Table1_Application of Defined Approaches for Skin Sensitization to Agrochemical Products.XLSX

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

皮肤致敏试验是多国开展农药安全评估的法定要求。纳入化学体外法（in chemico）与体外生物法（in vitro）的全球协调试验指南，可减少动物实验的使用，但目前尚无单一试验方法可完全替代动物试验。整合多种非动物试验数据的定义性方法（Defined approaches, DAs）已获监管认可，但此类方法的验证均基于单一成分物质，而非混合物或制剂。为填补这一数据空白，本研究针对27种农药制剂开展了直接肽反应性试验（direct peptide reactivity assay, DPRA）、角蛋白致敏试验（KeratinoSens™ assay）与人细胞系激活试验（human cell line activation test, h-CLAT）检测。将上述检测数据作为输入，本研究评估了3种用于皮肤致敏潜力危害分级的DAs，以及2种用于致敏强度分级的DAs。与历史动物试验结果对比后可见，用于预测体内（in vivo）皮肤致敏危害（即致敏剂与非致敏剂）的DAs，其平衡准确率介于56%至78%之间。表现最优的DA为“2 out of 3（2o3）”方法，其危害分级基于DPRA、KeratinoSens或h-CLAT三项试验中两项结果一致的判定标准。采用h-CLAT与DPRA结果的KE 3/1序贯试验策略（sequential testing strategy, STS），以及整合h-CLAT、DPRA与经济合作与发展组织QSAR工具箱（OECD QSAR Toolbox）计算机模拟（in silico）危害预测结果的整合试验策略（integrated testing strategy, ITSv2），其危害分级的平衡准确率分别为56%~57%。在单项试验方法中，KeratinoSens对体内危害结局的预测性能最优，其81%的平衡准确率与2o3 DA的78%相当。针对联合国全球化学品统一分类和标签制度（United Nations Globally Harmonized System of Classification and Labelling of Chemicals, GHS）定义的致敏强度分级标准，STS的正确分类率为52%，ITSv2为43%。本研究结果表明，相较于动物参考数据，非动物试验方法可有效评估农药制剂的皮肤致敏潜力。尽管尚需补充更多试验数据，但以人体生物学与机制信息为核心的试验策略（如上述DAs）为农药制剂的皮肤致敏性检测提供了极具前景的解决方案。