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101]

Original Article

Comparative evaluation of ethylenediaminetetraacetic acid,

maleic acid, and peracetic acid in smear layer removal
from instrumented root canal system: A scanning electron
microscopic analysis study
Roshni Butala, Pradeep Kabbinale, Vasudev Ballal
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India

Abstract Introduction: The aim of this study was to assess the ability of 7% maleic acid, 0.5% peracetic acid (PAA),
and 17% ethylenediaminetetraacetic acid (EDTA) in removing smear layer from root canal system of human
teeth using scanning electron microscopic analysis (SEM).
Materials and Methods: Thirty‑five non-carious human anterior teeth with single roots were selected for the
study. Chemo‑mechanical preparation was done using crown down technique with irrigation of 2.5% NaOCl
after every instrument use. Depending on the final irrigation solution, the samples were divided randomly
into three experimental groups and one control group: (1) The maleic acid group: 07% (n = 10), (2) the
PAA group: 0.5% (n = 10), (3) the EDTA group: 17% (n = 10), and (4) the control group: 0.9% saline (n = 5).
These teeth were then evaluated using SEM analysis for the absence or presence of smear layer, thereby
analyzing their cleaning effectiveness in the coronal, middle, and apical thirds of the root canal system.
The inter examiner’s reliability was verified with the use of Kappa test. The data of the score for intragroup
comparison and intergroup comparison for evaluation of the presence or absence of smear layer were
statistically analyzed by Pearson Chi‑square test. The level of statistical significance was set at P < 0.05.
Results: In the coronal thirds of the root canal, there was no statistically significant difference between
the EDTA and the maleic acid groups when evaluated for their efficacy at smear layer removal. Whereas,
maleic acid performed significantly better than PAA and EDTA in removing smear layer from middle and
apical thirds of the root canal system.
Conclusion: A final irrigation with 7% maleic acid is more efficacious than 17% EDTA and 0.5% PAA when
used as a smear layer removal agent in the apical third of the root canal system.

Key Words: Ethylenediaminetetraacetic acid, maleic acid, peracetic acid, root canal instrumentation, smear
layer

Address for correspondence:

Dr. Pradeep Kabbinale, Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal University, Manipal ‑ 576 104,
Karnataka, India. E‑mail: endopradeep@gmail.com

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How to cite this article: Butala R, Kabbinale P, Ballal V. Comparative

DOI: evaluation of ethylenediaminetetraacetic acid, maleic acid, and peracetic
10.4103/1658-5984.213484 acid in smear layer removal from instrumented root canal system: A scanning
electron microscopic analysis study. Saudi Endod J 2017;7:170-5.

170 © 2017 Saudi Endodontic Journal | Published by Wolters Kluwer ‑ Medknow

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Butala, et al.: Smear layer removal using peracetic acid
INTRODUCTION
Microorganisms in the root canal system are considered to
play a major role in the pathogenesis of apical periodontitis.[1]
The complete obliteration of root canal space with an inert
filling material and creation of a fluid tight seal are the goals
for successful endodontic therapy. It is imperative for the
endodontic filling material to adhere closely to the tooth
structure for the creation of this seal. This, however, is
impaired by the formation of smear layer after mechanical
instrumentation of the root canal. [2,3] The endodontic
smear layer which is amorphous and irregular in structure
contains organic components such as microorganisms and
their metabolic products, necrotic debris, pulp tissue, and
odontoblastic processes as well as inorganic components like
dentin debris.[4]
There have been controversial reports over the maintenance
or removal of this endodontic smear layer. While some
suggest that its persistence will alter dentinal permeability by
blocking the dentinal tubules thus limiting bacterial or toxin
penetration,[5‑7] others believe that it can harbor bacteria and
cause leakage thus necessitating its entire removal from the root
canal walls.[8‑10] This loosely adherent structure is also known
to prevent adaptation of endodontic sealers to canal walls as
well as interfere with penetration of irrigants and intracanal
medicaments into the dentinal tubules.[11‑14]
Several techniques have been employed for the removal of smear
layer ranging from the use of chelating agents and ultrasonics to
lasers during root canal therapy.[15] Current methods to remove
the smear layer might involve the use of a chelating agent during
irrigation or as a final rinse in combination with other irrigants
having tissue dissolving properties.[16]
Ethylenediaminetetraacetic acid (EDTA), a calcium chelating
agent is used routinely in endodontics for the removal of smear
layer.[17] The combination of sodium hypochlorite (2.5%–5%)
and EDTA (10%–17%) has been proven to be successful in
the removal of organic and inorganic debris.[18,19] Studies have
stated that a final irrigation of the root canal with EDTA can
open up the dentinal tubules thereby increasing the number of
lateral canals to be filled eventually by the sealer.[20]
Maleic acid is used as an acid conditioner in adhesive
dentistry.[21] This mild organic acid is found to remove smear
layer from the surface of teeth.[22] Ballal et al. have shown that
smear layer removal from the apical third of the root canal was
performed better using 7% maleic acid than EDTA.[23]
Peracetic acid (PAA) is one of the most potent disinfectants.
It has been used as a single endodontic irrigant in the former
Saudi Endodontic Journal | Volume 7 | Issue 3 | September-December 2017 171
German democratic republic.[24] It has antibacterial, sporicidal,
antifungal, and antiviral effects.[24] It has been used for the
elimination of biofilm formation in various areas.[25,26] The
acetic acid content seems to cause inorganic material dissolution
while also forming water soluble complexes with calcium. It
has been shown that 2.25% PAA solution is comparable with
17% EDTA at removing the smear layer.[19] The caustic effect
of 2.25% PAA on oral mucosa has led to its use in lower
concentrations such as 0.5%.[26] In vitro studies done by
Lottanti et al. and De‑Deus et al. have proven that 0.5% PAA
is effective in removal of smear layer.[19,27]
The aim of the present study is to evaluate and compare the
efficiency of 7% maleic acid, 0.5% PAA, and 17% EDTA to
remove smear layer following instrumentation of root canal
system.
MATERIALS AND METHODS
Ethical clearance was attained for the use of human extracted
teeth (IEC 342/2016). Thirty‑five non-carious anterior teeth
with type I canal anatomy and straight roots were selected for
the study. Radiographs were taken to ascertain the presence
of single, non-calcified canal with mature apex. Teeth showing
the presence of resorption or obturation of root canal system
were excluded from this study. The teeth were cleaned with a
brush to remove the superficial soft tissues following which
they were stored in 0.2% sodium azide (Sigma Chemical Co.,
St. Louis, MO, USA) at 4°C.
A standardized root length of 14 mm was achieved by
decoronation of the samples at the cementoenamel junction.
Following this, the samples were randomly divided into three
experimental groups (n = 10) and one control group (n = 5). A
no. 10 K file (Mani Inc., Tochigi Ken, Japan) was used to assess
the working length. It was inserted into each canal until it was
just visible at the apical foramen (observed under magnifying
loupes) and 1 mm was subtracted from this point. Thereafter,
the apices were sealed using sticky wax. The canals were prepared
using ProTaper nickel titanium rotary instruments (Dentsply/
Tulsa Dental, Tulsa, OK, USA) to size F4 in a crown‑down
technique. Throughout the preparation, canals were irrigated
with 2.5% NaOCl (KMC Pharmacy, Manipal, Karnataka,
India) solution for 1 min using 29‑gauge Navi Tip side vented
needle (Ultradent products Inc., South Jordan, UT, USA) after
every instrument change. The needle was introduced 1 mm short
of the working length for all samples by adjusting the rubber
stopper on the needle to the desired length.
The final irrigation sequence in each group was as follows:
• Group  1: 5 ml of 7% maleic acid was used for 1 min
(KMC Pharmacy, Karnataka, India)
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Butala, et al.: Smear layer removal using peracetic acid
• Group 2: 5 ml of 0.5% PAA was used for 1 min (National
Peroxide Ltd., India)
• Group 3: 5 ml of 17% EDTA was used for 1 min (Presvest
Denpro, India)
• Group 4: 5 ml of 0.9% saline was used for 1 min (Fresenius
Kabi Pvt. Ltd., India).
Thereafter, drying of canals was carried out using sterile paper
points (Dentsply‑Maillefer, Ballaigues, China). Longitudinal
grooves were prepared on the buccal and lingual surfaces
of each root by using a diamond disc (Horico, Germany)
at a slow speed. Care was taken not to penetrate the root
canal. A chisel was used to split the root into two halves.
The specimens were dehydrated using 100% ethyl alcohol
and placed in furnace at 60°C for 24 h. The samples were
manually marked at the coronal (10–12 mm from apex),
middle (6–7 mm from apex), and apical (1–2 mm from
apex) thirds of each specimen before the scanning electron
microscopic analysis (SEM) analysis. The samples were
mounted on metallic stubs followed by gold sputtering using
an ion sputter. These samples were then examined under
scanning electron microscope (JEOL Ltd., USA) for the
presence or absence of smear layer. Several photomicrographs
were taken at ×1500, 20 kV magnification to observe the
surface morphology of the canal walls. These were evaluated
by two independent examiners unaware of the experimental
groups to which the samples belonged.
The images were scored according to the criteria given by
Torabinejad et al.:[28]
• 1 = No smear layer (no smear layer on the surface of the
root canal; all tubules were clean and open)
• 2 = Moderate smear layer (no smear layer on the surface
of the root canal, but tubules contained debris)
• 3 = Heavy smear layer (smear layer covered the root canal
surface and the tubules).
The inter examiner’s reliability was verified using the Kappa
test. The data of the score for intragroup comparison and
intergroup comparison to evaluate the presence or absence of
smear layer were statistically analyzed by Pearson Chi‑square
test. The level of statistical significance was set at P < 0.05.
RESULTS
Kappa results displayed a fair (0.41) agreement between the two
examiner values for scoring smear layer in the coronal, middle,
and apical thirds of the root canal.
Figure 1 represents the comparative percentage of smear layer
removal at the coronal, middle, and apical thirds of the root
canal by the test irrigants.
172 Saudi Endodontic Journal | Volume 7 | Issue 3 | September-December 2017
There was a highly significant difference between the maleic
acid, PAA, and EDTA groups in removing the smear layer in
the apical third of the root canal (P < 0.05). No significant
difference was noted between the EDTA and the maleic acid
groups in smear layer removal from the coronal third. Both of
these irrigants along with sodium hypochlorite removed smear
layer efficiently in the coronal third of the instrumented root
canal walls. PAA removed smear layer equivalent to that removed
by saline in the coronal third but in the apical and middle third;
it performed better than saline. However, in the middle and
apical third, maleic acid performed significantly better when
compared to EDTA or PAA [Figure 2]. In EDTA and PAA
treated specimens there was moderate smear layer present,
and in some areas, peritubular dentinal erosion was observed
[Figures 3 and 4]. In the control (saline) group, there was the
presence of excessive smear layer in the coronal, middle, and
apical thirds of the root canal walls.
DISCUSSION
Endodontic smear layer for med during root canal
instrumentation has been shown to significantly increase
apical leakage, thus necessitating its removal to facilitate close
adaptation of gutta‑percha to the root canal wall.[12] In addition,
this non-homogeneous structure may disintegrate around leaky
filling margins resulting in the formation of voids between root
canal wall and the filling material.[8]
In this study, 2.5% NaOCl was used for the chemo‑mechanical
preparation between each instrument, which could also remove
the organic contents of smear layer. This is in accordance to
a study which stated that diluted NaOCl is as effective as a
greater concentration of NaOCl for the removal of an organic
component of smear layer.[28]
The outcome of this research revealed that 7% maleic acid
was better in the removal of smear layer than 17% EDTA and
0.5% PAA in the middle and apical thirds of the root canal.
In the coronal third, both maleic acid and EDTA were equally
effective without any statistical difference between them. This
is in agreement with other studies.[28,29] In this study, the apical
part of canal preparation was done up to ISO size no. 40 which
is in consensus with other studies stating that larger apical
preparation as compared to a smaller preparation produces a
greater reduction in remaining bacteria and dentin debris.[30]
Even though the apical preparation was performed up to ISO
size no. 40, EDTA and PAA were not able to remove smear
layer effectively when compared with 7% maleic acid.
This might be related to the differences in surface tension
between 1 7% EDTA (0.0783 N/m) and 7% maleic
acid (0.06345 N/m).[23] EDTA is a chelating agent effective
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Butala, et al.: Smear layer removal using peracetic acid

Figure 1: Comparison of the percentage of smear layer removal among the test irrigants at coronal, middle, and apical thirds of the root canal system

irrigants.[19] De‑Deus et  al. indicated that PAA solutions

a b c PAA is relatively cytotoxic. Nevertheless, it is considered to
Figure 2: Photomicrographs of root canal walls instrumented with 7%
maleic acid (×2000, 20 kV). (a) The coronal, (b) middle, and (c) apical
thirds in which dentinal tubules are wide open without any debris
at a neutral pH and thus is independent of a high hydrogen
ion concentration to cause decalcification. A decrease in pH
in dentin is due to the exchange of calcium by hydrogen which
is responsible for a reduced efficacy of EDTA over time.[31]
Maleic acid has a better demineralizing effect within a shorter
period as it is highly acidic.
It has been reported by Paqué et  al. that the apical dentin
of root canal is sclerosed[31] thus, minimizing the action of
EDTA. Recent studies have stated a reduction in the mineral
and noncollagenous proteins (NCPs) component of dentin by
EDTA facilitating removal of calcium ions as well as calcium
bonded to NCPs. This decreased content of NCPs causes a
lower degree of decalcification effect of EDTA in the apical
part of the root canal.[32]
In this study, 0.5% PAA was inefficacious in removal of the
smear layer when compared to maleic acid and EDTA. This
was in contrast to a study by Lottanti et al. who investigated
the effects of 2.25% PAA and EDTA on the smear layer
in their study and found comparable results between the
in various concentrations could dissolve the smear layer as
quickly as 17% EDTA solutions. It may be due to acetic acid
present in the PAA. They also found dentin erosion after
the use of 2.25% PAA solutions in the root canals. They
found that PAA in low concentration (0.5%) did not cause
irritation of oral mucosa and was able to eliminate the smear
layer, hence in this study, we preferred to use 0.5% PAA.[27]
be an alternative to sodium hypochlorite for drinking water
disinfection.[33]
The irrigating needle was introduced 1 mm short of working
length for all the samples by adjusting the stopper on Navi
Tip irrigation needle[34] and is dependent on fluid flow rate.
However, it is difficult to standardize and control the fluid
flow rate during syringe needle irrigation.[35]
Other than conventional SEM, the smear layer can also
be evaluated using digital image analysis, micro‑computed
tomography, atomic force microscopy, environmental SEM,
and co‑site optical microscopy.[27] However, SEM was opted in
this study because it is a commonly available tool for evaluating
the smear layer.
The results obtained in this study may be confined to
only single‑rooted teeth with almost straight root canal
configuration. Curved canals can be more challenging and
make effective cleaning of the root canal system more difficult.
Deeper penetration of the needle takes place in the single‑rooted
anterior tooth because of wider canals; therefore, results may
vary in posterior teeth with narrow canals. Nevertheless, further
long‑term clinical studies are necessary to confirm these results
and evaluate their relevance to treatment outcome.

Saudi Endodontic Journal | Volume 7 | Issue 3 | September-December 2017 173

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Butala, et al.: Smear layer removal using peracetic acid
a b
Figure 3: Photomicrographs of root canal walls instrumented with 17% ethylenediaminetetraacetic acid (×2000, 20 kV). (a) The coronal, (b)
middle, and (c) apical thirds showing a moderate amount of debris
a b c sodium hypochlorite: A scanning electron microscope evaluation. J Endod
Figure 4: Photomicrographs of root canal walls instrumented with
0.5% peracetic acid (×2000, 20 kV). (a) The coronal, (b) middle, and
(c) apical third which show moderate amount of debris
CONCLUSION
The apical third is deemed as an important area for the complete
disinfection of the root canal system. Within the limitations
of this study, it can be stated that a 1‑min application of
7% maleic acid as a final irrigant is highly efficacious for the
removal of smear layer when used in the apical third of the
root canal system.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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