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Reliability of In Vitro methods used to measure intrinsic clearance of hydrophobic organic chemicals by rainbow trout: Results of an international ring trial
Citation:
Nichols, J., K. Fay, M. Bernhard, I. Bischof, J. Davis, M. Halder, J. Hu, K. Johanning, H. Laue, D. Nabb, C. Schlechtriem, H. Segner, J. Swintek, J. Weeks, AND M. Embry. Reliability of In Vitro methods used to measure intrinsic clearance of hydrophobic organic chemicals by rainbow trout: Results of an international ring trial. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 164(2):563-575, (2018). https://doi.org/10.1093/toxsci/kfy113
Impact/Purpose:
The use of in vitro data in a regulatory setting requires that methods used to generate this information are shown to be reliable. In this context, “reliability” refers to both repeatability (the ability of one user to generate the same result) and reproducibility (the ability of multiple users to generate the same result). This paper describes the results of an international ring trial involving 6 laboratories, each of which evaluated 6 test chemicals using two in vitro metabolizing systems: cryopreserved trout hepatocytes (RT-HEP) and trout liver S9 fractions (RT-S9). The results show that assays performed using RT-HEP and RT-SR are highly reliable, and suggest that either system may be used with confidence to inform modeled bioaccumulation predictions for fish. Importantly, this ring trial highlights a number of issues related to the demonstration of assay reliability, including aspects of study design, assessment of starting biological material, and the analysis of experimental findings. As such, this effort provides a template that could be employed to evaluate other in vitro assays, including those used to measure intrinsic clearance in humans. Standardized OECD test guidelines for in vitro determination of intrinsic hepatic clearance using RT-HEP and RT-S9 are currently under review. The results of this ring trial provide critical information needed as part of the guideline approval process.
Description:
In vitro assays used to measure intrinsic clearance are widely employed to provide information needed for toxicokinetic modeling efforts. However, the reliability of these methods is seldom evaluated. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in fish. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved rainbow trout hepatocytes and rainbow trout liver S9 fractions. Intra- and inter-laboratory variability were characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells). Inter-laboratory CVs for each chemical ranged from 14.9% to 41.6% for hepatocytes and from 9.5% to 44.3% for S9 fractions, while intra-laboratory CVs tended to be lower. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO,INT; L/d/kg fish), both assays yielded similar levels of activity (< 4-fold difference for all chemicals). Hepatic clearance rates (CLH; L/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to support bioaccumulation modeling efforts with fish. This study highlights a number of issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.
URLs/Downloads:
DOI: Reliability of In Vitro methods used to measure intrinsic clearance of hydrophobic organic chemicals by rainbow trout: Results of an international ring trial