Antagonistic Interactions between Sodium Hypochlorite,
Chlorhexidine, EDTA, and Citric Acid
Giampiero Rossi-Fedele, DDS, MClinDent,*†Esma J. Do? gramacı, BDS, MFDS (RCS Eng),‡
in the elimination of microorganisms, tissue dissolution,
and the removal of debris and smear layer. No single
solution is able to fulfill these actions completely; there-
fore, their association is required. The aim of this inves-
tigation was to review the antagonistic interactions
occurring when sodium hypochlorite (NaOCl), chlorhex-
idine (CHX), EDTA, andcitric acid(CA) are usedtogether
during endodontic treatment. Methods: A search was
performed in the electronic database Medline (articles
published through 2011; English language; and the
following search terms or combinations: ‘‘interaction
AND root canal irrigant or endodontic irrigant or sodium
hypochlorite or chlorhexidine,’’ ‘‘sodium hypochlorite
AND EDTA or ethylenediaminetetraacetic acid or citric
acid or chelating agent or chlorhexidine,’’ and ‘‘chlo-
rhexidine AND EDTA or ethylenediaminetetraacetic
acid or citric acid or chelating agent’’) to identify publi-
cations that studied unwanted chemical interactions
between NaOCl, CHX, and EDTA and CA. Results: The
search identified 1,285 publications; 19 fulfilled the
inclusion/exclusion criteria of the review. Their research
methodology was classified as either in vitro or
ex vivo. Conclusions: Antagonistic interactions
included the loss of free available chlorine for NaOCl
when in contact with chelators, which consequently
reduced the tissue dissolution capability and to a lesser
extent antimicrobial activities. When CHX and NaOCl
are mixed, a precipitate forms that can present detri-
including a risk of discoloration and potential leaching
of unidentified chemicals into the periradicular tissues.
CHX and EDTA mixtures cause a precipitate, whereas
CHX and CA do not exhibit interaction. (J Endod
Chlorhexidine, citric acid, EDTA, endodontic irrigant,
interaction, root canal irrigants, sodium hypochlorite
Irrigants should ideally have antimicrobial and tissue-dissolution actions as well as
other advantageous properties, such as lubrication, demineralization, and the ability
to remove debris and the smear layer (1).
Sodium hypochlorite (NaOCl) is recommended as the main endodontic irrigant
because of its ability to dissolve organic matter together with its broad antimicrobial
action (2). NaOCl is commercially available as aqueous solutions with concentrations
ranging from1% to 15%and havinganalkalinepH withvaluesaround 11 (3). Among
other salts, they also contain sodium hydroxide salts in order to increase their stability
disclosed by the manufacturer (4).
dissolving organic tissue simultaneously (5). Therefore, the adjunctive use of chelating
formation of the smear layer associated with root canal instrumentation (2).
(7) to valuesbetween 7 and 8 to increaseits chelating capacity (2, 6). Like many well-
both protonated and unprotonated forms. CA is an organic acid normally used in
endodontics at concentrations between 10% and 50% (2) with a pH value between
1 and 2 (8).
Although the role of smear layer removal has been widely debated, endodontic
literature concerning the antimicrobial action of irrigants suggests that the combined
use of EDTA and NaOCl is more efficient than NaOCl alone when measuring bacterial
survival after multiple appointments (9); bacterial survival analysis is a surrogate
measure of treatment outcome. A recently published outcome investigation indicated
that 2.5% to 5% NaOCl followed by 17% EDTA had a profoundly beneficial effect on
secondary nonsurgical root canal treatment success while having a marginal effect
on the original treatment (10).
tissue-dissolutionactivities (11). A reduction of the pH to values around6.0 to 7.5 has
been found to improve the antimicrobial efficacy (11–13) but hinders tissue-
dissolution action (11, 13–15). If the pH is lowered to values below 4, then the
and therefore unstable (17). If NaOCl is mixed with other irrigants possessing low pH
values, there is a possibility of altering its properties.
oot canal cleaning and disinfection during chemomechanical preparation relies
heavily on irrigants because of the anatomic complexities of the pulp canal system.
From *Warwick Dentistry, The University of Warwick, Coventry, United Kingdom;†Post-Graduate Program in Dentistry, Pontifical Catholic University of Rio Grande
do Sul, Porto Alegre, Rio Grande do Sul, Brazil;‡Orthodontic Department, Guy’s Hospital, King’s College London Dental Institute, London, United Kingdom; and
§Chemical Engineering Department, University of Barcelona, Barcelona, Spain.
Address requests for reprints to Dr Giampiero Rossi-Fedele, 10 Station Path, Staines, Middlesex, UK TW18 4LW. E-mail address: firstname.lastname@example.org
0099-2399/$ - see front matter
Copyright ª 2012 American Association of Endodontists.
Rossi-Fedele et al.
JOE — Volume 38, Number 4, April 2012
Chlorhexidine (CHX), a bisguanide, is stable as a salt although it
dissociates in water at a physiologic pH, releasing the CHX component
(18). It is frequentlyused at concentrations between0.2% and 2% (2)
and exhibits an optimal antimicrobial activity at a pH of 5.5 to 7.0 de-
most common preparation is CHX gluconate (20). It has been recom-
mended that CHX be used as either an alternative or an adjunct root
canal irrigant because of its antimicrobial qualities. Studies comparing
its antimicrobial action versus NaOCl solutions present conflicting
results (10, 21–29).
Some investigations suggest that NaOCl is more effective as an
antimicrobial agent compared with CHX. One in vivo study showed
2.5% NaOCl was a more effective antimicrobial agent compared with
0.2% CHX (21). However, an in vitro study (22) using a bovine root
model showed that CHX had a similar antimicrobial effect as NaOCl,
whereas another investigation into bovine dentinal tubule disinfection
comparing NaOCl and CHX 0.2% to 2% found no difference in antimi-
crobial efficacy between either solution at these concentrations (23).
ence when comparing 5.25% NaOCl and 2% CHX (24). Contemporary
to reduce the numbers of cultivable bacteria (25) and the presence of
bacteria, archaea, and fungi on teeth with apical periodontitis using
molecular microbiology procedures (26) suggest no difference in
effectiveness between the solutions. On the contrary, an in vivo inves-
tigation into the percentage of growing bacterial species after irrigation
with 5.25% NaOCl or 2% CHX in teeth with pulpal necrosis, apical pa-
thosis, or both found the latter to be significantly more effective at
are summarized in Table 1.
A seminal investigation comparing the use of 0.2% CHX and 2.5%
NaOCl individually and in combination in human teeth presenting with
periapical radiolucencies suggested that their combined use produced
of 2%CHXto 1%NaOClin teethwithinfectednecroticpulpswasfound
to enhance the disinfection of the root canal system because of the
reduction of cultivable bacteria in those cases (29). Ng et al (10) in
ment (10). CHX lacks tissue dissolution capacity (30), an important
quality desired from root canal irrigants.
cation of adhesives prevents resin-dentin bond degradation because of
its ability to inhibit collagenolytic enzymes (31). Concerns about the
longevity of bonding to root canal dentin have been raised for bonded
taken into consideration when resin-based sealers are used even when
gutta-percha is used as the core material.
The chemical interactions of NaOCl with EDTA or CHX are redox reac-
basereactionoccurswhen CHXandNaOClare mixedbecauseCHXhas
the ability to donate protons as a positive component, whereas NaOCl
can accept them (20, 28, 34). In regard to EDTA associated with
CHX, it may potentially degrade CHX, forming a salt (35). CA and
CHX apparently pose no antagonistic reactions (36). Therefore, the
purpose of this article was to review the undesired effects after interac-
tions between NaOCl, CHX, and the commonly used chelating agents
EDTA and CA.
Materials and Methods
A literature search using electronic database Medline was con-
ducted on June 15, 2011, for articles published through to the date
using the following search terms and combinations: ‘‘interaction AND
root canal irrigant or endodontic irrigant or sodium hypochlorite or
chlorhexidine,’’ ‘‘sodium hypochlorite AND EDTA or ethylenediamine-
tetraacetic acid or citric acid or chelating agent or chlorhexidine,’’ and
‘‘chlorhexidine AND EDTA or ethylenediaminetetraacetic acid or citric
acid or chelating agent.’’ Publications were included if they studied
antagonistic interactions between NaOCl, CHX, EDTA, and CA by
comparing 1 of the solutions against a mixture of them and were pub-
cations were included for full-text evaluation by 1 reviewer (G.R.F.) if
the content of the abstracts met the inclusion criteria. Full-text assess-
cations were excluded if they did not meet the inclusion criteria (ie, if
or EDTA by comparing 1 of these alone and when combined with
a substance mentioned previously) or if they were not published in
English. Of 1,285 publications identified, 19 were included in the
Interactions between NaOCl and Chelating Agents
The addition of chelators to NaOCl reduces its pH in a ratio and
time-dependent manner (37–39). This affects the forms of free
chlorine in the solution and causes an increase in hypochlorous acid
and chlorine gas, which subsequently reduces the amount of the
hypochlorite ion (3, 11). When 1% NaOCl was mixed with 17%
EDTA (pH = 8) in ratios of 1:1, 1:5, and 5:1, the pH of the solutions
in the same ratios resulted in pH values between 1.8 and 4.3 (37).
an elapse of 48 hours. However, when mixed in a 1:3 ratio, although
with a larger volume of EDTA, the pH value was stable during the 48-
hour experimental time, probably because of an immediate interaction
between the solutions (38).The reduction of pH values in the NaOCl
solution causes the release of chlorine gas, which has potentially
icantly more chlorine is detectable and present at a further distance.
This is according to a laboratory-based investigation that studied the
reactions between NaOCl (5.25%, pH = 12.12) and CA (50%,
TABLE 1. Characteristics of Some Root Canal Irrigants
of solution (%)
0.5 to 15
10 to 17
0.2 to 2
10 to 50
9 to 12
7 to 8
5.5 to 7
1 to 2
EDTA disodium salt
JOE — Volume 38, Number 4, April 2012 Irrigant’s Antagonistic Interactions
pH = 1.28) or EDTA (15%, PH = 7.51). Portions of the chelator were
added to the NaOCl at regular time intervals for a total time period of 2
hours; the release of chlorine gas was measured at 6 inches and 6 feet
from the container (39).
The consequences of chemical interactions between chelating
agents and NaOCl result in a loss in the free available chlorine (FAC)
of the mixtures (6, 37, 40). The effects on FAC contents in a 1%
NaOCl solution were assayed by mixing it with either 17% EDTA or
water (1:1) and measured via an iodine/thiosulfate titration method.
NaOCl’s FAC was substantially modified by the presence of EDTA with
a reduction to 0.06% when compared with 0.5% of the water
dilution control (6). This research group subsequently looked into
the impact on available chlorine in 1% NaOCl from the interaction
with 17% EDTA (pH = 8) and 10% CA using the same methodology
and taking into account the time factor. Their results indicated that
level (37). These results were confirmed by a different research group
that looked into time-related effects (between 5 and 18 minutes) on
active chlorine content because of dilution with a EDTA solution
(17%, pH = 7.5, and containing a surfactant). Different NaOCl prepa-
NaOCl:EDTA ratios (9:1, 3:1, and 1:1) were tested via iodometric titra-
tion. Apart from modifications because of dilution, the available chlo-
rine loss was extreme (ie, up to 80% even when adding small
amounts of EDTA at the early stages of the process and then becoming
more gradual, indicating that a chemical reaction occurs between the
original concentration for chlorine loss (4). When gel-type prepara-
tions of chelators containing 15% EDTA and 10% Urea peroxide
(RC-Prep; Premier Dental, Philadelphia, PA, and Glyde; DeTrey Dents-
ply, Konstanz, Germany) were tested for interaction with 1% NaOCl
using spectroscopy, it was shown that both compounds depleted the
solution from its chlorine content after 5 minutes (40).
The dramatic reduction of FAC in NaOCl mixtures caused by
chemical interactions appears to explain the inability of NaOCl and
EDTA mixtures to dissolve soft tissues. An investigation looking into
bovine tissue dissolution of NaOCl (1%-2.5%) alone and combined
with 17% EDTA in different ratios (2:2 and 1:3) showed that after
48 hours only unmixed NaOCl was able to completely dissolve the
tissue (38). Similarly, the tissue dissolution effects of the interactions
between NaOCl and EDTA were tested on porcine palatal mucosa by
assessing the percentage of original tissue weight after different expo-
sure periods up to 120 minutes; 8.5% EDTA, 0.5% NaOCl, and a 1:1
mixture of 17% EDTA together with 1% NaOCl were the test solutions.
This investigation suggested that NaOCl alone was substantially more
efficient than the other groups, with no statistically significant differ-
ences among them (6).
The degradation and consequent deactivation of EDTA after its
interaction with NaOCl is extremely slow, and, therefore, it does not
has been analyzed via nuclear magnetic resonance with no reactions
detected in the first 7 minutes, and the process was not complete
after 120 minutes (33).
of EDTA and CA (6, 7, 37). An investigation, using standardized dentin
or 17% EDTA and 0.5% NaOCl, found greater calcium chelation
occurring in the solution containing NaOCl (7). Similarly, the chelating
was compared using a calcium titration method in order to assess the
amount of chelated calcium per mole of EDTA. The results indicated
that NaOCl had little effect on EDTA’s calcium chelating ability (6).
Another investigation from the same research group studied calcium
chelation and smear layer removal from root canals after irrigation
calcium content using atomic absorption spectrophotometry. No statis-
tically significant differences for EDTA or CA were found between the
combinations containing water or NaOCl. The teeth were subsequently
split and observed using a scanning electron microscope for the pres-
enceorabsence of smear layer in a semiquantitative manner;nodiffer-
ences were found among the irrigant combinations described earlier
(37). The addition of NaOCl to EDTA does not alter EDTA’s ability to
decalcify human dentin, and this has been shown through studies as-
sessing Vickers microhardness after adding either NaOCl or distilled
water to EDTA in a 1:1 ratio and observing for 7 minutes (7).
Chelators can eliminate NaOCl’s antimicrobial efficacy if the orig-
inal FAC values are modest, whereas EDTA and CA performance does
not seem to be jeopardized because of interactions with NaOCl (6,
37). The effects on antimicrobial ability, related to the interactions
between EDTA and NaOCl, have been analyzed using an agar diffusion
test against Enterococcus faecalis and Candida albicans using
0.5% NaOCl, 8.5% EDTA, and a mixture with 1% NaOCl and 17%
EDTA (1:1 mixture) (6). Pure NaOCl produced smaller zones of inhi-
bition when compared with pure EDTA or the mixture of EDTA/NaOCl,
and there were no statistically significant differences among the EDTA-
same group. E. faecalis was suspended in phosphate buffered saline
and then added (1:1) to tubes containing chelating agent mixtures
with 1% NaOCl and their 1:10 and 1:100 dilutions; after incubation,
bial action at the 1:100 dilutions because growth was present (37).
Interactions between NaOCl and CHX
From the review of the literature, it transpires that mixing NaOCl
itate (41–49). Basrani et al (41) looked into the minimum NaOCl
concentration required to form a precipitate when mixed with 2%
CHX (41). Concentrations ranging from 0.023% to 6% were tested,
and an instant color change occurred in all samples from dark brown
to light orange. A precipitate was induced with 0.19% NaOCl with
varying amounts of material formed in the different mixtures (41).
Several investigations have been undertaken to elucidate the
chemical composition of the flocculate produced by the association
of NaOCl with CHX (41–45, 47–49). Different proportions and
concentrations of NaOCl (0.5%, 2.5%, and 5%) and CHX (0.2%–
2%) have been mixed, which results in the formation of
a brownish flocculate evident when the solutions make contact with
each other; atomic absorption spectrophotometry showed the
presence of Ca, Fe, and Mg (42). Although most investigations report
the presence of parachloroaniline (PCA) in the precipitate, 1 failed to
detect its presence. The precipitate was analyzed using X-ray photo-
electron spectroscopy and time-of-flight secondary ion mass spec-
trometry, which detected that PCA was present at concentrations
directly related to the NaOCl concentration (41). The same
researchers used gas chromatography-mass spectrometry in order
to further identify the precipitate composition after the mixture of
6% NaOCl with 2% CHX; PCA was detected again although no further
Rossi-Fedele et al.
JOE — Volume 38, Number 4, April 2012
aniline derivatives or chlorobenzene were found (43). Krishnamurthy
and Sudhakaran (44) mixed 2.5% NaOCl with 2% CHX and were able
to detect PCA in the precipitate by using Beilstein and HCl solubility
tests followed by nuclear magnetic resonance. Despite Thomas and
Sen (45) using nuclear magnetic resonance spectroscopy, they failed
to detect PCA in the precipitate after combining 5.25% NaOCl with 2%
CHX acetate. PCA has been suggested to be a toxic and carcinogenic
substance, hence the significance of this subject (46).
Three studies have evaluated the cleaning efficacy after irrigation
with CHX containing solutions (44, 47, 48). Bui et al (47) investigated
using 5.25% NaOCl and 2% CHX ex vivo and analyzed it with an envi-
NaOCl and then aspiration and drying with paper points. There was no
difference in remaining debris and a reduction in number of patent
dentinal tubules in the coronal and middle third between the 2 test
groups. A scanning electron microscopic investigation into the
on human teeth using 2.5% NaOCl and 2% CHX in liquid or gel forms,
intercalated by physiologic saline, with half of the experimental groups
receiving a final flush with 17% EDTA was performed by Valera et al
(48). Their results indicated that 2% CHX gel produced the largest
amount of open dentinal tubules, whereas 2% CHX liquid presented
the worst result. The addition of EDTA and physiologic saline as a final
flush improved cleaning and debris removal. The presence and thick-
ness of the precipitate formed after irrigation with 17% EDTA followed
by 2.5% NaOCl and a final flush with 2% CHX (test group) was
compared against the same sequence, but they were intercalated
of the precipitate(44).This wasperformedon exvivo root canalsand
examined with a stereomicroscope (44). Isopropyl alcohol resulted in
completely clean canals, whereas the use of saline or distilled water
produced a sparse precipitate. The test group presented deposits all
along the canal wall with a mean thickness 2 to 3 times greater than
mainly in the coronal and middle thirds of the canals.
This precipitate has an effect on dentinal permeability (34) and
dye penetration after root canal obturation (49). An ex vivo investiga-
tion compared the effects of combining 1% NaOCl and 2% CHX on
(34). When compared against a ‘‘no irrigation’’ control, the mixture of
NaOCl and CHX caused a reduction of permeability only in the apical
third. This was explained by the formation of a brown mass suspended
in the liquid that becomes a flocculate precipitate, which acts as
a ‘‘chemical smear layer.’’ Another ex vivo investigation assessed dye
penetration in clear teeth after preparation using different irrigants
and obturation (49). The results suggested that a precipitate formed
when combining 1% NaOCl and 2% CHX gel, which stained the dentin
and adhered to the canal walls. Therefore, this group presented the
largest values of linear dye penetration. Statistically significant differ-
ences were found with the other groups, which included NaOCl alone,
NaOCl and EDTA, CHX gel alone, and distilled water.
Interactions between CHX and Chelating Agents
CHX is easily mixed with CA, and no modification of its deminer-
alizing ability or precipitation occurs (34, 36). An in vitro study on
bovine dentin slices using atomic absorption spectrophotometry
looked into the effect of adding 1% CHX and 10% to 20% CA on the
TABLE 2. Reported Interactions between Root Canal Irrigants
NaOCl + EDTA
Loss on active chlorine content
Breakdown of NaOCl al low pH
Glioxilic acid; EDTA
Degradation of EDTA molecule
Oxidation of EDTA (after 7
minutes of reaction)
NaOCl + CHX
Potentially Toxic compound;
Int End J
Oral Surg Oral
Med Oral Pathol
Oral Radiol Endod
NaOCl + CA
Loss on active chlorine content
Breakdown of NaOCl al low pH
CHX + EDTA
Salt; white precipitate
Chemical degradation of CHX
Salt formed by neutralization
between CHX and EDTA
CHX + CA
No reaction known
JOE — Volume 38, Number 4, April 2012Irrigant’s Antagonistic Interactions
(36). Another study looking into modification of dentinal permeability
after the irrigation of human teeth found no statistically significant
differences when compared against the ‘‘no irrigation’’ group although
statistically significant differences were found when compared against
the NaOCl and CHX group in the apical third of the root canal (34).
It has been shown that 15% CA followed by 2% CHX causes the forma-
CHX; no precipitation occurs (34).
When mixing CHX with EDTA, it is difficult to obtain a homoge-
neous solution; a precipitate composed chiefly of the original compo-
a homogenous solution by mixing 17% EDTA and 1% CHX because
using reverse-phase high-performance chromatography analyzed the
precipitate that forms after the combination of 17% EDTA with 2% or
20% CHX in equal volumes and 3 different mixing conditions (35).
Over 90% of the precipitate mass was either EDTA or CHX although
PCA was not detected. It was suggested that the precipitate was most
likely a salt formed by neutralization of the cationic CHX by anionic
This literature review highlights the importance of clinicians
Because these solutions are used in succession, theycome into contact
with each other inside the endodontic space. This might impact treat-
ment due to the modifications to tissue dissolution, antimicrobial and
cleaning efficacy, seal, the risk of discoloration, and most importantly
the potential adverse effects to a patient’s general health as a conse-
quence of leaching chemicals in the periradicular tissues. Table 2
to prevent or reduce the occurrence of the detrimental reactions
described, the following strategies have been suggested:
1. NaOCl and EDTA: rinse out with copious amounts of NaOCl (37),
making sure that fluid exchange occurs at all levels in the canal to
prevent stratification of the solutions through the canal, which will
natively, evacuation or drying before the placement of the next irri-
gant (4) can also help.
2. NaOCl and CHX: to prevent the formation of a precipitate associated
after NaOCl has been suggested. They include saline (48); water
izing solution, which can be CA (34) or EDTA (42). Finally, if the
flocculate is formed, then acetic acid can be used to dissolve the
3. CHX and chelating agents: CA can be used in association with CHX
because no interactions occur (34, 36). Alternatively, maleic acid
can be used because it has been shown that this combination
does not cause the formation of a precipitate, and only a marginal
reduction of CHX availability occurs (50).
In summary, chelating agents have a dramatic effect on the free
a reduction of their chelating ability in mixtures containing NaOCl. CHX-
and NaOCl-containing solutions develop a precipitate that might contain
CHX and EDTA, it is difficult to obtain a homogenous solution, and
a precipitate composed mainly of those substances is formed. CA is
not influencedbyCHX,and noprecipitateisformedwhenmixed withit.
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JOE — Volume 38, Number 4, April 2012Irrigant’s Antagonistic Interactions