Assessment of the cytotoxic effects and chemical composition of the insoluble precipitate formed from sodium hypochlorite and chlorhexidine gluconate

Abstract

Aim To investigate (1) the cytotoxic potential of the brown precipitate (BP) formed with sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX), using both a small animal model of Caenorhabditis elegans (C. elegans) and cultured human gingival fibroblasts; and (2) the chemical composition of BP using Time‐of‐Flight Secondary Ion Mass Spectrometry (ToF‐SIMS). Methodology Brown precipitate was obtained by mixing equal volumes of 6% NaOCl and 2% CHX and separating the BP from clear supernatant by centrifugation. The brown precipitate was weighed and solubilized in dimethyl sulfoxide for cytotoxicity experiments. The cytotoxic effect of BP was assessed using C. elegans larvae and primary immortalized human gingival fibroblasts‐hTERT (hTERT‐hNOF) cells. Various dilutions of BP (25 ng/µL–150 ng/µL), supernatant (0.15% v/v), NaOCl (1:100–1:1000 dilutions of 6% NaOCl) or CHX (1:500–1:1000 dilutions of 2% CHX) along with vehicle control (0.5% v/v ethanol and 0.15% v/v DMSO) or untreated control (growth medium) were tested on C. elegans larvae and hTERT‐hNOF cells. Viability was assessed in C. elegans larvae using stereomicroscopy and in hTERT‐hNOF cells using dehydrogenase‐based colorimetric assay. ToF‐SIMS was used to assess the chemical composition of BP in comparison with CHX and para‐chloroaniline (PCA). The C. elegans and cell line data were analysed using Log‐Rank test and Student's t‐test, respectively (p < .05). Results BP‐75 ng/µL and BP‐150 ng/µL were significantly more toxic to C. elegans larvae than the untreated, vehicle, supernatant or CHX treatment groups (p < .0001). Similarly, in hTERT‐hNOF cells, BP‐50 ng/µL, BP‐75 ng/µL and BP‐150 ng/µL induced significant cytotoxicity within 2 h compared with untreated, vehicle, supernatant and CHX treatments (p < .05). ToF‐SIMS analysis of BP revealed ion composition characteristic of both CHX and the carcinogen PCA. Conclusions Brown precipitate was toxic in both C. elegans larvae and hTERT‐hNOF cells. The ToF‐SIMS analysis of BP revealed ions characteristic of CHX and PCA that could account for the toxicities observed in C. elegans larvae and human gingival fibroblasts. Because of the insoluble and toxic nature of BP, consecutive use of CHX and NaOCl irrigants should be avoided in root canal treatment.

Antibacterial efficacy of peracetic acid in comparison with sodium hypochlorite or chlorhexidine against Enterococcus faecalis and Parvimonas micra

“…A further potential advantage of PAA, when compared with CHX is a clinical disadvantage when using CHX in combination with NaOCl. The possible production of a precipitate, thus, concomitant effects, demand surplus supplementary handling precautions during endodontic irrigation [ 39 ]. A clinically oriented investigation comparing the overall efficacy/advantages of PAA, CXH and EDTA should be the subject of further investigations.…”

What does not form after accidental sodium hypochlorite contact with chlorhexidine?

“…Concordantly, we have noticed that there is a methodological error in the study by Jeong et al (2021). Although it has been shown in a multimethod-analysis-controlled study that the free PCA did not form in the brown precipitate (Orhan et al, 2016), Jeong et al (2021) have reignited the concern with their report regarding the PCA-related toxicity of precipitate. Thus, we hope to consider the chemical analysis of the brown precipitate with a methodological perspective.…”

“…The authors declare that there is no expectation of a response to this letter from Jeong et al (2021). The authors have stated explicitly that there are no conflicts of interest or financial interest in connection with chlorhexidine manufacturing or chlorhexidine‐related products.…”

“…How can the toxicity reported in Jeong et al (2021) be explained when there is the absence of free PCA in brown precipitate formed after the NaOCl and CHX interaction?…”

Response to letter by Orhan et al. 2021, regarding Jeong et al. (2021) ‘Assessment of the cytotoxic effects and chemical composition of the insoluble precipitate formed from sodium hypochlorite and chlorhexidine gluconate’

“…This kind of spatial heterogeneity cannot be deduced from classical MS techniques. Based on our results and the attribute of ToF-SIMS in providing data from fragmented ions, we conclude that the brown precipitate contains ions representative of both CHX and PCA (Jeong et al, 2021). We would like to emphasize that whilst the appropriate methodologies to determine the exact composition of the brown precipitate need to be further explored, it is important to assess the cellular/biological effects of the brown precipitate irrespective of the chemical composition.…”