Musengi Yemurai, Suciu Ilinca, Tralau Tewes, Bloch Denise
Department of Pesticides Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Straße 8-10, 10589, Berlin, Germany.
Department of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Alle 114-116, 14558, Nuthetal, Germany.
Arch Toxicol. 2025 Apr 28. doi: 10.1007/s00204-025-04071-7.
Plant protection product (PPP) risk assessment predominantly focuses on the active substances, at least with respect to the regulatory data requirements. The reason is that for threshold values for the ready-to-use formulations one would need significantly more animal data. Given the high number of formulations, this is not a realistic option, be it for capacity, cost, or animal welfare. Thus, any extension of the data for evaluating formulations should ideally first make maximum use of other approaches. Using an adverse outcome pathway (AOP)-guided approach, we, therefore, investigated the potential mixture effects of two active substances (difenoconazole and mandipropamid) by focusing on the qualitative and quantitative toxicokinetic effects on metabolism. Since difenoconazole is a potential liver steatogen, cytotoxicity and liver triglyceride accumulation in HepaRG cells were used as primary endpoints. In addition, transcriptomics and biochemical analyses were conducted to investigate potential effects on gene expression and inhibition of cytochrome P450 (CYP) enzymes. CA was observed for cytotoxicity of the formulated product-equivalent active substance mixture (1:1); the mixture with a higher concentration of the CYP3A4 inhibitor demonstrated a more than additive effect. Furthermore, modulation of the expression and activity of CYP and steatosis-related nuclear receptors, such as PXR, AhR, and CAR, was observed in the mixtures and the formulated product. Overall, the findings highlight that toxicokinetic interactions between active substances play a vital role in PPP mixture effects and that co-formulants also contribute to this effect. Moreover, this study demonstrates that more than additive effects from kinetic interaction require the enhancer to be within the concentration ranges that potentially saturate CYP enzyme activity. In conclusion, the current findings suggest that a comprehensive PPP risk assessment must consider additive effects between active substances as well as the contribution of co-formulants to adverse effects.
植物保护产品（PPP）风险评估主要聚焦于活性物质，至少在监管数据要求方面是这样。原因在于，对于即用型制剂的阈值，人们需要大量更多的动物数据。鉴于制剂数量众多，无论是从能力、成本还是动物福利角度来看，这都不是一个现实的选择。因此，评估制剂的数据的任何扩展，理想情况下首先应最大程度地利用其他方法。因此，我们采用不良结局途径（AOP）指导的方法，通过关注对代谢的定性和定量毒代动力学效应，研究了两种活性物质（苯醚甲环唑和霜霉威）的潜在混合效应。由于苯醚甲环唑是一种潜在的肝脂肪生成剂，因此将HepaRG细胞中的细胞毒性和肝脏甘油三酯积累用作主要终点。此外，还进行了转录组学和生化分析，以研究对基因表达和细胞色素P450（CYP）酶抑制的潜在影响。观察到配制产品等效活性物质混合物（1:1）的细胞毒性存在协同作用；含有较高浓度CYP3A4抑制剂的混合物表现出超相加效应。此外，在混合物和配制产品中观察到了CYP以及与脂肪变性相关的核受体（如PXR、AhR和CAR）的表达和活性的调节。总体而言，研究结果突出表明，活性物质之间的毒代动力学相互作用在PPP混合效应中起着至关重要的作用，并且辅剂也对此效应有贡献。此外，本研究表明，动力学相互作用产生的超相加效应要求增强剂处于可能使CYP酶活性饱和的浓度范围内。总之，当前研究结果表明，全面的PPP风险评估必须考虑活性物质之间的相加效应以及辅剂对不良反应的贡献。
