Dominant lethal assays in male mice: evaluation of experimental design, statistical methods and the sensitivity of Charles River (CD1) mice

Male CD1 mice were dosed by intraperitoneal injection of 20, 40 or 80 mg of methyl methanesulphonate (MMS) per kg body weight or with the solvent, water. After dosing, each male was caged with (a) 2 or (b) 3 females per week for 8 consecutive weeks and 13 days after mating the females were killed and examined for evidence of dominant lethal mutations. Results were analysed to determine (1) the sensitivity of CD1 mice to the mutagenic effect of MMS. (2) the influence of the number of females mated with each male. (3) the adequacy of the experimental design and (4) the adequacy of the statistical methods. A dose-related increase in early foetal deaths was induced in females mated to male mice in the first week after dosing with 20, 40 and 80 mg/kg MMS. A reduction in foetal implants was demonstrated in females in the 40 and 80 mg/kg MMS groups but not after dosing males with 20 mg/kg. Thus dominant lethal mutations were induced in male CD1 mice dosed IP with 20, 40 and 80 mg/kg giving a degree of sensitivity which compares favourably with that obtained with other strains of mice in other laboratories. For a given number of male mice, the greater precision obtained with 3 rather than 2 females arose directly from the numbers used. For a given number of female mice, the allocation of 2 per male (rather than 3 per male) is a more efficient design in that 50% more treated animals are available for estimation of the relevant error. Also more information would be available as a result of the probable increase in pregnancy rate. The experiment did not suggest the need for any modification in the statistical approach.