**4. The cytokinesis blocking micronucleus assay (CBMN)**

The cytokinesis blocking micronucleus assay in lymphocytes (CBMN) was developed by a Ph.D. student more than 30 years ago [40], who anecdotally relates that while reviewing a biochemistry textbook [41]. He noticed that cytochalasin-B had the ability to block the action of actin *in vitro* cultures of human lymphocytes and thus obtain binucleated cells capable of recording clastogenic or aneugenic events resulting from exposure to xenobiotics. This biochemical principle is a fundamental aspect, enabling binucleated cells to remain in telophase (**Figure 1**). CBMN ensures that binucleated cell have undergone a single cell duplication in culture 72 hours after its initiation (**Figure 1**), making it possible to record cytotoxic or genotoxic events before blocking cytokinesis based on the following biomarkers: micronuclei (MNi), nuclear buds (NBUDs), nucleoplasmic bridges (NPBs), as well as mononucleated, binucleated, trinucleated and tetranucleated cells, which are used to calculate the cell duplication index (NDI); also the number of cells in necrosis and apoptosis can be recorded to perform a complete analysis of genomic instability [42–44].
