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Rodent Cytogenetics

In Vivo Mouse1 Bone Marrow Chromosal Aberrations Test Protocol

In the absence of adequate pre-existing toxicity information, a dose range-finding study was performed. The highest dose selected was limited by solubility, clinical signs of toxicity, and/or induction of cell cycle delay, and was not to exceed 5,000 mg/kg. The test chemical was evaluated for induction of chromosomal aberrations in mouse bone marrow using two different protocols. The first experiment used a standard harvest time of 17 hrs and the second used a delayed harvest time of 36 hrs. Male B6C3F1 mice (10 animals/dose group) were typically administered test chemical via intraperitoneal injection (IP) with the chemical dissolved in corn oil or PBS (injection volume = 0.4 ml.). Other routes were possible, including drinking water and gavage. Solvent control mice received equivalent injections of the solvent alone (typically saline or corn oil). A positive control group, treated with dimethylbenzanthacene, mitomycin C, or cyclophosphamide, was included. The mice were subcutaneously implanted with a BrdUrd tablet (McFee et al., 1983) 18 hrs before the scheduled harvest (for the standard protocol, this required BrdU implantation to precede injection with test article by one hour). The use of BrdU allowed selection of the appropriate cell population for scoring. (Chromosomal aberrations induced by chemical administration are present in maximum number at the first metaphase following treatment; they decline in number during subsequent nuclear divisions due to cell death.) Two hrs prior to sacrifice, the mice received an IP injection of colchicine in saline to arrest cells in metaphase and facilitate visualization of chromosomes. The animals were killed 17 or 36 hrs after test chemical administration (18 hrs after BrdU dosing). One or both femurs were removed and the marrow was flushed out with phosphate-buffered saline (pH 7.0). Cells were treated with a hypotonic salt solution, fixed, and dropped onto chilled slides. After a 24 hr drying period, the slides were stained with Giemsa and scored. Fifty first-division metaphase cells were scored from each of eight animals per treatment. The types of aberrations observed (gaps, breaks, rearrangements, and chromatid vs. chromosome) were recorded separately for each animal. The mean total number of aberrations and the mean percentage of cells with aberrations (excluding gaps) were determined for each treatment group. The values for percent cells with aberrations were analyzed by a one-tailed trend test (Margolin et al., 1986) and significance was set at P = 0.025.

In Vivo Mouse1 Bone Marrow Sister Chromatid Exchange Test Protocol

A dose range-finding study was performed in the absence of pre-existing toxicity information. The highest dose was limited by clinical signs of toxicity, solubility, and/or induction of cell cycle delay, and, in the absence of any of these factors, was limited to 5,000 mg/kg. The test chemical was assessed for induction of sister chromatid exchanges (SCE) in mouse bone marrow using two protocols. Male B6C3F1 mice (five animals/dose group) were typically administered test chemical via intraperitoneal (IP) injection with test agent dissolved in corn oil or PBS (injection volume = 0.4 ml). Other routes were possible, including drinking water and gavage. Solvent control mice received equivalent injections of solvent alone (typically saline or corn oil). The positive control was dimethylbenzanthacene (or another compound as specified). The first experiment had a (standard) harvest time of 23 hrs, and the second had a (delayed) harvest time of 42 hrs. The mice were implanted subcutaneously with a BrdU tablet (McFee et al., 1983) 24 hrs before harvest (one hour prior to treatment in the case of the standard protocol). The use of BrdU allowed selection of the appropriate cell population for scoring (cells in the second metaphase following chemical treatment). Two hours prior to sacrifice, the mice received an IP injection of colchicine in saline to arrest cells in metaphase and facilitate visualization of chromosomes. The animals were killed 23 or 42 hrs after treatment. One or both femurs were removed and the marrow was flushed out with phosphate-buffered saline (pH 7.0). The cells were treated with a hypotonic salt solution, fixed, and dropped onto chilled slides. After a 24 hour drying period, the slides were stained using fluorescence-plus-Giemsa and scored. Twenty-five second-division metaphase cells were scored from each of four animals per treatment group. Responses were evaluated as SCE/cell, and the data were analyzed by trend test (Margolin et al., 1986), with significance set at P = 0.025.

1 For one test article only a mixture of 25 water contaminants administered for 14 days in drinking water both mice and rats were evaluated. The procedure was the same for each species. Animals were evaluated only at the 17 hr time point in this drinking water study.