This in vitro study was conducted to assess the cytotoxicity of the QMix™ irrigating solution on human bone marrow MSCs. MSCs have been suggested as a good model for toxicological testing . The MSCs that were used in this study were immortalized by the ectopic expression of human telomerase reverse transcriptase (h-TERT), which increased the life span of the cells  and maintained their stem-like properties . Earlier studies have reported that immortalized cells can be used as a test model for dental materials .
The observations from the study showed that both solutions (QMix™ and NaOCl) are toxic to human bone marrow MSCs and cause cellular damage. This is consistent with the results of previous studies that reported on NaOCl toxicity [23, 32, 33]. NaOCl toxicity can be attributed to its high pH (hydroxyl ion action), which interferes with cytoplasmic membrane integrity . Furthermore, our results are in agreement with those of a previous in vivo study, which found that QMix™ is toxic and can induce an inflammatory response . CHX is a toxic agent that binds to the cell’s plasma membrane and increases its permeability, allowing the leakage of lysosomal enzymes . EDTA, which is the second QMix™ component, is also known to be cytotoxic, perhaps due to its chelating effect and the accentuated drop in pH that it causes .
Cell viability decreased significantly when the cells were exposed to NaOCl for all time periods examined. Cell viability decreased significantly after being exposed to the QMix™ solution for 2 or 4 hours. Moreover, after 24 hours, cell viability was significantly decreased compared to 2 and 4 hours of exposure. These findings show that the toxic effect of an agent gradually increases with time. This observation is in agreement with those of previous studies, confirming that toxicity is time dependent . In contrast, the MTT assay results showed a significant decrease in the cell viability of cells that were exposed to NaOCl at all time periods examined. Compared with QMix™-exposed cells, NaOCl decreased viability at 2 and 4 hours .
Previous studies have reported that the AB assay is slightly more sensitive than the MTT assay. However, both assays rely on enzymatic metabolism, which may be inhibited or induced by the testing agent, thus producing a false-positive or false-negative result. Therefore, careful interpretation of the results is always recommended . Our observations suggest that the AB assay is a better choice for cell viability testing because it is easy to perform, more consistent than the MTT assay, and recommended by previous studies [38, 39]. However, it is always recommended to use more than one assay to assess cytotoxicity. Therefore, previous studies that relied solely on the MTT assay should be re-evaluated and interpreted with caution.
Microscopic morphological investigations are necessary to confirm cellular toxicity . This is because a cell can undergo toxic changes, such as detachment, while still continuing to metabolize MTT to formazan, resulting in an over estimation of cell viability in the MTT assay compared with the AB assay . Therefore, cellular morphological characteristics were investigated for both solutions.
Cells that were exposed to QMix™ displayed fewer morphological alterations. This observation is in agreement with Faria et al., who reported that higher concentrations of CHX would preserve the shape of the L929 cells  due to its cell fixation effect .
The SEM images for the QMix™ group showed cytoplasmic shrinkage with partially fragmented but intact cell walls. These characteristics are typical of apoptosis, as reported previously . In addition, the EB/AO staining showed bright green fragmented nuclei, indicating early apoptosis . The cells exposed to NaOCl had cytoplasmic shrinkage or ruptured membranes, which are typical characteristics of necrosis . The EB/AO staining of the NaOCl group displayed red and green, reflecting remnant material that may be the result of cell lysis, and no cell structure could be observed. The overall analysis of these results suggests that the cells in the QMix™ groups are in the early stages of apoptosis but are not yet dead, in contrast to the NaOCl group. Both NaOCl and QMix™ are cytotoxic; however, it seems that their mode of cell killing differs as a result of their different compositions. According to Galluzzi et al. , cell death can be classified into four different types based on the morphological characteristics of the dying cells: apoptosis (Type 1), autophagy (Type 2), necrosis (oncosis, Type 3), and mitotic catastrophe. Each mode of cell death has its own function; necrosis induces an inflammatory response when it is needed, whereas apoptotic cells in vivo are rapidly phagocytosed without inducing an inflammatory response, which is considered a mechanism for avoiding immune activation. According to our findings, this mode of cell death could be associated with a high concentration exposure to NaOCl.
Apoptosis is an active form of cell death known as programmed cell death, and it is characterized by cell shrinkage and the nuclear chromatin condensation followed by nuclear fragmentation, with the normal morphological appearance of cytoplasmic organelles and the maintenance of an intact plasma membrane . According to our findings, this mode of cell death could be associated with QMix™ exposure. However further markers such as detection of caspases, cleaved substrates, regulators and inhibitors are necessary to substantiate this mode of cell death speculated with QMix™ . Cell death through apoptosis is known to occur through two primary pathways: an extrinsic pathway that involves death receptors, and an intrinsic pathway that is modulated by members of the Bcl-2 family , which consists of outer mitochondrial membrane components . Therefore, mitochondria are considered key organelles in the pathways to cell death in addition to its normal metabolic activity [48, 49]. However, these proteins may also function in some normal metabolic pathways. Thus, mitochondrial metabolic activity investigations, such as the MTT assay, must consider these potential overlaps between pre-apoptotic cell activity and normal cell metabolism . This overlap could explain the contradictory results of the AB and MTT assays.
In vitro cytotoxicity investigations reported that CHX had a higher toxicity in cell cultures than NaOCl [33, 41]. However, in vivo studies suggest that CHX or QMix™ is less aggressive than NaOCl [35, 50]. This difference could be attributed to host defense mechanisms that operate in the in vivo environment.
Our in vitro study has the following limitations: it was conducted on cultured cells, and the results represent only the response of these cells in isolation, without taking into account the host defense mechanism for detoxification. Furthermore, in a clinical setting, the solutions are always delivered to root canals, which are surrounded by dentine, and are then extruded to the periapical area. Previous studies have reported that the cytotoxic effect of irrigants can be neutralized by dentine [33, 51]. We placed the solutions directly onto the cells, and no attempts were made to deliver these solutions through root canals to mimic the clinical scenario.