The development of in vitro testing strategies may achieve a cost-effective generation of comprehensive datasets on a large number of chemicals, according to the requirements of the European Regulation REACH. Much emphasis is placed on in vitro methods based on subcellular mechanisms (e.g., nuclear receptor interaction), but it is necessary to define the predictive value of molecular or biochemical changes within an adverse outcome pathway (AOP). AOP pivots on the description of the flow from a

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... molecular initiating event through a cascade of intermediate events needed to produce a specific adverse effect at organism level: downstream responses at cell level are, therefore, essential to define an AOP. Several in vitro assays are based on human cell lines representative of endocrine-targeted tissues (e.g., prostate) and on functional biomarkers of clinical relevance (e.g., PSA secretion in human prostate epithelial cells). We discuss the implementation of such functional biomarkers in the AOP context.