Origi na l A r tic le DOI: 10.

17354/ijss/2015/233

Evaluation of the Interaction between Sodium

Hypochlorite and Chlorhexidine used as Root Canal
Irrigants: An In-Vitro Stereomicroscopic Study
Anil Ricchawal1, Vathsalya Shetty2, Sandeep K Pimpale3, Pravin Patil4, Ashish Mandwe2, Santosh Kiswe5
1
Assistant Professor, Department of General Dentistry, Grant Medical College & J.J Group of Hospitals, Mumbai, Maharashtra, India,
2
Associate Professor, Department of Conservative Dentistry and Endodontics, Nair Hospital Dental College, Mumbai, Maharashtra, India,
3
Assistant Professor, Department of Periodontics, Nair Hospital Dental College, Mumbai, Maharashtra, India, 4Assistant Professor, Department
of Conservative Dentistry and Endodontics, Nair Hospital Dental College, Mumbai, Maharashtra, India, 5Senior Lecturer, Department of
Conservative Dentistry and Endodontics, Dhanwantari Dental College, Parbhani, Maharashtra, India

Abstract
Introduction: The combination of sodium hypochlorite (NaOCl) and the chlorhexidine (CHX) results into formation of an insoluble
precipitate. The aim of this in-vitro study was: (1) To evaluate the amount of precipitate formed and chemical composition of
this precipitate which is formed due to interaction between NaOCl and CHX and, (2) to prevent the formation of precipitate by
using absolute alcohol as an intermediate flush.
Methodology: Totally, 20 extracted single-rooted human teeth were selected, cleaning and shaping has been done with all root
canals. After instrumentation, all teeth were decoronated at the cemento-enamel junction. All teeth were randomly divided into
two groups containing 10 teeth each. In the test group (GroupA), canals were irrigated with 15% ethylenediaminetetraacetic
acid and 5.25% NaOCl followed by 2% CHX. In the GroupB, absolute alcohol was used as intermediate flush between 5.25%
NaOCl and 2% CHX. All teeth were sectioned longitudinally and subjected to stereomicroscopic examination.
Results: Test group (GroupA) samples showed orange-brown precipitate, concentrated more in the coronal and middle thirds,
whereas the GroupB showed no evidence of precipitate. The thickness of the precipitate was measured using the ProgRes
computer software (Olympus, Hamburg, Germany). Bonferroni test was implicated for statistical analysis to compare the
significant difference of precipitate formation at three different levels in GroupA.
Conclusion: An insoluble orange-brown precipitate formed due to the interaction between NaOCl and CHX. This precipitate
can be prevented by using absolute alcohol flushing between 5.25% NaOCl and 2% CHX. Dentinal tubules are blocked and
occluded due to this NaOCl/CHX precipitate. Utmost care has been taken while irrigating with NaOCl and CHX.

Key words: Chlorhexidine, Interaction of irrigants, Parachloroaniline, Precipitate, Sodium hypochlorite

INTRODUCTION the infected root canal and to prevent reinfection. Due to

Bacteria are the etiological agents of pulp necrosis and
apical periodontitis and bacteria in the root canal system
can initiate and cause periapical inflammatory lesions.1 He
aims of endodontic treatment is to eliminate bacteria from
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the complexity of the root canal system, bacteria, and tissue
from root canal surfaces cannot be removed by mechanical
instrumentation.2 Mechanical instrumentation results
into the formation of a smear layer on the canal surface.3
Irrigation along with the mechanical instrumentation is
required to remove debris, tissue remnants, microbes, and
the smear layer from root canal surfaces.

Month of Submission : 03-2015 A combination of sodium hypochlorite (NaOCl) and

Month of Peer Review : 04-2015 ethylenediaminetetraacetic acid (EDTA) is an effective
Month of Acceptance : 04-2015 combination of removal of both tissue and smear layer.4
Month of Publishing : 05-2015 NaOCl is known for its tissue dissolving property and
www.ijss-sn.com
is antimicrobial.5 However, disadvantage of NaOCl if
Corresponding Author: Dr.Sandeep K Pimpale, Nair Hospital Dental College, Dr.A. L. Nair Road, Near Mumbai Central Railway Station,
Opposite Maratha Mandir, Mumbai-400008, Maharashtra, India. Phone: +91-9833553072. E-mail:sandeepudentist3@gmail.com

159 International Journal of Scientific Study | May 2015 | Vol 3 | Issue 2

 Ricchawal, et al.: Interaction between Sodium Hypochlorite and Chlorhexidine used as Root Canal Irrigants
extruded out of the root canal apex and into the periapical
tissues is destructive tissue damage. 6 NaOCl has a
disagreeable odor also corrodes and weakens endodontic
instruments.7 Thus, 2% chlorhexidine (CHX) gluconate
have been considered as an alternative irrigant.8
CHX is a broad-spectrum antimicrobial agent that disrupts
the membranes of microbes.9 CHX has lower toxicity as
compared with NaOCl so, it has been suggested for use as an
irrigant and intracanal medicament.10 CHX has comparable
antibacterial efficacy to NaOCl11 and has the advantage of
having substantivity.12 However, CHX does not show the
tissue dissolution capabilities that of NaOCl. Hence, it has
been suggested that CHX cannot be used as a replacement
to NaOCl as a main irrigant but used as supplemental in
final irrigation step after NaOCl and EDTA irrigation.13 The
suggested protocol for irrigation,14 is NaOCl (which dissolves
the organic components during instrumentation), followed
by EDTA (which used as final flush of canals to remove the
smear layer), then NaOCl and finally CHX (which imparts
substantive antibacterial activity). Although combination
of NaOCl and CHX may enhance their antimicrobial
properties, a major concern about this irrigation regimen is
that irrigation with CHX in the presence of NaOCl in the
root canal produces an orange-brown precipitate known as
parachloroaniline (PCA).15
Basrani et al.16 used X-ray photoelectron spectroscopy and
time of flight secondary ion mass spectrometry to identify
this precipitate. The precipitate contains a significant
amount of PCA; a hydrolysis product of CHX.17 PCA
can further degrade to 1-chloro-4-nitrobenzene.18 Even
without the presence of NaOCl, CHX might spontaneously
hydrolyze into PCA in the presence of heat and light.19
PCA has industrial uses in pesticides and dyes and has
been demonstrated to be carcinogenic in animals.20 Its
degradation product, 1-chloro-4-nitrobenzene, is also a
carcinogen.21
In the root canal system, a major concern is that this formed
precipitate might attach to the root surface and leaches
slowly into the periapical tissues. It could also compromise
the seal of an obturated root canal. Therefore, the main
objective of this in vitro study was to evaluate the maximum
thickness of precipitate formed along the canal wall caused
by the interaction between NaOCl and CHX and to prevent
the formation of precipitate by using an intermediate flush
of absolute alcohol.
METHODOLOGY
A total of 20 single-rooted, extracted human teeth were
selected for this study. Calculus, bone, residual soft tissues
International Journal of Scientific Study | May 2015 | Vol 3 | Issue 2
were removed from the teeth. Storage of selected teeth
were done in refrigerated 0.9% saline and used within
1month (Baxter Health Care Corp, Round Lake, IL).
Access cavity was prepared by using a high-speed #2
round bur. Pulp extirpation done with each tooth, coronal
flaring was achieved by using #2, 3, and 4 Gates Glidden
drills. Working length determination has been done with
the help of surgical operating microscope by using #10
K-file, which was introduced into the canal until the tip of
the file was visible at the apical foramen. Aglide path was
established by using #15 and #20 K-files.
A fresh mix of aquasil vinyl polysiloxane impression
material (Dentsply/Caulk, Milford, DE) was used to fill
a small glass vial (VWR Scientific, West Chester, PA).
The root end of the prepared tooth was inserted into the
impression material and allowed to set. This resulted in the
prevention of extrusion of irrigant solution out of the apex
and allowed ease of handling during instrumentation. All
root canals were instrumented by using the rotary engine
(AsepticoInc, Dentsply, Woodinville, WA) with a 1:8 gear
reduction handpiece. The hand piece was set at 300rpm and
torque level two, which is within the range recommended
by the rotary file manufacturer (Tulsa Dental, Dentsply,
Tulsa, OK). Canal enlarged with Profile NiTi rotary files
(Tulsa Dental, Dentsply) to a size 40/0.06 in a crown-down
manner. The apical matrix was further enlarged to a 60/0.04
by using Profile NiTi hand files (Tulsa Dental, Dentsply).
Needle irrigation 1 mm short of the working length was
delivered with a Monojet syringe (Sherwood Medical Co.,
St. Louis, MO) and a 27-gauge needle. Irrigation was done
with a total of 5ml of 5.25% NaOCl between instrument
changes. A#15 K-file was intermittently used to maintain
working length. After instrumentation, the teeth were
irrigated with 5ml of buffered (pH7.4) 15% EDTA
(Sigma-Aldrich Inc., St. Louis, MO).
All teeth were decoronated at the cemento-enamel junction
and then randomly divided into two groups containing
10 teeth each. In the test group (GroupA), canals were
irrigated with 15% EDTA and 5.25% NaOCl followed by
2% CHX. In the GroupB, absolute alcohol was used as
intermediate flush between 5.25% NaOCl and 2% CHX.
GroupA: In this test group5ml of 5.25% NaOCl was
used to irrigate, and the canal was left filled with NaOCl.
Then 5ml 2% CHX was used as a final irrigant followed
by immediately drying of the canal with paper points.
GroupB: In this group absolute alcohol was used as an
intermediate flush between 5.25% NaOCl and 2% CHX.
Teeth in GroupA and GroupB were sectioned longitudinally
and subjected to stereomicroscopic examination. The
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 Ricchawal, et al.: Interaction between Sodium Hypochlorite and Chlorhexidine used as Root Canal Irrigants

sections were then examined under stereomicroscope Bonferroni test was implicated to find out significant
for the precipitate formed. At 100 magnification, differences between pair of levels.
representative areas for each third of the root canals were
chosen for analysis. The maximum thickness of precipitate No significant difference between coronal and middle
from its surface to the dentinal wall in each chosen area was levels (P > 0.05).
measured using ProgRes computer software program A significant difference between coronal and apical levels
at the uniplanar level. The measurements obtained were (P < 0.001).
subjected to statistical analysis. A significant difference between middle and apical levels
(P < 0.01).
RESULTS
DISCUSSION
Figures1a and b and 2a-c represents the variable thickness
of precipitations formed due to the interaction between Biomechanical cleaning and shaping, using files and
NaOCl and CHX in GroupA. Precipitation thickness was antibacterial irrigants reduces the bacterial count in the root
measured by using ProgRess computer software program.
Figure3a and b represents no evidence of precipitation
formation in GroupB. Intermediate flushing with absolute
alcohol was done in between 5.25% NaOCl and 2% CHX
throughout the root surface area and showing no evidence
of precipitation formation also dentinal tubules appears
to be patent with no obliteration. Table1 summarizes
precipitation thickness at coronal, middle, and apical third a b c
of root canal walls in GroupA (test group). There was
Figure2: Thickness of precipitate measured using progress
no significant difference in precipitate thickness between software at 100 magnification. (a-c) 100
coronal and middle levels. There was a significant difference
seen between coronal and apical levels. Similarly, there was
a significant difference between middle and apical levels.
Graph 1 represents mean precipitation thickness recorded
at coronal, middle, and apical aspect of root canal walls
in GroupA.

Table 1: Comparison of the precipitation thickness

at three levels on root surfaces
Level N Mean Std. Dev Min Max F P value
Coronal 10 646.7 133.51 437.04 790.38 25.877 <0.001
Middle 10 511.3 127.13 389.67 715.09
a b
Apical 10 167.2 37.92 115.78 197.79
Figure3: No evidence of precipitate formation in group B which
received an intermediate flush of absolute alcohol in between
5.25% NaOCl and 2% CHX. (a) 40 (b) 100

a b
Figure1: Thick brownish precipitate which was observed
immediately on interaction of NaOCl and CHX, concentrated at the
middle and coronal third in group A (test group). (a) 40 (b) 100 Graph 1: Mean precipitation thickness recorded at three levels

