In vivo Mammalian Alkaline Comet Assay
The test is used to detect the influence of potentially genotoxic substances in vivo under experimental conditions. Comet analysis is based on the ability of denatured DNA fragments to migrate during electrophoresis. In this case, damaged DNA creates a “comet tail”, while undamaged DNA remains inside the cell, creating a “comet head”. Alkaline comet analysis allows detecting single- and double-stranded DNA breaks, alkaline labile sites, and other damage. The intensity of the comet’s tail relative to the head reflects the number of DNA breaks.
Service details: In a typical design, five groups of female mice (or rats) are dosed with 3 selected doses of a test article (MTD, and two descending doses). The test compounds are applied at least 2, or more days depending on compound toxicity level (14-28 days optimal) at 24-hour intervals. Each compound-treated group as well as the vehicle dosing group, and the positive control group, contain 5 animals. Animals are observed for mortality and clinical signs of toxicity at 30 min, 2, 4, and 6 hours after the administration and thereafter daily for a period of 14 consecutive days. Individual bodу weights of animals are determined on the day of the compound administration and every two days thereafter. Food consumption is measured weekly. Tissue sampling is performed for all animals at the endpoint of study 2-6 h after the last treatment. Tissue (liver is optimal for PO route of treatment) is removed after In situ perfusion and placed into a mincing buffer. Comet assay is performed in accordance with the OECD 489 recommendations.
Micronucleus Test In Vivo
The micronucleus test is used to determine the genotoxicity of a test compound. The test is based on the assessment of the presence of micronuclei in the cytoplasm of interphase cells, or erythrocytes. Micronuclei may contain fragments of chromosomes that are devoid of centromeres and therefore are excluded from cell nuclei at the time of cell division (DNA breakage), or whole chromosomes produced by disruption of the mitotic apparatus (aneugens).
Service details: In a typical design, five groups of female mice (or rats) are dosed with 3 selected doses of a test article (MTD, and two higher doses, up to 1000 mg/kg). The test compounds are applied at least 2, or more days depending on compound toxicity level (3-14 days optimal) at 24-hour intervals. Each compound-treated group as well as the vehicle dosing group, and the positive control group, contain 5 animals. Animals are observed for mortality and clinical signs of toxicity at 30 min, 2, 4, and 6 hours after the administration and thereafter daily for a period of 14 consecutive days. Individual bodу weights of animals are determined on the day of the compound administration and every two days thereafter. Food consumption is measured weekly. Tissue sampling is performed for all animals at the endpoint of study 18-24 h after the last treatment. Tissue (blood or bone marrow) is removed and stained in accordance with the OECD 474 recommendations.