The micronucleus test with peripheral reticulocytes from phenacetin-treated mice
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N Higashikuni1, T Baba, T Nakamura
1Itoham Central Research Institute, Ibaraki, Japan.
Abstract:
The in vivo micronucleus test is conventionally performed using mouse bone marrow cells (BM assay). Using phenacetin as a test chemical, an alternative method using reticulocytes (RET assay) was examined to determine if this could be substituted for the BM assay. Single doses of 400, 600, and 800 mg/kg gave negative results 24 h after i.p. administration, but positive results were obtained with 600 and 800 mg/kg after 48 h. Responses were weak at 72 h. Double treatment enhanced the responses; 400 mg/kg gave a positive result. Maximum responses were generally reached 24 h after the second treatment, 48 h if doses were highly toxic. When the BM and RET assays were compared, the BM assay seemed to be slightly more sensitive than the RET assay; double treatment was superior to a single treatment in both BM and RET assays. Both assays can be used routinely but in the RET assay, sequential samples can be obtained from the same individuals without killing them, providing a firm basis to substitute it for the BM assay. Taking advantage of this characteristic of the RET assay, a regimen of double treatments and double sampling at 24 and 48 h is recommended for a wide range of doses. These data were obtained with CD-1 mice; MS/Ae mice gave a higher incidence of micronuclei than did the CD-1 strain.
Insights
The reticulocyte (RET) assay offers a viable alternative to the traditional mouse bone marrow (BM) assay for genotoxicity testing. This non-lethal method allows for sequential sampling, enhancing its utility in in vivo micronucleus tests.
Area of Science:
- Toxicology
- Genotoxicity Testing
- In Vivo Assays
Background:
- The in vivo micronucleus test is a standard genotoxicity assay.
- Traditionally, this test uses mouse bone marrow (BM) cells, requiring animal sacrifice.
Purpose of the Study:
- To evaluate the reticulocyte (RET) assay as a non-lethal alternative to the BM assay.
- To compare the sensitivity and effectiveness of the RET assay versus the BM assay using phenacetin as a test chemical.
Main Methods:
- Comparison of mouse bone marrow (BM) and reticulocyte (RET) assays.
- Administration of phenacetin at single doses (400-800 mg/kg) and double doses.
- Sampling at 24, 48, and 72 hours post-administration.
- Evaluation of micronuclei incidence in CD-1 and MS/Ae mice.
Main Results:
- The RET assay detected genotoxicity with phenacetin at 48 hours after single doses (600-800 mg/kg) and with double doses (400 mg/kg).
- The BM assay showed slightly higher sensitivity than the RET assay.
- Double treatments improved responses in both assays compared to single treatments.
- Maximum responses in the RET assay were typically observed 24 hours after the second dose.
Conclusions:
- The RET assay is a suitable alternative to the BM assay for in vivo micronucleus testing.
- The non-lethal nature of the RET assay permits sequential sampling from the same animals.
- A recommended regimen for the RET assay involves double treatments and sampling at 24 and 48 hours post-second treatment.