Evaluation of the need for a late harvest time in the assay for chromosome aberrations in Chinese hamster ovary cells.

Harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In CHO cells we have found that for a variety of chemicals one harvest time near 20 h is optimal following a 3-h treatment (Bean et al., 1992). However, some guidelines for testing for regulatory purposes recommend an additional late harvest time 24 h after the first. We tested 10 diverse chemicals in CHO-WBL cells harvested 20-21 h and 42-44 h from the beginning of a 3-hr treatment. We added BrdUrd after treatment and recorded the total% of aberrant cells, and the proportions of aberrations (abs) in first (M1), second (M2) or later metaphases. The chemicals fell into 3 categories: ab yield greatly decreased at 44 h: benzo[a]pyrene, cadmium sulfate, chlorambucil, 2,6-diaminotoluene, 4-nitroquinoline N-oxide and mitomycin C (e.g., 37.0% cells with abs at 20 h and 1.0% at 44 h); ab yields similar at 20 and 44 h: 2-aminobiphenyl, eugenol and 8-hydroxyquinoline (e.g., 8.5% at 20 h and 7.0% at 44 h); and one, dimethylnitrosamine (DMN), which was detected at both times but gave a stronger response at 44 h than at 20 h (e.g., at 10 mM: 6.2% at 20 h and 25.0% at 44 h). This DMN effect was not seen in normal diploid human cells. For DMN the higher ab levels at 44 h than at 20 h were contributed by abs in M3 cells. Thus, while for some chemicals ab yields decrease with successive division, further increases can be seen in CHO in later metaphases, notably for DMN. Overall, however, after a 3-h pulse treatment of CHO cells a positive ab result could be obtained at the early harvest time (20 h) for all 10 chemicals.