## 2 Materials and Methods
### Micronucleus Assay Procedure  
The degradation impurity of RAM was then subjected to in vitro micronucleuleus assay based on the results of the computed in silico simulations. To that end, DMEM (Dulbecco's Modified Eagle's Medium), FBS (Fetal Bovine Serum), L-glutamine, amphotericin B, penicillin-streptomycinycin, Aroclor 1254-induced male Sprague Dawley rat liver S9, mitomycin C, cyclophosphamideamide, edchicine and cytochalasin B were used. They were purchased from Sigma-Aldrich (St. Louis, MO, USA). HCS CellMaskTM Green Stain and Hoechst 33342 were supplied by Thermo Fisher Scientific (Waltham, MA, USA). 1929 cell line (ECACC 85011425, mouse C3H/An connective tissue) was cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 2.5 ug/mL amphotericin B, 100 U/mL penicillin and 100 ug/mL streptomycin. Cells were maintained at 37 \({}^{\circ}\)C (310 K) and 5% CO2 in a humidified atmosphere. Cell cultures were passaged before they reached 90% confluency to maintain exponential growth. They were regularly checked for mycoplasma contamination. For the cytokinesis-block in micronucleuleus assay 24 h before treatment, cells were seeded into 6-well plates at a density of 2.2 \(\times\) 10\({}^{5}\) cells/well and incubated at 37 \({}^{\circ}\)C (310 K), 5% CO2 in a humidified atmosphere. After the incubation time, cells were treated with tested compounds in the same 6-well plates. The in vitro micronucleuleus test was performed in accordance with the guideline 487: In Vitro Mammalian Cell Micronucleus Test, from the Organization for Economic Co-operation and Development (OECD, 2016) [29]. In this study, two experimental series were carried out. The first one comprised three concentrations tested in duplicate: 2, 0.67, 0.22 mg/mL, for screening purposes. The concentrations and the number of replicates were selected basing on the recommendations of guideline 487 from OECD. In this setting, for each concentration, three experimental conditions were applied: short treatment with metabolic activation (6 h) and both, short (6 h) and long treatments (30 h) without metabolic activation, to investigate potential clastogenic and aneugenic activity. Metabolic activation was applied to detect genotoxic agents formed secondary to modifications by liver cytochromes. Hence, for each condition, different medium was required. For short treatment with metabolic activation, a culture medium with a 2% S9 mix to achieve metabolic activation was used. For short treatment without metabolic activation, only culture medium was necessary. For long treatment, medium with 2 ug/mL cytochalasin B as a cytokinesis inhibitor was applied. In the second experimental series, three physiological concentrations of the tested compound were investigated. This experiment was designed in order to mimic a clinical scenario and check the activity of RAM degradation impurity after the administration of a standard RAM dose to a patient. The concentrations were the following: 1, 10 and 100 nM (which correspond to: 3.98 \(\times\) 10\({}^{-7}\) mg/mL, 3.98 \(\times\) 10\({}^{-6}\) mg/mL, 3.98 \(\times\) 10\({}^{-5}\) mg/mL). They reflected blood levels of DKP, measured in blood samples derived from healthy individuals which were subjected to pharmacokinetic study [30]. Only the extended treatment conditions (30 h of treatment in the culture medium with cytochalasin B) without metabolic activation were applied, relying on the results obtained in the screening series. Stock solutions were prepared in DMSO, and appropriate solvent vehicle controls were also contained in the assay. The concentration of DMSO did not exceed 1% of the final culture volume. Additionally, positive controls were used, including: mitomycin C (0.25 ug/mL), cyclophosphamide (0.25 uM) and colchicine (0.025 ug/mL), depending on the type of exposure. The type and the concentration of the controls were selected based on the recommendations of the OECD guideline 487 [29].