Methanol acetic acid solution

The CBMN assay followed the protocol by Fenech [28] (link) and was previously described in detail [9] (link). For the culture setup, 500 μL of whole blood was added to the appropriate nutrient medium (F-10, RPMI 1640, or Euroclone) supplemented with foetal bovine serum (FBS; Gibco), phytohemagglutinin (Remel, USA), and antibiotics (penicillin and streptomycin; Sigma, USA), and incubated at 37 • C and 5 % CO 2 for 72 h. Cytochalasin-B (Sigma) was added to each sample at a final concentration of 6 μg/mL after 44 h of incubation to prevent cytokinesis, and the cells were harvested after three days of culturing. The lymphocytes were fixed in a methanol-acetic acid solution (Kemika, Croatia), air-dried, and stained with 5 % Giemsa (Merck, Germany). Optical microscopes with a final magnification of 400× were used for slide analysis. Well-trained scorers did the analysis by counting the total number of MNi, NPBs, and NBUDs per 1000 binucleated cells (Fig. 1), while the proliferation kinetics was calculated on the same slides by counting either 500 or 1000 cells.