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.