The effect of aging on the response to 2-acetamidofluorene in the repeated-dose rat-liver micronucleus assay.

The repeated-dose liver micronucleus (RDLMN) assay is being developed toward an International Workshop on Genotoxicity Testing (IWGT)-recommended protocol, for potential adoption as an Organisation for Economic Co-operation and Development (OECD) guideline. During this development, the possible effect of animal age has been identified as a key issue, since, in general rodent toxicity studies, agent administration is typically initiated at 6-8 weeks of age (OECD Test Guideline 407, "Repeated-Dose 28-Day Oral Toxicity Study in Rodents"). To address this issue, we have conducted an RDLMN assay in rats treated with 2-acetamidofluorene (2-AAF), with dosing initiated at either 6 or 8 weeks of age. 2-AAF was selected as a representative compound known to produce weak positive responses in genotoxicity studies. The results demonstrated comparable increases in micronucleus frequency between the two age groups, indicating no clear impact of age. These findings, obtained with a weakly positive compound, provide further supporting evidence that the RDLMN assay can be reliably applied to animals when dosing is initiated at either 6 or 8 weeks of age, thereby supporting its integration into repeated-dose toxicity studies. Using PCNA- and Ki-67-positive cell counts as indicators of cell proliferative activity, we examined the suppressive effect of 2-AAF on cell proliferation; such suppression may affect the sensitivity of the assay. Our findings support the use of both PCNA and Ki-67 as indicators of cell proliferative activity in the RDLMN assay.