doi:10.1111/iej.

13129

Safety assessment of an etidronate in a sodium

hypochlorite solution: randomized double-blind
trial

N. V. Ballal1 , P. Gandhi1, P. A. Shenoy2, V. Shenoy Belle3, V. Bhat3, D.-K.

Rechenberg4 & M. Zehnder4
1
Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal Academy of Higher
Education, Manipal, Karnataka; 2Department of Microbiology, Kasturba Medical College, Manipal, Manipal Academy of Higher
Education, Manipal, Karnataka; 3Department of Biochemistry, Kasturba Medical College, Manipal, Manipal Academy of Higher
Education, Manipal, Karnataka, India; and 4Department of Preventive Dentistry, Periodontology and Cariology, University of
Z€
urich, Z€
urich, Switzerland

Abstract Postoperative pain levels were assessed 24 h after

treatment. Categorical data were compared between
Ballal NV, Gandhi P, Shenoy PA, Shenoy Belle V,
groups using the Fisher’s exact test, continuous data
Bhat V, Rechenberg D-K, Zehnder M. Safety
using the Wilcoxon signed-rank test, a = 0.05.
assessment of an etidronate in a sodium hypochlorite
Results Irrigation with pure NaOCl rendered 40%
solution: randomized double-blind trial. International
canals free of culturable microorganisms, compared to
Endodontic Journal, 52, 1274–1282, 2019.
50% with the NaOCl/HEDP mixture (P = 0.60). As
Aim To assess whether Dual Rinse HEDP, an etidronate assessed by matrix-assisted laser desorption/ioniza-
that can be combined with NaOCl to create an endodon- tion-time-of-flight analysis (MALDI-TOF), no apparent
tic irrigating solution containing both hypochlorite and a selection of aerobic or anaerobic taxa occurred in
chelator in the form of 1-hydroxyethane 1,1-diphospho- either group. One patient in the NaOCl group experi-
nic acid (HEDP), alters the clinical efficacy of NaOCl or enced moderate pain, whilst two patients in the
adds any untoward clinical effects. NaOCl/HEDP group experienced mild postoperative
Methodology In this randomized controlled double- pain. MMP-9/TP levels in periapical fluid declined sig-
blind single-centre trial, a pure NaOCl solution was nificantly (P < 0.001) after 1 week with no medica-
compared to a HEDP-containing counterpart regarding tion in the root canal, without significant difference
antimicrobial efficacy, postoperative pain, and the host between treatment groups (P > 0.05).
response by means of changes in MMP-9 levels in peri- Conclusions This trial found no influence of HEDP
apical fluid. Sixty patients presenting with asymp- on clinical NaOCl effects.
tomatic apical periodontitis (one tooth each) were
Keywords: bacteria, clinical outcomes, endodon-
randomly divided into two groups (N = 30) based on
tics, evidence-based dentistry/health care, inflamma-
irrigation regime. Pre- and post-treatment microbial
tion, matrix metalloproteinases (MMPs).
aerobic and anaerobic cultures and MMP-9/total pro-
tein (TP) periapical fluid samples were collected. Received 12 December 2018; accepted 12 April 2019

Introduction
Apical periodontitis is characterized by a complex
Correspondence: Matthias Zehnder, Head, Division of interplay between microbial tissue invasion and host
Endodontology, Department of Preventive Dentistry, Peri-
defence (Hahn & Liewehr 2007). The aim of root
odontology and Cariology, University of Z€
urich, Plattten-
strassee 11, CH-8032 Z€ urich, Switzerland (e-mail: canal treatment in teeth with this condition is to
matthias.zehnder@zzm.uzh.ch). eliminate microbial biofilm and by-products from the

1274 International Endodontic Journal, 52, 1274–1282, 2019 © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Ballal et al. Dual Rinse HEDP clinical trial

root canal system to promote apical healing. Cleaning effectiveness of NaOCl in the clinical situation, or
and shaping the root canals using endodontic instru- whether any unintended side effects could occur. Con-
ments and irrigating solutions plays an essential role sequently, before any superiority studies are per-
in this context (Bystr€om & Sundqvist 1985). Sodium formed, a clinical safety assessment is required. It is
hypochlorite (NaOCl) solutions are the most widely conceivable that HEDP, which does react slowly with
used irrigants for root canal disinfection due to their the NaOCl in a combined irrigant, could reduce its
unique effectiveness against biofilms and their ability antimicrobial effectiveness (Zollinger et al. 2017).
to dissolve organic matter (Zehnder 2006). However, Moreover, HEDP, by means of its reduction of debris
NaOCl solutions lack the ability to remove the inor- accumulation in apical parts of the root canal, could
ganic components of the smear layer and accumu- induce over-irrigation and thus increase postoperative
lated hard tissue debris formed during mechanical pain and/or inflammatory changes in periapical tis-
instrumentation (Paque et al. 2011). Therefore, alter- sues (Gondim et al. 2010). Dual Rinse HEDP (Medcem
nating application of the sequestering (chelating) GmbH, Weinfelden, Switzerland) is the first HEDP pro-
agent ethylenediaminetetraacetic acid (EDTA) and duct approved for use in the root canal. It comes in a
NaOCl is commonly advocated (Baumgartner & Mader capsule containing 0.9 g of etidronate powder, which
1987). However, EDTA has been found to eradicate should be mixed immediately with 10 mL of a NaOCl
active chlorine when combined with NaOCl (Grawehr solution of choice directly before treatment, resulting
et al. 2003), and may erode dentine if used overzeal- in a combined irrigant containing both active chlo-
ously (Calt & Serper 2002). EDTA is therefore advo- rine and approximately 9% HEDP (Zollinger et al.
cated as a final irrigant before root filling or placing 2017).
the interim dressing. In the present safety trial on adult patients present-
Etidronic acid (more precisely: 1-hydroxyethane ing with teeth affected by primary asymptomatic api-
1,1-diphosphonic acid or HEDP) is a mild chelator cal periodontitis, root canals were either irrigated
that is compatible with NaOCl in the short term with a pure 2.5% NaOCl solution or a combined
(Zehnder et al. 2005). It can be used in combina- 2.5% NaOCl/ 9% Dual Rinse HEDP solution during
tion with a NaOCl solution, a concept that has the cleaning and shaping procedure. EDTA was not
been termed ‘continuous chelation’ (Neelakantan used in the control arm of this trial to not divert from
et al. 2012). The main clinical advantage of a com- the core question, which was: ‘are there any unto-
bined use of HEDP and NaOCl for root canal irriga- ward effects added to the NaOCl by combining it with
tion is the simplicity and time-saving application an etidronate?’ The evaluated outcomes were as fol-
compared to alternately flushing root canals with lows: (i) the percentage of root canals rendered free of
two irrigants. However, until recently, no clinically culturable bacteria; (ii) postoperative pain levels; and
approved material based on this chemistry was (iii) host response assessed by the levels of MMP-9
available, and all studies were based on laboratory (neutrophil gelatinase) in the periapical fluid.
experiments. These investigations have suggested
that a combined NaOCl/HEDP solution could have
Materials and methods
various beneficial effects compared to a pure NaOCl
solution, for example the prevention of a smear
Study design
layer (Lottanti et al. 2009) and the reduction of
hard tissue debris accumulation during root canal This was a randomized controlled double-blind single-
instrumentation (Paque et al. 2012). In addition, a centre clinical trial with two parallel experimental
possible reduction of torsional load on rotary instru- arms. The trial was approved by the institutional
ments has been described (Boessler et al. 2007). It ethics committee and registered at Clinical Trials Reg-
has also been shown that HEDP in fresh mixtures istry (CTRI/2017/08/009493). All patients were
with NaOCl does not reduce the antibacterial effect informed regarding the benefits, risks and alternative
of the latter (Arias-Moliz et al. 2014). It may be treatment choices before enrolment in the trial. They
that HEDP can improve the disinfection of NaOCl in were also informed that not participating in this study
the presence of a smear layer or hard tissue debris had no consequences regarding their treatment what-
(Morago et al. 2016). soever. Informed consent was obtained from all
With the lack of clinical investigations, however, it patients. The study was conducted in accordance with
is not known whether HEDP would reduce the the guidelines of the World Medical Association

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 1274–1282, 2019 1275
 Dual Rinse HEDP clinical trial Ballal et al.

Declaration of Helsinki, and the Institutional ethical was achieved using a block randomization technique
committee. The CONSORT guidelines (2010) for ran- (block size of six) with 1:1 allocation ratio. One
domized trials were followed. researcher (JV) picked a closed envelope containing
the instruction to either use the pure 2.5% NaOCl
solution or 2.5% NaOCl containing the freshly dis-
Inclusion and exclusion criteria
solved HEDP powder (Dual Rinse, Weinfelden,
The inclusion criteria were patients aged 18 years or Switzerland). This researcher prepared 30 mL of test
above attending the Department of Conservative Den- or control solution, and then handed it to the investi-
tistry and Endodontics, presenting with a tooth hav- gator performing the clinical procedures in an amber
ing a pulpal diagnosis of necrosis and an apical glass bottle. The irrigation solutions under investiga-
diagnosis of asymptomatic apical periodontitis (Ameri- tion cannot be discerned from each other, neither by
can Association of Endodontists 2009). The diagnosis smell nor by colour. Consequently, both the operator
was established according to the patient’s history, and the patient were blinded to the respective irrigant
clinical inspection, palpation, tenderness to percus- used.
sion, pulpal sensitivity testing, probing depth and Teeth in both groups were treated according to a
radiographic examination. Patients were not included standard protocol. Only one root canal per patient
if they were not willing, or able to give informed con- was evaluated. In case of multirooted teeth, the sam-
sent, or if they presented with: (i) pain before treat- ples for both microbial and MMP analyses were col-
ment, (ii) a chronic condition requiring the intake of lected from one individual root canal without an
anti-inflammatory/antibiotic drugs, (iii) a non-restor- isthmus, which was clearly associated with the peri-
able tooth, or (iv) root canals in which patency for apical radiolucency. Forty-three of the canals included
periapical fluid sampling could not be achieved. in this study were from single-rooted teeth, 12 were
buccal canals in maxillary premolars, 2 palatal canals
in maxillary molars, and 3 distal canals in mandibu-
Sample size estimation
lar molars. Canals were selected based on anatomical
Binary/dichotomized outcomes such as residual bacte- similarity and controllability of the sampling proce-
rial growth and pain after the cleaning and shaping dure. The teeth were anaesthetized (2% lidocaine
procedure typically require more than 100 cases per hydrochloride with epinephrine 1:80,000; Septodont,
group to test for equality or non-inferiority (Laster Saint-Maur-des-Fosses, France) and isolated with rub-
et al. 2006). The more sensitive outcome yielding ber dam (Hygenic; Coltene Whaledent, Altst€ atten,
continuous data assessed here was the change of peri- Switzerland). The operating field was disinfected by
apical MMP-9 levels according to treatment. However, swabbing with 30% hydrogen peroxide, followed by
no clinical trials were identified that would allow 5% tincture of iodine (M€ oller 1966). Subsequently,
deducing a power analysis (Wahlgren et al. 2002, the access cavity was prepared using a sterile dia-
Martinho et al. 2016). Hence, the number of patients mond-coated bur (Horico, Berlin, Germany), and
was set to be 30 per group, which is a sufficient num- working length was estimated using the preoperative
ber for a first tentative statistical analysis on continu- radiograph. Patency of the root canal was achieved
ous data (Krithikadatta 2014). However, since no using size 10 K-file (Dentsply Sirona Endodontics, Bal-
proper sample size estimation was performed, the P laigues, Switzerland). Working length was determined
values reported here should be interpreted with care. using an electronic apex locator (Root ZX; Morita,
Osaka, Japan). The canal was enlarged to size 20
using hand instrumentation. The pre-treatment
Clinical procedures
microbial sample (S1) was then collected by placing a
A CONSORT flow diagram outlining the treatment size 20 sterile paper point (Dentsply Sirona Endodon-
methodology is represented in Fig. 1. Sixty patients tics) to working length for 1 min. Subsequently, the
(males – 35; females – 25) from the age group 18– paper point was immediately placed inside a sterile
65 years meeting the inclusion criteria participated. centrifugation tube containing 20 mL of thioglycol-
Patients were randomly divided into two groups based late broth (Merck, Darmstadt, Germany). This proce-
on irrigation regimen. Random sequence generation dure was repeated with a second paper point, which
was performed using a computer-generated number was added to the same broth. Then, the root canal
(www.randomizer.org), and allocation concealment was apically enlarged to size 30 (F3) using ProTaper

1276 International Endodontic Journal, 52, 1274–1282, 2019 © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Ballal et al. Dual Rinse HEDP clinical trial

ENROLMENT Assessed for eligibility

Excluded:
(n = 65)
Did not turn up
for scheduled appointment
(n = 5)
Randomized
(n = 60)

Allocated to intervention ALLOCATION Allocated to intervention

(2.5% NaOCl) (n = 30) (2.5% NaOCl /9% Dual Rinse)
(n = 30)

No of patients analysed for MICROBIAL No of patients analysed for

microbial analysis (n = 30) ANALYSIS microbial analysis (n = 30)

No of patients analysed for No of patients analysed for

MMP ANALYSIS
MMP 9 (n = 30) MMP 9 (n = 30)

No of patients analysed for No of patients analysed for

postoperative pain (n = 30) postoperative pain (n = 30)
PAIN ANALYSIS
Did not respond after 24 hrs Did not respond after 24 hrs
(n = 0) (n = 0)

No of patients analysed for No of patients analysed for

MMP 9 after 1 week (n = 30) FOLLOW-UP MMP 9 after 1 week (n = 30)
Loss to follow-up (n = 0) Loss to follow-up (n = 0)

Total no of patients analysed FINAL Total no of patients analysed

(n = 30) ANALYSIS (n = 30)

Figure 1 The Consolidated Standards of Reporting Trials (CONSORT) flow diagram of patients included in this study.

Universal instruments (Dentsply Sirona Endodontics) sample was collected by introducing a fine sterile size
and irrigated for the first time. Irrigation was per- 20 paper point 2 mm beyond the canal terminus for
formed using a 29-gauge side-vented needle (Vista 1 min (Shimauchi et al. 1996). This procedure was
Dental Products, Racine, WI, USA), which was kept performed twice. The paper points were placed in a
1 mm short of the working length. Between each sterile micro-centrifugation tube (Merck) containing
instrument change, the root canal was irrigated with 2 mL of sterile physiological saline solution, and
the allocated irrigant (5 mL for 1 min). Hence, a total immediately transferred to a 80 °C freezer until fur-
of 25 mL of the irrigating solution were used. Once ther processing. In case of multirooted teeth, the
the shaping procedure was completed, the root canal canals which were not sampled were enlarged to size
was flushed with 5 mL of sodium thiosulfate (Merck) #30 (F3) using ProTaper Universal instruments and
for 1 min, followed by 5 mL of distilled water for were irrigated with the same group of irrigant which
1 min, to avoid potential carry-over effects by NaOCl was used for the canal from which the samples were
remnants. taken. The access cavity of the tooth was then tempo-
The root canal was dried using paper points (Dents- rized (Cavit G, 3M ESPE, Seefeld, Germany). The root
ply Sirona Endodontics), and the post-treatment canals were left empty for the interim in order to
microbial sample (S2) was collected as described avoid any possible effects by intra-canal medicaments.
before (see above). In addition, a periapical fluid All patients were recalled after 1 week. On the recall

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 1274–1282, 2019 1277
 Dual Rinse HEDP clinical trial Ballal et al.

