The Test Guideline (TG) describes the use of liver S9 sub-cellular fraction (RT-S9) of rainbow trout (Oncorhynchus mykiss) as a metabolising system to determine the clearance (CL, IN VITRO, INT ) of a test chemical using a substrate depletion approach. Introduction of the test chemical to the RT-S9 incubation medium initiates the reaction. In order to collect samples at various time points, the reaction is terminated by transferring an aliquot of the medium to a stopping solution. The decrease of the test chemical concentration from the incubation vial is measured with a validated analytical method and used to determine the CL, IN VITRO, INT. The value obtained can then be used to improve in silico predictions of the test chemical bioaccumulation in fish.