# Pulsed Electric Fields (PEF) to Mitigate Emerging Myotoxins in Juices and Smoothies
## 3 Results and Discussion
### In Silico Prediction Methods  
In silico prediction methods were used in this work to evaluate the toxicity of detected and identified degradation products by using the ProTox-II web server. Results of oral acute toxicity expressed as LD50 (mg/Kgbw) and the corresponding toxicity class for each identified compound are shown in Table 2. It should be highlighted that, according to the obtained predictions, both ENB degradation products showed a predicted LD50 of 3 mg/Kgbw, both with 100% average similarity and prediction accuracy. Thus, the assigned toxicity class was I. According to this result, special attention should be paid to those degradation products, as their predicted toxicity is comparable to that of ENB and also T-2 Toxin (Table 2), which is known to be a toxic fungal metabolite with the lowest tolerable daily intake (TDI) within the _Fusarium_ mycotoxins [32]. Using the ProTox-II web server, the organ toxicity can be also predicted, specifically the hepatotoxicity, which has been evaluated for different identified compounds, as the liver is the organ where mycotoxins are metabolized. The results obtained regarding the organ toxicity and also the calculated predictions for diverse toxicological endpoints using the ProTox-II web server are \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Mycotoxin**} & \multicolumn{4}{c}{**Oral Toxicity Prediction Results**} \\ \cline{2-5}  & **Predicted LD50** & **Predicted** & **Average** & **Prediction** \\  & **(mg/Kgbw)** & **Toxicity Class** & **Similarity (\%)** & **Accuracy (\%)** \\ \hline ENA degradation product & 1600 & IV & 76.28 & 69.26 \\ ENA1 degradation product & 1600 & IV & 76.28 & 69.26 \\ ENB degradation product & 3 & I & 100 & 100 \\ ENB degradation product (2) & 3 & I & 100 & 100 \\ ENB1 degradation product & 1600 & IV & 75.91 & 69.26 \\ BEA degradation product & 200 & III & 75.89 & 69.26 \\ \hline \hline \end{tabular} Class I: fatal if swallowed (LD50 \(\leq\) 5 mg/Kgbw); Class II: fatal if swallowed (5 mg/Kgbw \(<\) LD50 \(\leq\) 50 mg/Kgbw); Class III: toxic if swallowed (50 mg/Kgbw \(<\) LD50 \(\leq\) 300 mg/Kgbw); Class IV: harmful if swallowed (300 mg/Kgbw \(<\) LD50 \(\leq\) 2000 mg/Kgbw); Class V: may be harmful if swallowed (2000 mg/Kgbw \(<\) LD50 \(\leq\) 5000 mg/Kgbw).  
\end{table} 
Table 2: Acute oral toxicity prediction obtained by using ProTox-II web server. Figure 4: LC-ESI-qTOF-MS chromatogram of degraded product ENB1 m/z 443.1674 obtained in matrix samples after PEF treatment. reported in Table 3. These results showed that identified degradation products were predicted as inactive compounds for hepatotoxicity. However, the ENB1 degradation product was predicted as an active compound for an immunotoxicity endpoint, although the percentage of prediction accuracy (probability score) was low (51%). The prediction results obtained for the toxicological pathways, nuclear receptor signaling pathways, and stress response pathways are reported in Tables 4 and 5, respectively. According to the Tox21 Consortium, chemical compounds might have the potential to disrupt processes in the human body, which may lead to negative health effects [23]. Regarding the nuclear receptor signaling pathway, seven different pathways were assessed while for the stress response pathways, five diverse assays were evaluated. The computational estimations revealed that the degradation products identified in the present study were predicted as inactive for all of the analyzed pathways. However, this computational analysis is presented only as a useful support tool for the exploration of mitigation strategies; more detailed toxicological studies must be carried out to confirm the reduction of toxicity of the PEF mycotoxins degradation products obtained.