Validation of the in vitro rainbow trout (Oncorhynchus mykiss) liver S9 metabolic assay to assess bioaccumulation potential of chemicals

Methods currently in use to assess bioaccumulation are limited to the in vivo OECD 305 test, QSAR, and computer models, though for many chemicals these models are not valid and outside the domain for certain chemical classes. To improve bioaccumulation estimates in fish, methods to estimate metabolism are critical. An in vitro assay using rainbow trout liver S9 fractions has been developed to predict in vivo fish metabolism. With funding provided from the European Commission, Joint Research Center, Institute for Health and Consumer Protection, European Centre for the Validation of Alternative Methods (JRC/IHCP/ECVAM) and the European Chemical Industry Council (CEFIC), researchers from 5 laboratories are working to standardize the method. Six chemicals were chosen for this initial standardization work, all of which have measured in vivo BCF values: pyrene, 4-n-nonylphenol, dibenzylether, fluroxypyr-methylheptylester, chlorpyrifos and methoxychlor. In phase I, all laboratories utilized a single large pool of S9, with the objective of comparing inter and intra laboratory variability in using this methodology. Pyrene and fluoroxypyr-methylheptylester demonstrate consistent, measureable turnover rates among most of the participating laboratories, whereas results were more varied with the other 4 compounds. The second phase involves investigation of independently-prepared S9 fractions, examining metabolism of the same 6 chemicals, as well as the evaluation of a single S9 pool with 9 additional chemicals. Results to date suggest differences between S9 prepared in different laboratories using the same protocol but different rainbow trout strains. This presentation will provide a summary of the data obtained to-date, discussing factors that potentially account for variability.