## 3 Results
### Mutagenicity Assay  
To verify the QSAR prediction for the carcinogenicity and mutagenicity of DKP, two potential mechanisms of mutagenicity were checked by in vitro Ames test. Therefore, two experimental series were performed. The first experiment with DKP aimed at the verification of direct DNA-reactivity of the studied compound. The experiment with DKP nitrosation mixture (N-DKP), in turn, was to check the potential indirect mechanism of DKP carcinogenicity, involving nitrosamine formation after the NAP test. The highest tested concentration of DKP was 5 mg/mL and of N-DKP--4.5 mg/mL. None of these concentrations were cytotoxic against the TA98 strain. The number of revernants in treated cultures was achieved, and the results of plate scoring are provided in Tables 5 and 6. The detailed data are demonstrated in Supplement S7: Mutagenicity assay results. FIB and B-value were adopted as criteria for determining mutagenicity. The primary criterion was FIB, and the supporting criterion was B-value. As shown in Table 5, for pure DKP, the number of revernants at each concentration was not relatively increased compared to the control in any experiment performed (details in Supplement S7: Mutagenicity assay results). The FIB value below 2 indicates that the difference between the mean number of revernants in the control and exposition was not statistically different. The binominal B value below 0.99 suggests that the probability of spontaneous mutations was 99%. This means that DKP did not induce point mutations in either TA98 or TA100 strains at any of the tested concentrations. Therefore, the direct mutagenic effect of DKP by frameshift mutations or base-pair substitution was ultimately excluded. For N-DKP (Table 6), the induction of revernants in the TA98 strain was not statistically different from the control (FIB was below 2). For concentrations of 0.14, 0.28 and \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**N-DKP** & **TA98** & **–S9** & **TA98** & **+S9** & **TA100** & **–S9** & **TA100** & **+S9** \\ \hline
**c** (mg/mL) & **FIB** & **B-Value** & **FIB** & **B-Value** & **FIB** & **B-Value** & **FIB** & **B-Value** \\ \hline Baseline & 12.03 & & 4.72 & & 5.32 & & 4.87 \\
0.14 & 0.91 & 0.9911 & 0.14 & 0.0270 & 0.65 & 0.6613 & 0.37 & 0.3892 \\
0.28 & 0.94 & 0.9950 & 0.14 & 0.0270 & 0.53 & 0.4110 & 0.66 & 0.8961 \\
0.56 & 0.80 & 0.9372 & 0.49 & 0.5987 & 0.53 & 0.4110 & 1.18 & 0.9998 \\
1.125 & 0.94 & 0.9950 & 0.14 & 0.0270 & 0.95 & 0.9703 & 0.96 & 0.9953 \\
2.25 & 0.78 & 0.9062 & 0.28 & 0.1662 & 0.36 & 0.1018 & 1.69 & 1.0000 \\
4.5 & 0.80 & 0.9372 & 0.35 & 0.2943 & 0.47 & 0.2874 & 3.09 & 1.0000 \\ K+ & 3.97 & 1.0000 & 10.00 & 1.0000 & 7.30 & 1.0000 & 9.49 & 1.0000 \\ \hline \hline \end{tabular}  
* K\(\leftarrow\)positive control, FIB—Fold increase over baseline, B-value—indicates the probability that spontaneous mutations events alone.  
\end{table} 
Table 6: The results of bacterial reverse mutation test for DKP nitrosation mixture (N-DKP). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**DKP** & **TA98** & **–S9** & **TA98** & **+S9** & **TA100** & **–S9** & **TA100** & **+S9** \\ \hline
**c** & **FIB** & **B-Value** & **FIB** & **B-Value** & **FIB** & **B-Value** & **FIB** & **B-Value** \\ (mg/mL) & & & & & & & & & \\ \hline Baseline & 12.03 & & 4.72 & & 4.87 & & 5.32 \\
0.16 & 0.64 & 0.5854 & 0.42 & 0.4460 & 0.55 & 0.4069 & 0.69 & 0.9353 \\
0.31 & 0.47 & 0.1160 & 0.35 & 0.2943 & 0.41 & 0.1691 & 0.56 & 0.8000 \\
0.63 & 0.47 & 0.1160 & 0.28 & 0.1662 & 0.27 & 0.0399 & 0.31 & 0.2531 \\
1.25 & 0.58 & 0.4043 & 0.21 & 0.0767 & 0.21 & 0.0144 & 0.19 & 0.0612 \\
2.5 & 0.53 & 0.2382 & 0.42 & 0.4460 & 0.21 & 0.0144 & 0.63 & 0.8818 \\
5 & 0.55 & 0.3172 & 0.99 & 0.9954 & 0.27 & 0.0399 & 0.13 & 0.0207 \\ K+ & 3.97 & 1.0000 & 10.00 & 1.0000 & 8.43 & 1.0000 & 8.08 & 1.0000 \\ \hline \hline \end{tabular}  
* K\(\leftarrow\)positive control, FIB—Fold increase over baseline, B-value—indicates the probability that spontaneous mutations events alone.  
\end{table}