Effectiveness of 1 mol L1 citric acid and 15% EDTA
irrigation on smear layer removal
R. Di Lenarda1, M. Cadenaro1 & O. Sbaizero2
1
Institute of Odontology and Stomatology; and 2Institute of Applied Chemistry and Material Engineering, University of Trieste,
Trieste, Italy
Abstract
Di Lenarda R, Cadenaro M, Sbaizero O. Effectiveness
of 1 mol L1 citric acid and 15% EDTA irrigation on smear layer
removal. International Endodontic Journal, 33, 4652, 2000.
Aim The aim of this study was to evaluate in vitro
the cleansing and smear layer removal capability of
alternate canal irrigation with citric acid and
NaOCl.
Methodology Eighty-one teeth were divided into
three groups on the basis of the type of instrumenta-
tion, namely, manual stainless steel, Ni-Ti mechanized
ProFile .04 taper or MACXim. The groups were further
divided on the basis of irrigation protocol: 5% NaOCl
alone, NaOCl alternated with 1 mol L1 citric acid
solution or a combination of 15% EDTA and Cetrimide
solution. After longitudinal sectioning, dentinal walls
were microphotographed with scanning electron
microscopy at 300 and 1000 magnifications.
Qualitative and quantitative cleansing level evaluations
were performed using computerized image analysis
software. Data were statistically evaluated using
KruskalWallis analysis and t-test.
Introduction
One of the principal goals of root canal treatment is
the cleansing of the entire root canal system, through
removal of pulpal debris, smear layer and smear plugs
(Liolios et al. 1997, Taylor et al. 1997, Calas et al.
1998). Endodontic instrumentation, using either
Correspondence: Professor R. Di Lenarda, Erta dei Pruni 22/1, 34136
Trieste, Italy (fax: +39040912579; e-mail: dile_rdl@hotmail.com).
46 International Endodontic Journal, 33, 4652, 2000
Results Qualitative evaluation at 300 and 1000
showed no statistically significant differences in
cleansing ability between citric acid, EDTA and
NaOCl groups. Quantitative evaluation of smear layer
removal, measured as open tubules/total dentinal
surface ratio, showed that 1 mol L1 citric acid
solution was comparable to EDTA (11.97% vs.
10.36%) (NS); in samples treated with ProFile .04
taper instruments citric acid was most effective
(16.17%), whilst in the group treated with manual
instrumentation EDTA and Cetrimide were the most
effective (11.94%). Specimens irrigated with 5%
NaOCl demonstrated significantly more cleansing
than those obtained in the other two groups
(P < 0.001).
Conclusions 1 mol L1 citric acid solution was as
effective in removing smear layer as EDTA, but was
superior in specimens treated with ProFile .04 taper
instruments.
Keywords: citric acid, cleaning, EDTA, root canal
treatment, smear layer, sodium hypochlorite.
Received 13 August 1998; accepted 23 March 1999
manual or mechanized techniques, produces a smear
layer and plugs of organic and inorganic particles of
calcified tissue and organic elements such as pulp
tissue debris, odontoblastic processes, microorganisms
and blood cells in dentinal tubules (Sen et al. 1995).
There is no scientific consensus regarding the
efficacy of smear layer removal in root canal treatment
(Cergneux et al. 1987, Gettleman et al. 1991, Sen et al.
1995); it has been suggested that retaining the smear
layer may inhibit or delay bacterial colonization of the
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root canal by reducing dentine permeability. However,
currently the consensus is toward smear layer removal
in order to reduce the microflora and associated
endotoxins, enhance the sealing capability of
obturating materials and decrease the potential of the
bacteria to survive and reproduce.
At present, chemical conditioning is the only way to
obtain complete cleaning of dentinal walls. Smear layer
removal requires a combination of sodium hypochlorite
(an organic solvent) and substances active on
inorganic compounds, including chelating agents
(EDTA or REDTA) or acids (orthophosphoric,
polyacrylic, tannic, maleic or citric) to remove both
organic and inorganic components.
A review of the literature reveals significant
agreement on the alternate use of two different
substances for irrigation: sodium hypochlorite and
EDTA (Hanes et al. 1991, Sterrett et al. 1991,
