## 5 Conclusions  
In our study, we managed to describe the mechanism of RAM degradation under dry air. We proved that such conditions compromise RAM stability and promote its rapid degradation via intramolecular cyclization. The only degradant formed at RH0% was DKP. From QSAR simulations, we established that DKP was a potential carcinogen acting by a genotoxic mechanism. However, this prediction needed confirmatory studies due to its insufficient reliability. The QSAR model also suggested that DKP was a non-mutagen with high reliability. In in vitro studies, we found two potential mechanisms of DKP-induced carcinogenicity; i.e., the direct aneugenic action of excessive doses and indirect mutagenicity secondary to the in vivo formation of N-nitroos metabolites. From the in vitro micronucleus assay, we found that DKP was a cytotoxic and aneugenic agent at high concentrations. At normal blood concentrations, DKP was neither cytotoxic nor aneugenic. Hence, due to the potential aneugenicity, the safe level of DKP must be evaluated in follow-up in vivo studies, as excessive doses above normal blood concentrations could contribute to cancer as a supporting factor. Our results do not establish a threshold concentration of DKP. Concurrently, no clastogenic effect is expected from DKP based on in vitro micronucleus assay. Furthermore, from the in vitro Ames test, DKP was not a direct mutagen. However, it can form nitroso derivatives in vivo, which induce base-pair substitution mutations upon metabolic activation in the liver. Nitroso-metabolites of DKP were mutagenic in the Ames test; thus, it was concluded that no safe level of DKP nitroso-metabolites exists. This can be extrapolated to DKP as it is the only source of DKP-nitroso derivatives in vivo. Consequently, it is probable that RAM dry air degradation could be associated with increased cancer incidence, secondary to forming reactive, mutagenic nitroso-metabolites and by inducing aneugenic damage of genomes at high concentrations. For this reason, dry formulation methods and excessive heating must be avoided in RAM technological processing. The real-life significance of our findings must be verified in follow-up in vivo experiments.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13042358/s1](https://www.mdpi.com/article/10.3390/app13042358/s1)]([https://www.mdpi.com/article/10.3390/app13042358/s1](https://www.mdpi.com/article/10.3390/app13042358/s1)). Supplement S1: HPLC validation procedures. Supplement S2: Thermodynamic parameters calculation. Supplement S3: HPLC validation results. Supplement S4: Kinetic study results. Supplement S5: QSAR prediction for IMD and IMD-DKP carcinogenicity, genotoxicity and mutagenicity. Supplement S6: Micronucleus assay--cell scoring. Supplement S7: Mutagenicity assay results.  
K.R.: conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; supervision; validation; visualization; original draft writing; review and editing, A.M.-W.: resources, formal analysis, draft writing, funding acquisition, supervision, A.M.: resources, investigation; formal analysis, draft writing, B.J.S.: supervision. All authors have read and agreed to the published version of the manuscript.  
This research was funded by Greater Poland Cancer Center, grant No. 4/2020(235).  
Not applicable.  
The data presented in this study are available in DOI:10.3390/pharmaceutics13101600.  
HPLC-MS analysis was performed in the Laboratory of faculty of Chemistry, A Mickiewicz University, Poznan, Poland. The procedure of Ames test was performed in Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland. In vitro micronucleus test was performed in Screening of Biological Activity Assays and Collection of Biological Material Laboratory, Wroclaw Medical University, Poland.  
The authors declare no conflict of interest.