doi:10.1111/iej.

13213

From an assessment of multiple chelators,

clodronate has potential for use in continuous
chelation

P. P. Wright1 , C. Cooper2, B. Kahler1 & L. J. Walsh1

1
The School of Dentistry, The University of Queensland, Herston, Qld; and 2Central Analytical Research Facility (CARF),
Institute for Future Environments, Queensland University of Technology, Brisbane, Qld, Australia

Abstract sequences 5% NaOCl/17% EDTA/5% NaOCl and 5%

Wright PP, Cooper C, Kahler B, Walsh LJ. From an
assessment of multiple chelators, clodronate shows potential
for use in continuous chelation. International Endodontic
Journal.
Aim To identify chelators which when mixed with
sodium hypochlorite (NaOCl) are stable, exhibiting
minimal loss of free available chlorine (FAC) over
80 min and to further investigate potential mixtures
by assessing FAC over 18 h and the capacity to
remove smear layer.
Methodology 0.25 mol L 1 EDTA (10%),
0.25 mol L 1 EGTA (egtazic acid), 0.25 mol L 1 CDTA
(cyclohexanediaminetetraacetic acid), 0.25 mol L 1
DTPA (pentetic acid), 0.5 mol L 1 ATMP (aminotri
(methylene phosphonic acid)) and 1 mol L 1 HPAA,
(hydroxyphosphonoacetic acid), all at alkaline pH, were
mixed equally with 5% NaOCl. 0.5 mol L 1 alkaline
clodronate and 0.5 mol L 1 Na4etidronate (15%) were
mixed equally with 10% NaOCl. For all mixtures, the
pH and temperature were measured over 80 min and
additionally for the clodronate mixture over 18 h. Iodo-
metric titration was used to measure the FAC of all mix-
tures except for HPAA. The following were compared
with respect to their ability to remove smear layer:
1 mol L 1 clodronate + 10% NaOCl, 0.5 mol L 1 clo-
dronate + 10% NaOCl, 1 mol L 1 etidronate + 10%
NaOCl, 0.5 mol L 1 clodronate + 10% NaOCl and the
NaOCl/17% EDTA. The area fraction occupied by open
dentinal tubules as a percentage of the total area (%
AF) from scanning electron microscopy micrographs
was calculated using Image J. The results were statisti-
cally analysed with alpha set at 0.05.
Results Compared to its control, the mixture
0.5 mol L 1 clodronate + 10% NaOCl lost no FAC
over 18 h (P > 0.05). The FAC of 0.25 mol L 1
CDTA mixed with 5% NaOCl fell to 96%, 92%, 75%
and 4.9% at 20, 40, 60 and 80 min, respectively.
Temperature rises were observed in all cases except in
the etidronate and clodronate mixtures. Only in the
clodronate mixture did the pH remain above pH 12
for the whole experiment. Although smear layer was
removed, the % AF in 1 mol L 1 clodronate + 10%
NaOCl, 0.5 mol L 1 clodronate + 10% NaOCl,
1 mol L 1 etidronate + 10% NaOCl was less than for
0.5 mol L 1 etidronate + 10% NaOCl and 5% NaOCl/
17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA.
Conclusion Alkaline 0.5 mol L 1 clodronate mixed
equally with 10% NaOCl has potential for use in con-
tinuous chelation, based on this assessment of stabil-
ity and smear layer removal. Further research is
needed to establish its efficacy and safety.
Keywords: clodronate, continuous chelation, eti-
dronate, smear layer, sodium hypochlorite, stability.
Received 3 May 2019; accepted 28 August 2019

Correspondence: Patricia P. Wright, The School of Dentistry, The University of Queensland, 288 Herston Rd, Herston, Qld
4006, Australia (Tel.: +61 410 404 325; fax: +61 7 33658183; e-mail: p.wright1@uq.edu.au).

