USE OF CYCLOPHOSPHAMIDE AS A POSITIVE CONTROL IN DOMINANT LETHAL AND MICRONUCLEUS ASSAYS

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclo phosphamide (CP) has been widely used as a positive control in the sin gle-dose mouse MN assay; however, its utility as a positive control fo r the DL assay has not been fully studied. In the present study, CP wa s tested in both assays under similar experimental conditions and MN s een in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 ma le mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and b one marrow was harvested 24 h later for MN assay. CP induced a dose-re lated increase (7- and 11-fold over controls at 30 and 40 mg/kg) in mi cronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs ( to 60% and 54% of controls at 30 and 40 mg/kg, respectively), Based on this, a definitive DL and MN study was conducted using separate group s of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing reg imen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL ass ay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiat ion of breeding, the females were evaluated for the number of implanta tion sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a sligh t (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (5 5%, 71% and 34% PIL, respectively) over controls. These data clearly s how that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg C P can be used as a positive control compound in the DL assay and in th e multiple-dose bone marrow MN assay.