## 4 Discussion
This observed pH alteration following dilution could potentially explain the concentration-dependent cytotoxicity of EEP. In addition, both acetic acid and naringenin have been shown to cause the accumulation of and increases in reactive oxygen species (ROS) within the cell in a dose-dependent manner. Upon exposure to high concentrations of these substances, the level of ROS starts to build up within the cell, exceeding the natural antioxidant threshold for the reduction of ROS. This subsequently activates the signalling pathways, which leads to a significant alteration in the cellular morphology and, later, cell apoptosis [58; 59; 60; 61]. [PERSON] et al. [58] and [PERSON] et al. [61] suggested that the mitochondria could be the first organelle affected by acetic acid, triggering the release of cytochrome c and further activating the cell apoptosis process. These effects were evident and correlated well with the TEM analysis in our study, where the presence of the apoptotic body and degraded mitochondria were observed when the MC3T3-E1 cells were exposed to higher concentrations of EEP beyond 6.25% for both fermentation periods. Additionally, the presence of cell debris within the vacuolated cytoplasm, chromatin condensation and plasma membrane blebbing, as visualised through the TEM analysis, is associated with the activation of the autophagy process [62], a critical step in cell death. This further strengthens the role of ROS-induced cell death exerted by acetic acid and naringenin. The cytotoxic effects exerted by naringenin [63] and acetic acid [61] in a concentration-dependent manner also explained the increasing toxicity of EEP as observed in our study. Based on the two cytotoxic assays and TEM findings from the present study, we proposed the safe concentration of EEP at 6.25% with a 6-month fermentation period. EEP at this concentration was significantly less cytotoxic than the 2.5% NaOCl, the gold standard irrigant used in root canal treatment. Since NaOCl has been used widely as an irrigation solution for root canal treatment with a good success rate, EEP offers huge potential which can be explored further as an alternative irrigant to NaOCI. Furthermore, we proposed the abovementioned concentration at 6 months of fermentation based on the findings of our previous study [31]. In the study, using an antibiofilm assay, EEP at a lower concentration below 6.25% showed a positive percentage of _Enterococcus faecalis_ biofilm removal. The effects were comparable to those depicted by 2.5% NaOCI, suggesting its high antibiofilm potential. On the other hand, EEP at higher concentrations did not show antibiofilm activity; instead, an increase in the biomass of the biofilm was detected through crystal violet staining, leading to a negative percentage of biofilm removal. In addition, six-month-old EEP showed better antibiofilm effects than three-month-old EEP, where more biofilm was removed in the former fermentation period as compared with the latter. The assessment of the antibacterial activity of an endodontic irrigant through its antibiofilm removal efficacy is more clinically relevant since bacteria such as _Enterococcus faecalis_ and _Streptococcus mutans_ reside within the root canal system in the form of biofilm rather than in isolation [64]. These bacterial species have the ability to produce a sessile biofilm consisting of the exopolymeric matrix, which is attached to the dental surface [65]. The slow-growing bacteria within its exopolymeric matrix make it more resistant to host-defence mechanisms and other antimicrobial agents [66; 67]. Thus, it is not surprising that these bacteria are recognised as the primary etiological factor that is responsible for the development of pulpal necrosis, periapical infection and post-root-canal treatment reinfection [64; 68]. [PERSON] et al. [68] have shown that 5.25% NaOCI is effective at removing the _Enterococcus faecalis_ biofilm following 10 s of application. Nevertheless, the present study has some limitations.