2.5. Ames MPF™ Test Protocol

The Ames MPF™ test protocol was performed, according to the method’s supplier protocol (Xenometrix), with minor adaptations. As solvent control, DMSO was applied. As a positive control, for TA98 without S9 50 µg/mL 2-nitrofluorene (2NF), for TA100 without S9 2.5 µg/mL 4-nitroquinoline-1-oxide (4NQO) and for TA98 and TA100 with S9 50 µg/mL 2-aminoanthracene (2AA) was applied. Exposures were performed in triplicates in 24-well plates and 10 µL of the test substance or the controls were used per well. The pre-culture was mixed with exposure medium (10% bacteria v/v for TA98 and 5% v/v for TA100) and then 240 µL of this mix was added to each well. After 90 min of incubation at 37 °C at 250 rpm in an orbital shaker, 2.6 mL of indicator medium (Xenometrix) were added. The content of the 24-well plates was distributed into three 384-well plates. For the metabolic activation, a 15% S9 mix was prepared and kept on ice, until use and consisted of either PB/β-NF, or Aroclor 1254-induced rat liver S9 and the co-factor mix (see metabolic activation section). The 15% S9 and the co-factor mix were added as required, resulting in a final concentration of 2.25% S9 during the exposure.