The primary objective of this study was to determine if quantitative tPOD values could be derived from chemical exposure studies that followed OECD Test No. 249 (rainbow trout gill cell line) and expanding the assay to rainbow trout liver and intestinal epithelial cell lines. The secondary objective was to determine if tPOD values could be derived from similar studies on rainbow trout liver and gut cell lines. Methylmercury and fluoxetine were the test chemicals. From the resulting data, we compared tPOD data with literature-derived apical BMDs, characterized mechanisms of action through analysis of the transcriptomic data, and compared responses (cytotoxicity and transcriptomic) across the three cell types. By bringing together transcriptomics measurements and tPOD calculations with the OECD Test, and expanding the number of cell lines, we believe that this work can help establish a cost effective in vitro test method that can yield quantitative tPOD values that are protective of in vivo concentrations associated with adverse outcomes while also advancing knowledge of a test chemical’s mechanism of action. Overall design: RNA Sequencing analysis of rainbow trout gill, liver, and gut cell lines exposed to methylmercury (in duplicate) and fluoxetine (in single replicates)