Relevance of kinetic interactions and co-formulants for plant protection product liver toxicity in vitro.

Plant protection product (PPP) risk assessment predominantly focuses on the active substances, at least with respect to the regulatory data requirements. The reason is that for threshold values for the ready-to-use formulations one would need significantly more animal data. Given the high number of formulations, this is not a realistic option, be it for capacity, cost, or animal welfare. Thus, any extension of the data for evaluating formulations should ideally first make maximum use of other approaches. Using an adverse outcome pathway (AOP)-guided approach, we, therefore, investigated the potential mixture effects of two active substances (difenoconazole and mandipropamid) by focusing on the qualitative and quantitative toxicokinetic effects on metabolism. Since difenoconazole is a potential liver steatogen, cytotoxicity and liver triglyceride accumulation in HepaRG cells were used as primary endpoints. In addition, transcriptomics and biochemical analyses were conducted to investigate potential effects on gene expression and inhibition of cytochrome P450 (CYP) enzymes. CA was observed for cytotoxicity of the formulated product-equivalent active substance mixture (1:1); the mixture with a higher concentration of the CYP3A4 inhibitor demonstrated a more than additive effect. Furthermore, modulation of the expression and activity of CYP and steatosis-related nuclear receptors, such as PXR, AhR, and CAR, was observed in the mixtures and the formulated product. Overall, the findings highlight that toxicokinetic interactions between active substances play a vital role in PPP mixture effects and that co-formulants also contribute to this effect. Moreover, this study demonstrates that more than additive effects from kinetic interaction require the enhancer to be within the concentration ranges that potentially saturate CYP enzyme activity. In conclusion, the current findings suggest that a comprehensive PPP risk assessment must consider additive effects between active substances as well as the contribution of co-formulants to adverse effects.