This laboratory has reported that the combined use of MicroFlow and MultiFlow assays provides information regarding chemicals’ genotoxic mode of action (MoA). In an effort to go beyond MoA assessments, we incorporated a panel of biological response modifiers that elicit specific effects on the assays’ biomarker response profiles. This was done to pursue our hypothesis that such perturbation signatures would reveal information on clastogenic mechanisms and molecular targets. For this proof-of-concept study, we exposed TK6 cells to 20 previously identified clastogens. TK6 cells in 96-well plates in the presence and absence of each of four modifying agents at one optimized concentration: talazoparib (PARP inhibitor), MK-8776 (CHK1 inhibitor), AZD-7648 (DNA-PK inhibitor), or a cocktail of reactive oxygen species scavengers. In parallel, cells were also exposed to each of the test chemicals for 4 hr, at which time cells were washed and allowed to recover for an additional 20 hours. For each of these treatment conditions, sample processing and flow cytometric analyses were performed using standard MicroFlow and MultiFlow procedures to measure micronuclei, γH2AX, phosphohistone-H3 (p-H3), p53 activation, and relative nuclei counts. The resulting biomarker response data were processed with PROAST benchmark dose (BMD) software, with modifying agent as a covariate. Unsupervised hierarchical clustering of the collective potency metrics for various combinations of biomarkers showed that clastogens with similar genotoxic mechanisms grouped together. Overall, this study shows that in combination with biological response modifiers, MultiFlow and MicroFlow biomarkers can provide mechanistic insights into chemical-induced genotoxicity.

New approach methodologies (NAMs) used to assess chemicals for biological effects are typically designed to limit the amount of test article required, and to promote efficiencies such as compatibility with liquid handlers, etc. This is certainly true in the case of genetic toxicology, where many methods have been and continue to be developed with 96- or 384-well plate processing in mind. However, one recognized concern with microwell plates is that the volatility of test substances and/or their metabolites and/or their degradation products may affect adjacent wells. Here, we describe an approach that combines breathable membranes as well as activated carbon filters to mitigate volatility issues in 96 well plates. These experiments were performed with cyclophosphamide-exposed TK6 cells, and utilized the biomarkers micronuclei, p53, gammaH2AX, phospho-histone H3, and nuclei to counting bead ratios to both demonstrate volatility impact, and to assess the effectiveness of the solution described herein.