Whole mixture-based testing using in vitro new approach methodologies (NAMs) has been suggested to facilitate the hazard and risk assessment of complex environmental mixtures. Previous studies have shown that phosphorylation of DNA damage signaling proteins checkpoint kinase 1 (pChk1) and histone 2AX (γH2AX) are sensitive markers that can be used for estimating carcinogenicity potencies in vitro. Here, and with the aim to better validate the applicability, in vitro-based Mixture Potency Factors (MPFs) of Standard Reference Materials (SRMs) from environmental polycyclic aromatic hydrocarbon (PAH)-containing mixtures were determined and compared to published mutagenicity and tumorigenicity data. Also, genotoxicity was assessed by a flow cytometry-based micronucleus (MN) assay which showed that only benzo[a]pyrene (B[a]P) and coal tar SRM (SRM1597a) caused dose-dependent increases of MN formation, while extracts of diesel particulate matter (SRM1650b), diesel particulate extract (SRM1975), and urban dust (SRM1649b) did not. However, a dose-dependent activation of DNA damage signaling was observed for all PAHs and SRMs. The results demonstrated that all SRMs were more potent than B[a]P, at B[a]P-equivalent concentrations, to induce pChk1 and γH2AX, and that western blot was more sensitive than the In-Cell Western assay in detecting their activation in response to these complex mixtures. Relative MPFs, based on dose–response modelling of pChk1 and γH2AX, ranged 113 – 5270 for the SRMs, indicating several orders of magnitude higher genotoxic potential than B[a]P. Moreover, these MPFs were in good agreement with potency values based on published data from Salmonella mutagenicity and in vivo carcinogenicity studies. In conclusion, these comparisons further validate the feasibility of applying in vitro NAMs, such as whole-mixture based MPFs, in cancer risk assessment of complex mixtures.

建议使用体外新方法（NAM）进行基于混合物的整体测试，以促进复杂环境混合物的危害和风险评估。先前的研究表明，DNA 损伤信号蛋白检查点激酶 1 (pChk1) 和组蛋白 2AX (γH2AX) 的磷酸化是敏感标记，可用于估计体外致癌性。为了更好地验证其适用性，我们确定了来自环境多环芳烃 (PAH) 混合物的标准参考物质 (SRM) 的体外混合效力因子 (MPF)，并与已发表的致突变性和致瘤性数据进行比较。此外，通过基于流式细胞术的微核（MN）测定评估了遗传毒性，结果表明，只有苯并[ a ]芘（B[ a ]P）和煤焦油SRM（SRM1597a）引起MN形成的剂量依赖性增加，而提取物柴油颗粒物 (SRM1650b)、柴油颗粒提取物 (SRM1975) 和城市灰尘 (SRM1649b) 的含量没有变化。然而，所有 PAH 和 SRM 均观察到 DNA 损伤信号传导的剂量依赖性激活。结果表明，在B[ a ]P 等效浓度下，所有SRM 都比 B[ a ]P 更有效地诱导 pChk1 和 γH2AX，并且蛋白质印迹在检测它们的激活方面比 In-Cell Western 测定更灵敏响应这些复杂的混合物。基于 pChk1 和 γH2AX 的剂量反应模型，SRM 的相对 MPF 范围为 113 – 5270，表明其遗传毒性潜力比 B[ a ]P高几个数量级。此外，这些 MPF 与基于沙门氏菌致突变性和体内致癌性研究已发表数据的效力值非常一致。总之，这些比较进一步验证了体外NAM（例如基于全混合物的 MPF）在复杂混合物癌症风险评估中的可行性。