Basic Research—Technology
Effects of Final Irrigation with SmearOFF on the
Surface of Dentin Using Surface Analytical
Methods
Myrto Piperidou, DDS,* Rana N.S. Sodhi, BSc, MSc, PhD,† Kamil P. Kolosowski, HBSc, DDS,*
and Bettina R. Basrani, DDS, MSc, PhD*
Abstract
Introduction: SmearOFF (Vista Dental Products,
Racine, WI) is an irrigation solution containing chlor-
hexidine (CHX), EDTA, and a surfactant. This study
examined the chemical interaction of SmearOFF with
sodium hypochlorite (NaOCl) on the dentin surface,
specifically the formation of precipitate and/or para-
chloroanaline (PCA). Methods: Dentin blocks pre-
pared from human maxillary molars were mounted
in resin. Dentinal tubules were exposed in a perpen-
dicular orientation using an ultracryomicrotome. The
blocks were divided into 2 groups: the CHX group, irri-
gation with 6% NaOCl, 17% EDTA, 6% NaOCl, and
2% CHX, and the SmearOFF group, irrigation with
6% NaOCl and SmearOFF. The dentin surface was
analyzed with time-of-flight secondary ion mass spec-
trometry and X-ray photoelectron spectroscopy to
determine the formation of precipitate or/and PCA
on the surface of dentin. Results: Precipitation with
PCA and occlusion of the dentinal tubules were noted
on the dentin surface in the CHX group. No precipitate
and no PCA were detected on the surface of dentin in
the SmearOFF group. Conclusions: Interaction of
SmearOFF with NaOCl on the dentin surface did not
result in the formation of precipitate or PCA. (J Endod
2018;44:1714–1719)
Key Words
Chlorhexidine, EDTA, hypochlorite, parachloroaniline,
precipitate, TOFSIMS, XPS
From the *MSc Endodontic Program, Faculty of Dentistry and †Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario,
Canada.
Address requests for reprints to Dr Bettina R. Basrani, 124 Edward Street #348C, M5R1S9, Toronto, Ontario, Canada. E-mail address: bettina.basrani@dentistry.
utoronto.ca
0099-2399/$ - see front matter
Copyright ª 2018 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2018.07.019
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C hemical irrigation,
combined with me-
chanical instrumentation,
Significance
Precipitate formation can occlude the dentinal tu-
bules and stain the tooth, and it has been shown
is a fundamental step in
to be toxic. Therefore, if a new irrigant is indicated
endodontic therapy, aimed
to be used as a final flush after the use of NaOCl,
at achieving debridement
it is imperative that its interaction should be
of pulp tissue and disinfec-
studied.
tion of the root canal space
(1). Sodium hypochlorite
(NaOCl), the principal irrigation solution used, possesses antibacterial and organic
tissue–dissolving properties, but it does not efficiently remove the smear layer (2, 3).
Also, it is cytotoxic to the periapical tissues (4), and it lacks substantivity. Chlorhexidine
(CHX), a suggested adjunct irrigation solution (3, 5), may exert a substantive
antibacterial effect (6–8) and has low-grade toxicity (9), but it does not dissolve organic
tissue or remove the smear layer (2, 3). Removal of the smear layer is commonly
achieved by adjunctive irrigation with EDTA, which is a chelating agent (2, 10).
It has been established that the combination of irrigation solutions can improve
antibacterial activity (11); thus, the final rinse of root canals with CHX after irrigation
with NaOCl followed by EDTA has been proposed (3, 5). However, the application of
CHX after NaOCl results in the formation of a brown precipitate (12–21), shown
in several studies to contain the presence of toxic parachloroaniline (PCA)
(12–14, 22). Apart from potential toxic effects, the formation of precipitate can lead
to partial occlusion of dentinal tubules and prevention of lateral penetration of
irrigation agents, medicaments, and filling materials (23). Also, PCA can lead to methe-
moglobin formation in humans (24) and is carcinogenic in animals (25, 26).
Two standard surface analytical techniques were used to examine the effects of the
irrigation agents on the dentin surface, namely time-of-flight secondary ion mass spec-
trometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) (27, 28). In TOF-
SIMS, a primary ion beam is used to generate a secondary ion mass spectrum. It has
been used in dentistry to qualitatively analyze the dentinal surface in various studies
(17, 29–31). Complementary examination of the chemical composition of a surface
can be obtained with XPS. Irradiation of the sample by X-rays results in the emission
of photoelectrons characteristic of the elements present and, via chemical shifts,
their chemical environment. Unlike TOF-SIMS, XPS is readily quantifiable. Previous
studies using TOF-SIMS and XPS have examined the adsorption of CHX to hydroxyapatite
and analyzed as well as quantified the precipitate formed by combinations of NaOCl with
CHX (14, 32).
JOE — Volume 44, Number 11, November 2018

