2.5.3 Micronucleus (MN) test to assess chromosome damage
This test was performed according to the guidelines of the GeneTox program of the US Environmental Protection Agency (Mavournin et al., 1990; Krishna e Hayashi, 2000) and following the new OECD test guideline (TG) 474 for the in vivo mammalian erythrocyte micronucleus (MN) test (29 July 2016).
Bone marrow was extracted from animal femurs, and a smear was deposited directly onto a microscope slide using a drop of fetal calf serum. The slides were subjected to Giemsa staining (5%) then air-dried and coded for blinded analysis. To avoid false negative results and as a toxicity measure, the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was determined for 500 erythrocytes per animal. The presence of the MN was determined in 4000 erythrocytes per animal (2000 from each of the two slides) by bright field microscopy (1000× magnification with immersion oil). For individual animals, mean values of MnPCE were used as the experimental units, and variation was based on differences among animals within the same group.