Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial

Abstract

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. 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 hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was 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) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. 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 inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

Comparison of Alternative Methods for Bioaccumulation Assessment: Scope and Limitations of In Vitro Depletion Assays with Rainbow Trout and Bioconcentration Tests in the Freshwater Amphipod Hyalella azteca

“…In vitro metabolism assays using isolated primary hepatocytes or liver S9 subcellular fractions from fish have been introduced as promising and reliable tools to generate hepatic biotransformation rates of xenobiotics, which can be used for in vitro-in vivo extrapolation (IVIVE) of BCFs (Fay et al 2014b(Fay et al , 2017Nichols et al 2018). Standard protocols for the isolation of hepatocytes and S9 fractions from rainbow trout have been developed (Han et al 2007;Johanning et al 2012;Fay et al 2014a), and new OECD test guidelines for the performance of in vitro assays have recently become available (Organisation for Economic Co-operation and Development 2018a, 2018b).…”

“…The resulting intrinsic clearance rate values can then be extrapolated to the in vivo whole-body biotransformation rate constant (k MET ) of the test compound as part of an IVIVE approach (Nichols et al 2006(Nichols et al , 2013Cowan-Ellsberry et al 2008;Fay et al 2014a). Incorporating such information into established bioaccumulation models for fish was shown to substantially improve their performance, leading to predicted BCF values that are generally closer to measured values from in vivo studies than in silico-based predictions obtained assuming no metabolism (Han et al 2007;Cowan-Ellsberry et al 2008;Nichols et al 2018). In addition to the determination of metabolic rates, the in vitro hepatocyte assay may provide important information on the metabolite patterns of xenobiotics in fish.…”

Concentration dependence of in vitro biotransformation rates of hydrophobic organic sunscreen agents in rainbow trout S9 fractions: Implications for bioaccumulation assessment

“…This information is scaled to the intact liver to estimate in vivo hepatic clearance, which is then extrapolated to a whole‐body biotransformation rate constant (Han et al , ; Cowan‐Ellsberry et al ; Laue et al ; Trowell et al ). In vitro biotransformation assays developed for rainbow trout have been shown to be highly reliable (Nichols et al ), leading to recent adoption of these methods by the Organisation for Economic Co‐operation and Development in test guidelines 319A and 319B (2018a, 2018b). Results to date show that BCFs calculated by incorporating measured in vitro biotransformation rates into established BCF models are much closer to measured values than BCFs generated assuming no biotransformation (Han et al , ; Cowan‐Ellsberry et al ; Laue et al ).…”

Performance of Three‐Dimensional Rainbow Trout (Oncorhynchus mykiss) Hepatocyte Spheroids for Evaluating Biotransformation of Pyrene

“…The same study estimated CL in vivo for S9 fractions (2603 mL/h/g liver) to be 2.6 times higher than the hepatocytebased assays. This suggests that the S9 fraction is more efficient than the cryopreserved hepatocyte (Nichols et al 2018) and the spheroid-based assays presented herein.…”

“…Despite the substantial amount of pyrene adhering to the glass vial and thus being less available for the spheroids, this was not the rate-limiting factor for metabolism of pyrene ( Figure 3 and Supplemental Data, Figure S4). Other studies using cryopreserved primary hepatocytes (Nichols et al 2018) have not reported issues with recovery of dissolved pyrene during their exposures. This might be caused by the presence of higher dissolved protein content (hepatocytes 2.85 ± 0.34 mg/mL; Nichols et al 2018) in the exposure vial than in the present study (spheroids 0.0211 mg/mL; calculations based on Baron et al 2012).…”