New approach methodologies for in vitro detection of chemical-induced cholestatic liver injury

Cholestasis is a pathological impairment of bile acid homeostasis resulting in accumulation of bile acids in the liver and systemic circulation. Cholestasis is of major concern, in particular in pharmaceutical industry, substantiating the need for human-relevant mechanistic screening tools to identify cholestatic liability early in the drug developmental pipeline. New approach methodologies (NAMs) centred around adverse outcome pathway (AOP) networks are promising tools in this respect. Leveraging human-relevant in vitro models integrated in a battery of activity-based in vitro assays allows to target specific key events along the cholestatic pathway. This approach not only enhances human relevance, but also provides deep mechanistic insights essential for human-relevant hazard assessment. The present paper focuses on the practical development of an AOP network-driven in vitro testing platform tailored for cholestatic hazard assessment. In a first part, the mechanistic foundation of cholestasis is discussed based on state-of-the-art toxicological knowledge. In a second part, a standardized workflow for NAM establishment is proposed, detailing the selection of human-relevant in vitro models, and a curated library of activity-based in vitro assays as well as reference chemicals, alongside optimized exposure parameters.