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal 1
 Clodronate in continuous chelation Wright et al.
Introduction
Endodontic irrigation involves the use of sodium
hypochlorite (NaOCl) and a chelator. The application
of NaOCl both dissolves organic material and exerts
antimicrobial actions (Haapasalo et al. 2014). The
subsequent use of a chelator removes the instrument-
created smear layer. The prior application of NaOCl
assists the chelator in removing the inorganic compo-
nent of the smear layer because it removes associated
organic matter (Haapasalo et al. 2014). The removal
of smear layer is recommended as it can contain not
only bacteria and nutrients for bacterial growth, but
it impedes irrigant penetration into the dentinal
tubules (Violich & Chandler 2010). EDTA is the most
commonly employed chelator, although the use of
citric acid is also well documented (Sch€ afer 2007).
Typically, EDTA solutions are either pH neutral or
slightly alkaline (Wright et al. 2017). The sequence
NaOCl/EDTA/NaOCl is common, and although this
combination gives powerful antimicrobial actions, bio-
film removal in the root canal system is incomplete
(Neelakantan et al. 2015). Additionally, the risk of
eroding peritubular dentine is of concern, and some
have advised against the use of a final rinse with
NaOCl for this reason (Wang et al. 2016). Also of
concern is the inactivation of NaOCl, as it reacts
chemically with EDTA. This has led to the recommen-
dation that NaOCl and EDTA should not be used
sequentially, without first emptying and drying the
canal (Clarkson et al. 2011). The other common
sequence, NaOCl/EDTA, lacks a final rinse with an
antimicrobial agent, and its use results in less killing
of bacteria in biofilms when compared to NaOCl/
EDTA/NaOCl (Neelakantan et al. 2015).
In 2005, in the search for new improved irrigation
regimes to address some of the above issues, the concept
of continuous chelation was introduced. This involves
the simultaneous use of a chelator and NaOCl in a sin-
gle solution, thus simplifying the irrigation procedure
(Zehnder et al. 2005). Considerable research has
focused on the use of the weak chelator etidronate at
alkaline pH combined with NaOCl, and from this, a
commercial product, Dual Rinse HEDPâ has been devel-
oped (Zollinger et al. 2018). The form of etidronate used
is tetrasodium etidronate (Na4etidronate), also known
as Na4HEDP. The reported benefits include the
increased removal of dentinal debris (Paque et al.
2012), enhanced antimicrobial effects (Arias-Moliz
et al. 2016, Morago et al. 2016) and good compatibility
with dental materials (De-Deus et al. 2008a).
2 International Endodontic Journal
Even though these findings are promising, etidro-
nate-hypochlorite mixtures lack chemical stability and
lose their free available chlorine (FAC) over time. The
‘use life’ of solutions containing 18% Cublen etidro-
nate and 5% NaOCl (Biel et al. 2017), or 9% Dual
Rinse HEDP and 2.5% NaOCl (Zollinger et al. 2018) is
1 and 2 h, respectively, at room temperature.
Alkaline EDTA in the form of tetrasodium EDTA
(Na4EDTA), as opposed to the pH neutral EDTA used
in standard irrigation, has also been tested in contin-
uous chelation. Even at concentrations as low as 5%
EDTA and 2.5% NaOCl, the chemical stability is con-
siderably less than the etidronate mixtures, with a
use life of only 30 min after mixing (Biel et al. 2017).
The identification of a suitable chelator with
improved stability when combined with NaOCl would
extend the therapeutic window of continuous chela-
tion. It would streamline endodontic irrigation and
may give benefits similar to those described for etidro-
nate mixtures. Particular chemical properties may be
useful in the search for chelators compatible with
NaOCl. These are the ligand stability constant for the
formation of calcium ion (Ca2+) complexes, pH, chem-
ical structure and solubility.
The stability constant is a measure of how well a
chelator binds metal ions, with a higher value indi-
cating stronger binding. EDTA has a higher stability
constant than etidronate (Smith & Martell 1989).
This explains the better removal of smear layer by the
former under the same conditions (Deari et al. 2019).
A high stability constant is then a desirable quality
for a chelator. The alkalinity of chelating agents is
particularly important. Acidic chelators such as citric
acid react rapidly with NaOCl, with a concomitant
rapid loss of FAC (Zehnder et al. 2005). Alkaline
chelator solutions are needed to ensure that when
mixed with NaOCl, the pH remains high. Na4EDTA
and Na4etidronate, when mixed with water, give
solutions of over pH 11 (Deari et al. 2019). Solutions
of other chelators can be made alkaline by the addi-
tion of sodium hydroxide (NaOH). Structural differ-
ences in chelators, such as elemental variances where
chelators contain phosphorous rather than nitrogen,
or have altered stereochemistry, can also be expected
to contribute to differences in stability.
If a chelator with improved compatibility with
NaOCl were identified, its useful application to
endodontics would need to be demonstrated. As the
main function of a chelator is to remove smear layer,
the ability of any new chelator-NaOCl mixtures to
clear the dentinal tubules of debris would help
© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd

establish its clinical relevance. This can be achieved
by employing a variety of techniques including scan-
ning electron microscopy (SEM) (Tartari et al. 2017),
co-site optical microscopy (De-Deus et al. 2008b) and
laser microscopy (Deari et al. 2019), all of which
have assessed smear layer removal in endodontics.
Calcium removal from dentine can be determined by
atomic absorption spectroscopy (Zehnder et al. 2005,
Deari et al. 2019).
The aim of this study was to identify chelators that
have improved chemical compatibly with NaOCl, as
measured by FAC levels, when compared to those
currently identified for use in continuous chelation.
An additional goal of this research was to demon-
strate that such a chelator-NaOCl mixture can
remove smear layer. Thus, the hypothesis tested was
that a chelator with better stability with NaOCl than
either Na4EDTA or Na4etidronate can be identified
and that such a chelator when mixed with NaOCl is
capable of removing smear layer from dentine.
Materials and methods
Chemicals
The following chelators were used: Na4EDTA
(ethylenediaminetetraacetic acid tetrasodium salt)
(34104-500G; Merck, Darmstadt, Germany), EGTA
(egtazic acid) (BePharm, Shanghai, China), CDTA (cy-
clohexanediaminetetraacetic acid) (BePharm, Shang-
hai, China), DTPA (pentetic acid) (BePharm), ATMP
(aminotri(methylene phosphonic acid)) (BePharm),
HPAA (hydroxyphosphonoacetic acid) (iChemical,
Shanghai, China), Na4etidronate (tetrasodium etidro-
nate) (Cublen, Zschwimmer & Schwarz, Burgst€ adt,
Germany), Na2clodronate.4H2O (iChemical) for the
FAC experiment over 80 min and Na2clodronate
(Enke Pharma-tech, Cangzhou, China) for all other
experiments.
Chelator solutions were prepared freshly with dem-
ineralized water and the purity of the chemical
accounted for. All solid chelators were 98–99% pure
except for the Cublen product which contained 85%
Na4etidronate. ATMP and HPAA were aqueous solu-
tions with a concentration of 50%. All pH adjust-
ments were made with NaOH (SA178-500G; Chem-
supply, Gillman, SA, Australia). NaOCl (AJA-485-5L;
Ajax Finechem, Scoresby, VIC, Australia) was titrated
by iodometric titration and diluted to either 5% or
10%. The stock and diluted solutions were stored at
2–4 °C in the dark until used.
© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
Wright et al. Clodronate in continuous chelation
Mixtures tested for FAC and pH assessment over
80 min
(i) 0.25 mol L 1 EDTA (10%) (pH 11.1) mixed
equally with 5% NaOCl (pH 12.6). (ii) 0.25 mol L 1
EGTA adjusted to pH 11.0, then mixed equally with
5% NaOCl (pH 12.5–12.6). (iii) 0.25 mol L 1 CDTA
adjusted to pH 11.4, then mixed equally with 5%
NaOCl (pH 12.5–12.6) (iv) 0.25 mol L 1 DTPA
adjusted to pH 10.8, then mixed equally with 5%
NaOCl (pH 12.7). (v) 0.5 mol L 1 ATMP adjusted to
pH 10.3, then mixed equally with 5% NaOCl (pH
12.6). (vi) 1 mol L 1 HPAA adjusted to pH 11.1,
then mixed with 5% NaOCl (pH 12.5). Only the pH-
temperature experiment was performed. (vii)
0.5 mol L 1 etidronate (15%) (pH 11.6) mixed
equally with 10% NaOCl (pH 12.6–12.7). (viii)
0.5 mol L 1 clodronate adjusted to pH 11.4, then
mixed equally with 10% NaOCl (pH 12.6–12.7).
Each of solutions a–h above was matched with a
control of demineralized water mixed with equal vol-
umes of NaOCl. The final concentration of NaOCl was
the same as that used in the chelator-NaOCl mixtures.
Mixture tested for FAC and pH assessment over
18 h
0.5 mol L 1 clodronate was adjusted to pH 10.7,
then mixed equally with 10% NaOCl (pH 12.7) with
a control of 10% NaOCl mixed with an equal volume
of demineralized water.
Mixtures and sequences tested for smear layer
assessment
(i) 1 mol L 1 clodronate adjusted to pH 10.7, then
mixed equally with 10% NaOCl (pH 12.7). (ii)
0.5 mol L 1 clodronate adjusted to pH 10.7, then mixed
equally with 10% NaOCl (pH 12.7). (iii) 1 mol L 1
(30%) etidronate (pH 11.5) mixed equally with 10%
NaOCl (pH 12.7). (iv) 0.5 mol L 1 (15%) etidronate (pH
11.5) mixed equally with 10% NaOCl (pH 12.7). (v)
0.14 mol L 1 phosphate-buffered saline (PBS). (vi) 5%
NaOCl (pH 12.5) followed by 17% EDTA (pH 7.2). (vii)
5% NaOCl (pH 12.5) followed by 17% EDTA (pH 7.2)
and then followed by 5% NaOCl (pH 12.5).
pH-temperature and FAC recordings
All measurements were performed at a room tempera-
ture of 23 °C. A total of 10 replicates was used for
International Endodontic Journal 3
 Clodronate in continuous chelation Wright et al.
every group. The pH-temperature experiments were
conducted separately to FAC testing. At T = 0, 4 mL
of either the chelator solution or demineralized water
was mixed with 4 mL of NaOCl, and stored in a
closed 15-mL Falcon tube (Corning, Tewksbury, MA,
USA).
pH and temperature recordings were performed at
1, 20, 40, 60 and 80 min and additionally for the
clodronate solution at 1 min, 3 and 18 h after mix-
ing, using a pH electrode (Ionode IJ40CT; Ionode,
Brisbane, QLD, Australia) connected to a pH metre
(Orion Star 211; Thermo Scientific, Beverly, MA,
USA). The pH electrode, which incorporated an ATC
temperature sensor, was rinsed in demineralized
water between recordings and wiped dry. The pH
metre was calibrated daily.
Under the same conditions, FAC was measured by
iodometric titration with a burette. A variation of a
standard iodometry technique was employed (Stan-
dards Australia 2003). This variation entailed the
delivery of a 1 mL sample of the neat solution with a
1000 lL pipette, as opposed to a 10 mL sample of a
1 in 10 dilution. The variation was tested by compar-
ison of the variation with the standard technique.
Smear layer assessment
Disc preparation and treatment
The study was approved by the ethics committee of
both the University of Queensland, Brisbane, Australia
(approval number: 2018001868) and Metro South
Health, Brisbane, Australia (reference number: HREC/
17/QPAH/521). Following extraction, the external sur-
face of 71 human third molars was scraped to remove
soft tissue and cementum from the coronal aspect of
the root, stored in 0.05% thymol in PBS and then ster-
ilized by gamma irradiation at a dose of 25 kGy. A pre-
cision saw (IsoMet 1000; Buehler, Lake Bluff, IL, USA)
was used to cut a 1.8-mm thick cross-sectional slice
from immediately below the CEJ. The coronal aspect of
each slice was polished with a series of coarse, medium
and fine Softlex discs (3M ESPE, St Paul, MN, USA),
rinsed in demineralized water and subsequently stored
in 0.05% thymol in PBS at 2–4 °C until 24 h before
the commencement of the experiment when they were
transferred to demineralized water.
The discs were allocated randomly to the seven
groups, each group containing 10 discs except for the
PBS group which was comprised of 11 discs. All discs
were treated in 24-well cell culture plates (Costar
3738; Corning, Kannebunk, ME, USA), at room
4 International Endodontic Journal
temperature, without agitation. A total of 7.5 mL of
active irrigant over 15 min was used for all groups,
with additional demineralized water rinses between the
NaOCl and EDTA, in groups 6 and 7. All discs were
finally rinsed in a beaker containing 100 mL of dem-
ineralized water. For groups 1–5, the dentine discs
were moved to a new well containing 1.5 mL of fresh
irrigant every 3 min. This was also the pattern in
groups 6 and 7 for the first 9 min, where NaOCl served
as the irrigant. After this, in group 6, the discs were
immersed in 1.5 mL of NaOCl for 4 min and finally
1.5 mL EDTA for 2 min and for group 7, 1.0 mL
NaOCl for 3.5 min, then 1.5 mL EDTA for 2 min and
lastly 0.5 mL NaOCl for 30 s. These changes for
groups 6 and 7 ensured that equal irrigant times and
volumes for all groups were maintained.
Disc imaging and image analysis
The discs were air-dried, mounted on aluminium
stubs using conductive adhesive carbon tabs and car-
bon paint before being coated in 4 nm of platinum
using a Leica ACE EM600. The slices were imaged at
3 kV using the secondary electron detector on a Tes-
can Mira3 field emission SEM. The microscope opera-
tor, who was blinded to the experimental details, was
instructed to obtain five images/disc at low magnifica-
tion (30009, field of view 92.3 lm), from a region
roughly equidistant to the canal and root surface,
where the tubules were round and approximately
equal in number. Additionally, a representative image
from each group was obtained at high magnification
(20 0009, field of view 13.8 lm).
Nine discs in each group were chosen for image
analysis using Image J 1.52i (NIH, Bethesda, MD,
USA). Discs were excluded for variance in tubule
number or shape. The 16-bit images were batch anal-
ysed with a sequence of operations which included
image thresholding at 11 000/65 535 in order to
calculate the area occupied by open tubules as a per-
centage of the total area, that is, the percentage area
fraction (% AF). The output images were checked to
ensure that the Image J routine had not excluded any
open tubules. Of the 315 images analysed, three were
excluded for this reason.
Statistical analysis
Prism software version 7.00 (GraphPad, La Jolla, CA,
USA) was used for the statistical analyses. The
D’Agostino and Pearson test was used to assess nor-
mality. For the FAC data over 80 min, the Mann–
© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd

Whitney test was used to test each chelator-NaOCl
mixture against its control at each time point, after
which the Bonferroni correction was applied to adjust
for multiple comparisons. Unpaired t-tests with the
Bonferroni correction were employed for the 18-h FAC
experiment of the 0.5 mol L 1 clodronate + 10%
NaOCl mixture and its control at each time period. The
smear layer assessment was performed with a one-way
ANOVA. Alpha was set at 0.05 for each experiment.
Means of the pH and temperature data were calculated
to connect replicate values in the line plots.
Results
pH and FAC measurements over 80 min
Replicate measurements for the pH and temperature
experiments are shown in Fig. 1 (pH) and Fig. 2
(temperature). Only the clodronate mixture main-
tained a pH level of 12 or above for the entire experi-
ment. The etidronate mixture held this pH level for
60 min, dropping to pH 11.9 at 80 min, whilst the
CDTA mixture maintained a pH of 12 or above for
40 min, which then fell to pH 11.6 and pH 9.2 at 60
and 80 min, respectively. In contrast, the EDTA mix-
ture had a pH of 11.2 at 20 min, but by 40 min this
had fallen to a pH of just 9.0. The EGTA, DTPA,
ATMP and HPAA mixtures all showed dramatic falls
in pH, with the latter two dropping to pH 8.8 and pH
7.4, respectively, by 1 min. Large pH falls were
accompanied by temperature spikes, the highest of
which were for HPAA and ATMP. The temperature
in these solutions rose from the baseline of 23–42.2
and 37.1 °C, respectively, at 1 min.
The minor variation in the titration method used in
this study did not give results that were significantly
different from those obtained by using the standard
titration technique with larger volumes, and thus, the
altered method was accepted as valid. Individual FAC
data points for the chelator mixtures and correspond-
ing controls at all time points appear in Fig. 3. The
mixture of 0.5 mol L 1 clodronate + 10% NaOCl was
the only solution where there was no significant dif-
ference in FAC compared with the control at any time
period (P > 0.05). FAC values for all other mixtures
were significantly different (P < 0.05) to their controls
at every time point except for T = 1 min.
However, two of the other chelator mixtures showed
reasonable maintenance of FAC over time. In the eti-
dronate mixture, FAC fell to 93% of the control value
at 80 min, whilst for the CDTA mixture the FAC was
© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
Wright et al. Clodronate in continuous chelation
96%, 92% and 75% at 20, 40 and 60 min, but by the
end of the experiment this had diminished to just 4.9%.
The EDTA mixture maintained a reasonable level of
FAC at 20 min (82%), but with large variations in
the FAC values between replicates at this time point.
By 40, 60 and 80 min, the FAC had fallen to 6.1%,
4.5% and 2.9%, respectively.
FAC levels in the remaining mixtures were low. In
the ATMP, EGTA and DTPA mixtures, the FAC had fal-
len to 15%, 7.6% and 1.5%, respectively, at 20 min.
pH, temperature and FAC measurements of
0.5 mol L 1 clodronate + 10% NaOCl over 18 h
There were no significant differences in the FAC levels
between the test and control solutions at each of the
time periods (P > 0.05). At 1 min, 3 and 18 h, for
0.5 mol L 1 clodronate + 10% NaOCl, the FAC,
respectively, was 47.5  0.1, 47.4  0.1 and
47.4  0.1 g L 1 whilst for 5% NaOCl control at the
same time points it was 47.6  0.1, 47.5  0.1 and
47.5  0.1 g L 1. All test and control solutions
maintained a pH of 12.5. Temperature variations in
both the test and control solutions were minor and
less than one degree Celsius.
Smear layer removal
Figure 4 illustrates the % AF results for 1 mol L 1
clodronate + 10% NaOCl, 4.39  1.27; 0.5 mol L 1
clodronate + 10% NaOCl, 4.18  0.72; 1 mol L 1
etidronate + 10% NaOCl, 4.07  1.06; 0.5 mol L 1
etidronate + 10% NaOCl, 5.09  1.21; 5% NaOCl/
17% EDTA, 5.39  1.47 and 5% NaOCl/17% EDTA/
5% NaOCl, 5.27  1.33. There were no significant
differences between the first three groups mentioned
(P > 0.05) nor between the second three groups
(P > 0.05), whilst each of the first three groups dif-
fered significantly to all of the second three groups
(P < 0.05). Every treatment group differed signifi-
cantly to the PBS control (P < 0.05), for which the %
AF was 0.23  0.16.
Scanning electron micrographs are shown for each
group taken at high magnification as well as, for the
1 mol L 1 clodronate + 10% NaOCl group, an image
at low magnification and its corresponding Image J
output file, illustrating the identification of open
tubules (Fig. 5). On a qualitative level, the high mag-
nification images (d)–(g) revealed a similar texture in
the intertubular dentine for mixtures containing
either clodronate or etidronate together with a lack of
International Endodontic Journal 5
 Clodronate in continuous chelation Wright et al.

