On November 27,2024, FDA released a draft guidance intended “to inform industry and the review staff in the Center for Drug Evaluation and Research (CDER) on how CDER views positive findings in the in vitro bacterial reverse mutation (Ames) test of a drug (active ingredient) or its metabolites and to provide recommendations on follow-up in vitro and in vivo mutagenicity testing of Ames-positive active ingredients to support the enrollment of healthy human subjects in first-in-human (FIH) clinical trials.”
Background
The Ames test is a standard assay used during drug candidate screening. Positive results in the Ames test are usually grounds for discontinuation of the development of molecules intended for noncancer indications. However, some Ames-positive candidates possess sufficiently beneficial pharmacological properties for the intended clinical application that there is a strong desire to continue development of a candidate drug into human trials. For those assets, the FDA has provided new guidance that lays out testing and analysis frameworks for deciding whether the candidate can proceed into Phase 1 testing in healthy volunteers.
FDA Recommendations for Confirming an Ames Positive Finding
The guidance, entitled “Recommended Follow-Up Testing for an Ames-Positive Drug (Active Ingredient) or Metabolite to Support First-in-Human Clinical Trials with Healthy Subjects” indicates that the first step upon receiving a positive Ames finding should be to confirm the veracity of the positive Ames findings. Did the assay proceed as expected? Did cytotoxicity or precipitation limit the number of analyzable concentrations such that a dose-response could be established? Did solvent controls perform as expected and did positive results clearly exceed concurrent and historical controls?
FDA Recommendations for Ames-Positive Drugs
If the positive Ames test is considered reliable, the FDA recommends evaluating potential mitigating factors. Mitigating factors may include the identification of causative bacteria-specific metabolic products (nitro-reduction) or rat-specific metabolites produced by S9 that are not relevant to humans. If information exists on the molecular functional group contributing to the positive finding, identification of uniformly negative carcinogenicity data for that functional group on other molecules with the same functional group could eliminate concerns for mutagenicity risks for healthy volunteers in Phase 1 trials.
If mitigating factors are not identified, then the FDA recommends that the potential for the drug to cause mutations in mammalian assays be conducted. In vivo assays such as the erythrocyte Pig-a gene mutation assay or a transgenic rodent somatic and germ cell gene mutation assay are recommended by the FDA. Negative results in either assay would eliminate concerns for mutagenicity risks for healthy volunteers in Phase 1 trials. However, Sponsors may wish to take a staged approach employing in vitro assays with quicker readouts such as the mouse lymphoma assay or the hypoxanthine-guanine phosphoribosyl transferase forward mutation assay to determine whether conducting the suggested in vivo assays are worthwhile as both in vivo assays use 28-day dosing regimens before assaying for mutations.
The same framework as is discussed above for active pharmaceutical ingredients applies to unique human metabolites that are also subject to Ames testing requirements.
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