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Reliable Assessment of Micronuclei for Genotoxicity Testing in Mice or Rats

iability of the kits FCM-based method to score clastogen-induced MN. Four treatment groups, consisting of five female and five male BALB/c mice, were given 0.9% saline and a dose response of potassium chromate (K2CrO4) (12.5, 25, and 50 mg/kg body weight) via intraperitoneal injections. Chemicals were delivered in a volume of 25 ml/kg body weight at times 0, 24, and 48 hours. Twenty four hours after the last injection, peripheral blood samples were collected and fixed according to the manual.

Flow cytometric analysis was performed at Litron as previously described.7 When 10,000 total RETs were acquired, the MN-RET frequency was determined. Reticulocyte frequencies were also calculated as an indication of stem cell toxicity. The dual-labeling method used in this study resolved MN-RETs from other erythrocyte populations (Figure 1). Achieving this high resolution facilitated quantitative determination of MN-RET and RET frequencies.

Figure 3 shows a dose-dependent increase in MN-RET frequencies; the control group was low, compared to the K2CrO4-treated animals (0.28, 0.41, 0.43, and 0.91%, respectively). These data show similar trends in MN-RET frequencies reported by other laboratories using conventional microscopy-based methods.11 Reticulocyte frequencies were also quantitated to measure stem cell toxicity. No substantial toxicity was evident at any of the doses tested.

Easy and Reproducible Procedure

The flow cytometric procedure employed in the kit is used routinely at Litron Laboratories with excellent results. While the procedure critically depends on fixing the cells reliably and consistently, the method developed for the kit is both technically simple and highly repr
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