Liver steatosis induced by per- and polyfluoroalkyl substances exerts a limited influence on heterocyclic aromatic amine-mediated DNA damage at population-relevant exposure levels.

The human population is chronically exposed to complex mixtures of environmental contaminants. However, classical toxicological risk assessment still relies mainly on individual compound testing, often at high concentrations and over short treatment durations that poorly reflect real-life exposure. In this study, we investigated per- and polyfluoroalkyl substances (PFAS) and heterocyclic aromatic amines (HAA) mixtures at concentrations aligned with human serum measurements using mechanistic knowledge of each compound. To evaluate mixture effects, we employed Hepoid®, a 3D human liver model composed of polarized, proliferative, metabolically active, and highly differentiated HepaRG cells for long-term cultures. At human population internal exposure levels, PFAS and HAAs, alone or in combination, induced steatosis measured by automated lipid droplet quantification along with early transcriptional stress responses, without increased DNA damage under mixture conditions, as assessed by 3 complementary genotoxicity and mutagenesis assays (comet assay, γH2AX immunolabelling and micronucleus assay). Overall, this study highlights the importance of concentrations, treatment duration and human-relevant metabolic competence in determining toxic outcomes. By reproducing realistic exposure scenarios in an advanced human liver model sensitive to low-dose mixture effects, this work contributes to improving human relevance of chemical risk assessment.