Part 2. Assessment of genotoxicity after exposure to diesel exhaust from U.S. 2007-compliant diesel engines: report on 1- and 3-month exposures in the ACES bioassay.

Micronucleus (MN*) formation is a well-established endpoint in genetic toxicology; studies designed to examine MN formation in vivo have been conducted for decades. Conditions that cause double-strand breaks or disrupt the proper segregation of chromosomes during division result in an increase in MN frequency. Thus this endpoint is commonly employed in preclinical studies designed to assess the potential risks of human exposure to a myriad of chemical and physical agents, including inhaled diesel exhaust (DE). As part of the Advanced Collaborative Emissions Study (ACES) this investigation examined the potential of inhaled DE to induce chromosome damage in chronically exposed rodents. The ACES design included exposure of both rats and mice to DE derived from 2007-compliant heavy-duty engines. The exposure conditions consisted of air control and dilutions of DE resulting in three levels of exposure. At specified times, blood samples were collected, fixed, and shipped by the bioassay staff to Litron Laboratories for further processing and analysis. Significant improvements have been made to MN scoring by using objective, automated methods such as flow cytometry, which allows for the detection of micronucleated reticulocytes (MN-RET), micronucleated normochromatic erythrocytes (MN-NCE), and reticulocytes (RETs) in peripheral blood samples from mice and rats. By using a simple staining procedure coupled with rapid and efficient analysis, many more cells were examined in less time than was possible in traditional, microscopy-based MN assays. Thus, for each sample, 20,000 RETs were scored for the presence of MN. In the chronic-exposure bioassay, blood samples were obtained from independent groups of exposed animals at specific time points throughout the course of the entire study. This automated method is supported by numerous regulatory guidelines and meets the requirements for an Organization of Economic Cooperation and Development (OECD)-compliant assay for genotoxicity. Statistical approaches employed analysis of variance (ANOVA) to compare effects of sex, exposure condition, and duration, as well as their interactions. This initial assessment of MN was performed on both mouse and rat blood samples from the 1-month and 3-month exposures. The data from mice demonstrate the well established, sex-based difference in MN-RET and MN-NCE frequencies regularly observed in this species, with females exhibiting slightly lower frequencies. There were no sex-based differences observed in rats. An examination of the mean frequencies across the exposure groups and durations of exposure did not show an appreciable induction of MN at the 1- or 3-month exposures in either species. Further statistical analyses did not reveal any significant exposure-related effects. An examination of the potential genotoxic effects of DE is clearly valuable as part of a large-scale chronic-exposure bioassay. The data and observations from the 1-and 3-month exposure studies will eventually be combined with the results from the 1- and 2-year exposure studies to provide a comprehensive examination of chronic exposure to DE in a rodent model. This examination of chromosome damage serves an important role in the context of the entire ACES bioassay, which was designed to assess the safety of diesel combustion engines.