Non-Negligible Sourceof PFOS: Biochemical Fate andMetabolic Activation of Precursors

Abstract The widespread detection of PFOS precursors in the environment and human matrices raises a new wave of health concerns. However, their absorption, distribution, metabolism, and excretion (ADME) fates and toxicity profiles in mammals remain largely unexplored. Here, we investigated the toxicokinetic, metabolic transformation, and toxicity of three precursors, N-MeFOSA, N-EtFOSA, and N-EtFOSAA, by integrating toxicokinetic modeling in mice, rat liver microsome metabolism, and hepatocellular toxicity assays. N-MeFOSA and N-EtFOSA were rapidly eliminated in vivo and exhibited high efficiencies of conversion to legacy PFAS in liver microsomes, surpassing the typically low transformation rates in mammals reported for other precursors. Notably, N-MeFOSA showed the greatest metabolic transformation potential (25% legacy PFAS and 22% PFOS in 3 h). Moreover, in hepatocellular toxicity assays, both precursors exhibited weaker direct metabolic perturbations than their metabolites (PFOS and FOSA), indicating that their toxicity is primarily enhanced via metabolic activation. In contrast, N-EtFOSAA displayed persistence and stability in vivo and in vitro and induced lipid accumulation comparable to that of PFOS, together with stronger inhibition of cellular energy metabolism. Collectively, our study provides crucial ADME data identifying N-alkyl-FOSA-type precursors as non-negligible internal PFOS sources, underscoring a dual-risk paradigm where toxicity is driven either by metabolic activation or intrinsic parent compound toxicity.