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 e nsure that cells are sampled at an appropriate time for the detection of clastogenic effects. The sources of variation in the cell cycle tim e in routine cytogenetic assays have not been well studied, Consequent ly 13 laboratories, all members of the Industrial Genotoxicology Group , participated in a collaborative study to measure the variation in ce ll cycle time in cultured human peripheral blood lymphocytes under var ious conditions, The study was performed in two phases, spaced 6 month s apart. The average generation time (AGT) was measured by the incorpo ration of bromodeoxyuridine. Very similar AGTs were found in the prese nce 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 laboratorie s than within laboratories, A comparison of AGT values at 72 h perform ed in experiments at least 6 months apart indicated good reproducibili ty in most laboratories, The study indicates that a 24 h post-treatmen t harvest may result in the analysis of very few first division cells unless very significant cell cycle delay is induced by the test substa nce. 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 sec ond division cells and therefore should by suitable for assessing both the induction of chromosome aberrations and polyploidy.