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.