Determination of mutagenicity of the precipitate formed by sodium hypochlorite and chlorhexidine using the <scp>A</scp>mes test

Patil, Pradeep A.; Aminoshariae, Anita; Harding, Jarrod; Montagnese, Thomas A.; Mickel, Andre

doi:10.1111/aej.12100

Cited by 8 publications

(13 citation statements)

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“…In contrast, another study using human periodontal ligament and 3T3 fibroblast cell lines reported only slight cytotoxic effects of BP compared with moderate cytotoxic effects of supernatant (Nocca et al, 2017). A mutagenesis study reported no significant mutagenicity at a low testable concentration of BP because of toxicity observed with higher concentrations in S. typhimurium (Patil et al, 2016).…”

Section: Introductionmentioning

confidence: 95%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 95%

“…A mutagenesis study reported no significant mutagenicity at a low testable concentration of BP because of toxicity observed with higher concentrations in S . typhimurium (Patil et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…Patil. [ 31 ] investigated the mutagenic potential of the brown precipitate by Ames test and concluded that the precipitate is free from carcinogenic elements.…”

Section: Discussionmentioning

confidence: 99%

Decoding the perplexing mystery of para-chloroaniline formation: A systematic review

Khatib

Ameer²,

Mannur³

et al. 2020

J Int Soc Prevent Communit Dent

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A BSTRACT Objective: The objective of this article was to understand and decode the mystery of the formation of para-chloroaniline (PCA). The ingredient of the brown precipitate after mixing sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) is still in debate. Materials and Methods: Various studies adopt a different methodology to substantiate that it may contain PCA, which is a carcinogenic agent. The purpose of this systematic review is to evaluate the relationship between PCA and brown precipitate. Two reviewers independently conducted a comprehensive literature search. The MEDLINE, Embase, Cochrane, and PubMed databases were searched. In addition, the bibliographies were manually searched. There was no disagreement between the two reviewers. This review was reported and conducted in step with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results: Of 233 articles, only 13 articles met the inclusion criteria. Available scientific evidence was more supportive that the brown precipitate form after mixing NaOCl and CHX may form para-chloroamide moiety rather than free PCA, and PCA may be the by-product of CHX degradation. Conclusion: On the basis of the current evidence and data extracted from the various databases, it can be concluded that the mixture of sodium hypochlorite and chlorhexidine does not form PCA, and PCA may be the by-product of high concentrated chlorhexidine. Further studies are required to substantiate the evidence.

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“…• The brown precipitate has a sticky, insoluble and viscous nature may contribute to the inhibition of the cell proliferation (Patil et al, 2016). • CHX is more cytotoxic than NaOCl or EDTA (Vouzara et al, 2016); however, within the confines of the canal system and periapical tissues, its clinical toxicity could be considered well within the acceptable boundaries (Harrison et al, 1978).…”

Section: What Does Not Form After Accidental Sodium Hypochlorite Cont...mentioning

confidence: 99%

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

2022

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If the chemical structure of brown precipitate is aimed to analyse in any research and the hypothesis is tested by any mass spectroscopy method, the fragmented PCA main peak of CHX is always detected in mass spectra. After all these considerations, in summary, it is certain that mass spectrometry-based on fragmentation analysis of molecules is not suitable for PCA analysis from brown precipitate, and this has been experimentally proven. CONFLICT OF INTERESTThe 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. Also, the authors have stated that there are no conflicts of interest in connection with this manuscript.

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Determination of mutagenicity of the precipitate formed by sodium hypochlorite and chlorhexidine using the Ames test

Cited by 8 publications

References 37 publications

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

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

Decoding the perplexing mystery of para-chloroaniline formation: A systematic review

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

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