The Interaction of Two Widely Used Endodontic Irrigants, Chlorhexidine and Sodium Hypochlorite, and Its Impact on the Disinfection Protocol during Root Canal Treatment

In recent years, sodium hypochlorite and chlorhexidine digluconate have been the gold standard of irrigation solutions utilized within the disinfection protocol during root canal treatments. Nowadays, it is known that, during chemical disinfection of the root canal, consecutive application of sodium hypochlorite and chlorhexidine digluconate leads to the formation of an orange-brown precipitate. This precipitate is described as being chemically similar to para-chloroaniline, which is suspected to have cytotoxic and carcinogenic effects. Concerns also exist regarding its influence on the leakage of root canal fillings, coronal restorations, and tooth discoloration. The purpose of this article is to review the literature on the interaction of sodium hypochlorite and chlorhexidine digluconate on the tooth and its surrounding tissues, and to discuss the effect of the precipitate formed during root canal treatment. We further address options to avoid the formation of the precipitate and describe alternative irrigation solutions that should not interact with sodium hypochlorite or chlorhexidine digluconate.


Introduction
Complete cleaning and disinfection of the root canal system are considered mandatory for long-term success in root canal treatment [1,2]. However, even after thorough mechanical cleaning, residual pulp tissue, bacteria, and dentin debris can remain in the root canal system [3,4]. Therefore, a variety of irrigating solutions are used in combination with the mechanical processing, such as sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX) [5], 17% ethylenediaminetetraacetic acid (EDTA), citric acid (CA), BioPure ® MTAD ® (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA), and 37% phosphoric acid (PA) [6], as well as etidronate, alexidine (ALX), and Octenisept ® (Schülke & Mayr, Norderstedt, Germany) [7]. Following internationally accepted quality guidelines, the main goals of irrigation are: eliminating microorganisms, flushing out debris, lubricating root canal instruments, and dissolving organic debris. Therefore, the used irrigation solution should preferably have disinfectant and organic-debris-dissolving properties, whilst not irritating the periradicular tissues [8]. For this purpose, sodium hypochlorite and chlorhexidine digluconate are widely recommended and well accepted in endodontics [9,10].
Unfortunately, endodontic irrigation solutions may interact chemically with each other during an alternating irrigation technique, potentially forming unwanted by-products, which may be toxic or cause allergic reactions [7]. Sodium hypochlorite and chlorhexidine are the best known and, at least in recent years, most frequently recommended irrigating solutions used for eliminating residual bacteria in chemo-mechanical root canal processing [5,6]. The undesirable adverse effects, after sodium hypochlorite and chlorhexidine in- Sodium hypochlorite (NaOCl) is the most common irrigant used in root canal treatments. NaOCl is an effective tissue solvent and antimicrobial agent. It is usually used in a concentration range from 0.5 to 8.25% [18][19][20]. Its germicidal ability is related to the formation of hypochlorous acid when in contact with organic debris. In high concentrations, NaOCl is toxic and can cause inflammation in the periapical tissues [21], whereas in low concentrations, it is ineffective against specific microorganisms. NaOCl is not a substantive antimicrobial agent; it tends to discolor and corrode surgical instruments; and it has a very unpleasant odor [11].

Chlorhexidine (CHX)
Chlorhexidine digluconate (CHX) is the gluconate salt form of chlorhexidine, a biguanide compound used as an antiseptic agent with topical antibacterial activity ( Figure  2). Chlorhexidine digluconate is positively charged and reacts with the negatively charged microbial cell surface, thereby destroying the integrity of the cell membrane. Subsequently, chlorhexidine gluconate penetrates into the cell and causes a leakage of intracellular components, leading to cell death. Since gram-positive bacteria are more negatively charged, they are more sensitive to this agent [22].  Sodium hypochlorite (NaOCl) is the most common irrigant used in root canal treatments. NaOCl is an effective tissue solvent and antimicrobial agent. It is usually used in a concentration range from 0.5 to 8.25% [18][19][20]. Its germicidal ability is related to the formation of hypochlorous acid when in contact with organic debris. In high concentrations, NaOCl is toxic and can cause inflammation in the periapical tissues [21], whereas in low concentrations, it is ineffective against specific microorganisms. NaOCl is not a substantive antimicrobial agent; it tends to discolor and corrode surgical instruments; and it has a very unpleasant odor [11].

Chlorhexidine (CHX)
Chlorhexidine digluconate (CHX) is the gluconate salt form of chlorhexidine, a biguanide compound used as an antiseptic agent with topical antibacterial activity ( Figure 2). Chlorhexidine digluconate is positively charged and reacts with the negatively charged microbial cell surface, thereby destroying the integrity of the cell membrane. Subsequently, chlorhexidine gluconate penetrates into the cell and causes a leakage of intracellular components, leading to cell death. Since gram-positive bacteria are more negatively charged, they are more sensitive to this agent [22].
Antibiotics 2023, 12, x FOR PEER REVIEW 2 of 18 chlorhexidine are the best known and, at least in recent years, most frequently recommended irrigating solutions used for eliminating residual bacteria in chemo-mechanical root canal processing [5,6]. The undesirable adverse effects, after sodium hypochlorite and chlorhexidine interaction, of building precipitates are known, published and discussed controversially [11]. However, it is recommended that, until this precipitate is studied further, its formation should be avoided by removing the NaOCl before placing CHX into the canal [11]. Since 2006, the number of articles in PubMed concerning the interaction of NaOCl and CHX have grown significantly, and the topic was greatly debated [12][13][14][15][16]. Therefore, the aim of the present review is to summarize and discuss recently published papers focusing on the different outcomes regarding the interactions between sodium hypochlorite and chlorhexidine. Furthermore, based on the results of the review, the possible impact for the clinical disinfection protocol in endodontic therapy is summarized.

Sodium Hypochlorite (NaOCl)
Sodium hypochlorite (Figure 1) is the most used irrigating solution in endodontics, because its mechanism of action causes biosynthetic alterations in cellular metabolism and phospholipid destruction, the formation of chloramines that interfere in cellular metabolism, oxidative action with irreversible enzymatic inactivation of bacteria, and lipid and fatty acid degradation [17]. Sodium hypochlorite (NaOCl) is the most common irrigant used in root canal treatments. NaOCl is an effective tissue solvent and antimicrobial agent. It is usually used in a concentration range from 0.5 to 8.25% [18][19][20]. Its germicidal ability is related to the formation of hypochlorous acid when in contact with organic debris. In high concentrations, NaOCl is toxic and can cause inflammation in the periapical tissues [21], whereas in low concentrations, it is ineffective against specific microorganisms. NaOCl is not a substantive antimicrobial agent; it tends to discolor and corrode surgical instruments; and it has a very unpleasant odor [11].

Chlorhexidine (CHX)
Chlorhexidine digluconate (CHX) is the gluconate salt form of chlorhexidine, a biguanide compound used as an antiseptic agent with topical antibacterial activity ( Figure  2). Chlorhexidine digluconate is positively charged and reacts with the negatively charged microbial cell surface, thereby destroying the integrity of the cell membrane. Subsequently, chlorhexidine gluconate penetrates into the cell and causes a leakage of intracellular components, leading to cell death. Since gram-positive bacteria are more negatively charged, they are more sensitive to this agent [22].  Chlorhexidine digluconate (CHX) can be used as a complement to increase the antibacterial action of NaOCl solutions during root canal preparation. CHX shows similar antimicrobial effects to sodium hypochlorite [23,24] in vitro and possesses a lower toxicity [25,26]. A disadvantage compared to NaOCl is its lack of ability to dissolve vital and necrotic tissue [27].
Chlorhexidine digluconate is a broad-spectrum antibacterial agent with substantivity to tooth structures, i.e., it binds to the hydroxyapatite of the enamel and dentin or to anionic groups of glycoproteins, is slowly released and, due to the moderate concentration decrease, its antibacterial effects are prolonged for an extended period of time [28].

Proteolysis
When NaOCl and CHX are mixed, NaOCl dissociates into different ions (H + , O 2− , and Cl − ). The chloride group then reacts with the chlorhexidine molecule in the guanine group (NH). This leads to the formation of chlorhexidine chloride (N + and Cl − ). In this reaction, the formation of an orange-brown precipitate is described. This precipitate contaminates the dentin and adheres to the canal walls [29]. Furthermore, CHX is a dicationic acid and has the ability to donate protons, whereas NaOCl is alkaline and can absorb protons from the dicationic acid. This proton exchange leads to the formation of a neutral and insoluble precipitate [11,30,31]. A color change due to the reaction can already be seen from a concentration of 0.023% NaOCl and the formation of the precipitate from 0.19% NaOCl by means of X-ray Photoelectron Spectroscopy (XPS), and the absolute amount by means of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) [11].
While the undesirable effects of the initial developing substances have been well studied and are classified as acceptable [5], the precipitate with regard to its ingredients and undesirable effects still gives rise to discussions [32,33]. Figure 3 demonstrates microtubes filled with 2% CHX mixed with different concentrations of NaOCl. The first microtube is a control sample with 2% CHX alone. From left to right, a color change, which becomes brighter as the concentration of NaOCl decreases, can be observed.
Chlorhexidine digluconate (CHX) can be used as a complement to increase the antibacterial action of NaOCl solutions during root canal preparation. CHX shows similar antimicrobial effects to sodium hypochlorite [23,24] in vitro and possesses a lower toxicity [25,26]. A disadvantage compared to NaOCl is its lack of ability to dissolve vital and necrotic tissue [27].
Chlorhexidine digluconate is a broad-spectrum antibacterial agent with substantivity to tooth structures, i.e., it binds to the hydroxyapatite of the enamel and dentin or to anionic groups of glycoproteins, is slowly released and, due to the moderate concentration decrease, its antibacterial effects are prolonged for an extended period of time [28].

Proteolysis
When NaOCl and CHX are mixed, NaOCl dissociates into different ions (H + , O 2− , and Cl − ). The chloride group then reacts with the chlorhexidine molecule in the guanine group (NH). This leads to the formation of chlorhexidine chloride (N + and Cl − ). In this reaction, the formation of an orange-brown precipitate is described. This precipitate contaminates the dentin and adheres to the canal walls [29]. Furthermore, CHX is a dicationic acid and has the ability to donate protons, whereas NaOCl is alkaline and can absorb protons from the dicationic acid. This proton exchange leads to the formation of a neutral and insoluble precipitate [11,30,31]. A color change due to the reaction can already be seen from a concentration of 0.023% NaOCl and the formation of the precipitate from 0.19% NaOCl by means of X-ray Photoelectron Spectroscopy (XPS), and the absolute amount by means of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) [11].
While the undesirable effects of the initial developing substances have been well studied and are classified as acceptable [5], the precipitate with regard to its ingredients and undesirable effects still gives rise to discussions [32,33]. Figure 3 demonstrates microtubes filled with 2% CHX mixed with different concentrations of NaOCl. The first microtube is a control sample with 2% CHX alone. From left to right, a color change, which becomes brighter as the concentration of NaOCl decreases, can be observed.

Materials and Methods
An unlimited search in all fields of the PubMed database (https://pubmed.ncbi.nlm.nih.gov/, accessed on 4 October 2022) was carried out through the website of the National Center for Biotechnology Information (NCBI), utilizing the combination of the Medical Subject Headings (MeSH terms) "sodium hypochlorite" (NaOCl) AND "chlorhexidine" (CHX) and yielded 955 results from the years 1974-2022. Specifying the

Materials and Methods
An unlimited search in all fields of the PubMed database (https://pubmed.ncbi.nlm. nih.gov/, accessed on 4 October 2022) was carried out through the website of the National Center for Biotechnology Information (NCBI), utilizing the combination of the Medical Subject Headings (MeSH terms) "sodium hypochlorite" (NaOCl) AND "chlorhexidine" (CHX) and yielded 955 results from the years 1974-2022. Specifying the search term to "chlorhexidine AND sodium hypochlorite AND interaction", 64 publications remained from the original result. By individually reviewing the references and abstracts of these 64 publications the keywords "precipitate" and "para-chloroaniline" were regularly found in the keywords of the relevant articles ( Figure 4). search term to "chlorhexidine AND sodium hypochlorite AND interaction", 64 publications remained from the original result. By individually reviewing the references and abstracts of these 64 publications the keywords "precipitate" and "para-chloroaniline" were regularly found in the keywords of the relevant articles ( Figure 4). Finally, the search term was further expanded to include "chlorhexidine AND sodium hypochlorite AND (interaction OR precipitate OR chloroaniline)", which resulted in a selection of 88 articles that included the manually determined references. The abstracts of all articles of the final online search result were evaluated and 25 articles that showed no relevance to the question were sorted out (Table 1, Figure 4).

Inclusion Criteria
Exclusion Criteria -representing all search terms used -articles without apparent relevance -original articles, reviews, scientific short communications -case reports, case series, editorials, case reviews

Results
The 63 papers included in this review are listed in Table 2, where the title and objective were summarized. These publications were read in full and evaluated.
58 publications (49 studies, 8 reviews, 1 short communication) were relevant to the topic; another 5 were excluded after reading the full texts. Sources to which the research publications referred were included if they were relevant to the topic, even if the date of their publication was before 1994. Finally, the search term was further expanded to include "chlorhexidine AND sodium hypochlorite AND (interaction OR precipitate OR chloroaniline)", which resulted in a selection of 88 articles that included the manually determined references. The abstracts of all articles of the final online search result were evaluated and 25 articles that showed no relevance to the question were sorted out (Table 1, Figure 4).

Results
The 63 papers included in this review are listed in Table 2, where the title and objective were summarized. These publications were read in full and evaluated.
58 publications (49 studies, 8 reviews, 1 short communication) were relevant to the topic; another 5 were excluded after reading the full texts. Sources to which the research publications referred were included if they were relevant to the topic, even if the date of their publication was before 1994. Effectiveness of several solutions to prevent the formation of precipitate due to the interaction between sodium hypochlorite and chlorhexidine and its effect on bond strength of an epoxy-based sealer AIM: To evaluate the effectiveness of isopropyl alcohol, saline or distilled water to prevent the precipitate formed between sodium hypochlorite (NaOCl) and chlorhexidine (CHX) and its effect on the bond strength of an epoxy-based sealer in radicular dentine.

Laboratory study
Bernardi & Teixeira, 2015 [28] The properties of chlorhexidine and undesired effects of its use in endodontics The purpose of this article was to review the literature on the properties of chlorhexidine (CHX) and the adverse effects that may occur from its use in endodontics. Evaluation of orange-brown precipitate formed in root canals after irrigation with chlorhexidine and QMix and spectroscopic analysis of precipitates produced by a mixture of chlorhexidine/NaOCl and Qmix/NaOCl AIM: To compare chlorhexidine and Qmix™ in terms of orange-brown precipitate generation in root canals and (ii) to analyse the precipitate produced by mixing chlorhexidine and Qmix(™) with NaOCl to determine whether para-chloroaniline was produced.

Laboratory study
Kolosowski et al., 2014 [60] Qualitative analysis of precipitate formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a final rinse of chlorhexidine or QMiX The aim of this study was to qualitatively assess the formation of precipitate and PCA on the surface and in the tubules of dentin irrigated with NaOCl, followed either by EDTA, NaOCl, and CHX or by saline and QMiX. Evaluation of the interaction between sodium hypochlorite and several formulations containing chlorhexidine and its effect on the radicular dentin-SEM and push-out bond strength analysis The aim of the current study was to evaluate the presence of debris and smear layer after endodontic irrigation with different formulations of 2% chlorhexidine gluconate (CHX) and its effects on the push-out bond strength of an epoxy-based sealer on the radicular dentin.

Laboratory study
Cintra et al., 2014 [63] The use of NaOCl in combination with CHX produces cytotoxic product The aim of this study was to evaluate the tissue response to implanted polyethylene tubes filled with PPT-soaked fibrin sponge.
Laboratory study Interaction between chlorhexidine-impregnated gutta-percha points and several chlorine-containing endodontic irrigating solutions AIM: To evaluate if the immersion of chlorhexidine-impregnated gutta-percha points in chlorine-containing endodontic irrigants causes colour changes and precipitate formation.
Laboratory study Prado et al., 2013 [68] Interactions between irrigants commonly used in endodontic practice: a chemical analysis The aim of this work was to characterize the by-products formed in the associations between the most commonly used irrigants in endodontic practice.
Laboratory study Pasich et al., 2013 [69] Efficacy of taurine haloamines and chlorhexidine against selected oral microbiome species In this in vitro study we have compared antimicrobial activity of CHX with that of taurine chloramine (TauC1) and taurine bromamine (TauBr).
Laboratory study Gupta et al., 2013 [70] Evaluation of the sealing ability of two sealers after using chlorhexidine as a final irrigant: An in vitro study The aim of this study was to evaluate the effect of the precipitate formed by using sodium hypochlorite and chlorhexidine as a root canal irrigant on the sealing ability of different root canal sealers.
Laboratory study Gomes et al., 2013 [6] Chlorhexidine in endodontics The aim of this paper is to review CHX's general use in the medical field and in dentistry. The effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system AIM: To determine if the formation of para-chloroaniline (PCA) can be avoided by using an alternative irrigant following sodium hypochlorite but before chlorhexidine.
Laboratory study Kim, 2012 [31] Precipitate from a combination of sodium hypochlorite and chlorhexidine . . . Chlorhexidine can form a precipitate when used in combination with NaOCl during intra-canal irrigation. What is the adverse effect of this precipitate and how can I reduce the chance of precipitation? short communication Chemical interaction of alexidine and sodium hypochlorite This study determined by electrospray ionization mass spectrometry (ESI-MS) and scanning electron microscopy (SEM) whether the chemical interaction between ALX and NaOCl results in PCA or precipitates.
Laboratory study Gasic et al., 2012 [74] Ultrastructural analysis of the root canal walls after simultaneous irrigation of different sodium hypochlorite concentration and 0.2% chlorhexidine gluconate AIM: To determine whether sodium hypochlorite (NaOCl) with 0.2% chlorhexidine gluconate (CHX) leads to colour change and precipitate formation, and to ultrastructurally analyse the dentine surface after simultaneous irrigation with 0.5% NaOCl and 0.2% CHX.
Laboratory study Prado et al., 2011 [75] Effect of disinfectant solutions on the surface free energy and wettability of filling material The aims of this study were to evaluate the surface free energy of GP and Res cones after disinfection procedures and to investigate the wettability of endodontic sealers in contact with these surfaces.

Laboratory study
Nowicki & Sem, 2011 [76] An in vitro spectroscopic analysis to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite and chlorhexidine The purpose of this in vitro study was to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite (NaOCl) and chlorhexidine (CHX) and the relative molecular weight of the components.
Laboratory study de Assis et al., 2011 [77] Evaluation of the interaction between endodontic sealers and dentin treated with different irrigant solutions The aim of this study was to investigate the wettability of endodontic sealers in contact with dentin treated with 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) in the presence or absence of smear layer.
Laboratory study Thomas & Sem, 2010 [78] An in vitro spectroscopic analysis to determine whether para-chloroaniline is produced from mixing sodium hypochlorite and chlorhexidine The purpose of this in vitro study was to determine whether para-chloroaniline (PCA) is formed through the reaction of mixing sodium hypochlorite (NaOCl) and chlorhexidine (CHX).

Laboratory study
Krishnamurthy & Sudhakaran, 2010 [79] Evaluation and prevention of the precipitate formed on interaction between sodium hypochlorite and chlorhexidine The purpose of this study was (1) to evaluate maximum thickness the and chemical composition of the precipitate formed between sodium hypochlorite (NaOCl) and chlorhexidine (CHX) and (2) to evaluate effectiveness of absolute alcohol to remove residual NaOCl and thereby prevent the formation of the precipitate.
Laboratory study Basrani et al., 2010 [80] Determination of 4-chloroaniline and its derivatives formed in the interaction of sodium hypochlorite and chlorhexidine by using gas chromatography The aim of this study was to further identify the precipitate by using gas chromatography-mass spectrometry (GC-MS).

Laboratory study
Akisue et al., 2010 [12] Effect of the combination of sodium hypochlorite and chlorhexidine on dentinal permeability and scanning electron microscopy precipitate observation This study compared the combined use of sodium hypochlorite (NaOCl) and chlorhexidine (CXH) with citric acid and CXH on dentinal permeability and precipitate formation.

Laboratory study
Mohammadi & Abbott, 2009 [81] The properties and applications of chlorhexidine in endodontics The purpose of this paper is to review the structure and mechanism of action of CHX, its antibacterial and antifungal activity, its effect on biofilm, its substantivity (residual antibacterial activity), its tissue solvent ability, its interaction with calcium hydroxide and sodium hypochlorite, its anticollagenolytic activity, its effect on coronal and apical leakage of bacteria, its toxicity and allergenicity and the modulating effect of dentine and root canal components on its antimicrobial activity.
review Using diazotization to characterize the effect of heat or sodium hypochlorite on 2.0% chlorhexidine The aim of the present study was to use a diazotization technique to confirm the presence of an aromatic amine (such as PCA) in the NaOCl/CHX precipitate and also in the 2.0% CHX at different temperatures (37 degrees C and 45 degrees C).
Laboratory study Bui et al., 2008 [30] Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin The aim of this study was to evaluate the effect of irrigating root canals with a combination of NaOCl and CHX on root dentin and dentinal tubules . . .
Laboratory study Basrani et al., 2007 [11] Interaction between sodium hypochlorite and chlorhexidine gluconate The aim of this study was to determine the minimum concentration of NaOCl required to form a precipitate with 2.0% CHX.
Laboratory study Zehnder, 2006 [5] Root canal irrigants In this review article, the specifics of the pulpal microenvironment and the resulting requirements for irrigating solutions are spelled out.

Discussion
The PubMed search found 63 publications, 58 of which were relevant. After full text analysis, 49 were studies that have been published since 2006 in the medical and especially in the dental-endodontic field, which have dealt with the interaction of NaOCl and CHX. Eight reviews with different focuses giving an overview of the state of knowledge at the date of publication were also selected. Furthermore, one article made recommendations on how to avoid formation of the precipitate, and thus was included.

Methodology
The 1998 study by Kuruvilla and Kamath [83] indicated that the alternating use of NaOCl and CHX reduces the microbial flora to a greater percentage (84.6%) than the use of NaOCl (69.4%) or CHX (70%) on its own. In order to optimize the tissue-dissolving properties of NaOCl and the antiseptic properties of the CHX against gram-positive germs, it was considered to use a combination of both irrigation solutions. However, by mixing the two solutions, for example through consecutive use in the root canal, a peach-colored to brown precipitate is formed [7], which is difficult to remove [10,13,16,17]. It is undisputed in the literature that the precipitate forms due to the acid-base reaction of NaOCl and CHX. The exact composition and, in particular, the question of whether the precipitate contains para-chloroaniline (PCA), motivated studies in the period from 2007 to 2021. Controversial views on the suitability of test methods for the analysis of the precipitate [13,78,80] and partly contradicting test results from the same test methods [47,59,76] leave doubts as to whether free PCA arises from the reaction of NaOCl and CHX [32]. However, recent studies emphasize the use of multiple non-destructive test methods and always examine 98% PCA as a comparison group, and they could not detect any free PCA in the precipitate [47,55]. In a review carried out by two independent authors on the basis of 13 included articles from different databases, Khatib et al. [33] concluded that the brown precipitate, which forms after mixing NaOCl and CHX, may contain a proportion of para-chloramide rather than free PCA and that PCA may be the by-product of the breakdown of highly concentrated CHX. It is also disputed whether PCA has mutagenic potential. While Gomes et al. [6] were citing publications from 1986 and 1995 according to which PCA was found to be mutagenic in microorganisms, Patil et al. [54] found no significant difference in the mutagenicity of the precipitate and the comparison group.

Toxicity
Regarding the toxicity of the precipitate, Cintra et al. [63] found a short-term increased toxicity compared to the starting substances, while Vouzara et al. [53] identified a pre-dominantly antagonistic effect in the combination of NaOCl and CHX, indicating that the precipitate was less toxic than the starting substances. Surrender et al. [51] found the precipitate to be less toxic than either NaOCl or CHX alone. Furthermore, Jeong, Sarmast, Terlier, van der Hoeven, Holland and Parikh [35] all concluded that the precipitate has a toxic effect against human gingival fibroblasts, but highly concentrated NaOCl has an even greater cytotoxic effect. Nocca et al. [14] also observed a lower mortality of fibroblast cells to which the precipitate was applied than in those treated with the supernatant.
Marchesan et al. [84] evaluated the metals present in the precipitate of NaOCl and CHX by means of atomic absorption spectrophotometry and identified statistically significant proportions of copper (Cu), tin (Zn), iron (Fe), manganese (Mn), magnesium (Mg) and calcium (Ca). Siddique et al. [16] found selenium (Se) with inductively coupled plasma mass spectrometry. A discoloration of enamel and dentin was found by Souza et al. [65] on bovine anterior teeth that were placed in CHX gel and NaOCl consecutively. Therefore, it could be concluded that the combined use of NaOCl and CHX solution can cause dentin discoloration during endodontic treatment.

Recommended Irrigation Protocol
In order to prevent the formation of precipitates when using NaOCl and CHX, an irrigation protocol that includes intermediate rinses has frequently been recommended. For example, Zehnder [5] recommended rinsing the root canals exclusively with NaOCl during the mechanical preparation, which he ascribed to "unique tissue-dissolving properties". Before a final rinse with CHX recommended by him for chronic pulpitis and revisions, Zehnder [5] advised an intermediate rinse with EDTA or citric acid in order to prevent the formation of precipitates. It should be noted here that the root dentin can soften, if it is exposed to strong chelating agents, such as EDTA, for a long time [85]. Bueso et al. [34] used stereomicroscopic analysis to compare the effect of EDTA, distilled water and sodium thiosulfate (STS) as an intermediate rinse to prevent the formation of brown precipitates. In this context, 5% STS significantly reduced the intensity of brown precipitates, compared to no intermediate rinse. Alberto et al. [37] were able to demonstrate this effect ex vivo when CHX was added 10 min after the application of STS. Subsequent studies evaluated the endodontic irrigation regimen. The formation of precipitates was also demonstrated for the mixture of EDTA and NaOCl, but not for citric acid and CHX [72].
In addition, Mortenson et al. [13] found the least amounts of precipitate after intermediate flushing with 50% citric acid, compared to EDTA and saline. Intermediate rinsing with pure alcohol, distilled water, or saline solutions could also prevent or reduce the formation of precipitates [79]. However, a precipitate present in the root canal system represents a layer that occludes the dentinal tubules [12,30], is difficult to remove [29,79], and compromises the tightness of a root filling using AH 26 sealer (Dentsply Sirona, Konstanz, Germany) and gutta-percha proportionally to the amount of precipitate [61]. Whether the precipitate affects sealer adhesion has been controversially discussed: While Gupta et al. [70] came to the conclusion that the precipitate reduced the bonding capacity of an epoxy-based sealer (AH Plus ® , Dentsply Sirona, Konstanz, Germany) significantly, it was subsequently shown that the adhesion of Resilon ® -Epiphany SE obturation system (Pentron Clinical Technologies, Wallingford, CT, USA) was not affected by the precipitate. In addition, Magro et al. [58,62] found no correlation between the penetration depth of an epoxy sealer into the dentin and bond strength values between groups treated with or without CHX. However, the investigated CHX variants led to more precipitate in all root canal areas previously rinsed with NaOCl, though they did not reduce the bond strength of the sealer in the push-out test, which was traced back to the protocol for canal drying and covalent bonds between the sealer and the dentin surface [58,62].
Even by activating the rinsing solutions, the removal of the precipitate is only possible to a limited extent. However, it was found that activation of the chelating agents EDTA and citric acid, in particular using sonic (Eddy ® , VDW, Munich, Germany) or ultrasound devices, is superior to syringe rinsing [32,52,57].

Alternative Irrigation Solutions
Furthermore, CHX alternatives were also considered and examined. The substitution of CHX by the herbal antimicrobial substances neem, tulsi, aloe vera, and garlic was not successful, as the amount of precipitate resulting from these substances in combination with NaOCl was a factor of 4-7.5 higher than that with CHX [42]. ALX, a substance from the biguanide family, similar to CHX, developed only a slightly yellowish color, but no precipitate formation with NaOCl [46]. In studies by Thomas et al. [41] the effectiveness of the combination of ALX and NaOCl against Enterococcus faecalis was not significantly higher than that of NaOCl alone. Nevertheless, the authors propagated that it can be used in 1% concentration as an alternative to CHX in the endodontic irrigation protocol if used for a sufficiently long time (>5 min). In contrast to the combination of NaOCl and CHX, Kim et al. [86] found no PCA in the mixture of NaOCl and ALX and considered it to be a CHX alternative because it is just as effective against all bacteria and fungi. Czopik [36] described a yellowish precipitate when mixing NaOCl and ALX, which could be identified as aliphatic amines by using the UHPLC-MS (ultra-high-performance liquid chromatography-mass spectrophotometry) method. Surender et al. [51] found a significantly higher effectiveness of NaOCl with ALX against Enterococcus faecalis than with the combination of NaOCl and CHX. Octenisept ® , an octenidine-based preparation, which also contains 2% phenoxyethanol, led to a sparse, whitish deposits that partially closed the dentinal tubules and became transparent over time. Thaha et al. [50] saw potential for a combined application with NaOCl, but also a need for further investigations, for example, with regard to the effect on sealer adhesion to dentin. MTAD ® , which contains 3% doxycycline, 4.25% CA, and 0.55% polysorbate, forms a green-yellow precipitate with NaOCl, the color of which changes to brown when exposed to light. Intermediate rinsing with ascorbic acid can prevent precipitation [7]. SmearOFF TM (Vista Apex, Raxine, WI, USA) and QMix ® (Dentsply Sirona, Bensheim, Germany) are products that combine a biguanide and a chelator. After their application, the penetration depths of the sealer into the dentin were greater than those after sequential rinsing with 17% EDTA, saline solution, and CHX. While QMix ® is used after saline or distilled water (2-phase), SmearOFF TM combines the intermediate rinse and the final rinse, which simplifies and shortens the rinsing protocol. According to the manufacturer, the use of SmearOFF TM after NaOCl in the root canal does not lead to the formation of precipitates; this is also indicated by the sealer penetration depths. Since the manufacturers have not disclosed the formulation of the preparations, further studies on effectiveness and interactions are required [39].
The possibility of exchanging NaOCl in the combination of NaOCl and CHX was only considered possible by Buyukozer et al. [38]. Chlorine dioxide (ClO 2 ) can be utilized as an alternative means of root canal irrigation instead of NaOCl, due to its antimicrobial activity, biocompatibility, and ability to dissolve organic tissue [87][88][89].

Clinical Impact on Endodontic Therapy
In 2023, the best possible cleaning and disinfection of the root canal system by means of chemomechanical preparation is still an indispensable prerequisite for the success of endodontic treatment [2,90,91].
The desirable properties of the various irrigation solutions are: Since no irrigation solution is known that combines all the necessary properties and can be solely applied clinically, different solutions are used consecutively [92]. Certain combinations can interact with each other and result in undesirable effects or by-products. When in contact with each other in the root canal system, NaOCl and CHX interact in an acid-base reaction, forming an orange-brown precipitate, which has undesirable effects. The occlusion of the dentinal tubules [12,30] is indisputable and, depending on the sealer, can have a negative effect on its adhesive force or tightness [29,61,79]. The dyes of the precipitate can discolor the tooth substances, particularly the dentin.
Although it can be considered unlikely that the precipitate of NaOCl and CHX contains free para-chloroaniline, a substance that is suspected of being mutagenic, it is important to avoid precipitation in the root canal. After formation, the complete removal of the precipitate from the root canal system is difficult or impossible, even with advanced methods of activating irrigation solutions with sound, ultrasound, or laser pulses [32,57]. The safest method to avoid a precipitate from forming after use of NaOCl and CHX is to dispense only one of the two substances, which is the preferred option. Because of the sum of its properties, especially due to its ability to dissolve tissue, NaOCl is still the irrigation solution of choice during the mechanical preparation of the root canal. As long as the properties of potential alternatives, such as chlorine dioxide, have not been researched in more detail, it can still be regarded as the "gold standard" to use NaOCl exclusively in this phase [10].
In earlier studies, the combination of CHX and NaOCl was determined to have a better effect against Enterococcus faecalis and gram-positive germs compared to NaOCl alone [27,[93][94][95]. Therefore, it was seen as an ideal complement to NaOCl. Some studies have since denied that the effectiveness of this combination against Enterococcus faecalis is better than that of NaOCl alone. However, this point is actually discussed controversially in the international literature [27,[93][94][95][96].
A strict avoidance of the possible interaction between NaOCl and CHX in all its variants (including CHX solutions, CHX gels) is desired. According to the literature evaluated, this works best when an intermediate rinse with citric acid is utilized, as this removes the smear layer of the mechanical treatment without causing a precipitate with NaOCl or CHX and thereby triggering other complications.
In rinsing protocols that use NaOCl as the sole antimicrobial rinse, based on current knowledge, the final rinse to remove the smear layer should be carried out with citric acid or EDTA before the final use of NaOCl in an activated manner [10].

Conclusions
Since 2006, there has been a sharp increase in publications addressing the interactions between NaOCl and CHX. A total of 88 publications from the PubMed database were identified and evaluated. Of those, 58 publications were relevant to the topic. The results of the studies examined are often controversial, but certain aspects show a tendency over time.
The following findings relate to the endodontic irrigation protocol: -The chemo-mechanical preparation of the root canal system is currently the gold standard; -NaOCl should be used as the sole agent during mechanical reprocessing, due to its tissue-dissolving and antimicrobial properties; - The smear layer can be removed with CA or EDTA after the mechanical preparation. NaOCl should not be mixed with CA or EDTA, since chelators neutralize the tissuedissolving effect of NaOCl; - The consecutive use of NaOCl and CHX is obsolete due to the precipitate that forms;