We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay.

Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow V R assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): gH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow V R micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow V R assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow V R assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a labspecific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow V R . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow V R assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens.

The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound‐induced increases in MN frequency can be detected using the cytokinesis‐block (cytochalasin B) method in V79 cells after 24 h in a 96‐well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti‐kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound‐induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non‐genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227–242, 2019. © 2018 Wiley Periodicals, Inc.

The early detection of genotoxicity contributes to cutting‐edge drug discovery and development, requiring effective identification of genotoxic hazards posed by drugs while providing mode of action (MoA) information in a high throughput manner. In other words, there is a need to complement standard genotoxicity testing according to the test battery given in ICH S2(R1) with new in vitro tools, thereby contributing to a more in‐depth analysis of genotoxic effects. Here, we report on a proof‐of‐concept MoA approach based on post‐translational modifications of proteins (PTMs) indicative of clastogenic and aneugenic effects in TK6 cells using imaging technology (with automated analysis). Cells were exposed in a 96‐well plate format with a panel of reference (geno)toxic compounds and subsequently analyzed at 4 and 24 hr to detect dose‐dependent changes in PTMs, relevant for mechanistic analysis. All tested compounds that interfere with the spindle apparatus yielded a BubR1 (S640) (3/3) and phospho‐histone H3 (S28) (7/9) positive dose–response reflecting aneugenicity, whereas compounds inducing DNA double‐strand‐breaks were associated with positive FANCD2 (S1404) and 53BP1 (S1778) responses pointing to clastogenicity (2/3). The biomarker p53 (K373) was able to distinguish genotoxicants from non‐genotoxicants (2/4), while the induction of reactive oxygen species (ROS), potentially causing DNA damage, was associated with a positive Nrf2 (S40) response (2/2). This work demonstrates that genotoxicants and non‐genotoxicants induce different biomarker responses in TK6 cells which can be used for reliable classification into MoA groups (aneugens/clastogens/non‐genotoxicants/ROS inducers), supporting a more in‐depth safety assessment of drug candidates.