Irrigation solutions may be combined with detergents to reduce
surface tension and improve antibacterial efficacy (33). QMiX (Dents-
ply Tulsa Dental, Tulsa, OK) and SmearOFF (Vista Dental Products, Ra-
cine, WI) are 2 proprietary irrigation agents containing EDTA, CHX, and
detergent. They are both proposed for final rinse of the root canals with
the additional step of saline irrigation after NaOCl when QMiX is used
(17). Similar to QMiX (17), mixing SmearOFF with NaOCl (pilot
studies) resulted in a minor color change without detectable precipita-
tion; however, the manufacturer claims that SmearOFF can be applied
directly after NaOCl without the need for in-between saline irrigation.
Thus, the aim of this study was to examine the formation of precipitate
and PCA on the surface of dentin irrigated with NaOCl followed by
SmearOFF using TOF-SIMS and XPS analyses.
Materials and Methods
Specimen Preparation
This study protocol was approved by the University of Toronto,
Health Sciences Research Ethics Board, Toronto, ON, Canada (no.
00033125). Following a previously published protocol (17), 6 ex-
tracted human noncarious maxillary molars were stored in thymol so-
lution before use. Two horizontal slices were obtained from the cervical
part of the root from each tooth with a nominal thickness of 2 mm using
a diamond-coated saw (Leica EM TXP Target Sectioning System; Leica
Microsystems GmbH, Vienna, Austria). The 12 dentin blocks were
embedded in low-viscosity epoxy resin (Epo-Thin; Buehler, Lake Bluff,
IL) for 24 hours. In order to expose the dentinal tubules in a perpen-
dicular orientation to the surface, the surface was further ground with
either a diamond or glass microtome (Leica EM UC6/FC6 Ultra-
cryomicrotome, Leica Microsystems GmbH).
Exposure to Irrigation Agents
The dentin block specimens were randomly divided into 2 groups:
the CHX group, 6 specimens were completely immersed in 5 mL 6%
NaOCl (Lavo Inc, Montreal, Quebec, Canada) for 1 minute immediately
followed by 5 mL 17% EDTA (Vista Dental Products) for 1 minute, fresh
5 mL 6% NaOCl for 1 minute, and a final immersion in 5 mL 2% CHX
(Vista Dental Products) for 1 minute, and the SmearOFF group, 6 spec-
imens were immersed in 5 mL 6% NaOCl for 1 minute and then 5 mL
SmearOFF for 1 minute (as per manufacturer’s recommendation). After
completion of the immersion protocol, the samples were left on a
benchtop to dry overnight.
TOF-SIMS Analysis
Three specimens from each group were analyzed using an IonTOF
TOFSIMS V (IonTOF GmbH, M€unster, Germany) equipped with a bis-
muth (Bi) liquid metal ion gun. The Bi3++ cluster was used as the
primary ion source. The gun was operated in both high-mass and
high-spatial resolution modes. High-mass resolution spectra were ob-
tained on the initial surface (500 mm  500 mm, 100 seconds) fol-
lowed by high-mass resolution spectra on a 150 mm  150 mm area
within a 450 mm  450 mm cleaned area using an Ar cluster source.
Images (256  256 pixels, 20 scans) were also obtained from this re-
gion in high-spatial resolution mode. Both positive and negative spectra
were obtained.
XPS Analysis
XPS spectra from the remaining 3 specimens were obtained on a
Thermo Fisher Scientific (Waltham, MA) ESCALAB 250Xi. A monochro-
matic Al Ka source with a spot size of 400 mm was used. Photoelectrons
were collected at a takeoff angle of 90 relative to the specimen surface
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Basic Research—Technology
using a pass energy of 100 eV for the survey spectra and 20 eV for the
spectral regions. Charge compensation was applied using the combined
Ar+/e flood gun, and the peaks were shifted to place the main C 1s
peak at 285.0 eV. Composition was obtained from the latter spectra
by integrating the peaks after subtracting a Shirley-type background
(28) and applying the supplied sensitivity factors using the instrument’s
software (Avantage 5.926, Thermo Fisher Scientific). To ascertain the
depth of coverage, the surfaces were Ar cluster sputter cleaned followed
by more intense Ar+ depth profiling using the dual-mode Ar mono/clus-
ter source provided with the instrument. The beam was rastered over a
2 mm  2 mm area. The XPS spectra were obtained from a 400-mm
spot at the crater’s center.
Results
All the specimens were observed under a microscope for the pres-
ence of precipitate. Brown precipitate formation was detected in all
specimens of the CHX group, whereas no precipitate and no color
change were observed in any specimens of the SmearOFF group.
TOF-SIMS Analysis
The positive ion TOF-SIMS spectra for the CHX and SmearOFF
groups are shown in Figure 1A and B, respectively, for mass ranges
100–200 u and 500–510 u. The negative ion spectra for the CHX
and SmearOFF groups, for mass range 0–100 u, are shown in
Figure 1C and D, respectively. The position of the characteristic positive
fragments for CHX (127 u, 153 u, 170 u, and 195 u) and that of the
parent ion for CHX (505.24 u) are indicated in Figure 1A and B. It
should be noted that the peak at 127 u also corresponds to the main
peak of PCA. Characteristic peaks in the negative ion spectra, CN
and Cl for the additive (CHX or SmearOFF) and phosphate peaks
PO2 (63 u) and PO3 (79 u) for the substrate, are indicated in
Figure 1C and D. For the CHX group, the characteristic peaks for
CHX are clearly observed, whereas the substrate phosphate peaks are
greatly reduced, indicative of substantial coverage. The reverse occurs
for SmearOFF in that the substrate peaks are prominent and the peaks
assignable to CHX are greatly reduced.
This observation is confirmed in the high imaging resolution
mode for both the positive and negative ions. An irregular precip-
itate was observed occluding the dentinal tubules in specimens from
the CHX group (Fig. 2A and C), whereas no precipitate and patent
dentinal tubules were seen in specimens from the SmearOFF group
(Fig. 2B and D).
XPS Analysis
The XPS survey spectra for the SmearOFF group (Fig. 3B) show an
increase in the relative intensity of Ca+ and PO4 peaks arising from the
dentin substrate compared with the spectra for the CHX group (Fig. 3A),
suggesting that the dentin in the SmearOFF group is not covered by pre-
cipitate or thick (>10 nm) film.
From the molecular formula of CHX (C22H30Cl2N10), the ratio of N/
Cl is 5, and in PCA it is 1.96 (14). The ratio of N/Cl in the CHX group in
all samples was smaller than 5. Although not conclusive, this finding was
consistent with the presence of PCA. For the SmearOFF group, this ratio
was not assessed because nitrogen from the substrate would also be
present.
The Cl 2p spectra (Fig. 3C and D) show the presence of covalent Cl
and chloride (Cl ). For the SmearOFF group, the amount of Cl relative
to the covalent Cl was greater than that for the CHX group. This implied a
much thinner layer, as did the presence of Ca and P peaks in the
Effects of Final Irrigation with SmearOFF 1715
Basic Research—Technology

Figure 1. Selected TOF-SIMS high-mass resolution spectra of the treated dentin. Positive ion spectra for the (A) CHX and (B) SmearOFF groups and the corre-
sponding negative ion spectra for (C) CHX and (D) SmearOFF. Peaks assignable to PCA, CHX, and the substrate are indicated; see text for details.

spectrum, and indicated that the overlayer was less than the escape
depth of the photoelectrons (<10 nm) (32).
The C 1s envelope for SmearOFF (Fig. 3F) implied the presence of
CHX because a shake-up feature, characteristic of aromatic structures,
was observed, and the overall peak shape was similar to that of CHX
(Fig. 3E).
Discussion
Interaction between CHX and NaOCl has gained attention with the
controversial finding of the formation of PCA (12–14, 17–20). Even if
PCA does not form, mixing these 2 irrigation agents produces a visible
insoluble precipitate that has been shown to occlude dentinal tubules
and discolor teeth (23), in addition to the possibility of its being toxic
(25, 26). Therefore, it is imperative that any new CHX-containing irri-
gation agent applied as a final root canal flush after the use of NaOCl
should be assessed for its interaction with NaOCl. This study examined
the interaction of NaOCl and SmearOFF by assessing by-product forma-
tion on the dentin surface. SmearOFF is an irrigation agent containing
CHX gluconate (<1% wt), tetrasodium EDTA dihydrate (18% wt),
and a surface-active detergent as its active components (34). According
to the manufacturer, it is formulated to have an antibacterial effect and
to remove the smear layer of the root canal walls. An earlier report on its
interaction with NaOCl suggested that NaOCl lost the free available chlo-
rine after alternating with either SmearOFF or EDTA (34).
TOF-SIMS analysis provides the composition, distribution, and
molecular information of the analyzed surface. Analysis of the fragmen-
tation pattern can result in the identification of the various components

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 Basic Research—Technology

Figure 2. Selected TOF-SIMS high-spatial resolution images of dentin surfaces. Negative ion images for the (A) CHX and (B) SmearOFF groups and the corre-
sponding positive ion images for (C) CHX and (D) SmearOFF. The presence of precipitate is observed in the CHX group, whereas open and patent dentinal tubules
are observed in the SmearOFF group.

(27). In a previous study (35) from our group, the fingerprint of CHX
was the peaks of 127 u, 153 u, 170 u, 195 u, and its parent 505 u. The
characteristic peak of PCA was 127 u (35). Because the precipitate in
the present study showed peaks at 127 u, 153 u, 170 u, and 195 u, the
peak at 127 u could not solely be used as characteristic of PCA. There-
fore, comparison of the relative heights and ratios of these peaks was
used. In the precipitate, the peaks of 153 u, 170 u, and 195 u were
less intense than the 127 u, in contrast to CHX where the signal of
127 u was less intense than the rest of the peaks. Normally, from the
CHX spectra, the peak height ratio for 170 u/127 u is 3:1, and for
153 u/127 u it is 3:2. In the present study, the 153 u/127 u ratio was
more than 1:1, and the 170 u/127 u ratio was 3:2, indicating a stronger
signal coming from the 127 u component suggestive of PCA. This frag-
mentation pattern was absent from the SmearOFF, and the presence of
PO2 (63 u) and PO3 (79 u) was noted, suggesting that the dentin was
not covered by a thick layer or precipitate. Although Kolosowski et al
(17) analyzed cross sections of the dentin with TOF-SIMS to assess
penetration of precipitate into dentinal tubules, a similar analysis was
not performed for SmearOFF in the present study because no precipitate
was observed.
XPS analysis was used to further investigate the source of the chlo-
rine in the SmearOFF group, which could be either CHX or NaOCl. For
both PCA and CHX, chlorine should appear as the covalent species,
whereas the chloride reflects the presence NaOCl. Even though small
amounts of CHX exist in SmearOFF, the observed combination of
both forms of chlorine suggested that CHX was concentrated on the sur-
face. Specific profiling was performed using a combined mono/cluster
Ar source. The absence of detectable changes for the CHX group implied
the formation of a thick layer over the dentin surface; the quick reduc-
tion in the amount of Cl for the SmearOFF group implied that the surface
was covered with just a few monolayers. These results were consistent
with the presence of Ca and P peaks in the survey spectra for the Smear-
OFF group and not in the CHX group.
The lack of precipitate formation in the SmearOFF group could
be attributed to various reasons. The surfactant forms a tertiary
structure with CHX, which protects the CHX from hypochlorite anion
(OCl ) attack and eliminates the formation of a precipitate. More-
over, the layer that the detergent forms may be the one analyzed by
TOF-SIMS. However, the lack of detection of chlorine could also be
attributed to the possible small amount of CHX in the irrigation
agent.
The results of the present in vitro study suggest that SmearOFF can
be safely applied after NaOCl as a final irrigant without the need of an
extra step of saline rinse. However, the assessment of its antibacterial
properties is a research question that is prudent to be assessed in future
studies.

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Basic Research—Technology

Figure 3. Survey spectra with relative atomic percentages for (A) CHX and (B) SmearOFF. Also shown are the fitted high-resolution peaks for the (C and D) Cl 2p
region and (E and F) the C 1s region for CHX and SmearOFF, respectively; see text for details.

Conclusion 4. Hidalgo E, Bartolome R, Dominguez C. Cytotoxicity mechanisms of sodium hypo-

Within the limitations of this in vitro study, the exposure of dentin
surfaces to NaOCl followed by SmearOFF did not result in the formation
of precipitate or PCA.
Acknowledgments
We acknowledge the Ontario Centre for the Characterisation
of Advanced Materials for the conduction of the experiments as
well as Dr Anil Kishen for his valuable contribution to the study
design and Drs Shimon Friedman and Calvin Torneck for assistance
with the manuscript. This study was supported by a grant from the
Canadian Academy of Endodontics Endowment Fund.
The authors deny any conflicts of interest related to this study.
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Effects of Final Irrigation with SmearOFF 1719