doi:10.1111/iej.

12427

Evaluation of orange-brown precipitate formed in

root canals after irrigation with chlorhexidine and
QMix and spectroscopic analysis of precipitates
produced by a mixture of chlorhexidine/NaOCl and
QMix/NaOCl

H. Arslan1, A. D. Uygun1, A. Keskin2, E. Karatas1, F. Secßkin1 & A. Yıldırım2

1
Department of Endodontics, Faculty of Dentistry, Ataturk University, Erzurum; and 2Department of Chemistry, Faculty of
Science, Ataturk University, Erzurum, Turkey

Abstract orange-brown precipitate was evaluated using a

Arslan H, Uygun AD, Keskin A, Karatas E, Secßkin F,
Yıldırım A. Evaluation of orange-brown precipitate formed
in root canals after irrigation with chlorhexidine and QMix and
spectroscopic analysis of precipitates produced by a mixture
of chlorhexidine/NaOCl and QMix/NaOCl. International
Endodontic Journal, 48, 1199–1203, 2015.
Aims (i) To compare chlorhexidine and QMixTM in
terms of orange-brown precipitate generation in root
canals and (ii) to analyse the precipitate produced
by mixing chlorhexidine and QMixTM with NaOCl
to determine whether para-chloroaniline was
produced.
Methodology The root canals of 57 single-rooted
anterior teeth were prepared using ProTaper Univer-
sal rotary instruments up to size F4. Two millilitres of
2.5% NaOCl was used between each instrument
change. The specimens were then divided randomly
into three groups (n = 19) and irrigated as follows:
group 1, 5 mL of distilled water (control group) for
60 s; group 2, 5 mL of 2% chlorhexidine for 60 s;
and group 3, 5 mL of QMix for 60 s. The roots were
split longitudinally, and one of the halves was photo-
graphed using a stereomicroscope. The amount of
four-grade scoring system. The data were analysed
statistically using the Kruskal–Wallis and Mann–
Whitney U-tests (P = 0.05). To evaluate whether the
precipitates included para-chloroaniline, 5 mL of 2%
chlorhexidine and 5 mL QMix were separately mixed
with 5 mL of 2.5% NaOCl in two flasks. After centri-
fugation, precipitates were obtained and analysed
using 1H NMR spectra. The data were subjected to
statistical analysis using the Kruskal–Wallis and
Mann–Whitney tests at a 95% confidence level
(P = 0.05).
Results Chlorhexidine had significantly higher
scores than QMixTM in terms of orange-brown precipi-
tate formed in the root canals (P < 0.001). According
to the 1H NMR spectra, para-chloroaniline was pres-
ent in the mixture of chlorhexidine and NaOCl. How-
ever, the mixture of QMixTM and NaOCl did not result
in para-chloroaniline formation.
Conclusion QMixTM does not form para-chloroani-
line when associated with NaOCl.
Keywords: chlorhexidine, orange-brown precipi-
tate, para-chloroaniline, QMix, sodium hypochlorite.
Received 25 October 2014; accepted 23 December 2014

Correspondence: Hakan Arslan, Department of Endodontics,
Faculty of Dentistry, Ataturk University, Erzurum, 25620,
Turkey (Tel.: +90.442.325 4040- 2352; Fax: +90.442.325
2535; e-mail: dt_hakan82@hotmail.com).
Introduction
Successive canal irrigation with chlorhexidine and
NaOCl results in an orange-brown precipitate (Basrani
et al. 2007). This precipitate includes para-chloroani-
line, which is possibly carcinogenic to humans (Group

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 48, 1199–1203, 2015 1199
 QMix-para-chloroaniline Arslan et al.

