Three experiments are reported as a contribution to the validation of the multiple treatment protocol presently under discussion. (1) In the single treatment experiment with hydroquinone (HQ) the time of maximum micronucleus response was determined to be 24 h with a dose of 75 mg/kg given intraperitoneally. In the subsequent dose-response study at the 24-h interval the lowest positive dose was 50 mg/kg of HQ (P less than 0.01). The increase in micronucleus frequencies was non-linear. (2) Daily treatments with 15 or 75 mg/kg of HQ and bone marrow sampling on days 2-4 after the start of treatment resulted in an increased micronucleus yield for the lower dose (P less than 0.05) and a decrease in micronucleus frequencies at the higher dose (P less than 0.01) with increasing numbers of treatments. At the same time no change in the composition of the erythrocyte population was observed. (3) With 25 mg/kg of cyclophosphamide (CP) the micronucleus yields increased from 1 to 3 daily doses but dropped significantly from 3 to 4 daily doses (P less than 0.01). The polychromatic to normochromatic erythrocyte ratio was significantly decreased after single (P less than 0.01) and marginally lower than the control value after 4 daily treatments with CP. The present data suggest that the response of the bone marrow micronucleus assay to multiple treatments may depend on the dose employed and may differ from chemical to chemical. The specific clastogenic action or other cellular effects of a chemical may influence the micronucleus yields of an individual chemical and it may be difficult to generate a strict protocol recommendation.

Animals↗

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Induction of chromosome damage in mouse bone marrow by benzo[a]pyrene.

The cytogenetic effects, in vivo, of the polycyclic hydrocarbon benzo[a]pyrene were studied in adult male mice by chromosome analysis and the micronucleus test. To establish the appropriate time for bone-marrow sampling, the interval between treatment and preparation was varied. The maximal response in the micronucleus test was found 36 h after treatment versus 30 h when chromosomal aberrations were analyzed. In the micronucleus test the lowest effective dose was 25 mg/kg, whereas a significant effect (P less than 0.001) was observed at 6.25 mg/kg when aberrations were used as the mutagenic end-point. The results show that, with single treatments and different sampling intervals applied in both tests, aberration analysis had a higher resolving power than the micronucleus test at the time of maximal response. A suitable protocol for routine application of the 2 bone-marrow tests is discussed in view of the guidelines presently under preparation in the EEC and the OECD. Arguments are presented against a sub-acute treatment regimen and for a single treatment regimen with multiple sampling intervals.

Animals↗