Gwinnett 1994, Hennequin & Douillard 1995).
Although the efficacy of EDTA in removing the smear
layer has been proven, different mixtures, concentra-
tions and volumes of irrigation are often used. The
working time necessary to obtain complete removal of
the smear layer and plugs was reported as 23 min or
more for each irrigation, which prolongs the
endodontic procedure (Cantatore et al. 1996, Di
Lenarda et al. 1997).
Citric acid, a weak organic acid, has been applied
previously on root surfaces altered by periodontal
disease and instrumentation in order to increase cemen-
togenesis and to accelerate healing and regeneration of
a normal periodontal attachment after flap surgery
(Hanes et al. 1991, Sterrett et al. 1991, Wen et al.
1992, Hennequin & Douillard 1995). In operative
dentistry citric acid has been proposed as a mild etchant
for dental hard tissue, particularly for dentinal condi-
tioning, and enhanced smear layer and plug removal
(Gunday & Ibak 1990, Sharma 1992, Goracci et al.
1993, Gwinnett 1994, Hennequin et al. 1994, Salama
1994). In endodontic research, substitution of EDTA
with an aqueous citric acid solution as an endodontic
irrigant has recently been proposed (Yamaguchi et al.
1996). At present, in Italy, there is a commercial
formulation available (CanalClean, Ognapharma, Milan,
Table 1 Irrigation groups and instrumentation groups
Irrigation groups
I Sodium hypoclorite (SH) 5%
II SH 5% and Citric acid 1 mol L1 (19%)
III SH 5% and EDTA 15%-Cetrimide
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Di Lenarda et al. Citric acid irrigations
Italy) that consists of citric acid plus Cetrimide (a
surface-active agent) and phosphoric acid. A previous
clinical evaluation of this irrigating solution during
mechanical instrumentation highlighted a limited
capability to remove the smear layer, although it was
associated with excessive etching of the dentinal matrix
when used for 24 min (Cantatore et al. 1996).
The aim of this in vitro study was to evaluate using
scanning electron microscopy (Prati et al. 1992,
Goracci et al. 1993, Liolios et al. 1997) the cleansing
capability of alternate irrigation using citric acid and
NaOCl during root canal instrumentation.
The protocol used to investigate citric acid solutions
was based on the conclusions of Yamaguchi et al.
(1996) who compared the antibacterial activity and
decalcifying capability of citric acid solutions at
different concentrations (0.52 mol L1 ) with a 0.5-
mol L1 EDTA solution (15% aqueous solution).
Yamaguchi et al. (1996) noted citric acid antibacterial
capability at all tested concentrations; they also
observed that calcium extraction from a resin mixture
was actually more effective using citric acid solutions.
One of the main problems associated with using
citric acid is its very low pH, whilst an EDTA solution
is almost neutral. Irrigation of the canal with both
citric acid and sodium hypochlorite ensures neutraliza-
tion of the previous irrigant, with a drastic modification
of the pH inside the canal and the liberation of gaseous
chloride (Di Lenarda et al. 1997).
Materials and methods
Eighty-one single-rooted human teeth with healthy
pulp, with a Type 1 spatial configuration (Weine 1982)
and without significant canal curvature, extracted for
periodontal or orthodontic reasons, were selected. The
teeth were stored in 10% formalin until the root canal
treatment, which was performed within 7 days.
Study design
Samples were divided into nine groups on the basis of
instrumentation (three) and irrigation (three)
(Table 1). Within the instrumentation protocol two
Instrumentation groups
1 ProFile.04 taper Dentsply Maillefer, Ballaigues, Switzerland
2 MACXim and NT Engine file NT Company, Chattanooga, TN, USA
3 Manual Flexile file Mani, Machi Tochigiken, Japan
International Endodontic Journal, 33, 4652, 2000 47
 Citric acid irrigations Di Lenarda et al.
groups were treated with Ni-Ti rotating mechanical
instruments (ProFile. 04 taper, Dentsply Maillefer,
Ballaigues, Switzerland or MACXim and NT Engine
File, NT Company, Chattanooga, TN, USA), whilst the
third was treated with manual stainless steel
instruments (Flexile File, Mani, Machi Tochigiken,
Japan) following a crown-down preparation with
Gates-Glidden burs (size 14). Working length was
determined visually by using a file 0.5 mm shorter
than that observed to just perforate the apex. Each
canal was prepared by the same operator; specimens
were embedded in a `putty' silicone material during
irrigation to simulate the hydraulic counter-pressure
produced physiologically by periapical tissues.
The three groups were subdivided on the basis of the
method of irrigation: (i) 5% sodium hypochlorite was
used alone after every instrumentation step or (ii)
alternated with 19% citric acid solution (1 mol L1 );
(iii) a combination of 15% EDTA and Cetrimide
solution (Largal Ultra, Septodont, Saint Maur, France)
was used in the third group.
Preparation of specimens
Each irrigation was performed using 1.5 mL of
solution, which was left inside the canal for 20 s.
Canal irrigation was performed with ISO 30
atraumatic tip needles and was carried out for 30 s,
moving the syringe in a corono-apical direction inside
the canal, to reduce the irrigation pressure.
At the end of instrumentation the canal was
irrigated for 3 min with 3 mL of the test irrigant. A
final irrigation with sodium hypochlorite was always
performed in order to wash out the acid.
Within the samples subjected to manual instru-
mentation, the first part of the treatment was
performed with the progressive use of GatesGlidden
burs: in this way enlargement and initial preparation
of the coronal and middle third was obtained. The
apical third was then instrumented manually (Flexile
file) up to a size 35, with step-back as far as the
junction with the area treated with mechanical
instruments.
Mechanical instrumentation with Ni-Ti files was
carried out using a slow speed motor with continuous
rotation (Physio Dispenser 8000, Simit Dental,
Mantova, Italy), at 250 r.p.m., according to the manu-
facturer's recommendations.
After canal preparation, samples were irrigated with
5 mL of demineralizated water to avoid the precipita-
tion of salt crystals formed by irrigating solutions and
48 International Endodontic Journal, 33, 4652, 2000
dried with paper cones. The silicone putty was then
removed and the teeth were grooved longitudinally
using narrow conical diamond burs. The teeth were
then fractured along the groove.
Observation in SEM
One hundred and sixty-two samples were available for
SEM examination. These were progressively dehydrated
using increasing concentrations of alcohol and finally
sputter-coated with gold (Edwards S 150 A, Crawley,
UK). Both halves of the root were observed separately
using SEM (Leica Stereoscan 430i, Cambridge, UK).
After a complete visual examination, two examiners
chose multiple areas of the whole dentine wall (not less
than 70% of the total surface), to take microphoto-
graphs representative of the whole specimen. Where
there was disagreement, a consensus was obtained by
increasing the surface area examined. One SEM image
was obtained at both 300 and 1000 magnification
in the cervical, the middle and the apical third of the
root, as well as at the anatomical apex.
Qualitative SEM valuation
As proposed previously (Prati et al. 1992), all the
three examiners evaluated independently the `mean'
degree of cleansing of the selected area at 300 and
1000 magnification; in cases of disagreement, the
value recorded by at least two examiners was
accepted.
At 300 the presence of macro and microscopic
debris, pulpal or other residues was evaluated using a
qualitative scale from 0 (absence of debris) to 6 (severe
debris); a similar scale was used to evaluate the
1000 images for the presence of residual smear layer
and smear plug (Table 2).
Quantitative SEM evaluation: computerized image
analysis
Finally, a representative dentinal area of each
specimen was chosen using the above criteria, and the
1000 microphotograph was digitized and saved to
disk, in order to perform a computerized image analysis
(Image-Pro Plus 1.3, Media Cybernetics, Silver Spring,
MD, USA). This allowed an automatic calculation of
the number of dentinal tubules per unit area, their
mean diameter and the ratio between the area
represented by open tubules and the global dentinal
surface, expressed as a percentage.
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 Di Lenarda et al. Citric acid irrigations

Table 2 Qualitative scale of smear layer and smear plugs in SEM evaluation
Qualitative values Magnifications Criteria of classification

0 300 Complete absence of debris

1000 Complete absence of smear layer (SL) and smear plugs (SP)
1 300 Debris covering less than 10% of dentinal walls
1000 SL and SP covering less than 10% of dentinal walls
2 300 Debris covering 1035% of dentinal walls
1000 SL and SP covering 1035% of dentinal walls
3 300 Debris covering 3565% of dentinal walls
1000 SL and SP covering 3565% of dentinal walls
4 300 Debris covering 6590% of dentinal walls
1000 SL and SP covering 6590% of dentinal walls
5 300 Debris covering more than 90% of dentinal walls
1000 SL and SP covering more than 90% of dentinal walls
6 300 Dentinal surface completely covered by debris
1000 Dentinal surface completely covered by SL and SP

SL, smear layer; SP, smear plugs.

Statistical analysis
A statistical analysis was performed in order to
evaluate differences in treatment procedures. The
KruskalWallis test was used for the analysis of the
qualitative data obtained for cleansing at 300 and
1000; the t-test for independent data and one-way
analysis of variance was used to evaluate statistically
significant differences between two or more groups in
the quantitative determination of open tubules.

Results
Control groups
Figure 1 Dentinal surface of a sample irrigated with 5%
Sample groups irrigated with 5% sodium hypochlorite sodium hypochlorite (SEM 6.840).
constituted the control group and demonstrated an
inadequate level of cleansing of the dentinal walls that
was independent of the instrumentation technique Table 3 Mean scores for presence of organic debris at
used (Fig. 1). 1000 magnification for the three irrigation groups
Middle Apical Apex Total
third third
Qualitative SEM evaluation
EDTA 0.72 1.06 1.61 1.13
Data regarding a qualitative evaluation of the presence of Citric acid 0.78 0.78 1.61 1.06
organic debris made at 300 demonstrated that there NaOCl 0.9 0.98 1.58 1.15
were no statistically significant differences between citric
acid, EDTA and the NaOCl group (Table 3).
This observation was confirmed in the images at Table 4 Mean scores for presence of organic debris at 300
1000 (Fig. 2) and at 5000 (Fig. 3); qualitative magnification for the three irrigation groups
evaluation of the capability of removing microscopic
Middle Apical Apex Total
debris from dentinal wall at 1000 magnification third third
confirmed a clear lack of effect with NaOCl in every
EDTA 1.22 2.28 3.89 2.46
area (P < 0.001). In the apical third the best results
Citric acid 0.45 0.78 3 1.41
were obtained using the citric acid solution (Kruskal NaOCl 4.5 4.9 5.7 5.03
Wallis test P < 0.05) (Table 4).

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 Citric acid irrigations Di Lenarda et al.

Table 5 Irrigation groups: open tubules/total dentinal

surface ratio
Middle Apical Apex Total
third third

EDTA 16.1 9.91 5.05 10.36

Citric acid 16.11 14.49 5.31 11.97
NaOCl 3.79 2 1.2 2.33

Table 6 Instrumentation techniques versus irrigants: open

tubules/total dentinal surface ratio

Figure 2 Dentinal surface of a sample irrigated with EDTA Citric acid NaOCl

1 mol L1 citric acid and 5% sodium hypochlorite (SEM ProFile 9 16.17 2.4
1000). MACXim 10.13 12.93 1.5
Manual 11.94 6.8 3.1

