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DL-LACTONE CAS N°:79-50-5 - page 94 / 113





94 / 113



ID: 79-50-5 DATE: 18.01.2006


The test animals were killed by cervical dislocation 24 hours (groups A, B, D and E) respectively 48 hours (groups C and F) after dosing. In every instance, both femurs were removed and freed of blood and muscles. Then, both ends of the bone were shortened until a small opening to the marrow canal became visible. The prepared bones were flushed with foetal calf serum (FCS), the cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the pellets re-suspended in FCS. A drop of the suspension was placed on the end of a previously cleaned and marked (NOTOX study number, animal number) microscopic slide, spread using a clean slide and air-dried, fixed with 100% methanol and automatically stained in a HEMA-tek Slide Stainer (Miles, Bayer Nederland, The Netherlands) and covered with a glass coverslip. Before analysis, the unique marks of each slide were randomised by covering with an adhesive label bearing the NOTOX study number and a code. Slides were first screened at a magnification of x100 for suitable regions, then scored at x1000. The number of micronucleated polychromatic erythrocytes was counted in a total of 2000 polychromatic erythrocytes per slide. The ratio of polychromatic to normochromatic erythrocytes was determined in the first 1000 erythrocytes scanned. Micronuclei were only counted in polychromatic erythrocytes. Statistics After counting, the randomisation was unveiled and averages and standard deviations for the six groups were calculated. A test substance and/or dose would be considered positive if it induced a statistically significant (Wilcoxon Rank Sum test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes, at any dose or sampling time. Conversely, a test substance is considered negative if there is no such statistically signifcant difference at any dose or sampling time. Acceptability criteria A micronucleus test is considered acceptable if it meets the following criteria: 1) the positive control substance, cyclophosphamide, induces a significant increase in micronucleated polychromatic erythrocytes and the incidence of micronucleated polychromatic erythrocytes in the control animals is reasonably within the laboratory historical controls range (mean ± 3 SD). Dose range-finding study 4 males and 4 females were dosed with 2000 mg dl-lactone in physiological saline per kg bw. All treated animals showed abnormalities during an observation period of 3 days: 1 male died within 20 min, 2 more males and 1 female died within 1.5 hours. During the first 1.5 hours, all animals showed lethargy or convulsions, one male had tremors. At days 2 and 3 after gavage, all survivors showed no abnormalities. 3 males and 3 females were dosed with 1500 mg dl-lactone in physiological saline per kg bw. All treated animals except one female showed lethargy within the first 20 min; after 1.5 hours, 2 one male and female each showed no signs while thothers were lethargic and had a rough coat. At days 2 and 3 after gavage, all survivors showed no abnormalities. Therefore, 1500, 750 and 375 mg/kg bw were chosen as the doses for the main test, with two high-dose groups, plus a vehicle negative and a cyclophosphamide positive control group. All



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