Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane.

As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET(CD59-) and RBC(CD59-), respectively) in peripheral blood of male Sprague Dawley(®) rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET(CD59-) and RBC(CD59-) (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.

Summary of in vitro genetic toxicology assay results: expected and unexpected effects of recent study design modifications.

Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure-activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53-competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)-compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non-GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL-positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay.