In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT‐HEP) or hepatic S9 fractions (RT‐S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT‐HEP and RT‐S9 assays for difficult test chemicals, and the in vitro–based predictions were compared to in silico–based predictions and in vivo–measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design‐of‐experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro–in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF