In a second experiment, using the same strain and S9, reference sample 2R4F again gave the highest revertant yield, but there was no clear
concentration-related increase in mutagenicity for any PM. Two further experiments confirmed weak concentration-related increases in revertants for all of the LDK378 PMs, with reference sample 2R4F giving the clearest response. The mutagenic potencies of the extracts in TA1537 were generally lower than they were in TA100, and showed some variation between experiments. In one of the three experiments with a concentration-related increase in revertants, conducted with TA1537, W862 and W863 were significantly less mutagenic than W860, W861 and W864; and W863 and W864 also exhibited significantly lower potencies than W861 in two experiments. In conclusion, there were no qualitative differences between PMs in any strains. The PMs were also the same in terms of S9 dependence. Quantitatively, PMs with 80% BT tobacco Compound Library were less mutagenic than the other PMs in strain TA98 with S9 activation. All PMs induced
dose-related increases in cytotoxicity, and also induced genotoxicity with and without S9 and at the different treatment times. PMs increased the frequency of micronucleated binucleate cells by more than 3-fold. In terms of dose and %MnBn/μg NFDPM, the 20 h treatment without S9 was more sensitive than the 3 h treatments. At 20 h without S9, W862 induced fewer micronuclei than W860 and W861 in both experiments
(Fig. 2). This was statistically significant in both experiments for W860 and in one experiment for W861 (Table 6). At 3 h ± S9, W862 induced fewer micronuclei than W861, in two experiments (Table 6). The assay’s resolving power was limited by Branched chain aminotransferase relatively large variability within and between experiments, non-linear responses and >50% cytotoxicity at the higher doses in the 3 h treatments. This may have contributed to the inconsistent differences observed between PMs. Concentrations of test and reference PMs were selected in order to provide as many points as possible lying on the linear part of concentration–response curves, and to provide as many concentrations as possible that were common to each PM treatment, whilst allowing treatment up to toxicity limiting dose levels. In some cases the highest concentration levels were not selected for plating to determine viability and TFT resistance, or were excluded from analysis, due to excessive toxicity (based on cell count data). Statistically significant increases in mutation frequency (MF) of 3- to 4-fold were observed with each of the PMs, on each experimental occasion and with each of the treatment conditions employed (e.g. Fig. 3). In terms of dose and MF/μg NFDPM, the 20 h treatment without S9 was the most sensitive, and the 3 h treatment with S9 was the least sensitive.