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

Bioaccumulation assessment predominantly relies on the bioconcentration factor (BCF) as the sole decisive metric. The test guideline 305 by the Organisation for Economic Co‐operation and Development (OECD) provides the standard procedure for deriving this in vivo fish BCF, which is not only expensive and labor‐intensive, but also requires many animals. Accordingly, there is a great need for and interest in alternative methods that can help to reduce, replace, and refine vertebrate tests, as described in the 3R principles. Two alternative approaches have been developed: the bioconcentration test with the freshwater amphipod Hyalella azteca and the OECD test guideline 319 which provides a method to determine experimentally derived in vitro metabolism rates that can then be incorporated into in silico prediction models for rainbow trout BCF calculation. In the present study both alternative methods were applied to 5 substances of different physicochemical characteristics. The results were compared with literature values of fish in vivo BCFs and additional BCFs obtained with the alternative methods, if available. Potential differences between the results of the test methods are discussed utilizing information such as in vivo metabolism rates. The currently available data set suggests that these 2 alternative methods pose promising alternatives to predict bioaccumulation in fish, although defined applicability domains have yet to be determined. Environ Toxicol Chem 2020;39:1813–1825. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC

Section: Research Needs Of the Ivive Methodsmentioning

confidence: 99%

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

Kosfeld

Ebersbach

et al. 2020

“…In vitro metabolic stability assays with human or rodent liver preparations became a standard system in human toxicology and pharmacology for the prediction of hepatic clearance of drugs. However, these in vitro systems tend to underpredict in vivo rates of biotransformation (Wood et al 2017), and this may apply for piscine in vitro systems as well (Han et al 2009; Laue et al 2014; Saunders et al 2019). One possible mechanism behind the underprediction could be technical issues.…”

Section: Resultsmentioning

confidence: 99%

“…The metabolic rate values of in vitro metabolic stability assays are sensitive to the assay conditions. Avoiding underestimation of in vitro intrinsic clearance rates requires careful selection of substrate concentration (low enough not to saturate the metabolic enzymes but high enough to meet analytical sensitivity requirements), of biological material concentration (low enough to minimize nonspecific substrate binding to protein but high enough to ensure adequate substrate depletion), and of incubation time (short enough to ensure the stability of enzyme activities but long enough to be able to adequately measure loss of substrate) (Lo et al 2015; Nichols et al 2018b; Saunders et al 2019).…”

Section: Resultsmentioning

confidence: 99%

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

Kropf

Begnaud

Gimeno³

et al. 2020

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.

“…To our knowledge, the effect of PMSF on liver carboxylases from fish has not been evaluated. We note, however, that several chemical classes of interest with respect to bioaccumulation assessment contain ester bonds, including pyrethroid pesticides (Nishi et al 2006), phthalate esters (Mentlein and Butte 1989), and some UV‐blocking agents (Saunders et al 2019). Interactions of PMSF with CYP enzymes are also possible (Sevrioukova 2019).…”

Section: Discussionmentioning

confidence: 99%

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates

Nichols

Hoffman

Swintek

et al. 2020

Self Cite

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish.