## 4 Discussion
Because genotoxic substances usually have a threshold mechanism, two experimental series of concentrations were conducted. The first series involved high concentrations of DKP, and it was employed to screen for the exact mechanism of genotoxicity. The second series covered physiological concentrations of DKP that corresponded to the level ordinarily present in the blood of patients treated with RAM due to cardiological indications. The second series enabled us to show the real-life activity of DKP after standard RAM dosing. Firstly, the cytotoxicity of DKP was established, as this effect is a known confounding factor for micronucleic scoring. Notably, under the conditions of our test, the investigated degradation impurity exhibited a significant cytotoxic activity, exceeding 55.5%, as shown in Table 4, especially in the extended treatment setting in the screening series. Hence, only one screening concentration (0.22 mg/mL) was subjected to micronucleic scoring. For this concentration, in both short treatments, the percentage of micronucleic relative to the control was not increased, suggesting no clastogenic potential of DKP (\(p\) > 0.05, Table 4). On the contrary, based on the statistically significant, three-fold increase in micronucleic formation observed in the extended treatment mode, DKP was suggested to act as an aneugen (% MN/BMN = 33.33, \(p\) = 0.0184). This means that DKP could interact with proteins involved in the segregation of chromosomes during mitotic cell division, rather than DNA, leading to chromosome loss via micronucleic formation. Notably, it is typical for aneugenic substances that their effect only occurs in a narrow range of concentrations with a sublinear dose-response relationship; thus, their threshold dose can be estimated, as suggested by [PERSON] et al. [34]. As a consequence, low doses of aneugens induce zero effect, which was actually evidenced for DKP in our study, as shown in Table 4; at its physiological (micromolar) concentrations, the micronucleic were not formed (\(p\) < 0.05). This means that standard dosing of RAM should not pose a carcinogenic risk in humans with respect to the suspected genotoxicity of DKP. However, the threshold level of DKP, above which the aneugenic effect is manifested, remains unknown. It falls between the tested series, and it should be established, preferably in follow-up in vivo studies; for example, using an in vivo micronucleus assay. This knowledge is necessary to set the safe level of human exposure to this impurity. Furthermore, the observed significant cytotoxicity of DKP is also representative of a typical aneugen [34]. In fact, high doses of aneugens cause severe genomic imbalance and launch cell death mechanisms, leading to cell elimination, as was the case in our study. The high cytotoxic potency of DKP can be also explained by its structural features. Its changeable chiral and rigid skeleton favors its easier binding to molecular targets; for example, Aurora kinases or tubulin, both of which participate in chemical-induced aneugenicity in vitro [35]. Interestingly, this property of other DKP analogs, i.e., unsaturated 2,5-diketopiperazine derivatives, was examined in a number of human tumor cell lines, in which a 2,5-diketopiperazine ring was set as an optimal scaffold for exploring anticancer drug candidates [36]. Nonetheless, in the context of our studies, it must be emphasized that aneuploidy caused by aneugens is an important, but not independent, contributor to cancer development [37]. For this reason, a direct genotoxic/aneugenic activity of excessive doses of DKP combined with other carcinogenic factors is necessary to induce cancer in humans. Consequently, we further investigated other mechanisms of carcinogenicity of DKP; i.e., its mutagenic activity and the mutagenic activity of its N-nitrososo metabolite.