(a) (b) (c)

12 12 12

pH 10 pH 10 pH 10

8 8 8

6 6 6
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80

Time, min Time, min Time, min

EDTA + NaOCl EGTA + NaOCl CDTA + NaOCl

NaOCl control NaOCl control NaOCl control

(d) (e) (f)
12 12 12

pH 10 pH 10 pH 10

8 8 8

6 6 6
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Time, min Time, min Time, min
DTPA + NaOCl ATMP + NaOCl HPAA + NaOCl

NaOCl control NaOCl control NaOCl control

(g) (h)
12 12

pH 10 pH 10

8 8

6 6
0 20 40 60 80 0 20 40 60 80
Time, min Time, min
Etidronate + NaOCl Clodronate + NaOCl

NaOCl control NaOCl control

(superimposed)

1
Figure 1 Replicate pH values connected by their mean at 1 min, then at 20 min intervals for groups: (a) 0.25 mol L
1 1 1
EDTA + 5% NaOCl, (b) 0.25 mol L EGTA + 5% NaOCl, (c) 0.25 mol L CDTA + 5% NaOCl, (d) 0.25 mol L Pentetic
acid (DTPA) + 5% NaOCl, (e) 0.5 mol L 1 ATMP + 5% NaOCl, (f) 1.0 mol L 1 HPAA + 5% NaOCl, (g) 0.5 mol L 1
Etidronate + 10% NaOCl and (h) 0.5 mol L 1 Clodronate + 10% NaOCl.

erosion of the peritubular dentine. For sequences con-
taining 17% EDTA, (h) and (i), canicular branches
are seen both within the tubules and in the peritubu-
lar dentine. The peritubular dentine is eroded in the
sequence 5% NaOCl/17% EDTA/5% NaOCl, (i), whilst
in the sequence 5% NaOCl/17% EDTA, (h), it has a
moth-eaten appearance.
Discussion
This study clearly identifies NaOCl mixtures of CDTA
or clodronate at high pH as being more compatible
with NaOCl than alkaline EDTA or etidronate,
respectively. At room temperature, solutions of
0.25 mol L 1 CDTA mixed equally with 5% NaOCl
have a use life of up to 60 min and 0.5 mol L 1 clo-
dronate mixed equally with 10% NaOCl loses no FAC
over 18 h. This latter result compares with a drop in
the effective NaOCl concentration from 5% to 3.7% in
1 h for etidronate mixtures of the same concentration
(Zollinger et al. 2018). All other potential chelators
when combined with NaOCl lost their FAC rapidly,
with a concurrent spike in temperature. In the case of
HPAA, the 1-min fall to pH 7.4 coupled with a tem-
perature rise to 42.2 °C precluded its inclusion in the
FAC experiment.

6 International Endodontic Journal © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Wright et al. Clodronate in continuous chelation

(a) (b) (c)

45 45 45
40 40 40
°C °C °C
35 35 35
30 30 30
25 25 25
20 20 20
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Time, min Time, min
Time, min
EDTA + NaOCl EGTA + NaOCl CDTA + NaOCl

NaOCl control NaOCl control NaOCl control

(d) 45 (e) 45 (f) 45
40 40 40
°C °C °C
35 35 35
30 30 30
25 25 25
20 20 20
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Time, min Time, min Time, min
DTPA + NaOCl ATMP + NaOCl HPAA + NaOCl

NaOCl control NaOCl control NaOCl control

(g) (h)
45 45
40 40
°C °C
35 35
30 30
25 25
20 20
0 20 40 60 80 0 20 40 60 80
Time, min Time, min
Etidronate + NaOCl Clodronate + NaOCl

NaOCl control NaOCl control

(superimposed) (superimposed)

Figure 2 Replicate temperature values connected by their mean at 1 min then at 20 min intervals for groups: (a)
0.25 mol L 1 EDTA + 5% NaOCl, (b) 0.25 mol L 1 EGTA + 5% NaOCl, (c) 0.25 mol L 1 CDTA + 5% NaOCl, (d)
0.25 mol L 1 Pentetic acid (DTPA) + 5% NaOCl, (e) 0.5 mol L 1 ATMP + 5% NaOCl, (f) 1.0 mol L 1 HPAA + 5% NaOCl, (g)
0.5 mol L 1 Etidronate + 10% NaOCl and (h) 0.5 mol L 1 Clodronate + 10% NaOCl.

Table 1 lists the nomenclature and the Ca2+ stabil-
ity constant for the chelators evaluated in this study:
EGTA, CDTA, DTPA, ATMP, clodronate and HPAA.
Na4EDTA and Na4etidronate are included for compar-
ison. Except for ATMP, none of these chelators have
previously been tested in continuous chelation. ATMP
has been previously examined (Zehnder et al. 2005),
but in the current study, the pH was alkaline rather
than acidic.
The EDTA analogues, EGTA, CDTA and DTPA all
have high stability constants, with the values for
CDTA and EGTA being higher than that of EDTA.
HPAA and clodronate were included because they are
structurally similar to etidronate, containing phospho-
rous rather than nitrogen. ATMP is a known chelator
containing only one nitrogen atom. All the chelators
salts chosen for examination are water-soluble at the
desired pH and concentration. ATMP and HPAA are
both liquids and can be diluted with water to the
desired concentration.
The reason for the observed chelator compatibility
of CDTA and clodronate with NaOCl relates to their

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal 7
 Clodronate in continuous chelation Wright et al.

(a) 30 (b) 30 (c) 30

20 20 20
FAC FAC FAC
g L–1 g L–1 g L–1
10 10 10

0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
Time, min Time, min Time, min
EDTA + NaOCl EGTA + NaOCl CDTA + NaOCl
NaOCl control NaOCl control
NaOCl control
(d) 30 (e) 30 (f)

20 20 No HPAA in the FAC

FAC FAC experiment
g L–1 g L–1 10
10

0 0
0 20 40 60 80 0 20 40 60 80

Time, min Time, min

DTPA + NaOCl ATMP + NaOCl

NaOCl control NaOCl control

(g) (h)
40 40
FAC FAC
g L–1 g L–1
20 20

0 0
0 20 40 60 80 0 20 40 60 80

Time, min Time, min

Etidronate + NaOCl Clodronate + NaOCl

NaOCl control NaOCl control
(superimposed)

1
Figure 3 Replicate values connected by their medians at 1 min then at 20 min intervals for FAC in g L for groups: (a)
1 1 1
0.25 mol L EDTA + 5% NaOCl, (b) 0.25 mol L EGTA + 5% NaOCl, (c) 0.25 mol L CDTA + 5% NaOCl, (d)
0.25 mol L 1 Pentetic acid (DTPA) + 5% NaOCl, (e) 0.5 mol L 1 ATMP + 5% NaOCl, (f) the HPAA group was not used in
the FAC experiment, (g) 0.5 mol L 1 Etidronate + 10% NaOCl and (h) 0.5 mol L 1 Clodronate + 10% NaOCl.

chemical structure. Comparing EDTA and its ana-
logues, CDTA is the only chelator that contains a cyc-
lic hexane group, and this would impose
stereochemical restrictions, limiting molecular move-
ment. This is likely responsible for its lower reaction
rate with NaOCl as compared to EDTA. Clodronate,
like etidronate, is a non-nitrogenous chelator contain-
ing phosphorous instead. In NaOCl, the chlorine
essentially carries a positive charge and will attack
electrophilic centres such as nitrogen atoms (Fukuzaki
2006). Phosphorous is less electronegative than nitro-
gen (CRC Press 2017) so is less likely to react with
NaOCl. Additionally, as clodronate already contains
two chlorine atoms bonded at the central carbon
atom, chlorine addition here is unlikely. Indeed,
NaOCl is used in the synthesis of clodronate, with the
end-point of the reaction being the formation of clo-
dronate (Veps€ ainen et al. 1991), and this fact helps
al€
explain its lack of reaction with NaOCl.
The actual concentrations employed in the FAC
experiments were based on previous research. For eti-
dronate, much of the literature has used the
Zschwimmer & Schwarz product Cublen K8514 GR,
in concentrations of 18% Cublen (0.5 mol L 1 (15%)
etidronate) mixed equally with 5% NaOCl, giving a
final concentration of 2.5% NaOCl and 9% Cublen

8 International Endodontic Journal © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Wright et al. Clodronate in continuous chelation

Mean and SD of the area fraction 0.5 mol L 1 etidronate + 10% NaOCl, with differ-
of open tubules ences being of the order of 1% AF. Both clodronate
ns mixtures and 1 mol L 1 etidronate + 10% NaOCl
8
ns showed a similar % AF. In continuous chelation,
increased chemical reactions occur between the chela-
6
tor and NaOCl as the concentrations increase (Biel
%AF

4
et al. 2017) and the somewhat surprising result of
the 1 mol L 1 etidronate mixture compared to the
2
0.5 mol L 1 mixture is possibly explained by this fact.
Of concern in using chelators concurrently with a
0 proteolytic agent such as NaOCl is the potential for
S peritubular erosion because solutions remain in con-

l
l

TA

C
l
C

0% Cl

C
C

aO
aO

aO
aO

aO

ED
P

tact with dentine for prolonged periods. Thus, the

N
N

N
N

7%

%
0%

0%

current study did not use short chelator rinses follow-

/5
l/1
0
+1

+1
+1

+1

TA
C
te

te
te

te

aO

ED

ing the application of NaOCl as some other etidronate

na

na
na

na

N
ro

ro

7%
ro

L –1 o

5%
r
od

id
od

id

smear layer studies, possibly with other focuses, have

l/1
et
et
cl

cl

C
–1

L –1
L –1

aO

done (De-Deus et al. 2008b, Deari et al. 2019).

L

N
ol

ol

ol
ol

5%
m

m
m

Instead, it followed a methodology where the chelator

0

5
5
1.

1.

0.
0.

is admixed with NaOCl (Zehnder et al. 2005, Lottanti

Figure 4 The area fraction of open dentinal tubules as a per-
centage of the total area (% AF) for the groups: 1 mol L 1
clodronate + 10% NaOCl, 0.5 mol L 1 clodronate + 10%
NaOCl, 1 mol L 1 etidronate + 10% NaOCl, 0.5 mol L 1
etidronate + 10% NaOCl, PBS, 5% NaOCl/17% EDTA/5%
NaOCl and 5% NaOCl/17% EDTA. The statistical analysis
was performed with a one-way analysis of variance (ANOVA)
and Tukey’s multiple comparison tests, with alpha set at
0.05. ns = no significant differences.
etidronate (Neelakantan et al. 2012, Tartari et al.
2015, Arias-Moliz et al. 2016). However, because 5%
NaOCl is a better antimicrobial agent than 2.5%
NaOCl (Vianna & Gomes 2009), the former was cho-
sen for the current study. The clodronate concentra-
tion copied that used commonly for etidronate. The
same concentrations that were previously shown to
result in a use life for Na4EDTA mixtures of 30 min
were used for Na4EDTA, EGTA, CDTA and DTPA
(pentetic acid) solutions. For ATMP and HPAA,
0.5 mol L 1 (15%) and 1.0 mol L 1 (15%) solutions
were used, respectively, to match those used in a pre-
vious experiment using ATMP (Zehnder et al. 2005).
The superior stability of the clodronate mixture
warranted further examination of its clinical poten-
tial, and its ability to remove smear layer was
assessed. Two concentrations of clodronate were
tested and compared with corresponding etidronate
solutions and standard endodontic sequences. Smear
layer was removed in the clodronate mixtures; how-
ever, the % AF was found to be lower when com-
pared with the standard sequences and the mixture
et al. 2009). Because when chelator pH increases, the
chelation ability in dentine is reduced (Deari et al.
2019), the method used has the additional benefit of
making smear layer assessments at the pH of the
actual mixture, rather than at the lower pH of the
chelator. An important finding of the present study
was that even though dentine discs were exposed to
the chelator-hypochlorite mixtures for 15 min, nei-
ther the clodronate nor etidronate mixtures caused
peritubular erosion. This pattern was seen not only in
high magnification micrographs but also upon
enlargement of the low magnification images. This is
consistent with previous research which reported neg-
ligible decalcifications, but smear layer free dentine,
following irrigation with a mixture of 2% NaOCl and
18% etidronate over 18 min (Lottanti et al. 2009).
The time period of 15 min could be regarded as a
minimum time for endodontic treatment, and future
evaluations should assess longer timer frames.
Traditionally, the analysis of smear layer removal
has employed a scoring system and a team of scorers
using SEM images (H€ ulsmann et al. 1997). More
recently, image analysis software has facilitated and
refined this evaluation (George et al. 2008). Nonethe-
less, the assessment of smear layer removal from
dentine can be problematic due to variations in den-
tine tubules both from within a single sample and
between samples (Deari et al. 2019). Dentine sclerosis
can also lead to erroneous results (Lottanti et al.
2009). The technique known as co-site optical
microscopy has been successfully employed to auto-
mate image analysis and to follow the evolution of

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal 9
 Clodronate in continuous chelation Wright et al.

(a) (b) (c)

2 µm
20 µm

(d) (e) (f)

2 µm 2 µm 2 µm

(g) (h) (i)

2 µm 2 µm 2 µm

Figure 5 Representative scanning electron microscopy micrographs of dentine slices. Low magnification (a) and high magnifi-
cation (c)–(i) micrographs collected with a secondary electron detector. (a) From the group 1 mol L 1 clodronate + 10%
NaOCl and (b) the corresponding Image J analysis. (c) PBS control. (d) From the group 1 mol L 1 clodronate + 10% NaOCl.
(e) From the group 0.5 mol L 1 clodronate + 10% NaOCl. (f) From the group 1 mol L 1 etidronate + 10% NaOCl. (g) From
the group 0.5 mol L 1 etidronate + 10% NaOCl. (h) From the group 5% NaOCl/17% EDTA/5% NaOCl. (i) From the group 5%
NaOCl/17% EDTA. Scale bars: (a)–(b) = 20 lm, (c)–(i) = 2 lm.

the opening of a defined set of tubules over time (De-
Deus et al. 2007). This method was not employed in
the current study due to difficulties in adapting the
technique to a large number of test groups. Addition-
ally, the SEM analysis used allowed for high magnifi-
cation micrographs and additional insights. An
endeavour was made to address some of the above-
mentioned problems first by choosing coronal slices
of dentine, where sclerosis is known to be less com-
mon (Lottanti et al. 2009). Next, as much as possi-
ble, image acquisition was standardized during
microscope operation and although large standard
deviations in the data were seen, significant differ-
ences were recorded.

10 International Endodontic Journal © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Wright et al. Clodronate in continuous chelation

Table 1 Chelator common names and Ca2+ stability constants

Chelator common names and IUPAC nomenclature Ca2+ stability constant

Na4EDTA, ethylenediaminetetraacetic acid tetrasodium salt, sodium edetate tetrasodium, 10.65 (Smith & Martell 1989)
tetrasodium; 2-[2[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate*
EGTA, ethylene glycol tetraacetic acid, egtazic acid, 2-[2-[2-[2-[bis(carboxymethyl)amino] 11.0 (Owen 1976)
ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid*
CDTA, cyclohexanediaminetetraacetic acid, 2-[[-2-[bis(carboxymethyl)amino]cyclohexyl]- 12.5 (Kroll & Gordon 1960)
(carboxymethyl)amino]acetic acid*
DTPA, diethyltriaminepentaacetic acid, pentetic acid, 2-[bis[2-[bis(carboxymethyl)amino] 10.63 (Kroll & Gordon 1960)
ethyl]amino]acetic acid*
ATMP, aminotri(methylene phosphonic acid), [bis(phosphonomethyl)amino] 6.7 (Smith & Martell 1990)
methylphosphonic acid*
HPAA, hydroxyphosphonoacetic acid, 2-[hydroperoxy(hydroxy)phosphoryl]acetic acid* Not found
Na4 Etidronate, 1-hydroxyethane 1,1-diphosphonic acid tetrasodium salt, Na4HEDP, 6.04-6.08 (Smith & Martell 1989)
tetrasodium;1-diphosphonatoethanol*
Na2Clodronate; disodium clodronate, clodronic acid disodium sat, disodium;[dichloro- 5.95 (Smith & Martell 1989)
[hydroxy(oxido)phosphoryl]methyl]-hydroxyphosphinate*

IUPAC, International Union of Pure and Applied Chemistry.

*indicates IUPAC compliant nomenclature.

Clodronate has many desirable attributes. As a
first-generation bisphosphonate, it has been used for
the treatment of osteoporosis, Paget’s disease,
osteoarthritis and malignant hypercalcaemia for over
30 years (Frediani & Bertoldi 2015). Extensive medi-
cal research exists on its safety and effectiveness
(Frith et al. 2001, Atula et al. 2003, Frediani & Ber-
toldi 2015, Rodrigues et al. 2015). Clodronate inhibits
osteoclastic activity, and it is anti-inflammatory
(Atula et al. 2003), with both these properties being
potential advantages for an endodontic irrigant.
Recently, in the field of orthodontics, an in vivo study,
employing injections of clodronate solutions around
rat teeth, investigated the usefulness of the antiresorp-
tive property of clodronate for augmenting anchorage
(Nakas et al. 2017). Furthermore, clodronate itself
possesses some antibacterial activity against Pseu-
domonas aeruginosa (Kruszewska et al. 2002). A final
point in its favour is that the use of clodronate is not
associated with osteonecrosis of the jaw (ONJ), a com-
plication that has only been associated with nitrogen
containing bisphosphonates (Rodrigues et al. 2015).
Nonetheless, areas of concern exist, notably the rec-
ommendation for the avoidance of the use of bisphos-
phonate drugs such as clodronate and etidronate in
pregnancy or in women of childbearing age (Ioannis
et al. 2011). This cautionary approach has been
advised because of a paucity of human safety studies,
but the topic is considered open to debate (Losada
et al. 2010). Indeed, even for NaOCl itself, possible
adverse effects during pregnancy remain undefined
(Agency for Toxic Substances and Disease Registry
2002, UK Teratology Information Service 2017).
Other issues include the high cost of disodium clo-
dronate and the lack of a commercially available
tetrasodium salt. However, tetrasodium clodronate
can be synthesized, and this has been used as the
starting material for the manufacture of clodronate
pro-drugs (Kunnas-Hiltunen et al. 2010). If it were
necessary to use solutions of clodronate to mix with
NaOCl, experimentation would need to establish an
appropriate storage temperature and pH in order to
achieve long-term stability.
The hypothesis tested was accepted. A chelator, clo-
dronate, was identified with improved stability in
NaOCl mixtures compared with Na4EDTA and Na4eti-
dronate and which when mixed with NaOCl was able
to remove smear layer. Although clodronate has
potential for use in continuous chelation, other irrig-
ant requirements need investigation. Such aspects
include the following: the ability to dissolve organic
material and kill microorganisms, penetration into
the intricacies of the root canal system, safety, com-
patibility with dental materials and the fracture resis-
tance of tooth structure.
Conclusion
Alkaline 0.5 mol L 1 clodronate mixed equally with
10% NaOCl, resulting in final concentrations of
0.25 mol L 1 clodronate and 5% NaOCl, has poten-
tial for use in continuous chelation. This mixture does
not lose FAC over 18 h. It is capable of removing
smear layer, although the opening of dentine tubules

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal 11
 Clodronate in continuous chelation Wright et al.
is to a lesser extent when compared with standard
endodontic sequences and the mixture of 0.5 mol L 1
etidronate + 10% NaOCl.
Acknowledgements
Patricia Wright is supported by a UQ Graduate School
scholarship. The authors acknowledge the facilities
and the scientific and technical assistance from the
Australian Microscopy & Microanalysis Research
Facility (AMMRF) at the Central Analytical Research
Facility (CARF), Institute for Future Environments,
Queensland University of Technology, Brisbane, Aus-
tralia.
Conflict of interest
The authors have stated explicitly that there are no
conflicts of interest in connection with this article.
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