Development of an in vitro method for simultaneous evaluation of androgenic activity of chemicals and their metabolites using S9: Application in biocides.

EDCs such as pesticides and biocides may enter the food supply and be ingested by humans. Once absorbed, these substances are metabolized in the liver and subsequently distributed systemically. However, conventional in vitro assays assess only parent compounds, failing to consider the biological activity of metabolites formed through hepatic metabolism. This study integrated the S9 fraction, an enzyme extract from the liver tissue, into the OECD TG 458 assay to analyze Phases I and II metabolic processes. We introduced the non-metabolizable compound STZ as a new positive control, set a 6 h metabolism based on BPA and bicalutamide metabolism, and established metabolic activity criteria using STZ variability. Using this improved method, we evaluated AR activity and metabolic changes of 57 biocides regulated in Korea. The parent and metabolite forms of all biocides were negative for AR agonist activity. In the AR antagonist assays, 24 parent compounds were positive, 7 lost activity after metabolism, and 17 retained activity. This study demonstrated that incorporating hepatic metabolism into in vitro assays can classify EDCs whose toxicity is reduced through metabolism from those that retain endocrine-disrupting potential. Therefore, this approach may improve the identification of EDCs that pose a greater risk to human health.