Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

中文翻译：

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一种基于不良结果途径的方法，用于评估肝毒性农药在体外的脂肪混合作用。

接触复杂的化学混合物需要有效的混合物风险评估的分层策略。作为EuroMix项目的一部分，我们开发了一种基于不良结局途径（AOP）的分析工具箱，以研究诱导肝脂肪变性的化合物伊马替林，噻虫啉和可比丁对人HepaRG肝癌细胞的联合作用。使用基准剂量方法确定化合物特异性的相对效能因子。根据脂肪变性AOP中提出的分子引发事件和关键事件，测试了等价混合物的核受体活化，基因和蛋白质表达以及甘油三酸酯积累。所有这三种化合物都会影响核受体的活性，但不会影响提议的关键基因/蛋白质。用三种不同的方法观察到甘油三酸酯的积累。对于所有测试的组合和终点，混合物的效果与剂量加和的假设一致。在AOP下游研究的不同终点上，化合物特异性RPF保持相似。因此，有可能将测试减少到较小数量的关键测试。结果表明，我们的体外分析工具箱适用于分析化学混合物的脂肪变性效果，该工具箱集成在AOP框架内并与RPF方法结合使用。但是，mRNA结果表明，脂肪变性AOP仍需要改善。可以将测试减少到较小的关键测试范围。结果证明了我们体外分析工具箱的适用性，该工具箱集成在AOP框架内并与RPF方法结合，可用于分析化学混合物的脂肪变性效果。但是，mRNA结果表明，脂肪变性AOP仍需要改善。可以将测试减少到较小的关键测试范围。结果证明了我们体外分析工具箱的适用性，该工具箱集成在AOP框架内并与RPF方法结合，可用于分析化学混合物的脂肪变性效果。但是，mRNA结果表明，脂肪变性AOP仍需要改善。