In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals

Abstract

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties.

In vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes

“…The variability of CL in vitro, int across different laboratories was determined with up to 61% in a recent study using the trout hepatocyte assay (Fay et al, 2014b). Using freshly isolated hepatocytes of individual rainbow trout to measure in vitro metabolic rate values of difficult test compounds, Kropf et al (2020) obtained inter-assay variabilities of 11-59%. In our study, measured total coefficients of variation (CV) of CL in vitro, int ranged from 17.8 to 60.5% for MXC and from 17.4 to 52.5% for BaP.…”

“…However, there is a need to expand the applicability domain of the QSARs by adding more diversity to the chemical training and testing sets, as well by evaluating inter-species differences in biotransformation rate constants. An approach to broaden the database on chemical biotransformation rates under avoidance of animal testing is the use of in vitro assays based on isolated liver cells or liver subcellular (post-mitochondrial) fractions (hepatic S9 fractions) of fish (Segner and Cravedi, 2001;de Wolf et al, 2007;Han et al, 2007;Nichols et al, 2007;Weisbrod et al, 2009;Kropf et al, 2020). These assays determine the depletion of a test compound over time (substrate depletion approach), and provide in vitro intrinsic clearance rates (CL in vitro, int ).…”

Validation of Methods for in Vitro–in Vivo Extrapolation Using Hepatic Clearance Measurements in Isolated Perfused Fish Livers

“…Newer modeling approaches and prediction methods seek to eliminate the use of whole fish on both ethical and economic grounds while enabling the incorporation of experimental data from alternative test methods, including those from recently standardized in vitro assays using isolated rainbow trout ( Oncorhynchus mykiss ) hepatocytes (RT-HEP) or liver subcellular fractions (RT-S9). − Such alternative approaches require extrapolation from a low level of biological organization ( in vitro ) to that of the whole organism ( in vivo ); this process has been termed in vitro–in vivo extrapolation (IVIVE). , The goal of these methods is to create data of sufficient quality for use in environmental risk assessment. However, previous studies have focused on a relatively narrow and well-characterized chemical space (specifically polycyclic aromatic hydrocarbons, PAHs), which is not entirely representative of current risk assessment needs. − Recent studies have sought to expand this understanding using in vitro assays to investigate pesticides, fragrance chemicals, and other emerging contaminants. − Thus, there is a need to expand the validation of IVIVE approaches to a broader chemical space, potentially by using assays of intermediate biological organization to reduce uncertainty.…”

Predicting Hepatic Clearance of Psychotropic Drugs in Isolated Perfused Fish Livers Using a Combination of Two In Vitro Assays

“…This system has been used successfully to study biotransformation of various individual chemicals, including 7-ethoxycoumarin and several polycyclic aromatic hydrocarbons. , However, fish can be exposed to a variety of chemical stressors co-occurring in mixtures, such as pharmaceuticals and personal care products. Due to recent advances in analytical chemistry, it is now possible to operate at low nanomolar concentrations, where interactions among chemicals or saturation of the liver’s biotransformation capacity are not expected . Thus, there is a prospect of potentially measuring the hepatic clearance of dozens of chemicals in isolated perfused livers simultaneously, thereby drastically increasing the throughput and practical value of this model.…”