Open tubules/total dentinal surface ratio

More precise, repeatable and standardized data were

Figure 3 Dentinal surface of a sample irrigated with
1 mol L1 citric acid and 5% sodium hypochlorite (SEM
5000).
obtained with the computerized image analysis: the
software, discriminating between the different nuances
of grey within the 1000 images obtained with SEM
and discerning automatically the orifices of open
dentinal tubules, with no smear layer and smear plugs,
as darker areas (Fig. 4).
Total evaluation of tubule cleansing achieved with
the three protocols showed there was a statistically
significant difference between NaOCl and other groups
(P < 0.001). Results obtained with citric acid and
those obtained with EDTA at the apical third of the
treated canals, suggested that citric acid was a signifi-
cantly better cleanser (Student's t-test P < 0.05)
(Table 5).
Within the different instrumentation methods used,
the EDTA and citric acid irrigants gave significantly
better results than NaOCl (P < 0.001). Within the
group treated with manual instrumentation, EDTA
produced significantly cleaner walls (P < 0.05); with
the ProFile instrumentation, citric acid produced signif-
icantly cleaner walls (P < 0.05) (Table 6). In the
group treated with citric acid a significant difference
was observed between samples prepared manually
compared to those treated with ProFile: significantly
poorer results were obtained with the manual instru-
mentation (one-way ANOVA P < 0.01).

Risk associated with citric acid use

Figure 4 Computerized image analysis of a sample irrigated
with 1 mol L1 citric acid and 5% sodium hypochlorite (SEM In order to evaluate the risk of excessive dentinal
1000). etching linked with 1 mol L1 citric acid use, two

50 International Endodontic Journal, 33, 4652, 2000 q 2000 Blackwell Science Ltd

Figure 5 Dentinal surface of a sample over-treated with
1 mol L1 citric acid.
samples were selected from each instrumentation
technique (six samples in total) and were treated, at
the end of canal preparation, with the irrigating
solution left in the canal for 6 min. In these cases,
areas of disintegration of peri-and intratubular dentine
were observed with a substantial enlargement of the
middle diameter of the tubules (Fig. 5).
Discussion
Several authors have demonstrated the cytotoxicity of
EDTA solutions at concentrations similar to those used
in endodontics (Gambarini et al. 1997) and the
relatively lower toxicity of citric acid (Di Lenarda et al.
1997). Yamaguchi et al. (1996) has demonstrated that
0.5 mol L1 and 1 mol L1 citric acid solutions have a
capability of calcium extraction from a resin matrix
and an antibacterial activity comparable to or better
than 10% EDTA, which is often considered to be the
reference standard in endodontics.
The time of dentine exposure to citric acid must be
limited, in order to obtain complete removal of the smear
layer but control excessive etching of dentinal walls. In
our experiment we employed the irrigating solutions for
20 s, in addition to the instrumentation phase, when
irrigant and instrument were simultaneously present in
the root canal (Gunday & Ibak 1990).
Sodium hypochlorite used in sequence with citric
acid and EDTA significantly reduced the risk of etching
on radicular dentine through chemical neutralization
of both agents.
Under the conditions of the present study the
1 mol L1 citric acid solution revealed, on average, an
action comparable to EDTA and was superior in the
group of canals treated with ProFile .04 taper
instruments.
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Di Lenarda et al. Citric acid irrigations
Ten per cent EDTA and 1 mol L1 citric acid, both
alternated with sodium hypochlorite, had an efficacy
that varied with the duration of application. This was
evident in the present protocol because the global time
of action of the irrigating solution inside the canal was
dependent on the number of instrumentation phases;
this varied in the three groups. Within the manually
instrumented group (with a greater number of
instrument steps and a longer exposure to the
irrigating solution) EDTA was superior to citric acid.
However, in the group treated mechanically with
ProFile .04 taper instruments, citric acid was signifi-
cantly more effective.
The surface analysis failed to reveal significant
differences between manual and mechanical instrumen-
tation. It is interesting to note that the smear layer,
when present in large amounts as in the case of canal
irrigation with sodium hypochlorite alone, was uniformly
distributed by the mechanical instrument, but distribu-
tion was more irregular after manual instrumentation.
Conclusions
The simple preparation of citric acid solutions, their
low cost, good chemical stability if correctly used, and
their effectiveness even with short application times
(compatible with normal clinical usage) suggest this
irrigant is suitable for clinical use, indeed, initial
clinical results are encouraging (Cernaz et al. 1998).
Acknowledgements
We are indebted to Dr Alessandro Cernaz, a dear friend
and colleague passed away too soon.
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