Basic ResearchTechnology
Free Active Chlorine in Sodium Hypochlorite
Solutions Admixed with Octenidine, SmearOFF,
Chlorhexidine, and EDTA
Unni Krishnan, BDS, MDS, MSc, FRACDS (GDP), FRACDS (Endo),* Sreeja Saji, BSc, MSc,
Roger Clarkson, BDSc, MDSc,* Ratilal Lalloo, BChD, BSc Med(Hons), MChD, PhD,*
and Alex J. Moule, BDSc, PhD*
Abstract
Introduction: The therapeutic effects of sodium hypo-
chlorite (NaOCl) solutions are dependent on the levels
of free available chlorine (FAC). Mixing these solutions
with irrigants can result in significant reductions in FAC.
Although the effect of some irrigants on FAC is known,
the effect of other commonly used irrigants is not. Thus,
the therapeutic ramifications of the concurrent use of
these on the efficiency of NaOCl solutions is not known.
Methods: Aliquots of 5.2% (w/v) NaOCl solutions were
admixed in proportions of 90:10, 80:20, and 50:50 with
the following irrigants: octenidine dihydrochloride (OCT);
SmearOFF (Vista Dental Products, Racine, WI), 17%
EDTA; and 0.2%, 2%, and 5% chlorhexidine (CHX) solu-
tions. Changes in FAC were measured by iodometric
titration. Statistical differences between means were
determined using a post hoc Tukey analysis test after an
analysis of variance. Results: OCT appeared not to affect
FAC and was significantly different than all other irrigants,
except for 90:10 and 80:20 mixtures of low concentration
(0.2%) CHX. CHX solutions showed a marked concentra-
tion- and mixture proportiondependent detrimental ef-
fect on FAC. The reduction of FAC between different
concentrations of CHX was statistically significant in
80:20 and 50:50 proportions, with 50:50 mixtures of
5% CHX having the greatest influence. Mixtures contain-
ing even small proportions of SmearOFF or EDTA exhibited
significant losses in FAC. Conclusions: OCT has little ef-
fect on FAC and can be used concurrently with NaOCl so-
lutions. Higher concentrations of CHX significantly affect
FAC. Their combined use with NaOCl solutions should
be avoided. EDTA and SmearOFF should not be mixed
with NaOCl solutions. (J Endod 2017;-:16)
Key Words
Chlorhexidine, ethylenediaminetetraacetic acid, free
active chlorine, octenidine, SmearOFF, sodium
hypochlorite
From the *School of Dentistry, University of Queensland, Brisbane, Australia; and School of Dentistry, Kuwait University, Jabriya, Kuwait.
Address requests for reprints to Dr Unni Krishnan, University of Queensland School of Dentistry, 288 Herston Road, Brisbane 4006, Australia. E-mail address: drunni@
yahoo.com
0099-2399/$ - see front matter
Copyright 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2017.03.034
JOE Volume -, Number -, - 2017
S odium hypochlorite
(NaOCl) is an oxidizing
agent that exists in solution
Signicance
Octenidine can be used in combination with NaOCl
because it has a minimal effect on FAC. Chlorhex-
as hypochlorous acid and
idine has a concentration- and proportion-
hypochlorite ion in various
dependent decrease on FAC. SmearOFF and
proportions according to
EDTA should not be mixed with NaOCl.
the pH of the solution.
Together they constitute
the free active chlorine (FAC) content (1). It is this FAC that is responsible for protein
breakdown and inhibition of bacterial enzymes (2). The efficacy of NaOCl solutions
quickly decreases in contact with organic matter, especially in the presence of inflam-
matory exudate and dentin (3). NaOCl is widely accepted as an irrigant in endodontics
because of its tissue dissolution potential and antibacterial effect (3). Recently, howev-
er, concerns have been raised that NaOCl cannot predictably eradicate biofilm (4) or
diffuse completely into biofilm even at a 2% concentration (5) and that the potential
exists for subantimicrobial concentrations of NaOCl to actually increase biofilm forma-
tion (6). This may be of clinical significance, considering variations in the use of NaOCl
between countries and between practitioners within the same country (7, 8). Although
in vitro tests using higher concentrations of NaOCl have mostly demonstrated its
antibacterial effect, the presence of dentin collagen, exudate, and microbial biomass
inside root canals rapidly reduce its efficacy, resulting in bacterial persistence (3).
The collateral tissue damage seen with high concentrations of NaOCl (eg, a proportional
increase in cytotoxicity and a decrease in the mechanical properties of dentin) is an
additional concern (9, 10). The use of additional irrigation with secondary
antibacterial irrigants is 1 of the proposed strategies to enhance bacterial eradication
(3). Using some of the currently available irrigants in combination with NaOCl has
its drawbacks, such as the formation of toxic reaction products and antagonistic inter-
actions (11). Our group has previously shown that FAC is dramatically reduced when
NaOCl and EDTA are mixed together, even in small proportions (1). However, there are
no data that support the use of any particular secondary irrigant solution. Nevertheless,
there is a need to identify secondary irrigants with antibacterial and antibiofilm prop-
erties that can be used safely and effectively in root canal systems in combination with
NaOCl without reducing the FAC. This research investigates one such irrigant, octenidine
dihydrochloride (OCT), measuring its effect on FAC and comparing it with 3 other
commercially available irrigants.
OCT is a potentially useful root canal irrigant and antimicrobial agent with antibio-
film properties (12, 13). It has been extensively used in wound disinfection and in the
Free Active Chlorine in NaOCl Solutions 1
Basic ResearchTechnology
treatment of diabetic foot ulcers, burn injuries, and orthopedic implant
infections. It has excellent tissue tolerability on open wounds (14).
Although OCT has been shown to be specifically effective against end-
odontic pathogens and is capable of disinfection of dentin tubules, it
has limited pulp dissolution ability (1517). Its use as an irrigant in
combination with NaOCl, which has excellent tissue dissolution
properties, has potential. A recent study reported that no apparent
deleterious products are formed when OCT and NaOCl solutions are
mixed together (18). However, there is limited information on the effect
of the combination of these 2 irrigants on the active chlorine content in
NaOCl solutions.
Chlorhexidine (CHX) is a positively charged divalent biguanide
with the ability to bind to dentin and negatively charged bacterial sur-
faces, resulting in prolonged antibacterial and antifungal effects when
used in a root canal (19). Even though it is a popular root canal irrigant,
CHX lacks tissue dissolving property. It is also inactivated by serum
albumin and has a relatively limited effect on gram-negative bacteria
(19, 20). CHX forms a precipitate when mixed with NaOCl, the
composition and toxicity of which are still controversial (21, 22).
The effect of CHX on the FAC when it is mixed with NaOCl is unknown.
SmearOFF (Vista Dental Products, Racine, WI) is a new irrigant
containing, among other things, CHX gluconate <1% wt and tetraso-
dium ethylenediaminetetraacetate dihydrate 18% wt. It is marketed as
an irrigant that does not produce a precipitate with NaOCl (23). The
manufacturer suggests it can be used with NaOCl as a 2-step procedure
instead of the 3-step procedure usually involving the removal of NaOCl
from the canal by irrigation with saline or alcohol before other irrigants
are used (20, 23, 24). Tartari et al (25) have recently shown that alka-
line tetrasodium salts of EDTA do not have a substantial effect on the
FAC, even in 1:1 proportions of mixture with NaOCl, but they do have
an effect when the pH is lowered.
For NaOCl solutions to be effective in dissolving proteins and inhib-
iting bacterial enzymes, a high level of FAC is necessary. Thus, it is
important to know what effect mixing other solutions with NaOCl has
on FAC. The aim of this investigation was to measure the effect of
OCT, CHX, SmearOFF, and EDTA on the FAC content of NaOCl solutions
when mixed at varying proportions. Although the effect of EDTA on the
FAC when added to NaOCl solutions has been well reported previously
(1, 20, 26), this solution was retested as a positive control to confirm
that the results were consistent with previous studies.
Materials and Methods
The NaOCl solution used (primary solution) was Clorox regular
bleach (The Clorox Company, Oakland, CA), which had a nominal
free chlorine content of 5.2% (w/v).
The 6 secondary solutions tested were the following:
1. 0.1% OCT (Octenisept, Schulke & Mayr, Nordersdedt, Germany)
2. SmearOFF (Vista Dental Products, Racine, WI), a proprietary
mixture of CHX and tetrasodium salt of EDTA
3. Three CHX gluconate solutions prepared from concentrate (J&J Med-
ical Pty Ltd Australia, North Ryde, NSW, Australia) at 5%, 2%, and 0.2%
4. 17% EDTA (Vista Dental Products)
Preparation of Mixtures
The test solutions were made by diluting the primary solution with
the secondary solution to make up 25-mL aliquots of different propor-
tions. The proportions and their composition were as follows:
 90:10, 22.5 mL 5.2% NaOCl was mixed with 2.5 mL secondary
solution
2 Krishnan et al.
 80:20, 20 mL 5.2% NaOCl was mixed with 5 mL secondary solution
 50:50, 12.5 mL 5.2% NaOCl was mixed with 12.5 mL secondary
solution
The baseline FAC of the primary solution was established by aver-
aging 3 iodometric titrations immediately before mixing with the sec-
ondary solution. The FAC content of the mixtures was also
established immediately after mixing. Three independent assessments
were made for each 25-mL aliquot of each mixture in each of the 3 pro-
portions for each test solution and averaged.
Data Analysis
Statistical analysis was performed using SPSS 22.0 (SPSS Inc, Ar-
monk, NY). A post hoc Tukey test analysis was performed after analysis
of variance to compare the mean FAC between mixtures of NaOCl with
different secondary solutions at different proportions. The level of sig-
nificance was P < .05.
Results
The mean FAC in 5.2% (w/v) NaOCl at the commencement of each
experiment was 4.3% (w/v). The mean FAC of each mixture and the per-
centage of the remaining FAC in each mixture are graphically illustrated
(Figs. 13). Mixing NaOCl with other test solutions resulted in a
decrease of FAC in all mixtures. There was a statistically significant
difference (P < .05) in the mean FAC between different proportions
of the same secondary solution. With OCT, the loss of FAC was almost
identical to what would be expected because of dilution, indicating
that little if any reaction occurred between solutions. With all other
mixtures, the reduction in FAC was much greater than would have
been expected from dilution, indicating that a chemical reaction
occurred between the solutions. The percentages provided later in
parentheses represent the remaining percentage of the original FAC
at proportions of 90:10, 80:20, and 50:50.
OCT
When NaOCl was mixed with OCT, the FAC was 3.91% (w/v)
(90.9%), 3.44% (w/v) (81%), and 2.15 % (w/v) (51.2%), respectively,
almost the same as would be expected from dilution only.
CHX 0.2%
The FAC after mixing was 3.8% (w/v) (88.4%), 3.3% (w/v)
(76.8%), and 1.95% (w/v) (45.4%), respectively.
CHX 2%
The FAC after mixing was 3.68% (w/v) (85.6%), 3.05% (w/v)
(70.9%), and 1.23% (w/v) (28.2%).
Chlorhexidine 5%
The FAC after mixing was 3.38% (w/v) (78.6%), 2.5% (w/v)
(58.4%), and 0.39% (w/v) (9.1%) for the 90:10, 80:20, and 50:50
proportions, respectively. Although the addition of OCT resulted in
the least amount of FAC loss, this loss was not statistically significantly
different compared with NaOCl/0.2% CHX at 90:10 and 80:20 propor-
tions. However, the loss of FAC with NaOCl/OCT was statistically signif-
icant compared with the 50:50 proportion of NaOCl/0.2% CHX mixture
and with all other concentrations of CHX in all proportions. The greatest
loss (90%) of FAC resulted when NaOCl was mixed with 5% CHX at a
50:50 proportion and was statistically significantly lower than all other
secondary solutions, except the NaOCl/SmearOFF mixture.
Comparing the results among the CHX groups, FAC loss between
the different concentrations of CHX was statistically significant, except
JOE Volume -, Number -, - 2017

Figure 1. The mean (standard deviation) values of the FAC content of mixtures of NaOCl with SmearOFF (orange), OCT (gray), EDTA (yellow), and 2% CHX
(blue) in different proportions because of the combined effect of dilution and the chemical reaction. The calculated reduction caused by dilution alone is
also shown (green).
between 90:10 and 80:20 proportions of NaOCl/0.2% CHX and NaOCl/
2% CHX mixtures.
SmearOFF
The FAC after mixing was 1.29% (w/v) (30%), 1.04% (w/v)
(24.2%), and 0.53% (w/v) (12%) for the 90:10, 80:20, and 50:50 pro-
portions, respectively.
EDTA
The FAC after mixing was 1.47% (w/v) (35.2%), 1.07% (w/v)
(24.9%), and 0.69% (w/v) (14.7%), respectively. The loss with Smear-
OFF in all proportions was much greater than was expected from dilution
and more than that measured from EDTA, but the difference between the
2 was not statistically significant. However, the loss of FAC with SmearOFF
and EDTA was statistically significant compared with all the other groups,
except between SmearOFF and the 50:50 proportion of NaOCl/5% CHX
mixture, which were not statistically significant.
Discussion
In considering FAC in mixtures of irrigants, it must be remembered
that any reduction is a combined effect of dilution and a chemical reac-
tion (1). The effect of dilution alone on the FAC of NaOCl has been pre-
JOE Volume -, Number -, - 2017
Basic ResearchTechnology
viously confirmed by Clarkson et al (1). The expected reduction in
values caused by dilution alone is indicated in Figures 1 and 2. The
effect of dilution would be more clinically significant if solutions are
added to NaOCl solutions of lower strengths (1). Any reduction in
FAC beyond that expected from dilution can be assumed to be a result
of a chemical reaction. The decline of FAC in all proportions of the
NaOCl/OCT mixture was almost identical to this effect of dilution, indi-
cating that a minimal chemical reaction occurred between the 2 com-
pounds. This confirms a proposal in a recent article that identified the
cloudy precipitate formed when NaOCl and OCT were mixed to be phe-
noxyethanol (PE) and not a reaction by-product. PE is already present
in the commercially available OCT (18). Interestingly, PE has also been
shown to be an antibacterial agent known to have a synergistic effect
with OCT (27). The fact that FAC is not affected in the NaOCl/OCT com-
bination and the finding of the previous article (18) suggest that OCT
can be safely used in combination with NaOCl with potentially synergistic
activity.
Our results showed that the reduction of FAC with all concentra-
tions and combinations of CHX was more than the effect of dilution, con-
firming that NaOCl and CHX reacted with each other. The effect of 0.2%
CHX in smaller proportions on the FAC was not much greater than that
expected with dilution and not significantly greater than with OCT. This
is likely because at this concentration the CHX content is so low that it
Free Active Chlorine in NaOCl Solutions 3
Basic ResearchTechnology

Figure 2. The effect of dilution and chemical reaction of different concentrations of CHX in various proportions on the mean (standard deviation) FAC of 5.2%
NaOCl.

Figure 3. The percentage of the remaining FAC with different combinations and proportions of irrigants.

4 Krishnan et al. JOE Volume -, Number -, - 2017


would be almost immediately exhausted when mixed and thus would
have little effect on the overall FAC. At this low concentration, CHX is
also ineffective as a root canal irrigant (20, 28). Because larger
proportions and higher concentrations of CHX produced a significant
reduction in FAC, the poor clinical outcome reported when CHX was
used concurrently with NaOCl (29) is likely because of the combined
effect of the reduced FAC nullifying any antimicrobial effect of the
mixture and in part because of the precipitate (22), which could affect
the obturation seal (30).
Dentin tubule disinfection is an important step, especially in re-
treatment cases associated with persistent apical periodontitis, because
bacteria have been found to penetrate into deep dentin closer to the
cementum (31). At room temperature, 3% NaOCl is ineffective in
achieving dentin tubule disinfection, with 60% of live bacteria remaining
within the tubules at 300 mm (32). Even if 6% NaOCl is used at 45 C for
20 minutes, the intratubular penetration of NaOCl is limited to 300 mm
in dentin slab models in vitro (33). On the other hand, OCT has been
shown to be able to significantly disinfect dentinal tubules (17, 34) and
reduce the colony-forming units at a depth of 400 mm compared with
controls and CHX (35). Considering the retention of still more than 50%
of FAC when NaOCl and OCT are mixed in equal proportions, the poten-
tial for combined irrigation of NaOCl with OCT exists, with a likelihood
of better dentin tubule disinfection. Further studies are underway to
evaluate the effect of activation and temperature on the disinfection po-
tential of combined irrigation with OCT and NaOCl.
Previous studies have shown that EDTA can reduce up to 80%
88% of available chlorine from NaOCl solutions, which was mirrored
in the results of this study (1, 26). What was surprising was the
substantial reduction in FAC observed with SmearOFF. Recently,
Tartari et al (25) showed that alkaline tetrasodium salts of EDTA did
not have a substantial effect even in 1:1 proportions at a pH of 12. Never-
theless, tetrasodium salts of EDTA in Tartari et als pilot study showed a
significant reduction in tissue dissolution at a neutral pH, which is likely
because of the reduced presence of hypochlorite ion or free chlorine
(25). The marked reduction of FAC observed with admixtures of Smear-
OFF may be caused by the reaction of NaOCl with CHX and because the
product contains 4% wt sodium bisulfite (23). This is a reducing agent
known to rapidly react with NaOCl to form sodium bisulfate and salt
(36). Thus, the fact that admixtures of SmearOff and NaOCl do not
form a precipitate is likely caused by the rapid exhaustion of available
chlorine before a precipitate can occur.
Although a case could be made for the concomitant use of tetra-
sodium salt of EDTA with NaOCl at a pH of 12 to perform organic matter
dissolution and smear layer removal simultaneously (25), the commer-
cial product SmearOFF, which has a pH of only 8 to 9, appears not to
retain any significant active chlorine when mixed with NaOCl. Hence,
the intended use of SmearOFF should be clear; it should only be used
alone as a final irrigant. Even though it does not produce a precipitate
when mixed with NaOCl, SmearOFF should not be used concurrently
with NaOCl because of the rapid displacement of available chlorine.
On the other hand, the potential for OCT as a final irrigant requires
serious consideration because of its excellent tissue tolerability; its lack
of reaction with NaOCl; and its ability to retain antimicrobial effective-
ness even in the presence of albumin, blood, and mucin (27, 37).
Further studies are underway combining OCT and various salts of
EDTA at different pH levels to explore the antimicrobial and smear
layer removing ability of such combinations.
This article is not without limitations. The chlorine levels deter-
mined using the iodometric titration methods are generally higher
than those that are clinically achievable because of the inherent insta-
bility of NaOCl and the neutralizing effect of dentin and organic matter
(3). In addition, the inclusion of chlorate, chlorite, perchlorate, and
JOE Volume -, Number -, - 2017
Basic ResearchTechnology
bromate ions in FAC measurements in the iodometric titration makes
it less accurate compared with FAC measurement with liquid chroma-
tography and tandem mass spectrometry (38). Because these ions do
not have any therapeutic effect, there may be an overestimation of clin-
ically active chlorine with the iodometric method (39). Thus, the
measured levels of FAC may have been higher than if other more com-
plex titration methods had been used (1).
Because solutions vary in their density, a wt/wt mixture of the irri-
gants would have been ideal. However, a volume-based measurement of
the mixture as previously reported by Clarkson et al (1) was used
because it is a clinically relevant way of testing irrigant interaction. Water
was not used as a positive control. This additional step was considered
unnecessary because dilution of NaOCl with water is proven to reduce
the FAC proportionally (1).
In conclusion, when mixed with NaOCl solutions, SmearOFF ap-
pears to cause a marked reduction in FAC in a similar manner to that
of EDTA, and, thus, its combined use with NaOCl cannot be recommen-
ded. CHX showed a marked concentration- and mixture proportion
dependent effect on FAC. On the other hand, OCT has no demonstrable
effect on the available chlorine levels in all mixed proportions other
than the effect of dilution. Its use as an irrigant in combination with
NaOCl is a promising development. Further studies are required to eval-
uate the extent of synergism achievable using OCT as a root canal irri-
gant in combination with NaOCl and also with EDTA at various pH levels.
The ideal proportion and minimal concentration of NaOCl with OCT to
achieve optimal antimicrobial and tissue dissolution actions require
further investigation.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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