visit, the tooth was isolated with rubber dam and the protein extraction. Absorbance was read at 450 nm
operating field was disinfected as described above. The using a microplate reader (LISA Plus, Mumbai, India).
previously sampled root canal was re-entered, flushed The standard curve was generated using a four-pa-
with saline, and a second periapical fluid sample was rameter logistic curve fit for each set of samples
collected similar to the first one (see above). assayed.
After the final sampling procedure, root canals MMP levels were normalized to total protein (TP) in
were irrigated with 2.5% NaOCl followed by 17% each sample. TP was determined using a modification
EDTA (Vista Dental Products, Racine, WI, USA). Root of Lowry’s method (Peterson 1977) against a stan-
canals were filled with AH Plus sealer and ProTaper dard series of bovine serum albumin. Readings were
combined with size 20 gutta-percha points (Dentsply taken after 30 min using the microplate reader at
Sirona Endodontics) using a lateral compaction tech- 540 nm.
nique. Subsequently, a post-endodontic restoration
was placed.
Data presentation and analysis
Categorical data related to the presence/absence of
Microbiological analysis
infection after the cleaning and shaping procedure
To test for the presence of culturable bacteria, cul- and postoperative pain were compared between
tures were inoculated in thioglycollate broth. For the groups using Fisher’s exact test. Data related to abso-
identification of main anaerobic taxa, the root canal lute MMP-9/TP levels after cleaning and shaping and
samples were plated on 5% sheep blood agar, neomy- at the recall visit were skewed (Shapiro–Wilk test)
cin blood agar and phenyl ethyl alcohol agar with and are thus presented as medians and inter-quartile
metronidazole (5 lg, Oxoid, Basingstoke, UK) disc. ranges (IQRs). These data were compared between
Cultures were incubated at 37 °C for 72 h in an and within (first versus second visit) groups using
anaerobic chamber (Whitley A35 Anaerobic worksta- Wilcoxon signed-rank test. The alpha-type error for
tion, Don Whitley Scientific, Shipley, UK). In addition, all these comparisons was set to 5%.
specimens were also cultured aerobically on 5% sheep
blood agar and MacConkey’s agar (BD, Becton Dickin-
Results
son, Heidelberg, Germany). Bacterial morphotypes
were identified employing matrix-assisted laser desorp-
Microbiology
tion/ionization-time-of-flight (MALDI-TOF, BioMer-

ieux, Marcy-L’Etoile, France) analysis. All initial samples collected from the root canals
(N = 60; S1) had positive microbial growth (Table 1).
Of these canals, 46 had a mixed aerobic/anaerobic
Assessment of postoperative pain
infection, whilst 14 showed had only aerobic growth.
Postoperative pain was assessed 24 h after the first Irrigation with pure NaOCl rendered 12 of 30 (40%)
visit using a numerical rating scale (NRS-11, WGMC canals free of culturable microorganisms. The corre-
Center, 2003), with ‘0’ representing no pain and ‘10’ sponding treatment with the NaOCl/ Dual Rinse
being the worst pain imaginable as anchors. The HEDP mixture resulted in 15 of 30 canals being free
patients were interviewed over the telephone, and of microorganisms (P = 0.60). There was no apparent
their pain levels were tabulated. selection of bacterial species by either treatment, with
similar taxa predominating between groups and sam-
pling times (Tables 1 and 2). Streptococcus mitis/oralis
Assessment of inflammatory host response
and Enterococcus faecalis predominated amongst the
Apparent neutrophil activity in periapical tissues was facultative, and Veillonella spp. amongst the strictly
quantified using a commercially available ELISA kit anaerobic taxa.
targeting human MMP-9 in its pro- (92 kDa) and
active (82 kDa) form (Quantikine ELISA, R&D Sys-
Postoperative pain
tems, Minneapolis, MN, USA). The kit was used
according to the manufacturer’s recommendations. All 60 patients responded to the postoperative pain
Paper points were placed in the substrate solution inquiry. One of the 30 patients in the pure NaOCl
and agitated for one hour on a platform shaker for group experienced moderate postoperative pain (‘6’

1278 International Endodontic Journal, 52, 1274–1282, 2019 © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Ballal et al. Dual Rinse HEDP clinical trial

Table 1 Microbiological results at S1 (before Instrumenta- compared to 85 (8, 166) observed with NaOCl/ Dual
tion/Irrigation) Rinse HEDP. This tendency of the combined irrigant
Growth/no Main speciesa (frequency of to reduce MMP-9 levels in periapical tissues more
Group growth identification) than the pure NaOCl solution did not reach statistical
significance either (P = 0.25). MMP-9/TP values
2.5% NaOCl Aerobic: Streptococcus mitis/oralis (21)
purum 30/0 Enterococcus faecalis (6) dropped in all but 12 of the 60 individual cases. Cases
Streptococcus anginosus (2) with high initial MMP-9/TP values tended also to
Neisseria mucosa (1) have higher MMP-9/TP values at the recall visit
Staphylococcus epidermidis (1) (Spearman’s Rho = 0.51, P < 0.001).
Staphylococcus hominis (2)
Staphylococcus lugdunensis (1)
Streptococcus constellatus (1) Discussion
Streptococcus gordonii (1)
Streptococcus parasanguinis (1) The current trial revealed no difference between irri-
Anaerobic: Veillonella atypica (9) gation using a pure 2.5% NaOCl solution and a coun-
24/6 Veillonella parvula (9)
terpart containing 9% HEDP regarding microbial
Fusobacterium nucleatum (5)
Bifidobacterium spp (1) reduction during the cleaning and shaping procedure,
2.5% NaOCl Aerobic: Streptococcus mitis/oralis (13) postoperative pain or periapical levels of an enzyme
/9% DR HEDP 30/0 Enterococcus faecalis (10) related to neutrophil activity (MMP-9).
Streptococcus parasanguinis (4) This was a first clinical study on a chemical that
Streptococcus gordonii (2)
has not been available for endodontic usage until
Staphylococcus hominis (2)
Bacillus cereus (1) recently. It was not the goal to mimic a clinical sce-
Klebsiella pneumoniae (1) nario (i.e. effectiveness) but rather to check whether
Staphylococcus epidermidis (1) there could be any unexpected or un-wanted effects
Staphylococcus hominis (1) on the NaOCl solution by admixing Dual Rinse HEDP.
Streptococcus anginosus (1)
This is why EDTA was not used in the pure NaOCl
Streptococcus constellatus (1)
Anaerobic: Veillonella atypica (12) group, as this would have interfered with the NaOCl
23/7 Veillonella parvula (9) and also with blinding, and did not place an interim
Fusobacterium nucleatum (3) dressing, which would have obscured the assessment
Bifidobacterium spp (1) of the periapical marker of inflammation. This study
Parvimonas micra (1)
is further limited by the fact that only short-term
Prevotella buccae (1)
treatment outcomes were evaluated. The potential
a
In growth-positive samples, the 1-2 main aerobic and anaero- advantages of using HEDP in an NaOCl irrigant,
bic species were identified by MALDI-TOF.
which relate to ease of usage, the possibility to save
chair time, and conditioning of the root canal wall for
on the NRS-11), whilst 2 patients in the NaOCl/ Dual a subsequent root filling procedure, were not investi-
Rinse HEDP group experienced mild postoperative gated in this safety trial. The current results are in
pain (‘2’ on the NRS-11). None of the other patients line with a recently performed in vitro study, which
reported postoperative pain or discomfort related to reported no increase in NaOCl cytotoxicity when add-
the tooth that had received the cleaning and shaping ing the etidronate powder under investigation (Ballal
procedure reported here (P = 1.0). et al. 2019). Future clinical studies should be
designed as superiority trials, and EDTA should be
included in the control arm, as the sequence NaOCl-
Inflammatory host response
EDTA (or a chelating solution that is combined with
At the initial treatment visit, median and IQR MMP-9 an antiseptic) represents the current gold standard in
values were 118 (69, 215) pg mg 1 TP in the pure root canal irrigation (Ma et al. 2011, Neelakantan
NaOCl group and 169 (90, 295) in the NaOCl/ Dual et al. 2012). However, it should be considered that
Rinse HEDP group (P > 0.05). These values dropped the studies leading to such irrigating protocols were
significantly (P < 0.001) when the teeth were re-ac- done on extracted teeth, and a true evidence-base
cessed and sampled 1 week after the cleaning and remains elusive.
shaping procedure. Pure NaOCl induced a median Root canals with simple anatomy were selected
change in MMP-9 of 57 ( 1, 124) pg mg 1 TP, because bacterial sampling from fins and isthmus

© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 1274–1282, 2019 1279
 Dual Rinse HEDP clinical trial Ballal et al.

