5.5. Ames Microplate Format (MPF™) Assay

To assess the mutagenic properties of AOH (0.1, 10, and 50 µM), AA (0.5 and 50 mM), and their respective binary combinations, a commercial Ames kit with aroclor-induced rat liver S9 fraction was employed (Ames MPF 98/100 semisolid kit + Ames MPF S9 Cofactor Kit; Xenometrix^® AG, Allschwil, Switzerland). In particular, the test was conducted following the provider’s protocol and by employing the S. typhimurium strains TA98 (sensitive to frameshift mutations) and TA100 (base pair substitution). Briefly, overnight cultures of bacteria were used only after reaching an optical density at 600 nm (OD₆₀₀ value) ≥ 2. For incubations without S9 fraction, overnight cultures were diluted to 1:10 (for TA98) or 1:20 (TA100) with exposure media containing the various test chemicals. For incubations with S9 fraction, an S9 mix containing buffer salts, glucose-6-phosphate, NADP, and S9 fraction was prepared according to the provider’s protocol and diluted 1:6 with exposure media containing booster solution (final concentration: 0.16%) and the various test chemicals. For treatments with TA98 and TA100 strains in absence of S9 fraction, a mixture of 2 µg/mL 2-nitrofluorene and 0.1 µg/mL 4-nitroquinoline oxide was used as a positive control. For treatments of TA98 and TA100 in the presence of metabolic activation, 1 and 2.5 µg/mL 2-aminoanthracene were used as positive controls. DMSO (4%) was used as a solvent control (with the same % in all test conditions). Bacterial suspensions (250 µL/well in 24-well plates) were therefore incubated with the various test conditions for 90 min at 37 °C while shaken. At the end of the incubation time, 2.8 mL of indicator medium that lacks histidine was added to each well. Afterwards, 50 µL of each bacterial suspension (treated and diluted in indicator medium) was transferred into 48 wells of a 384-well plate, which was subsequently placed into a resealable plastic bag and incubated for 48 h at 37 °C (without shaking). The plates were scored by counting the wells that turned yellow. Results were expressed as fold increase over threshold, which was calculated as two times the sum of the mean value of the solvent control plus one standard deviation (in accordance with the provider’s instructions). The test was considered positive when the fold induction was higher than the threshold.

To assess the impact of single and combined treatments on the viability of bacteria, a cell viability test (in exposure medium) was performed on bacterial strains exposed to the various test conditions (for 90 min) by measuring the optical density at 600 nm. Furthermore, to avoid misinterpretation of the results due to poor solubility of the compounds, the presence of possible crystals in the wells was assessed by using an inverted microscope.

The concentration range chosen for AOH and AA aligned with OECD guideline n. 471, which delineates the evaluation of the mutagenic properties of chemicals via bacterial reverse mutation tests [40]. Specifically, the highest concentrations tested in this study were those that did not interfere with plate scoring due to precipitation of the compounds.