2.6. Agar-Based Ames Test Protocol

The pre-incubation Petri dish agar-based Ames test protocol was conducted according to the methods described by [31], with minor adaptations. The bacteria were grown as described above and the exposure was done in 24-well plates, in triplicates, containing 100 µL of pre-culture, 500 µL of phosphate buffer (0.2 M, pH 7.4) and 50 µL of the test substance dissolved in DMSO. For the negative control, pure DMSO was applied. As a positive control, for TA98 without S9 50 µg/mL 2NF, for TA100 without S9 2.5 µg/mL 4NQO and for TA98/100 with S9 25 µg/mL 2AA were applied. After 90 min of exposure the mixture was pipetted into 2 mL molten top agar (5 µM histidine and biotin), which was melted and kept at 48 °C in a water bath. The agar was then poured onto Petri dishes containing histidine free minimal glucose agar (MGA; 0.4% glucose). For the metabolic activation, a 1% S9 mix was prepared and kept on ice, until use. It consisted of PB/β-NF-induced rat liver S9 and the co-factor mix (see chapter metabolic activation). The 1% S9 and the co-factor mix were applied instead of the phosphate buffer as required, resulting in a final concentration of 0.77% S9 during the exposure.