Report No DWI0734

Nov 1995

Bromoform 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 324 and 1080 mg/kg bodyweight. (A small preliminary toxicity test indicated that a dose level of 1080 mg/kg bodyweight would be approximately the maximum dose level tolerable, this 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 10 µCi/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).

Bromoform did not cause any statistically significant increases in either the gross nuclear grain count or the net nuclear grain count (ie the gross nuclear grain count minus the cytoplasmic grain count) at any dose level at either sampling time.

Positive control group animals showed a highly significant increase in the net nuclear grain count which was accompanied by a large increase in the gross nuclear grain count.

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

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