## 4 Discussion  
The present study was our preliminary work on the development of a novel natural-based root canal irrigant by utilising the clinical benefits of EEP. Cytotoxic evaluation of EEP and the establishment of a safe and effective concentration is of paramount importance  
Figure 9: Representative images via transmission electron microscopy of the effect of 3 M EEP 12.5% and 6 M 12.5% on MC3T3-E1 cells: **(a)** 3 M 12.5% EEP (\(\times\)2400); **(b)** an enlargement of image **(a)** (\(\times\)3000); **(c)** 6 M 12.5% EEP (\(\times\)2400); **(d)** an enlargement of image **(c)** (\(\times\)3000). ApBs: apoptotic bodies; N: nucleus; NM: nuclear membrane; Chr: chromatin; V: vacuolisation; white arrow: cytoplasmic aggregates; yellow arrow: nuclear aggregates; red arrow: cell fragments. Scale bar of **(a,c)**: 2 \(\upmu\)m; scale bar of **(b,d)**: 1 \(\upmu\)m.  
since the existing synthetic chemical irrigants present with many potential hazardous side effects. A combination of different cytotoxic assays was used in the present study to assess the cytotoxic effects of EEP towards the cells. Alamar Blue assay was used as a quantitative measurement of cell viability, calculated as the percentage of reduction of the non-fluorescence blue resazurin to the fluorescent pink resorufin through the oxidation-reduction reaction provided by the mitochondrial redox enzyme [44]. The Live and Dead Cell assay measures the cellular membrane integrity and the activity of intracellular esterase enzymes. The assay provides a qualitative assessment of the cell viability, which can be visualised as green and red fluorescence under a microscope, representing the live and dead cells, respectively [45]. The use of both quantitative and qualitative assessment methods provides a comprehensive evaluation of the cytotoxic effects of EEP from different points of view. This enables the cross-validation of results, which enhances the reliability and validity of the study and ensures greater confidence in the conclusions drawn [46]. We did not conduct any MTT/XTT cytotoxic assays since both assays measure similar mitochondrial enzymatic activity as the Alamar Blue assay, but at a lesser sensitivity. Furthermore, the MTT cytotoxic assay requires an organic solvent to dissolve the crystals prior to the absorbance measurement, which may itself cause cytotoxicity towards the cells [45]. The results from our study showed that the cytotoxicity of EEP is concentration-dependent, regardless of the fermentation period. EEP at a concentration below 6.25% were not toxic to the cells, with cell viability of more than 70% [38] as depicted by the Alamar Blue cytotoxic assay. Further, there was no statistically significant difference in terms of toxicity between the EEP at 3.125% and the negative control. These findings are consistent with the qualitative assessment of the cells done through the Live and Dead Cell assay. Cells exposed to EEP at a concentration below 6.25% were densely packed, with green fluorescence cells predominating, while the red-stained cells were barely seen. The intensity of the green fluorescence increased as the concentration of EEP decreased, suggesting the concentration-dependent toxicity of the solution. Further, at similar concentrations, TEM analysis of the cells showed intact cell and nucleolar membranes and preservation of the organelle structure comparable to the untreated negative control. In contrast, the presence of autophagic vacuolisation and organelle derangement, a feature that is associated with cell apoptosis [47] and autophagy [48], were detected in cells exposed to EEP at a 12.5% concentration. This explains and validates the gradual reduction in cell viability following exposure to EEP at a concentration higher than 6.25%, as shown by the Alamar Blue cytotoxic assay. We also compared the toxicity of EEP with NaOCl, the gold standard irrigation solution in root canal treatment. Briefly, 2.5% NaOCl is toxic to the cells, with less than 7% of cell viability as measured through the Alamar Blue assay.