## 2 Materials and Methods
### In Silico Prediction of Genotoxicity, Mutagenicity and Carcinogenicity  
The kinetic study was followed by an in silico safety evaluation of the identified RAM degradation impurity in order to set the scope of further toxicological research in vitro. An open access software VEGA-GUI version 1.2.0 from www.vegahub.eu (accessed on 4 January 2023) was used for carcinogenicity, mutagenicity and genotoxicity prediction. The structure of the IMD derivative (a structural analogue of RAM) was tested in parallel in order to make a comparative analysis.  
The employed VEGA platform accessed QSAR models, which correlated its internal dataset of structurally-related chemicals with target molecules. In the version 1.2.0, it has four models for mutagenicity, ten models for carcinogenicity, one model for chromosomal aberration and two for micronucle activity. The models available for the mutagenicity endpoint (ISS, SARpy, CAESAR, Mutagenicity Read-Across/KNN) were built based on experimental data derived from in vitro studies (e.g., the Ames Test in _Salmonella typhimurium_ strains), while the carcinogenicity models (ISS, ISSCAN-CGX, CAESAR, ANTARES) used data from in vivo studies in different species, mainly mice and rats. All the VEGA models differ in their analytical approach (rule-based expert system, correlation between relevant fragments with toxicological endpoint, regression models, hybrid models), all of which had been validated prior to their commercialisation and none of which was considered inferior [27]. Thus, in this study, all of them were used to increase the predictive power of the stimulation.  
The identifiers of the tested molecules in SMILES format were inserted into the task list of the analysis tool. Then, the QSAR models for toxicity endpoints were selected and the prediction reports for each model were computed. The VEGA output was exported in.pdf file with 'yes' or 'no' prediction relative to each endpoint. The results were supported by the information on the reliability of prediction, measured by VEGA by the use of an in-built applicability domain evaluation tool. It was expressed as the Applicability Domain Index (ADI), which was computed to compare the tested molecules with the model training set. For ADI > 0.85, the prediction was considered strong. For ADI < 0.65, the prediction was considered weak. The values in between were considered as moderate. This parameter was automatically analyzed by VEGA and it was integrated into a final wording result expressed as \"low reliability\", \"moderate reliability\", \"good reliability\" [27]. To compare the results obtained for different QSAR models, the scoring approach developed by [PERSON] et al. [28] was employed. It translated VEGA wording results into numeric values (endpoint scores, ES) ranging from 0 to 1 where \"1\" represents high toxicity and \"0\" is low toxicity with respect to model endpoint. Then, the average ESav was calculated and expressed as the arithmetical mean of the ES values for respective endpoints. To interpret these data, the cut-off values were adopted as follows: ESav > 0.66 for true-toxicity claim; ESav < 0.33 for true non-toxicity claim. Scores between 0.33 and 0.66 were considered as inconclusive.