2-Hydroxy-1,4-naphthoquinone, the natural dye of Henna, is non-genotoxic in the mouse bone marrow micronucleus test and does not produce oxidative DNA damage in Chinese hamster ovary cells

Abstract

2-Hydroxy-1,4-naphthoquinone (HNQ) has been found positive in a previous chromosome aberration test in Chinese hamster ovary (CHO) cells and in a mouse bone marrow micronucleus test at 72h after oral administration (vehicle: DMSO). However it was negative at 24 and 48h sampling times, and in subsequent micronucleus tests that used 0.5% aqueous methyl cellulose (MC) as vehicle. We performed a bone marrow micronucleus test in male and female NMRI BRL/BR mice at oral doses of 75, 150 and 300mg/kg in two vehicles (DMSO and 0.5% aqueous MC), evaluated micronuclei at 24, 48 and 72h, plasma levels of HNQ at 0.5, 1 and 4h, and haematology parameters at 72h after administration. The mechanism of in vitro clastogenic activity of HNQ was investigated by evaluation of the potential of HNQ to produce oxidative DNA damage after treatment of CHO with 10mM HNQ, followed by quantification of DNA fragments using the comet assay. In the micronucleus test, HNQ at 300mg/kg produced mortality and clinical signs at similar incidence and severity for both vehicles. Levels of HNQ in the plasma of treated mice were dose-related, of similar magnitude for both vehicles, but higher in females than in males. Maximum concentrations were found at 0.5 or 1h. At 300mg/kg, HNQ slightly affected RBC parameters suggesting haematotoxicity. No increase in the frequency of micronuclei was observed for any dose, vehicle or time point, whereas the positive control substance (CPA) produced a clear positive response. No evidence of HNQ-induced oxidative DNA damage was found at clastogenic concentrations in vitro, whereas the positive control substance (H(2)O(2)) produced a clear increase. In conclusion, HNQ was negative for induction of bone marrow micronuclei in mice up to 72h after administration in two different vehicles, and its in vitro clastogenicity was not due to oxidative damage. These results confirm that HNQ poses no or negligible genotoxic risk.