**5. Metabolite safety testing**

Following Phase 1 FIH studies, the blood samples at or near the Cmax from the high dose cohort of the multiple ascending dose study undergo metabolite profiling. The resulting metabolite abundancies are then compared to the preclinical metabolite profiling studies in order to identify human only metabolites or metabolites that are present at higher plasma concentrations in humans than in the animals used in the preclinical studies. These metabolites are referred to as disproportionate drug metabolites. Per the FDA's March 2020 guidance, "In general, these metabolites are of interest if they account for plasma levels greater than 10 percent of total drugrelated exposure, measured as area under the curve at steady state." In the instance that a disproportionate meets these criteria, futher *in vitro* (genotoxicity) and *in vivo* (general toxicity and embryo-fetal development toxicity) studies are required to assess the safety of the metabolite. Typically, these studies can be conducted concurrently with Phase 2 studies prior to the large-scale Phase 3 trials.
