Single versus multiple dosing in the micronucleus test: the summary of the fourth collaborative study by CSGMT/JEMS.MMS. Collaborative Study Group for the Micronucleus Test, the Mammalian Mutagenesis Study Group of the Environmental Mutagen Society, Japan (CSGMT/JEMS.MMS).

The effects of multiple dosing in the micronucleus test were studied by treating male CD-1 mice 1, 2, and 4 times i.p. with various doses of 11 chemicals and by sampling bone marrow cells 6, 24, 48, and/or 72 h after the final dosing. The chemicals used were 2-acetylaminofluorene (2-AAF), 1-beta-D-arabinofuranosylcytosine (ARA-C), 7,12-dimethylbenz[a]anthracene (DMBA), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), 6-mercaptopurine (6-MP), phenacetin (PHEN), 5-fluorouracil (5-FU), methotrexate (MTX), benzene (BEN), and potassium chromate (K2CrO4). MTX, an inhibitor of nucleotide synthesis, was a weak micronucleus inducer after single dosing, but clearly positive after multiple dosing. Although ARA-C, 5-FU, and 6-MP became positive after single dosing, the multiple dosing enhanced the effects of these base analogues. ENU, DMBA, and BEN, at some doses and sampling times, also showed increased responses by multiple dosing, but a severe trade-off occurred enhanced toxicity tended to reduce the bone marrow cells, especially at 4 dosings, making the examination of micronuclei difficult. PHEN showed the multiple-dosing effect after 2 doses, but the effect disappeared at quadruple dosing. No apparent multiple-dosing effects were observed with 2-AAF, EMS, and K2CrO4, which gave similar incidences of micronuclei after single and double dosing. The original double dosing and sampling 6 h after the second injection method of Schmid (1975) was less sensitive than the modified method, i.e., sampling 24 h after the second dosing. The latter regimen is recommended for the general screening for the following reasons. (1) It was the most sensitive protocol among those tested here and detected all test chemicals as clastogens. (2) Since bone marrow specimens were prepared at or near the steady state of micronuclei, one sampling covered most chemicals with different actions. (3) For chemicals having multiple-dosing effects the recommended regimen was better than the protocol using 4 doses, because specimens could be prepared before the suppression of the bone marrow became excessive. (4) Chemicals having no multiple-dosing effect showed similar incidences of micronuclei after single and double dosing. (5) Dose selection is easier than with more multiple dosing, because, e.g., 50, 25, and 12.5% of LD50 values determined on single dosing will usually be tolerated by test animals. The detection spectrum would become wider if specimens are also prepared 48 h after the second dosing for metabolic inhibitors, base analogues, and the other chemicals which might show delayed activities.