The comet assay or the single-cell gel electrophoresis assay is a well-established direct method to detect a broad spectrum of DNA damage. It enables the detection of DNA single and double strand breaks, alkali-labile sites, oxidatively damaged DNA bases, apurinic and apyrimidinic sites, and DNA-DNA and DNA-protein crosslinks [79]. The comet assay or the single-cell gel electrophoresis assay also predicts the genotoxicity of potentially mutagenic or carcinogenic substances [80]. With regard to other genotoxicity tests, such as micronucleus assay, chromosomal aberrations and sister chromatid exchanges, the comet assay has sufficient sensitivity for detecting low levels of DNA damage, and has been applied in the evaluation of the possible genotoxicity of different pesticides [81,82,83].In the present study, values of tail intensity measured in the studied cell types of exposed rats, were significantly different from negative control at almost all of the applied doses. When interpreting the results of the comet assay, we should consider that they reflect the damage levels detected at the final point of the experiment, which lasted for 28 days. During that period, due to the constant delivery of the tested compound, cells are faced with the induction of many types of DNA lesions which they try to counteract via various repair mechanisms. Therefore, the net damage measured using the comet assay represents an array of the damage levels recorded in the single cells, pointing both to the cell sensitivity and the efficiency of the repair. In line with the results obtained for oxidative stress, the highest degree of DNA damage was observed in the brain of α-cypermethrin-treated rats, confirming that the increase in lipid peroxidation followed by an activation of antioxidant enzymes, primarily GPx, plays a major role in cypermethrin-induced genotoxicity. Increased levels of 8-hydroxy-2′-deoxyguanosine, which is one of the predominant forms of free radical-induced oxidative stress in nuclear and mitochondrial DNA, have been measured in rat liver and urine following 28 day oral exposure to cypermethrin [76,84]. It has also been reported that cypermethrin may cause DNA damage in rats’ brains, as evidenced by an increase in the percentage of DNA damage level, percentage of DNA in tail, and tail moment at doses representing 1/10 and 1/30 LD50 applied for 28 days [85]. Similarly, Hammad and Ziada [75] reported DNA damage in the brain and liver of male albino rats indicating α-cypermethrin-induced genotoxicity after oral exposure to dose corresponding to 1/10 LD50. Hepatic DNA damage after exposure to sub-lethal doses of cypermethrin has been observed in several other in vivo studies [26,76,86,87]. Đikić et al. [86] reported that cypermethrin damaged DNA at alkali-labile sites, and that the observed genotoxic alterations in hepatocytes demonstrated its clastogenic properties. Small hydrophobic molecules, such as cypermethrin, can easily pass through the cell membrane and reach the cell nucleus. Once within the nucleus, cypermethrin may bind to DNA through the reactive groups of its acid moiety and form DNA monoadducts and DNA inter strand crosslinks [27,88]. Moreover, vinyl and dimethylcyclopropane groups, which are an integral part of the cypermethrin structure, can be oxidized. The formed active metabolites (methyl butanol and vinyl) may induce DNA damage, leading to destabilization and unwinding of the DNA [89]. However, the data regarding genotoxicity after exposure to low doses of cypermethrin are lacking. The genotoxic action of cypermethrin has recently been demonstrated in the offspring of females exposed to doses considered safe (0.05 mg/kg/day) [90]. Cypermethrin induced a significant increase in the micronucleus frequencies and comet assay parameters in the blood and liver cells of the dams as well as in the blood, liver, and brain cells of the pups. As the authors suggest, genetic damage caused by cypermethrin in dams and in the offspring, may be the result of an increase in ROS levels, which directly affects the DNA molecule causing breaks and mutations [87,90]. Considering that, in the present study, changes in oxidative stress response were observed in rat brains and livers, we can presume that both direct and indirect toxic effects were responsible for the genome instability as measured by the alkaline comet assay. Although a dose-dependent hepatotoxicity was observed in previous experiments [21,87], this was not the case in our study. The expected half-life of double-strand breaks repair is slower in low-dose exposure than in cases of higher/high dose exposure [91], which may explain the absence of a dose–response relationship in the present study. Additionally, endocrine disruptive chemicals and pesticides such as pyrethroids are a classic example of compounds with a nonmonotonic dose response effect, exerting severe effects even at low doses while the same severity may not be anticipated at higher doses [10,92]. Our results suggest that kidney cells expressed lower levels of primary DNA damage than the other studied cell types. This could be explained by a possibly lower uptake of the tested chemical and/or their reactive metabolites, capable of producing measurable DNA instability. Another explanation could be that processing of agarose microgels itself contributes to the loss of the most damaged cells, which thus escape or avoid measurement, and the final value obtained for the tail intensity descriptor refers only to the nucleoids with lower levels of DNA damage. Nevertheless, cypermethrin toxicity at the level of kidney in rats must be further evaluated, considering that Liu et al. [93] recently showed that β-cypermethrin has nephrotoxic characteristics. Exposure of male rats to β-cypermethrin abnormally altered renal histomorphology and ultra-structures, induced renal DNA damage, and impaired renal functions. Exposure to β- cypermethrin activated the JNK/c-Jun pathway by inducing ROS and oxidative stress. Taken together, data obtained using the alkaline comet assay indicate that, at the applied doses, cypermethrin caused genomic instability in different rat tissues, which obviously depended on the inherited susceptibility of each cell type and its capacity to repair DNA lesions inflicted during the period of 28 day repeated exposure.