## 1 Introduction
Therefore, the World Health Organization experts board developed \"Nitrosation Assay Procedure\" (NAP test) to screen for the liability of pharmaceuticals to endogenous nitrosation. The conditions of the NAP test involve a fourfold excess of nitrite in an acidic solution which mimics in vivo nitrosation in the stomach. Unfortunately, this procedure has never been included in registration dossiers. Hence, the potential for endogenous nitrosation of pharmaceuticals remains unknown unless verified by scientific research [6]. It is therefore evident that the area of carcinogenic drug impurities and metabolites safety profiling still remains insufficiently supported by experimental data, which obviously poses a safety concern and explains the need for relevant toxicological studies, mainly for well-established pharmaceuticals of key clinical relevance and widespread use. Ramipril (RAM) is a dicarboxylate-containing angiotensin-converting enzyme inhibitor (ACE-I) of major clinical importance due to its indications for long-term treatment of severe cardiovascular conditions [12; 13]. It was patented in 1981 and approved in 1989 [14]. Its impurity assessment was not required by the ICH M7 (R1) guideline during the post-approval period, and therefore it is hardly to be expected from the industry. In 2019, its total number of prescriptions in the United States was nearly 4 million, while the estimated number of treated individuals was 854,000, indicating its broad patient exposure and significant impact on public health [15]. Concurrently, there has been a fair number of epidemiological studies demonstrating clear associations between ACE-Is (including RAM) and the development of malignancy, such as multiple myeloma, breast and lung cancer [16; 17; 18; 19; 20; 21; 22]. Furthermore, in lung cancer, high cumulative ACE-Is doses were associated with modestly increased odds of disease, while lower doses only showed neutral associations. The mechanism behind these effects has never been elucidated [20]. Based on the above-discussed insufficiency of safety profiling procedures in the group of well-established pharmaceuticals, a possible interpretation of these observations could be the carcinogenic impact of drug degradation impurities or N-nitroso metabolites, which have evaded the regulatory safety screening prior to drug approval, as was the case with valsartan and ranitidine. Such cause-effect relationships would be, however, hard to observe in the setting of observational case-control studies due to distant endpoints and multiple distracting factors. Therefore, the suggested correlation between RAM stability and its oncological safety must be verified by means of experimental methods. According to our previous report, RAM is chemically unstable as its degradation is rapid, and within the pharmacological class of ACE-Is it is the most vulnerable to temperature and humidity changes. Its degradation impurities formed under humid conditions include: biologically-active ramiprilat and a diketopiperazine derivative (DKP) [15]. The available stability data for RAM are, however, incomplete. Its mechanism of degradation under dry air still remains unknown. Probably, the only degradation impurity formed under such conditions is DKP, as with other structurally-related ACE-Is [23; 24]. This degradation pathway, in turn, is highly unfavorable from the clinical point of view, since DKP is pharmacologically inactive against RAM biological targets, its presence is of no clinical benefit and its toxicity to humans is unknown [15]. DKP also contains a diketopiperazine structural alert that is potentially susceptible to N-nitrosation in vivo, with further mutagenic hazard to patients, as discussed above [8]. Thus, we hypothesized that DKP formation in RAM dosage forms could be a possible reason for the reported increased cancer incidence, associated with ACE-I use. To verify this, we decided to perform this multistage study correlating the stability and safety features of RAM.