In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring

An expert working group on the in vivo micronucleus assay, formed as part o f the International Workshop on Genotoxicity Test Procedures (IWGTP), discu ssed protocols for the conduct of established and proposed micronucleus ass ays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction w ith the annual meeting of the Environmental Mutagen Society. The working gr oup reached consensus on a number issues, including: (1) protocols using re peated dosing in mice and mts; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omissi on of concurrently-treated positive control animals from the assay; (4) aut omation of micronucleus scoring by flow cytometry or image analysis; (5) cr iteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) of her than bone marrow. This report summar izes the discussions and recommendations of this working group. In the clas sic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-ter m toxicology studies, were considered acceptable as long as certain study c riteria were met. When the micronucleus assay is integrated into ongoing to xicology studies, relatively short-ierm repeated-dose studies should be use d preferentially because there is not yet sufficient data to demonstrate th at conservative dose selection in longer term studies (longer than 1 month) does nor reduce the sensitivity of the essay. Additional validation data a re needed to resolve this point. In studies with mice, either bone marrow o r blood was considered acceptable as the tissue For assessing micronucleus induction, provided that the absence of spleen Function has been verified i n the animal strains used. In studies with rats, the principal endpoint sho uld be the frequency of micronucleated immature erythrocytes in bone marrow , although scoring of peripheral blood samples gives important supplementar y data about the time course of micronucleus induction. When dose con centr ation and stability are verified appropriately, concurrent treatment with c t positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in mts or mice, treatment/sampling regimens should include treatment at intervals of no mor e than 24 hr (unless the test article has a half-life of more than 24 hr) w ith sampling of bone marrow or blood, respectively, within 24 or 40 hr afte r the last treatment. The use of a DNA specific stain is recommended For th e identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desira ble that systemic exposure to the test article is demonstrated. The group c oncluded that successful application of automated scoring by both flow cyto metry and image analysis had been achieved, and defined criteria that shoul d be met if automated scoring is employed. It Nas not felt appropriate to a ttempt to define specific recommended protocols for automated scoring at th e present time. Other issues re viewed and discussed by the working group i ncluded micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful resea rch tools that could also be used to elucidate mechanisms in certain regula tory situations, but that these assays had not yet been standardized and va lidated for routine regulatory application. (C) 2000 Wiley-Liss, Inc.