## 4 Discussion  
The alkaline comet assay can be used to study genotoxicity and genetic damage [15]. It is based on the measurement of the tail length of the comet and the measurement of migrated DNA percentage [13]. Even though multiple adaptations have been made to this assay in order to detect specific genetic damage, it is still considered as a test that only detects unspecific genetic damage [5,20] characterized by a high heterogeneity among the observed comets [24,26,28]. In this study, we used the tail length descriptor because despite of being the simplest criteria and it is possible to observe great heterogeneity in the magnitude of the migration of comets which allows cells with similar tail length size to be grouped. In an attempt to make the comet test more specific for the detection of genetic damage, human lymphocytes were exposed to MMS, MH, 2,4-D, and NDEA in different concentrations and an increase in genetic damage was observed, depending on the genotoxic agent and the concentration, which allowed organizing the data arbitrarily in migration groups of. Thus, differences between single comets (differences in genetic damage) are framed in a MG so that way, they do not lose relevance as a consequence of the \"average\" effect, which allows us to determine and differentiate genetic damage between individual comets. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c} \hline \hline \multicolumn{1}{c}{\multirow{2}{*}{ \begin{tabular}{c} **Migration** \\ **Groups** \\ **in** \\ \end{tabular}  }} & \multirow{2}{*}{**NC**} & \multirow{2}{*}{ \begin{tabular}{c} **MMS** \\ **2.5 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **MMS** \\ **5 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **MMS** \\ **10 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **MH** \\ **5 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **MH** \\ **20 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **2,4-D** \\ **45 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **2,4-D** \\ **0.5 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **2,4-D** \\ **1 mM** \\ \end{tabular}  } & \multirow{2}{*}{\ \begin{tabular}{c} **2,4-D** \\ **2 mM** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **2,4-D** \\ **2 mM** \\ \end{tabular}  } & \multirow{2}{*}{\begin{Each migration group indicates what we call a CGD, the number of MG represent distinct CGDs and the three MG with the largest number of comets represent the most frequent CGD (modes). Some of the groups showed the largest number of comets indicating that these types of damage are the ones that occur more frequently, and each of them depend on the genotoxic agent as it was shown in Table 1 where the most frequent CGD in the negative control corresponds to the \(6>4>5\) uc groups. Regardless of the concentration, the CGD for MMS and MH was at 6, 7, and 8 uc. Other CGD: 4, 5, 6, and 7 uc were observed for 2,4-D and 5, 6, 7, and 8 uc for NDEA. Nuclei can be classified as damaged or undamaged according to the percentile of the length of the tail [29]. However, the GM can be used as an additional indicator of genetic damage. Although previous reports have shown comet groups with different degree of damage [24; 26; 30], far too little attention has been paid to the role of migration groups in the comet assay. In this study, MG were formed. Each group was different from one another for 1 uc, therefore, observing CGD, could be a method that broadens the sensibility of the comet assay but still without being considered a method that detects specific genetic damage. Alkylating agents MMS and NDEA cause mutational events on the DNA [31] that may be related with the increase of migration groups and a higher frequency of some of them, which consequently indicates that these genotoxic agents induce certain CGD more frequently. A similar situation may occur for 2,4-D and MH. However, the genotoxic mechanism of action of these agents is still not clear [32].