Report No DWI0713

Sept 1995

Bromodichloromethane was assessed for induction of DNA repair in hepatocytes following acute oral administration to Specific Pathogen Free outbred albino Hsd/Ola Sprague-Dawley male rats at dosages of 135 and 450 mg/kg bodyweight. (A small preliminary toxicity test indicated that a dose level of 450 mg/kg bodyweight was approximately the maximum level tolerable under the conditions of this test, this dose level was therefore chosen as an appropriate maximum for use in the DNA repair test.)

A negative control group was treated with the vehicle, aqueous 1% methylcellulose, and a positive control group was treated with dimethylnitrosamine at 4 mg/kg (for the 2 hour expression) or 2-acetylaminofluorene at 50 mg/kg (for the 14 hour expression). Hepatocytes were isolated by enzymatic dissociation at 2 or 14 hours after exposure of the animals to the test substance. Four animals were assessed at each experimental point, with the exception that only two animals from the positive control group were assessed at each expression time.

The isolated hepatocytes were allowed to attach to glass coverslips and were cultured in vitro with (methyl-3H)thymidine at 10µCi/ml for four hours to 'radiolabel' replicating DNA. The hepatocytes were 'chased' for 24 hours with unlabelled thymidine then they were fixed and processed for autoradiography .

DNA repair was assessed by comparing the labelling levels of hepatocyte nuclei from treated animals with control values and with the accompanying cytoplasmic labelling levels (usually a total of 150 cells per animal were examined).

At the 14 hour expression time, at the high dose level of 450 mg/kg bodyweight, a statistically significant increase in net nuclear grain count was obtained in comparison with the concurrent control. Since this increase was small and was not accompanied by any increase in the gross nuclear grain count and the net nuclear grain count obtained was well within the laboratory historical control range, it is not considered to be indicative of unscheduled DNA synthesis. Bromodichloromethane did not cause any other significant increases in the net nuclear grain count in this test. Bromodichloromethane did not cause any substantial increases in the gross nuclear grain count at any dose level at either sampling time.

Positive control group animals showed a large and highly significant increase (P<0.001) in the net nuclear grain count which was accompanied by a large increase in the gross nuclear grain count.

It is concluded that bromodichloromethane did not elicit any evidence of DNA-damage in the rat liver in this in vivo test system.

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