161 International Journal of Scientific Study | May 2015 | Vol 3 | Issue 2

 Ricchawal, et al.: Interaction between Sodium Hypochlorite and Chlorhexidine used as Root Canal Irrigants
canal system. However, no irrigation solutions eliminate the
organic and inorganic debris completely and at the same
time impart a substantive antibacterial property. Hence, the
combination of irrigation solutions such as NaOCl and
CHX have been suggested.14,22 In the present study, the
combination of NaOCl and CHX resulted in the formation
of a thick brownish precipitate (Figure 1) this precipitate
on stereomicroscopic examination revealed a subjective
change in morphology on the root surface. The thickness
of precipitate was analyzed along the canal wall using
ProgRes computer software at uniplanar level (Figure 2).
Examination of the environmental scanning electron
microscopy micrographs showed a subjective change in
the morphology of the root surface. The use of NaOCl
and CHX appears to coat and cover the root surface. The
substance coating the root surface and obliterating the
dentinal tubules was not identified. The analysis revealed,
a maximum thickness of precipitate at the cervical and
middle third (Table 1) which could be related to the surface
area harboring the irrigant permitting enhanced interaction
of the irrigants. The reduced thickness of the precipitate
at the apical third because, the apical third is more difficult
to irrigate. Results at the apex might have been different if
irrigation has done sonic, ultrasonic or negative pressure
irrigation.15
The formation of the precipitate could be explained by
the acid-base reaction when NaOCl and CHX are mixed
(Illustration 1). CHX is a dicationic acid (pH5.5-6) can
donate protons to NaOCl. On the other hand, NaOCl is
alkaline in nature (pH7-9) and can accept protons from
CHX. This results in the formation of a neutral and
insoluble substance referred to as the precipitate.23
To prevent the formation of precipitate, it is advocated to
use an intermediate flush of EDTA or absolute alcohol.24-26
Residual EDTA-continued demineralization (canal
erosion) requiring neutralization by NaOCl. In the present
study, 90% alcohol was used. The clinical relevance of the
formation of this precipitate is staining of teeth, blocks
the dentinal tubules, and reduces the effectiveness of
medicaments used; interfere with the seal of the obturating
material to the canal wall dentin. Toxicity of this precipitate
involves short-term exposure of humans to PCA results
in cyanosis and methemoglobinemia, carcinogenic in rats-
sarcomas of a spleen. PCA shows transforming activity in
cell transformation assays-indicating that PCA is possibly
genotoxic.
To prevent the formation of a precipitate, 90% alcohol was
used which completely removed any residual NaOCl. This
deprived CHX of a reactant to form the precipitate with
NaOCl. On stereomicroscopic examination, clear canals
International Journal of Scientific Study | May 2015 | Vol 3 | Issue 2
were observed with no evidence of precipitate formed
(Figure 3). In addition, to eliminating the formation of
PCA, alcohol being volatile evaporates completely, thus
paving the way for the better penetration of CHX.
CONCLUSION
The interaction between NaOCl and CHX resulted in an
insoluble orange-brown precipitate. This precipitate can
be prevented by using absolute alcohol flushing between
5.25% NaOCl and 2% CHX. Dentinal tubules are blocked
and occluded due to this NaOCl/CHX precipitate. Utmost
care and precaution have been taken while irrigating with
NaOCl and CHX.
Hence, the suggested protocol while using NaOCl and
CHX would be:
NaOCl17% EDTANaOClAb AlcoholCHX.
REFERENCES
1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures
of dental pulps in germ-free and conventional laboratory rats. Oral Surg
Oral Med Oral Pathol 1965;20:340-9.
2. Bystrm A, Sundqvist G. Bacteriologic evaluation of the efficacy of
mechanical root canal instrumentation in endodontic therapy. Scand J Dent
Res 1981;89:321-8.
3. Baumgartner JC, Mader CL. Ascanning electron microscopic evaluation of
four root canal irrigation regimens. JEndod 1987;13:147-57.
4. Orstavik D, Haapasalo M. Disinfection by endodontic irrigants and
dressings of experimentally infected dentinal tubules. Endod Dent
Traumatol 1990;6:142-9.
5. Estrela C, Estrela CR, Barbin EL, Span JC, Marchesan MA, Pcora JD.
Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113-7.
6. Ehrich DG, Brian JD Jr, Walker WA. Sodium hypochlorite accident:
Inadvertent injection into the maxillary sinus. JEndod 1993;19:180-2.
7. Peters OA, Roehlike JO, Baumann MA. Effect of immersion in sodium
hypochlorite on torque and fatigue resistance of nickel-titanium instruments.
JEndod 2007;33:589-93.
8. Vivacqua-Gomes N, Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-
Filho FJ. Influence of irrigants on the coronal microleakage of laterally
condensed gutta-percha root fillings. Int Endod J 2002;35:791-5.
9. Leonardo MR, Tanomaru Filho M, Silva LA, Nelson Filho P, BonifcioKC,
Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root
canal irrigating solution. JEndod 1999;25:167-71.
10. Yesilsoy C, Whitaker E, Cleveland D, Phillips E, Trope M. Antimicrobial
and toxic effects of established and potential root canal irrigants. JEndod
1995;21:513-5.
11. Jeansonne MJ, White RR. Acomparison of 2.0% chlorhexidine gluconate
and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants.
JEndod 1994;20:276-8.
12. Dametto FR, Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, de Souza-Filho
FJ. In vitro assessment of the immediate and prolonged antimicrobial action
of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:76872.
13. Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium
hypochlorite and 0.2% chlorhexidine gluconate separately and combined,
as endodontic irrigants. JEndod 1998;24:472-6.
14. Zehnder M. Root canal irrigants. JEndod 2006;32:389-98.
15. Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction
between sodium hypochlorite and chlorhexidine gluconate and its effect on
162
 Ricchawal, et al.: Interaction between Sodium Hypochlorite and Chlorhexidine used as Root Canal Irrigants

root dentin. JEndod 2008;34:181-5.
16. Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between
sodium hypochlorite and chlorhexidine gluconate. JEndod 2007;33:966-9.
17. Thomas JE, Sem DS. An in vitro spectroscopic analysis to determine
whether para-chloroaniline is produced from mixing sodium hypochlorite
and chlorhexidine. JEndod 2010;36:315-7.
18. Basrani BR, Manek S, Mathers D, Fillery E. Determination of
4-chloroaniline and its derivatives formed in the interaction of sodium
hypochlorite and chlorhexidine by using gas chromatography. JEndod
2010;36:312-4.
19. Van der Bijl P, Gelderblom WC, Thiel PG. On the mutagenicity of
parachloroaniline, a breakdown product of chlorhexidine. JDent Assoc S
Afr 1984;39:535-7.
20. Chhabra RS, Huff JE, Haseman JK, Elwell MR, Peters AC. Carcinogenicity
of p-chloroaniline in rats and mice. Food Chem Toxicol 1991;29:119-24.
21. Matsumoto M, Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al.
Carcinogenicity and chronic toxicity of para-chloronitrobenzene in rats and
mice by two-year feeding. JEnviron Pathol Toxicol Oncol 2006;25:571-84.
22. Prado M, Santos Jnior HM, Rezende CM, Pinto AC, Faria RB, Simo RA,
et al. Interactions between irrigants commonly used in endodontic practice:
A chemical analysis. JEndod 2013;39:505-10.
23. Nowicki JB, Sem DS. An in vitro spectroscopic analysis to determine
the chemical composition of the precipitate formed by mixing sodium
hypochlorite and chlorhexidine. JEndod 2011;37:983-8.
24. Choi MS, Park SH, Cho KM, Kim JW. The comparison of different
canal irrigation methods to prevent reaction precipitate between sodium
hypochlorite and chlorhexidine. JKorean Acad Conserv Dent 2010;35:807.
25. Krishnamurthy S, Sudhakaran S. Evaluation and prevention of the
precipitate formed on interaction between sodium hypochlorite and
chlorhexidine 2010;36:1154-7.
26. Mortenson D, Sadilek M, Flake NM, Paranjpe A, Heling I, Johnson JD,etal.
The effect of using an alternative irrigant between sodium hypochlorite and
chlorhexidine to prevent the formation of para-chloroaniline within the root
canal system 2012;45:878-82.

How to cite this article: Ricchawal A, Shetty V, Pimpale SK, Patil P, Mandwe A, Kiswe S. Evaluation of the Interaction between Sodium
Hypochlorite and Chlorhexidine used as Root Canal Irrigants: An In-Vitro Stereomicroscopic Study. Int J Sci Stud 2015;3(2):159-163.

Source of Support: Nil, Conflict of Interest: None declared.

163 International Journal of Scientific Study | May 2015 | Vol 3 | Issue 2