AOP-informed qIVIVE modelling for liver steatosis using triazoles

• Combining in vitro distribution and PBK models refines qIVIVE prediction. • Insights from liver steatosis AOP can inform the qIVIVE approach. • AOP-based qIVIVE prediction fits observed in vivo results for fat vacuolation. • IVIVE and in vivo dose–response curves result in similar Benchmark Doses. Due to increasing scientific, societal and regulatory demands as well as ethical considerations there is an urgent need for improved animal-free strategies for chemical testing. A promising development in this context is the increased application of in vitro testing and in silico tools. This study aimed at integrating quantitative in vitro to in vivo extrapolation (qIVIVE) with the adverse-outcome pathway (AOP) for liver steatosis. Liver steatosis is an important (toxicological) endpoint which constitutes the first step of metabolic-dysfunction associated steatotic liver disease (MASLD), a growing challenge in the public health sector. Focus was set on the late key event of triglyceride accumulation measured in vitro after exposure of cells to the fungicides propiconazole and tebuconzole, and the corresponding key event of liver fat vacuolation observed in vivo . The qIVIVE approach was facilitated by physiologically based kinetic (PBK) and in vitro distribution models. Concentrations predicted by PBK modelling corresponded well with experimentally determined in vivo plasma and liver concentrations of the fungicides. The in vitro concentration–response data for triglyceride accumulation, when translated to equivalent oral doses, showed good correlation to rodent in vivo data on liver fat vacuolation after oral exposure to propi- and tebuconazole. qIVIVE-derived benchmark dose values were similar to values obtained from the in vivo experiments. This case study confirms the usefulness of integrating AOPs and qIVIVE for adversity prediction particularly with regard to the “replacement” aspect of the 3R principle.