The potential of 4-chloro-ortho-toluidine (4-CloT), an aromatic amine substituted on the ortho- and para-position of the amine function, to induce DNA damage in male Wistar rats was evaluated with the micronucleus test (peripheral blood), Pig-a (peripheral blood), and comet assay (peripheral blood, liver, urinary bladder, jejunum) at several time points. In addition to those markers of DNA damage, ie, gene mutation and clastogenicity, standard hematology, including methemoglobin, histopathology and immunohistochemistry of γ-H2AX and Ki-67 in liver, jejunum, and urinary bladder were performed. 4-CloT was administered orally over 28 consecutive days (days 1-28), followed by a 28-day treatment-free (days 29-56), and a second dosing phase of 3 days (days 57-59). 4-CloT showed some effects on the integrity of the DNA as measured by the comet assay in liver and urinary bladder but not in peripheral blood or jejunum. However, for liver and urinary bladder histopathological changes were observed. An increase in the frequency of micronuclei in peripheral blood was seen in parallel to a dose-dependent increase of reticulocytes and methemoglobin. Therefore, impact from a compensatory erythropoiesis on micronucleation cannot be excluded. Interestingly, no increase in the frequency of RETCD59- and RBCCD59- was observed in the Pig-a assay.