A flow cytometric method for analyzing suspension cell cultures for micronucleus content has been previously reported [Environ. Molec. Mutagen. 47 (2006) 56–66]. The experiments described herein were undertaken to evaluate the compatibility of this method (In Vitro MicroFlow®) with attachment cells. Initially, CHO-K1 cells were studied in nine independent experiments using mitomycin C and cyclophosphamide. The results demonstrated the effectiveness of the cell processing procedure, and also provided historical control data that were useful for setting criteria for making positive calls. Subsequently, CHO-K1 cells were treated with methyl methanesulfonate, mitomycin C, etoposide, vinblastine sulfate, dexamethasone, and sodium chloride. Whereas the four genotoxicants were each observed to increase micronucleus frequencies, the non-genotoxicants induced no such response up to cytotoxic concentrations. Following this initial work, inter-laboratory transferability was evaluated across three sites using a common cell staining and analysis protocol for CHO-K1 or V79 cells that had been treated with the ten chemicals listed in Annex 3 of the OECD Draft Proposal for a New Guideline 487: In Vitro Mammalian Cell Micronucleus Test. With the exception of benzo[a]pyrene at one site, each laboratory observed increased micronucleus frequencies for the genotoxicants, whereas no significant induction occurred with the non-genotoxicants. Interestingly, the method appeared to distinguish between genotoxic modes of action, as only aneugens increased the average micronucleus fluorescence intensity and the frequency of hypodiploid nuclei. Collectively, these data suggest that flow cytometry is capable of providing reliable micronucleus counts, and that additional information is obtained that appears to discern genotoxic modes of action.

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.

No abstract

We previously reported a high concordance of in vitro micronucleus (MNvit) results obtained by flow cytometry to the known cytogenetic activity often commercially available compounds mentioned as validation compounds in an early draft of the OECD MNvit TG487 [Bryce et al., 2010; Organization for Economic Co-operation and Development(OECD), 2007]. The current study investigated this method in Chinese hamster V79 cells with pharmaceutical compounds of unknown genotoxic potential. Twenty-five compounds from several therapeutic areas such as oncology, neuroscience and immunological research were tested in the flow cytometry assay, and for comparison using the cytokinesis-block microscopy assay. Five of these 25 compounds were considered positive for micronucleus induction by the microscopy assessment. In all cases, the results from the flow cytometry assess ment matched the results of the microscopy assay. Thus, flow cytometry is a viable method for assessing the aneugenic/clastogenic potential of pharmaceutical drug candidates. The flow method offered several advantages over traditional microscopy. For instance, the ratio of micronuclei (MN) to 10,000 nuclei was evaluated in less than 2 min vs.15 min to manually assess 600 binucleate cells. Evaluation by flow cytometry can be automated,freeing resources and eliminating scorer fatigue.The assay may also provide for mechanistic understanding of MN formation based on size and the ratio of nuclei with sub-2N DNA content, allowing for discrimination between aneugenic and clastogenic compounds.

Hydrazine has been described as a mutagenic, probable human carcinogen. It is mutagenic in in vitro systems such as bacterial reverse mutation (Ames) tests and some yeast systems, as well as in in vivo systems with drosophila. It was shown to cause chromosome damage both in vitro and in vivo but was negative in some wellvalidated mammalian mutation systems such as CHO HPRT assays. Importantly, there is only one in vivo gene mutation test reported, which was negative. Our objective was to determine if hydrazine is mutagenic in mammalian test systems. Thus, we conducted an in vitro gene mutation test in Muta™Mouse lung epithelial cells (FE1 cell assay) and a regulatory-compliant in vivo Big Blue ® mouse test. Consistent with previous reports, an additional six-well Ames assay showed that hydrazine was mutagenic to bacteria. The FE1 cell assay was negative in conditions with and without metabolic activation when tested to cytotoxicity limits. In the Big Blue ® mouse study, female mice received dosages of hydrazine up to 10.9 mg/kg via drinking water for 28 days. This dose is comparable to a dose used in a carcinogenicity study where female mice had significant increases in hepatocellular adenoma at 11.5 mg/kg. There were no increases in mutant frequency in liver and lung, two tissues sensitive to the carcinogenic effects of hydrazine in mice. Our research shows that hydrazine is not mutagenic in mammalian cells either in vitro or in vivo, indicating mutagenicity may not play a role in the carcinogenicity of hydrazine.