Evaluation of applicability of the repeated-dose liver micronucleus assay in rats initiated at８weeks of age as in vivo genotoxicity assessment for 2,6-dninitrotoluene (2,6-DNT), a compound with reported cytostatic potential

Abstract

The repeated-dose liver micronucleus (RDLMN) assay is an in vivo genotoxicity test that is useful for detecting hepatocarcinogens and is anticipated to be incorporated into general toxicity studies. Although many RDLMN studies have been reported, further investigation is necessary to demonstrate age-related sensitivity and to evaluate compounds with cytostatic properties. In this study, a repeated oral dose study of 2,6-dinitrotoluene (2,6-DNT), a substance known to have cytostatic properties, was conducted in Crl:CD(SD) rats starting at 6 or 8 weeks of age for 28 days. Liver micronucleus (LMN) frequency, mitotic index (MI), and cell proliferation markers, Ki-67 and PCNA, were subsequently evaluated. As a result, LMN frequency increased significantly at all dose levels, showing a reverse dose-response relationship, and there were little differences between age groups. The number of Ki-67-positive cells increased in both age groups, and it was considered compensatory cell proliferation following toxicity at higher doses. Histopathological evaluation revealed prominent anisokaryosis, particularly at 8 weeks of age at start of dosing, and DNA content analysis confirmed that these cells were polyploid, suggesting endoreduplication. This study confirmed that LMN detection sensitivity in the RDLMN assay did not differ between rats aged 6 or 8 weeks at the start of dosing, and that micronucleus-inducing ability was detected even in compounds with cytostatic properties. For appropriate interpretation of the results, histopathological assessment, the analysis of cell proliferation markers and DNA content analysis are useful.