## 2 Materials and Methods
### Mutagenicity Assay-Bacterial Reverse Mutation Test Procedure  
Ames test was performed to verify the results of QSAR assessment for mutagenicity and to check the mutagenic activity of the N-nitroos metabolite of the tested compound. This procedure was chosen since it is the first choice option for mutagenicity assessment of pharmaceuticals according to the ICH M7 (R1) guideline. A commercial Ames MPF 98/100 microplate format mutagenicity assay kit (Xenometrix, Allschwil, Switzerland) with _Salmonella typhimurium_ strains TA98 (containing frameshifts mutation h3D3052, rfa uvrB, pKM101) and TA100 (containing base-pair substitution mutation hisG46, rfa uvrB, pKM101) was used. It contained 2-nitrofluorene, 4-nitroquinolone-N-oxide (4-NQO), 2-aminoanthracene as positive controls, Aroclor 1254-induced rat liver fraction S9 as the activation system, sterile ampicillin (50 mg/mL), ready-to-use growth medium, exposure medium and indicator medium. For preparing the S9 mix, a ready-to-use kit from Xenometrix (Switzerland) containing Buffer Salts solution (phosphate buffer, MgCl\({}_{2}\), KCl, NADP solution, G-6-P solution) was employed. The tested samples were dissolved in sterile DMSO (Merck, Darmstadt, Germany). A completely degraded RAM sample (RAM content \(\sim\)0%) and the nitrosation mixture of the RAM degradation product (\(c\) = 112.6 mg/mL) were subjected to mutagenicity analysis. The degradation product was dissolved in DMSO to obtain a concentration of 125.0 mg/mL. Then, the test procedure provided by Ames MPF Instruction for use was followed [32,33]. The _Salmonella typhimurium_ strains TA98 and TA100 were grown overnight (15 h) in growth medium at 37 \({}^{\circ}\)C (310 K) on a shaker set at 250 rpm. The exposure concentrations were selected based on the pre-screening procedure for cytotoxicity and solubility using the TA98 strain, as per kit instructions [33]. The _Salmonella typhimurium_ strains TA98 and TA100 were then exposed to the tested substances in the presence and absence of rat liver S9 fraction. In the experiments without a metabolic activation system, the following positive controls were used: 2-nitrofluorene (2.0 mg/mL) for TA98 and 4-nitroquinolone-N-oxide (0.1 mg/mL) for TA100. In the experiments with a metabolic activation system, 2-aminoanthracene was used as a positive control at a concentration of 1.0 mg/mL for TA98 and 2.5 mg/mL for TA100. For the experiment with the degradation product, the baseline concentration was 125.0 mg/mL. For the experiments with the nitrosation mixture, the baseline concentration of the parent compound was 112.6 mg/mL. A serial \({}^{1/2}\)-log dilution was performed on a 96-well plate. To that end, in the experiments without metabolic activation, the standard solutions were diluted with histidine-rich exposure medium. In the experiments with a metabolic activation system, the standard solutions were diluted with histidine-rich exposure medium and microsomal S9 fraction mix. Six stock concentrations were achieved. Approximately 10\({}^{7}\) bacteria for each strain were treated in triplicate with the following test concentrations: 5.0 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.63 mg/mL, 0.31 mg/mL, 0.16 mg/mL for pure degradation product and 4.5 mg/mL, 2.25 mg/mL, 1.125 mg/mL, 0.56 mg/mL, 0.28 mg/mL, 0.14 mg/mL for the nitrosation mixture, in a 24-well plate. Treated bacteria were incubated at 37 \({}^{\circ}\)C (310 K) on a shaker (250 rpm) for 90 min. Then, pH indicator medium without histidine was used to dilute the exposure cultures. They were aliquoted into 48 wells of a 384-well plate (50 uL per well). This was followed by 48 h of incubation at 37 \({}^{\circ}\)C (310 K) to allow revertant bacteria to grow. The indicator medium contained a pH indicator dye, which changed colour from purple to yellow due to bacterial metabolism, if the mutation occurred. The scoring of positive wells (revernants) was visually performed. Mutagenicity was confirmed if at least a two-fold increase over the baseline was observed (FIB > 2).