2B-International Agency for Research on Cancer UT, USA) was used between each instrument change.
1993) and which has toxic and immunotoxic effects The needle was placed at a distance of 1 mm from
(Kacm ar et al. 1995). Moreover, para-chloroaniline the working length, and it was moved backwards and
occludes dentinal tubules (Bui et al. 2008) and forwards. The specimens were then divided randomly
induces colour change in teeth (Souza et al. 2013). into three groups (n = 19) and irrigated as follows:
Several irrigating solutions, including isopropyl alco- group 1, 2.5 mL of distilled water (control group) for
hol, saline and distilled water, were not able to pre- 60 s; group 2, 2.5 mL of 2% chlorhexidine for 60 s;
vent the precipitation of residue on canal walls and group 3, 2.5 mL of QMixTM for 60 s. The root
following the use of NaOCl and chlorhexidine (Magro canals were dried using paper points, and two longi-
et al. 2015). tudinal grooves were made along the buccal and lin-
Recently, QMixTM (Dentsply Tulsa Dental, Tulsa, gual surfaces of the roots using diamond disc. This
OK, USA), a novel irrigating solution for smear layer
removal and disinfection, was introduced. QMixTM (a)
contains EDTA, chlorhexidine and a detergent.
QMixTM was found to be more effective in killing
Enterococcus faecalis than chlorhexidine (Stojicic et al.
2012). A study by Dai et al. (2011) demonstrated
that QMixTM was as effective as 17% EDTA in remov-
ing smear layer after use of 2.5% NaOCl as the initial
rinse. To date, there have been no studies on whether
orange-brown precipitate is produced from a mixture (b)
of QMixTM and NaOCl. Therefore, the aims of this
study were (i) to compare chlorhexidine and QMixTM
in terms of orange-brown precipitate generation in
root canals and (ii) to analyse the precipitate pro-
duced by mixing chlorhexidine and QMixTM with NaO-
Cl to determine whether para-chloroaniline was
produced. The null hypothesis was that there would
be no differences between chlorhexidine and QMixTM.
(c)

Materials and methods

Fifty-seven single-rooted noncarious anterior teeth
with similar dimensions were selected from a collec-
tion of teeth extracted under conditions unrelated to
this study. The specimens were stored in distilled
water until they were used. Soft tissues and calculus
were removed mechanically from the root surfaces (d)
with a periodontal scaler. The teeth were decoronated
using a diamond disc to obtain a standardized length
of 14 mm. A size 10 K-file (Dentsply Maillefer, Ballai-
gues, Switzerland) was introduced, and the length
was recorded when the tip of the file was visible at
the apical foramen. The working length was deter-
mined by subtracting 1 mm from this length.
Figure 1 Representative images for the scores: (a) score 0,
The apices of the specimens were sealed with wax root canal third was free of orange-brown precipitate; (b)
to prevent overflow of irrigating solutions. The root score 1, orange-brown precipitate was present in less than
canals were instrumented up to size 40 using ProTa- half of the root canal third; (c) score 2, orange-brown precip-
per Universal rotary instruments (Dentsply Maillefer). itate covered more than half of the root canal third; (d) score
Two millilitres of 2.5% NaOCl injected with a size 3, the root canal third was completely covered with orange-
27-gauge notched-tip needle (Ultradent, South Jordan, brown precipitate.

1200 International Endodontic Journal, 48, 1199–1203, 2015 © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Arslan et al. QMix-para-chloroaniline

procedure was performed carefully to avoid perfora- graphed using a stereomicroscope (Novex, Arnhem,
tion of the root canals. The roots were then sectioned the Netherlands) at 159 magnification. The images
using a chisel, and one of the halves was photo- were transferred to a computer, and each third was

(a) (b)

Figure 2 (a) Precipitate produced from the mixture of chlorhexidine and NaOCl, and (b) precipitate produced from the mixture
of QMixTM and NaOCl. Arrows indicate the precipitates.

Figure 3 Distribution of scores according to the groups. Orange-brown precipitate was not seen in any of the specimens in the
distilled water group. According to the statistical analysis, chlorhexidine had significantly higher scores than QMixTM in terms
of orange-brown precipitate formed on the root canals (P < 0.001).

