Industrial Genotoxicology Group collaborative trial to investigate cell cycle parameters in human lymphocyte cytogenetics studies

Human lymphocyte cultures have been used for many years for assessing the in vitro clastogenic potential of test substances. In these assays the harvest time should be based on the cell cycle time in order to ensure that cells are sampled at an appropriate time for the detection of clastogenic effects. The sources of variation in the cell cycle time in routine cytogenetic assays have not been well studied. Consequently 13 laboratories, all members of the Industrial Genotoxicology Group, participated in a collaborative study to measure the variation in cell cycle time in cultured human peripheral blood lymphocytes under various conditions. The study was performed in two phases, spaced 6 months apart. The average generation time (AGT) was measured by the incorporation of bromodeoxyuridine. Very similar AGTs were found in the presence and absence of S9 mix. The mean AGT (mean of four donors) in each laboratory varied from 11.2 to 17.1 h, indicating there is significant variability in cell cycle times of human peripheral blood lymphocytes between laboratories. There was greater variation between laboratories than within laboratories. A comparison of AGT values at 72 h performed in experiments at least 6 months apart indicated good reproducibility in most laboratories. The study indicates that a 24 h post-treatment harvest may result in the analysis of very few first division cells unless very significant cell cycle delay is induced by the test substance. It was also found that a post-harvest time equivalent to 1.5 cell cycles will result in an approximately equal mixture of first and second division cells and therefore should by suitable for assessing both the induction of chromosome aberrations and polyploidy.