The possible biotransformation of a test chemical may strongly impact the extent to which it can accumulate in fish. Therefore, the incorporation of in vitro biotransformation data into established models of chemical accumulation is needed to improve model performance and thereby provide more realistic bioconcentration factors (BCFs).
One approach that can be used to generate this necessary in vitro metabolic information is the use of biotransformation systems derived from liver tissue such as liver S9 sub-cellular fractions (S9). These S9 fractions contain the enzymes responsible for both Phase I (e.g., cytochrome P450 (CYP)) and Phase II biotransformation (e.g., sulfotransferases (SULTs), uridine 5'-diphospho-glucuronosyltransferases (UGTs) and glutathione transferases (GSTs)) and are therefore ideal for biotransformation studies.
Based on procedures already applied in the pharmaceutical industry, general applicable methodologies have been developed to generate this in vitro biotransformation data and these methodologies make use of S9 obtained from rainbow trout (Oncorhynchus mykiss) and rely on the determination of the in vitro intrinsic clearance (CL, IN VITRO, INT) of a test chemical using a substrate depletion approach. The use of rainbow trout S9 (RT-S9) to determine the CL, IN VITRO, INT has been described in detail in OECD Test Guideline 319B.
Charles River has extensive experience performing in vitro metabolism studies in S9, and we now also offer these in vitro metabolism studies using RT-S9 to determine in vitro intrinsic clearance of test chemicals according to this OECD Test Guideline 319B. Our general setup for these types of studies is described below:
- An analytical method for the quantification of the test chemical in incubation mixtures will be developed and validated. Preliminary experiments (e.g., substrate range finding and protein-time-dependency) will be performed to optimize the reaction conditions to ensure that main experiments will be performed using first-order kinetics. At least two main incubation experiments will be performed during which the decrease of the test chemical in time will be evaluated to determine the t1/2 and CL, IN VITRO INT values.
- Negative control incubations with enzymatically inactive RT-S9 and positive control incubations with standard substrates for Phase I and Phase II metabolism will be included. If significant metabolism is observed, additional experiments and/or analyses can be performed to investigate which metabolites have been formed. Studies can be performed using both non-labeled and radiolabeled test chemicals.
By offering this service, we aim to help our clients to better predict the bioconcentration of test chemicals by rainbow trout and thereby offer a tool to use biotransformation data in the tiered approach for bioaccumulation assessment. For more information or questions about this service, contact us at [email protected].