How the methodology determines the outcome of the in vitro micronucleus assay (OECD TG 487): a comparison of the MicroFlow and the microscopic evaluation approach highlights the impact of cytotoxicity/cytostasis metrics in V79 cells for matrine

Abstract The in vitro micronucleus (MN) test (OECD TG 487) has become the gold standard in various regulatory silos for assessing compounds for their potential to cause clastogenicity. Its main advantages are the reproducibility and robustness, as well as the ability to investigate both structural and numerical chromosome damage, i.e. clastogenicity and aneuploidy. A disadvantage, however, can be seen in the resource-intensive microscopical evaluation. Consequently, faster, high-throughput methods, such as flow cytometry have been developed and integrated into the test guideline protocol. Regardless of the evaluation method applied, results must be consistent to justify the mutual acceptance of data established for OECD TG tests performed under GLP. This may be particularly challenging for substances of lower potency. Here, we present two case studies comparing the results of the MN test according to TG 487 with microscopic evaluation with those obtained using the MicroFlow kit (Litron Laboratories). The MN tests were performed with V79 cells without cytochalasin B, incubated for 4 h (with and without metabolic activation) or 24 h with 0.5, 1.0, 1.25, 1.375 and 1.5 mg/ml matrine or 0.5, 1.0, 1.5, and 2.0 mg/ml oxymatrine. These quinolizidine alkaloids, found in liquorice confectionary and organic green tea are suspected to be aneugenic or clastogenic. The MicroFlow test showed statistically significant increases in micronuclei formation from 1.0 mg/ml on, albeit with slightly reduced relative survival, the metric for cytotoxicity recommended for this method at the time of the assay implementation. Complementary testing using microscopical evaluation did not support these results due to excessive cytotoxicity observed at matrine concentrations of 1.0 mg/ml and higher. Additionally, mechanistic studies using the ToxTracker ACE assay indicated that matrine is not a direct clastogen. Herein, we illustrate and discuss the importance of appropriate metrics for cell proliferation and cytotoxicity which should account for the toxicological properties of the test compounds. The aim is to assure consistency between OECD TG-compliant evaluation methods and to avoid misleading positive findings when evaluating the biological relevance of increased micronuclei formation accompanied by cytotoxicity.