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 48, 1199–1203, 2015 1201
 QMix-para-chloroaniline Arslan et al.
Figure 4 For chlorhexidine, a peak between 7 and 8 ppm
demonstrated the presence of para-chloroaniline. However,
there was no peak between 7 and 8 ppm for QMixTM, reveal-
ing the absence of para-chloroaniline. All of the spectra were
obtained with a 400-MHz Bruker NMR System at 25 °C,
acquiring 32 scans, in d6-DMSO solvent.
examined using software (Adobe Photoshop CS5;
Adobe Systems, San Jose, CA, USA). Two calibrated
observers, blinded to the groups, scored the amount
of orange-brown precipitate, as follows:
0. The root canal was free of orange-brown precipi-
tate;
1. Orange-brown precipitate was present in less than
half of the root canal;
2. Orange-brown precipitate covered more than half
of the root canal; or
3. The root canal was completely covered with
orange-brown precipitate (Fig. 1).
The photographs were evaluated by the observers
1 week later to determine the intra-individual repro-
ducibility, and the Kappa test was used to analyse in-
terexaminer agreement. The data were subjected to
statistical analysis using the Kruskal–Wallis and
Mann–Whitney tests at a 95% confidence level
(P = 0.05). The statistical analyses were performed
using SPSSâ version 20 software (IBM SPSS Inc., Chi-
cago, IL, USA).
To evaluate whether the precipitate included para-
chloroaniline, 5 mL of 2% chlorhexidine and 5 mL of
1202 International Endodontic Journal, 48, 1199–1203, 2015
QMix were separately mixed with 5 mL of 2.5% NaO-
Cl in two flasks. Orange-brown discoloration formed
in both flasks (Fig. 2). The liquids were transferred
into two 1.5 microfuge tubes and centrifuged at
188.41 g for 2.5 min (Thomas & Sem 2010). The
precipitate solids were removed, dissolved and analy-
sed using 1H NMR spectra.
Results
The reliability between the examiners was good
(Kappa value = 0.983), and the difference between
the matched scores never exceeded one unit. Intra-
individual reproducibility was 99.4% (170/171) and
98.8% (169/171) for the observers.
The Kruskal–Wallis test revealed significant differ-
ences amongst the irrigating solutions (P < 0.001)
(Fig. 3). Orange-brown precipitate was not seen in
any of the specimens in the distilled water group. The
percentages of specimens free of orange-brown precip-
itate (Score 0) were 0% for distilled water, 5.2%
for chlorhexidine and 49.1% for QMixTM. The
Mann–Whitney U-test revealed that chlorhexidine
had significantly higher scores than QMixTM in terms
of orange-brown precipitate formed in the root canals
(P < 0.001). There were no significant differences
amongst the root canal thirds in terms of orange-
brown precipitate scores (P > 0.001).
According to the 1H NMR spectra, para-chloroani-
line was determined to be present from the mixture of
chlorhexidine and NaOCl (Fig. 1). However, the mix-
ture of QMixTM and NaOCl did not result in para-chlo-
roaniline formation (Fig. 4).
Discussion
The reactions of different irrigating solutions can be
detrimental. The reaction between chlorhexidine and
NaOCl is important because it results in a precipitate
that includes para-chloroaniline (Basrani et al. 2007).
Although the recently introduced irrigating solution
QMixTM contains chlorhexidine, there are no available
data regarding its safety. Thus, the aims of the pres-
ent study were (i) to compare chlorhexidine and
QMixTM in terms of orange-brown precipitate genera-
tion in root canals and (ii) to analyse the precipitate
produced by mixing chlorhexidine and QMixTM with
NaOCl to determine whether para-chloroaniline was
produced.
Krishnamurthy & Sudhakaran (2010) evaluated
the maximum thickness and chemical composition of
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd

the precipitate formed by the mixture of NaOCl and
chlorhexidine, and they found that the interaction
between NaOCl and chlorhexidine resulted in an
insoluble neutral salt as a precipitate. Similarly, the
presence of orange-brown precipitate was shown in
root canals irrigated with chlorhexidine and QMixTM
after NaOCl irrigation. However, chlorhexidine had
significantly higher scores than QMixTM. This result
might have been due to the concentration of chlorh-
exidine in QMixTM being so low that it could not be
detected. Basrani et al. (2007) determined the pres-
ence of para-chloroaniline in the precipitate obtained
by mixing chlorhexidine and NaOCl. Bui et al. (2008)
also showed that the precipitate resulted from the
interaction between chlorhexidine and NaOCl. Simi-
larly, in the present study, precipitate was obtained
by mixing chlorhexidine and NaOCl, and according to
the 1H NMR spectra, para-chloroaniline was present
in the precipitate.
The design of this study for the evaluation of pre-
cipitate was based on the study described by Thomas
& Sem (2010). Five millilitres of irrigating solutions
was mixed with 5 mL of 2.5% NaOCl, and the liquids
were centrifuged to form precipitate solids. The solids
were then analysed using 1H NMR spectra. According
to Thomas & Sem (2010), the reaction mixture of
NaOCl and chlorhexidine did not produce para-chlo-
roaniline in any measurable quantity. In contrast
with this finding, in the present study, para-chloroan-
iline was detected from the reaction mixture of NaOCl
and chlorhexidine. Clinically, it is important to avoid
the sequential use of chlorhexidine and NaOCl.
QMixTM might be less harmful than chlorhexidine.
However, further studies should be conducted to con-
firm the results of the present study.
Although the QMixTM and NaOCl mixture resulted
in precipitate formation, para-chloroaniline was not
detected in the precipitate. However, the contents of
this precipitate are not known. Thus, until the precip-
itate contents are revealed in further studies, NaOCl
should be completely removed from the root canals
prior to QMixTM irrigation.
Conclusions
Within the limitation of the present study, using
QMixTM did not form para-chloroaniline when associ-
ated with NaOCl. However, QMixTM was associated
with a precipitate of unknown chemical constituents.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Arslan et al. QMix-para-chloroaniline
Acknowledgements
This research was supported in part by an Ataturk
University Research Fund (Project No: 2014/31). The
authors deny any conflict of interests related to this
study.
References
Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A (2007)
Interaction between sodium hypochlorite and chlorhexi-
dine gluconate. Journal of Endodontics 33, 966–9.
Bui TB, Baumgartner JC, Mitchell JC (2008) Evaluation of
the interaction between sodium hypochlorite and chlorh-
exidine gluconate and its effect on root dentin. Journal of
Endodontics 34, 181–5.
Dai L, Khechen K, Khan S et al. (2011) The effect of QMix,
an experimental antibacterial root canal irrigant, on
removal of canal wall smear layer and debris. Journal of
Endodontics 37, 80–4.
Kacm ar P, Pistl J, Mikula I (1995) The effect of p-chloroani-
line on leucocytes of sheep peripheral blood under the
migration-inhibition test conditions. Immunopharmacology
and Immunotoxicology 17, 577–84.
Krishnamurthy S, Sudhakaran S (2010) Evaluation and pre-
vention of the precipitate formed on interaction between
sodium hypochlorite and chlorhexidine. Journal of Endodon-
tics 36, 1154–7.
Magro MG, Kuga MC, Aranda-Garcia AJ et al. (2015) Effec-
tiveness of several solutions to prevent the formation of
precipitate due to the interaction between sodium hypo-
chlorite and chlorhexidine and its effect on bond strength
of an epoxy-based sealer. International Endodontic Journal
48, 478–83.
Souza M, Cecchin D, Barbizam JVB et al. (2013) Evaluation of
the colour change in enamel and dentine promoted by the
interaction between 2% chlorhexidine and auxiliary chemi-
cal solutions. Australian Endodontic Journal 39, 107–11.
Stojicic S, Shen Y, Qian W, Johnson B, Haapasalo M (2012)
Antibacterial and smear layer removal ability of a novel irri-
gant, QMiX. International Endodontic Journal 45, 363–71.
Thomas JE, Sem DS (2010) An in vitro spectroscopic analy-
sis to determine whether para-chloroaniline is produced
from mixing sodium hypochlorite and chlorhexidine. Jour-
nal of Endodontics 36, 315–7.
World Health Organization (1993) World Health Organization.
International agency for research on cancer: monographs on
the evaluation of carcinogenic risks to humans—occupational
exposures of hairdressers and barbers and personal use of hair
colourants; some hair dyes, cosmetic colourants, industrial dye-
stuffs and aromatic amines. Lyon, France: International
Agency for Research on Cancer, pp. 1–436.
International Endodontic Journal, 48, 1199–1203, 2015 1203