For short-term treatment, cells were treated for 6 h at 37 °C in the presence or absence of 5% S9, after which, the medium was discarded, and the cells were rinsed with PBS(−) and cultured for another 18 h at 37 °C in fresh medium. For continuous treatment, cells were treated for 24 h in the absence of metabolic activation. At the start and end of the treatment period, and at the end of culturing, whether

or not the test sample had precipitated was checked macroscopically. At the end of culturing, the number of cells was counted by using a Microcell counter (CDA-500; Sysmex, Hyogo, Japan) after trypsinization, and the cell growth find more rate at each dose was calculated. Finally, the prepared specimens were stained with Giemsa solution (Merck, Darmstadt, Germany) and 200 metaphase cells per dose were evaluated for structural aberrations and numerical aberrations, respectively. 2) Evaluation of results The in vitro chromosomal aberration test was considered positive when the frequency of cells with structural aberrations or numerically aberrant cells was 10% or more and the increase was dose-related. All other cases were considered negative. No statistical analyses were used. A total of four SDs and five STs were detected in the test sample (Table 3 and Fig. 1). The total SD and ST content of the test sample was approximately 88%; approximately 12% of the styrene oligomers

in the test sample were of tetrameric or greater size (data not check details shown). Prior to conducting the Ames test, a dose range-finding study was conducted using six doses: 156, 313, 625, 1250, 2500, and 5000 μg/plate. No increase in the number of revertant colonies and no bacterial growth inhibition were observed at any of the doses examined (data not shown). Therefore, the same six doses were used for the first Ames test. To confirm the reproducibility of the results, a second Ames

test was conducted, but this time in consideration of the results of the first test, only five doses were used: 313, 625, 1250, 2500, and 5000 μg/plate. Because the dose–response curves obtained from the two Ames tests were comparable, only Terminal deoxynucleotidyl transferase the dose–response curves from the first test are presented (Fig. 2). The number of revertant colonies in each dose was similar to that in the negative control for all tester strains. No inhibition of bacterial growth and no precipitation of the test substance were observed for any of the test conditions in either the presence or absence of S9 mix. Therefore, because the number of revertant colonies in all treatment groups for all tester strains was less than twice that in each negative control in the presence or absence of S9 mix in both the first and second Ames test, the genotoxicity of the test solution was considered negative. The numbers of revertant colonies in the negative control and positive control were within the historical range for our laboratory (data not shown).