###### Abstract  
A new point of view for genetic damage assessment using the comet assay is proposed based on the number of migration groups, the number of comets in each group, and the groups with the highest number of comets. Human lymphocytes were exposed to different concentrations of Methyl Methane Sulfonate (MMS), Maleic Hydrazide (MH), 2,4-Dichlorophenoxyacetic (2,4-D), and N-nitroos diethylamine (NDEA). Using comet assay, the migration means of the comets were determined and later grouped arbitrarily in migration groups with no higher differences than 1 \(\upmu\)c. The number of migration groups, the number of comets in each group, and the groups with the highest number of comets (modes) were determined. All four of the genotoxic agents studied showed a significant increase (\(p<0.05\)) in the tail length and the number of migration groups compared to the negative control. The number of migration groups did not show a significant variation between the four-genotoxic agents nor within their different concentrations. However, the comparison of the modes did show differences between the genotoxic agents, but not within the concentrations of a same genotoxic agent, which indicated a determined chemical interaction on the DNA. These parameters can improve the detection of genetic damage associated with certain genotoxic agents.  
comet assay; bioassays; DNA damage; human lymphocytes; genotoxicity; tail length groups 4.0/. +
Footnote †: journal:types of specific genetic damage within the DNA, such as DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites [13; 14; 15; 16; 17; 18; 19]. Other variations of the test were developed by modifying it with enzymes for the detection of alkylated bases and the oxidation of purines and pyrimidines [11]. Despite its multiple modifications, this assay is still considered an unspecific technique [5; 20].  
Due to the great differences in tail migration among individual cells, the widely accepted criteria to determine genetic damage has been the use of migration averages of 25-100 comets per individual [15; 19]. This difference among the tail length is mainly given by the cell age [14], type of DNA damaged that occurred [21], and methodological variations of the assay, for instance, the low melting agarose percentage used, time of lysis, and electrophoresis [15], in addition to the differences within the observers [16] and statistical analysis used [17]. The large heterogeneity between the tail length of the comets observed in this test [14; 22; 23] indicates different levels of DNA damage given by broken loops losing its supercoiling. Using the migration average criteria, the main property of this assay is to determine and differentiate genetic damage between cells or individual nuclei losing relevance. Different comet groups with a similar tail length (migration groups) have been observed [24; 25; 26]. Therefore, the grouping of cells with equal tail lengths could indicate DNA damage associated specifically with different ages, genotoxic agents, or diseases avoiding the loss of data generated by averaging.  
In the present study, an analysis to characterize genetic damage caused by different agents using the alkaline comet assay based in three arbitrary parameters: number of migration groups, number of comets in each group, and the migration groups with the largest number of comets was proposed, with the aim of studying the differences in the genetic damage between individual cells and to extend the advantages of the use of the test in a wide variety of studies without increasing costs.