Norihide Asano

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and 2B): The summary report of the 6th collaborative study by CSGMT/JEMS·MMS

To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study. Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen). As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed. Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found. Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers. The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens. After merging, the positive rates for Groups 1, 2A and 2B were 68.6, 54.5 and 45.6%, respectively. Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals. Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds. Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones. After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 90.5, 65.2 and 60.0% for IARC Groups 1, 2A and 2B, respectively. Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity.

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS·MMS

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible.

An automated new technique for scoring the rodent micronucleus assay : Computerized image analysis of acridine orange supravitally stained peripheral blood cells

We developed an automated image analysis system to obtain objective data for the rodent peripheral blood micronucleus assay with acridine orange (AO) supravital staining. The system was able to identify micronucleated reticulocytes (MNRETs) and to evaluate inhibition of bone marrow cell proliferation by measuring the reticular area of reticulocytes (RETs). We also developed automated equipment to produce homogeneous acridine orange-coated glass slides. This study was designed to compare automated scoring with manual scoring using 4 model clastogens and 2 mouse strains. The MNRET incidence induced by each clastogen was similar for automated and manual scoring, and there was good correlation (r=0.92) between the methods. In addition, an index of bone marrow toxicity based on the reticular area of RETs was compared to the conventional index (% of polychromatic erythrocytes (PCE) to total erythrocytes; PCE ratio) and was similar. The results indicated that our technique for computer-assisted image analysis for the micronucleus assay with AO supravitally stained peripheral blood RETs was comparable to conventional microscopic scoring, and it was superior in objectivity and statistical power.

Micronucleus test with colchicine given by intraperitoneal injection and oral gavage

The effects of micronucleus induction in mouse bone marrow cells by intraperitoneal injection (i.p.) and oral administration (p.o.) were studied with the spindle poison colchicine in 2 strains of mice (MS/Ae and CD-1). The final micronucleus test was performed with a 24-h sampling time at doses of 0.25-2.0 mg/kg by the i.p. route and 2-16 mg/kg by the p.o. route based on a simple acute toxicity test and a pilot micronucleus experiment. Colchicine induced micronuclei in both strains and at all doses tested. Micronuclei were induced more effectively by the i.p. route.