## 4 Discussion
The second stage is deprotonation of the reacting amine, the addition of neutral nitrogen to the carbonyl of the neighboring carboxylic acid to form a tetrahedral intermediate, the escape of water molecules and a final new bond formation. Here, the rate-limiting step of this process is the _cis_/_trans_ transformation of RAM, associated with multiple bond rotations and a consequent high energy consumption, defined by high E\({}_{\text{a}}\) (Table 1, AE\({}_{\text{a}}\) = 174.12 kJ/mol). The subsequent water loss is thermodynamically favorable (as supported by positive AS, Table 1); hence, its rapid progression translates into a high value of the reaction rate constant. At RH 76%, no conformational changes are necessary for water molecules to access the ester bond in RAM; thus, the E\({}_{\text{a}}\) for RAM hydrolysis under humid conditions is relatively low. Our findings are significant from a manufacturing point of view. Employing dry formulation methods would lead to compromised RAM stability, secondary to dry air degradation, which would be faster than that at humid conditions. Furthermore, the impurity profile would be affected, with DKP becoming a major degradant instead of RAM diacid. As a result, on heating and dry-processing, RAM would rapidly cyclize to DKP with all downstream consequences on its clinical and toxicological performance. Therefore, the application of dry procedures for RAM in the industry should be avoided. In the next stage of this study, we decided to assess the impact of the RAM degradation mechanism on its safety. We only focused our interest on DKP since the toxicological data for both RAM and RAM diacid are available in the registration dossiers of commercially available RAM dosage forms. Based on the literature background, which suggested a possible carcinogenic activity of ACE-Is, we performed a preliminary in silico QSAR simulation assessing various oncologic endpoints, i.e., the general carcinogenicity, mutagenicity and genotoxicity of DKP. Mutagenicity refers to the capacity of chemicals to cause changes in DNA sequences, leading to mutations, while genotoxicity causes damage to genomes, i.e., DNA or chromosomes. The genotoxic agents that cause structural chromosomal aberrations are elastogens, while those that cause numerical chromosomal aberrations are aneugens. The main practical difference between mutagenicity and genotoxicity is that mutagens have no threshold level and any exposure to them is hazardous. Thus, they are not allowed in final dosage forms. Genotoxic agents, in turn, act via threshold mechanisms and their low exposure is not always associated with cancer outcome. Thus, their safe concentration must be established and maintained in drug formulations. The results obtained from our QSAR simulations allowed us to classify DKP as a non-mutagen, as the calculated endpoint score (ES\({}_{\text{av}}\) = 0.2) fell within the non-mutagenicity criteria. The reliability of this prediction was high. Despite this, the adopted in silico model suggested other mechanisms of toxicity, i.e., the carcinogenic and genotoxic activity of DKP, but these predictions were not sufficiently reliable (ES\({}_{\text{av}}\) = 0.5 and 0.6, respectively). On this basis, we assumed that DKP is a potential carcinogen that acts via a mechanism unrelated to direct DNA damage (non-mutagen), probably via chromosome damage (genotoxic agent). However, due to the insufficient reliability of the QSAR simulation for the genotoxicity endpoint, follow-up experiments were necessary, either by in vitro micronucleus or by chromosome aberration assay. The in vitro mammalian cell micronucleus test for the genotoxicity assessment of DKP was selected as a follow-up to our QSAR simulations. The study was designed so as to screen for both aneugenic (aneuploidy-inducing) or clastogenic (chromosome-damaging) activity of the investigated compound. To that end, different treatment modes were applied (a short one for elastogens and an extended one for aneugens).