ICH S9)

• [Marketing application] Genotoxicity studies should be performed to support marketing (see ICH S2).
• [Nonclinical/Marketing] For biopharmaceuticals, the principles outlined in ICH S6 should be followed for genotoxicity.

Source: ICH S2(R1)