Only cells that were not smeared, clumped or overlapped, and those who contained intact nuclei were included in the analysis. According to Tolbert et al.[18] and Gomez-Arroyo et al.,[20] exfoliated buccal cells undergo degenerative processes which can produce anomalies that are difficult to distinguish from MN (binucleates, pycnosis, karyorrhexis, and karyolysis). In our study, these were excluded from the micronucleus analysis and all the slides were coded to blind analysis. The MN frequency was estimated based on the number of normal exfoliated buccal cells counted using a bright-field Zeiss microscope at a magnification of 1000×. For each volunteer, 2000 buccal cells (i.e. 1000 from each of the duplicate slides) were scored.
Statistical analysis