Abstract
Nepetalactone (NL) is a volatile iridoid monoterpene widely used in biopesticidal and repellent applications, yet its toxicokinetic behavior and metabolic fate as a pure compound remain poorly characterized. This study aimed to provide an integrated toxicokinetic evaluation of NL by combining in silico absorption, distribution, metabolism, excretion and toxicity (ADMET) modeling with in vitro metabolism assays using rat and human liver microsomes, supported by UHPLC-MS/MS analysis for metabolite identification. The in silico biotransformation predicted extensive phase I oxidation followed by phase II conjugation, while ADMET predictions indicated low systemic persistence and limited toxicological concern for most metabolites. The performed in vitro microsomal assays confirmed the in silico prediction by a rapid and time-dependent NL metabolism via both oxidative (86% reduction in NL concentration after 120 min) and conjugative (89% reduction in NL concentration after 120 min) pathways in rat and human systems, with comparable depletion kinetics between species. UHPLC-MS/MS enabled the identification of multiple phase I and phase II metabolites, pointing to pronounced interspecies differences in conjugative metabolism. In this sense, while oxidoreduction and hydrolysis reactions were consistent with previously reported iridoid metabolism. This study suggests the possible formation of previously unreported amino acid-related derivatives, although these require further confirmation. Overall, these findings advance the understanding of NL biotransformation, propose a new, previously unknown, metabolic pathway for iridoids, and provide relevant data to support human health and environmental risk assessment frameworks.