An Analysis of Potential Limitations of in Vitro Cell Lines and in Silico Models in Current Chemical Risk Assessments for Mixtures: A Case Study on Azole Mixtures in Sweden

Chemical Risk Assessments (CRAs) are a well-established method to calculate safe levels of chemicals, determined using whole animal testing (in vivo). The European Chemicals Agency (ECHA) are working towards implementing chemical safety using non-animal testing methods, i.e. in vitro acute toxicity testing with fish embryos, fish cell-lines, and in silico mathematical models. In vitro methods are useful, yet they have limitations as fish embryos and cell-lines can exhibit dissimilar metabolism capacity compared to whole, and adult fish. Therefore, we have explored the correlation between in vitro and in vivo test types, where we used azoles as a case study. We studied the toxicokinetic interaction on the detoxification pathway when a fish liver cell-line Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) was exposed to the mixture of an azole and a polycyclic aromatic hydrocarbon (PAH) and created an in silico model to demonstrate the toxicokinetics using the detoxification enzyme Cytochrome P450 1A (CYP1A). In Study I (unpublished), we used Clotrimazole (CLO) and Benzo[a]pyrene (BaP) in our chemical mixture. This resulted in a 7.3-fold increase in CYP1A activity after a 24-hour exposure with mixture of 5nM BaP and 5µM CLO, and a 6.9-fold increase after 36-hour exposure with the mixture of10 nM BaP and 5µM CLO. This indicates a toxicokinetic interaction and inhibition of CLO on the biotransformation of BaP. In Study 2 (Paper I), we created a mathematical bottom-up model for a synergistic mixture effect, with CYP1A activity data of the mixture of the azole Nocodazole (NOC) and the PAH β-naphthoflavone (BNF). The model uses ordinary differential equations (ODEs) to describe the toxicokinetic pathway, and interaction. In Study 3 (Paper II), we show a moderate correlation between embryonic in vitro and in vivo LC50 values and that although detected azole concentrations indicate a risk in Sewage Treatment Plant (STP) effluent and surface water, their concentrations are well below the concentrations seen to have synergistic effects seen in fish cell-line in vitro studies