Table 2 Microbiological results at S2 (after Instrumentation/ antibacterial effect of NaOCl is not hampered by HEDP
Irrigation) when both reagents are freshly combined.
Growth/no Main speciesa (frequency of The microorganisms that were identified in the root
Group growth identification) canals of teeth presenting with primary non-symp-
tomatic apical periodontitis in the current study are
2.5% NaOCl Aerobic: Streptococcus mitis/oralis (12)
purum 18/12 Enterococcus faecalis (3) in line with published material. S. mitis and Veillonella
Staphylococcus epidermidis (2) spp., and F. nucleatum are consistently found as main
Staphylococcus hominis (2) taxa in such cases (Hommez et al. 2004, R^ oßcas &
Bacillus cereus (1) Siqueira 2018). However, 13 of the 60 initial root
Neisseria mucosa (1)
canal samples (S1) had only aerobic growth, which
Streptococcus parasanguinis (1)
Streptococcus constellatus (1) may reflect the difficulty in recovering and culturing
Anaerobic: Veillonella parvula (4) strict anaerobes. The relatively high occurrence of
6/24 Fusobacterium nucleatum (2) E. faecalis in the present study may be related to food
Veillonella atypica (1) and oral hygiene in the studied population (Zehnder
2.5% NaOCl/ Aerobic: Streptococcus mitis/oralis (10)
& Guggenheim 2009). In line with earlier reports,
9% DR HEDP 15/15 Enterococcus faecalis (4)
Bacillus cereus (1) there was no apparent selection of any ‘hard to elimi-
Neisseria mucosa (1) nate’ taxa such as E. faecalis observed in the current
Streptococcus parasanguinis (1) study (Zandi et al. 2016).
Streptococcus constellatus (1) Patients may experience discomfort and pain fol-
Anaerobic: Veillonella parvula (3)
lowing root canal treatment (Pak & White 2011).
6/24 Veillonella atypica (2)
Fusobacterium nucleatum (1) Several variables are associated with this outcome,
a
including factors related to root canal irrigation (da
In growth-positive samples, the 1-2 main aerobic and anaero-
bic species were identified by MALDI-TOF.
Silva et al. 2015). Various methods are described to
evaluate the intensity of postoperative pain (Ferreira-
Valente et al. 2011). In this study, a numeric pain
areas is doubtful (Siqueira & R^ oßcas 2009). Whether rating scale was used for evaluation (Warren Grant
these results can be extrapolated to teeth with more Magnusson Clinical Center 2003). The reason for this
complex root anatomy requires further investigation. was that the scale could be enquired in a telephone
Nevertheless, this report is first of its kind, confirming interview. Preoperative pain is a main predictor for
earlier in vitro studies that the desired effects of NaOCl the presence of pain after root canal treatment (Law
are not hampered by HEDP that is freshly admixed. et al. 2015). Therefore, patients presenting without
Various concentrations of NaOCl have been advo- pain were selected for this trial. Postoperative pain
cated for root canal treatment. However, it has been occurred rarely (1.8% of the patients, n = 3). Hence,
shown that at higher concentration, NaOCl has caus- an influence on postoperative pain of the irrigants
tic potential (Hauman & Love 2003). Hence, in the used here can be regarded as negligible under the
present study 2.5% of NaOCl was used. The antibacte- current conditions. This result is in accordance with a
rial effect of HEDP/NaOCl combinations against previous study, which reported a low postoperative
endodontic pathogens is well demonstrated in previ- pain incidence and, if occurring, merely moderate
ous in vitro studies (Arias-Moliz et al. 2014, 2015). pain levels after single-visit root canal treatment of
However, hitherto there has been no clinical trial teeth with asymptomatic apical periodontitis employ-
evaluating possible influences that cannot be simu- ing different irrigants (Almeida et al. 2012).
lated in vitro. In the present study, irrigation with the Analysis of periapical fluid was used to assess
combination of HEDP in 2.5% NaOCl irrigant caused change in the levels of the neutrophil-derived gelati-
the absence of culturable microorganisms in 50% of nase MMP-9. As has been shown in previous investi-
the root canals under investigation, compared to 40% gations, the change of neutrophil enzyme levels in
when 2.5% NaOCl was used alone. This result is in periapical fluid between the first and the second visit
accordance with previous clinical observations, which can provide information regarding the healing
have shown an incidence of negative cultures after propensity of periapical tissues (Wahlgren et al. 2002,
irrigation with NaOCl ranging from 40% to 60% Alptekin et al. 2005). In the present study, MMP-9
(Bystr€om & Sundqvist 1985, Siqueira et al. 2007). levels in periapical fluid declined significantly in
This result clearly shows that in clinics, the 1 week without significant differences between

1280 International Endodontic Journal, 52, 1274–1282, 2019 © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Ballal et al. Dual Rinse HEDP clinical trial

treatment groups. This reduction in the MMP-9 levels References

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1282 International Endodontic Journal, 52, 1274–1282, 2019 © 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd