TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 37 CONCLUSIONS AND FUTURE ASPECTS Under the conditions of the study, the test substance, OB, did not show promotion of the generation of micronuclei with and without enzymatic activation. The measurements of cellular viability showed that cyclosphosphamide + S9 mix (10 μg/ml), ethyl methane- sulfonate (EMS, 400 μg/ml), or OB (0.2 mg/ml and 0.3 mg/ml) alone or in combination with S9 did not inhibit the growth of cells (Figure 4) at the concentrations used. Conse- quently, we conclude that all tested substances do not promote cell death at the concen- trations used and that the positive controls may have generated a shift in cellular function that led to enhanced proliferation. While this method indentifi es the genotoxic potential of the tested compounds, there are ad- ditional end points that can be studied to better assess the effect of the compounds tested on cell survival. Such is the characterization of cell death by either apoptosis or necrosis. In addition, this study demonstrates a way to test a compound that is not fully solublized in the media. Stages of introduction of the OB to the cell culture included dissolution in Figure 3. Number of micronuclei per 1000 cells. CHO-K1 cells were incubated for 24 hours with the fol- lowing substances: (1) Media (negative control) (2) DMSO (negative control) (3) Cyclosphosphamide + S9 mix (positive control) (4) Ethyl methanesulfonate (positive control) (5) OB, 0.2 mg/ml in DMSO (6) OB, 0.3 mg/ml in DMSO (7) OB, 0.2 mg/ml in DMSO + S9 mix and (8) OB, 0.3 mg/ml in DMSO + S9 mix. Means ± S.D. are shown. *P0.05 when compared with negative control. Figure 4. Cellular viability. CHO-K1 cells were incubated for 24 hours with the following substances: (1) DMSO (negative control) (2) Cyclophosphamide + S9 mix (positive control) (3) Ethyl methanesulfonate (positive control) (4) OB, 0.2 mg/ml in DMSO (5) OB, 0.3 mg/ml in DMSO (6) OB, 0.2 mg/ml in DMSO + S9 mix and (7) OB, 0.3 mg/ml in DMSO + S9 mix. Means ± S.D. are shown (1).
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