Part 2. Assessment of micronucleus formation in rats after chronic exposure to new-technology diesel exhaust in the ACES bioassay.

The formation of micronuclei (MN*) 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 increases in MN formation frequency. This endpoint is therefore commonly used in preclinical studies designed to assess the potential risks to humans of exposure to a myriad of chemical and physical agents, including inhaled diesel exhaust (DE). As part of the Advanced Collaborative Emissions Study (ACES) Phase 3B, which examined numerous additional toxicity endpoints associated with lifetime exposure to DE in a rodent model, this ancillary 24-month investigation examined the potential of inhaled DE to induce chromosome damage in chronically exposed rodents. The ACES design included exposure of both mice and rats to DE derived from heavy-duty engines that met U.S. Environmental Protection Agency (EPA) 2007 standards for diesel-exhaust emissions (new-technology diesel exhaust). The exposure conditions consisted of air (the control) and three dilutions of DE, resulting in four levels of exposure. At specific times, blood samples were collected, fixed, and shipped by the bioassay staff at Lovelace Respiratory Research Institute (LRRI) to Litron Laboratories (Rochester, NY) for further processing and analysis. In recent years, significant improvements have been made to MN scoring by using objective, automated methods such as flow cytometry, which allows the detection of micronucleated reticulocytes (MN-RET), micronucleated normochromatic erythrocytes (MN-NCE), and reticulocytes (RET) in peripheral blood samples from mice and rats. By using a simple staining procedure coupled with rapid and efficient analysis, many more cells can be examined in less time than was possible using traditional, microscopy-based MN assays. Thus, for each sample in the current study, 20,000 RET were scored for the presence of MN. In the chronic-exposure (12 and 24 months) bioassay, blood samples were obtained from separate groups of exposed animals at specific time points throughout the course of the study. The automated method using flow cytometry has found widespread use in safety assessment and is supported by regulatory guidelines, including International Conference on Harmonisation (ICH) S2(R1) (2011). Statistical analyses included the use of analysis of variance (ANOVA) to compare the effects of sex, exposure condition, and duration, as well asthe interactions between them. Analyses of blood samples from rats combined data from our earlier 1- and 3-month exposure studies (Bemis et al. 2012) with data from our current 12- and 24-month exposure studies. Consistent with findings from the preliminary studies, no sex-based differences in MN frequency were observed in the rats. An initial examination of mean frequencies across the treatment groups and durations of exposure showed no evidence of treatment-related increases in MN at any of the time points studied. 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 results described in this report provide a comprehensive examination of chronic exposure to DE in a rodent model. Our investigation of chromosomal damage also plays an important role in the context of ACES, which was designed to assess the safety of emissions from 2007-compliant diesel engines.