Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line RTgutGC.

Hannah Schug

doi:10.14573/altex.1905032

Abstract

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we showed that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance specific relationship was established which reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by using the obtained in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

Highlights

Fish acute toxicity is an important criterion in chemical screening for product development and environmental hazard and risk assessment, whereby the fish acute toxicity test is conducted according to OECD TG 203 (OECD, 2019)
This study supports the concept of using cell-based assays as predictive tools for fish acute toxicity, in line with already published studies (Tanneberger et al, 2013; Natsch et al, 2018; Fischer et al, 2019)
Despite originating from different laboratories and different fish cell lines (Natsch et al, 2018; the present study), the two far available data sets for fragrances are in excellent agreement and, if combined, provide strong prediction models for fish acute toxicity without the need to use fish

Summary

Introduction

Fish acute toxicity is an important criterion in chemical screening for product development and environmental hazard and risk assessment, whereby the fish acute toxicity test is conducted according to OECD TG 203 (OECD, 2019). An in vivo test is conducted where juvenile or adult fish are exposed to the chemical for 96 h to determine the concentration that causes 50% fish lethality (LC50). This test is required for registration of any chemical produced or imported in amounts ≥ 10 tons per year in the European Union under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (EC, 2006) legislation and requires the sacrifice of at least 42 animals. Because the gill is the primary target of waterborne toxicants and a main site of interaction for shortterm chemical exposure, the RTgill-W1 cell line from rainbow trout (Oncorhynchus mykiss) (Bols et al, 1994) was selected for this approach. L-15/ex is ideal for chemical exposure because the ab-

Methods

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Conclusion