Unravelling the potential use of metabolomics and in vitro-in vivo extrapolation for plant protection product next generation mixture risk assessment.

Plant protection products (PPPs) contain one or more active substances (AS) and several co-formulants. While AS and PPPs are subjected to different approval requirements, the data requirements for PPPs are generally less comprehensive. New approach methodologies (NAMs) have the potential to detect early biological effects, provide mechanistic insight, and support the derivation of points of departure. In this study, in vitro metabolomics was used to assess a currently authorised PPP, its ASmix (i.e. tebuconazole (Teb) + prothioconazole (Pro)), and the ASmix combined with a co-formulant of concern (i.e. Teb + Pro + N,N-dimethyldecan-1-amide (NDA); ASmix+NDA). HepaRG cells were exposed for 24hours to seven concentrations and intracellular metabolites were analysed by liquid chromatography mass spectrometry. The targeted metabolomics dataset comprised of 236 unique intracellular metabolites. All treatments increased short-chain triacylglycerols (TAGs) and ceramides while decreasing carnitine, potentially indicating patterns associated with hepatic steatosis and apoptosis. Principal component analysis was used to derive multivariate metabolomics-based points of departure of 1.92mg/L (PPP), 9.32mg/L (ASmix+NDA), and 22.5mg/L (ASmix). These values were then translated into acceptable operator exposure levels (AOELs) using quantitative in vitro-in vivo extrapolation and uncertainty factors. ASmix-based mixture risk assessment resulted in a hazard index (HI) below 1 indicating no operator risk. However, when PPP-based AOELs were applied, HI exceeded 1, suggesting insufficient protection for operators even with protective equipment. Overall, this NAM-based approach successfully predicts AS and PPP-based mixture effects and can be a valuable tool to refine operator safety assessment in PPP regulation where indicated by a tiered testing strategy.