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The Effects of Temperature on Sodium Hypochlorite
Short-Term Stability, Pulp Dissolution Capacity, and
Antimicrobial Efficacy
George Sirtes, DMD, Tuomas Waltimo, DMD, PhD, Marc Schaetzle, DMD, and
Matthias Zehnder, DMD
Abstract
The purpose of this study was to test some effects of
preheating NaOCl solutions using a commercially avail-
able syringe heating device. Irrigating solution temper-
atures in 10-ml syringes were measured. Stability of
5.25, 2.62, and 1% NaOCl solutions for 60 min at 20,
45, and 60°C was assessed using iodine/thiosulfate
titration. Human pulp tissue dissolution capacity of a
1% NaOCl solution was gauged at the latter temper-
atures, and compared to corresponding values with a
5.25% solution at 20°C. Killing efficacy of diluted
NaOCl solutions against 48-h incubations of
Enterococ-
cus feacalis
ATCC 29212 was compared at 45°C and
20°C. Using the heating device, a 20°C solution
reached 45°C and 60°C in 7 and 20 min, respectively.
Solutions remained stable during the observation pe-
riod. The 1% NaOCl solution at 45°C dissolved pulp
tissues as effectively as the 5.25% solution at 20°C,
while the 60°C/1% solution was significantly more
effective (p ⬍0.05). A 100-fold increase in killing
efficacy was observed between corresponding NaOCl
solutions at 20°C and 45°C.
Key Words
Sodium hypochlorite, temperature, pulp dissolution,
antimicrobial
From the Department of Preventive Dentistry, Periodon-
tology, and Cariology, University of Zurich Center for Dental
Medicine Zurich, Switzerland (Drs Zehnder and Sirtes); Depart-
ment of Orthodontics and Pediatric Dentistry, University of
Zurich Center for Dental Medicine, Zurich, Switzerland (Dr
Schaetzle); and the Institute of Preventive Dentistry and Oral
Microbiology, University of Basel Center for Dental Medicine,
Basel, Switzerland (Dr Waltimo).
Address requests for reprints to Dr. Zehnder, Department of
Preventive Dentistry, Periodontology, and Cariology, University of
Zu¨ rich Center for Dental Medicine, Plattenstrasse 11, CH 8028
Zu¨ rich, Switzerland; E-mail: matthias.zehnder@zzmk.unizh.ch.
Copyright © 2005 by the American Association of
Endodontists
Sodium hypochlorite (NaOCl) was first recommended as an antiseptic solution by
Henry Dakin to irrigate open wounds in World War I (1). Several years later, the use
of chlorinated soda solutions was advocated for root canal therapy (2). Today, NaOCl
solutions are still the most favored root canal irrigants, based on their antibacterial,
tissue-dissolving, and lubricating properties (3). In addition, they have a good shelf life
if correctly stored, are inexpensive, and easily available from many sources (4).
The choice of concentration of NaOCl is still a matter of debate. Dakin originally
used an aqueous 0.5% NaOCl solution. Later, NaOCl solutions of higher concentrations
were advocated for root canal debridement (5). The antibacterial efficacy of hypochlo-
rite solutions is a function of their concentration (6), as is their tissue dissolution
capacity (7), and on the other hand, their caustic potential (8). Serious incidents have
been reported when concentrated hypochlorite solutions were inadvertently forced into
periodontal tissues (9), or when such a solution leaked through the rubber dam onto
the patient’s skin (10). Simply increasing hypochlorite concentrations in irrigating
solutions over 1% NaOCl to render them more effective may not be advisable.
One alternative approach to improve the effectiveness of hypochlorite irrigants in
the root canal system could be to increase the temperature of low-concentration NaOCl
solutions. This appears to improve their immediate tissue-dissolution capacity (11, 12).
At the same time, the systemic toxicity of preheated NaOCl irrigants, once they have
reached body temperature, should be lower than the one of more concentrated non-
heated counterparts with similar efficacy in the root canal. However, there is only little
data available on features of heated hypochlorite solutions relevant to the endodontist.
Previous studies provide some insight into chemical stability, dissolution action and
antimicrobial efficacy of NaOCl preparations, but the findings appear to be somewhat
contradictory. In addition, available data pertaining to heated hypochlorite solutions
were not always obtained using endodontically relevant tissues and/or microbiota, or at
temperatures higher than 37°C. Despite this lack of knowledge, heating devices for
endodontic irrigating syringes have recently been introduced to the dental market.
Consequently, the purpose of this in vitro study was threefold: (1) to evaluate the
short-term chemical stability of preheated NaOCl solutions using a commercially avail-
able heating device for irrigating syringes; (2) to compare dissolution capacities of
NaOCl solutions on necrotic human pulp specimens at different temperatures; and (3)
to assess the efficacy of preheated NaOCl solutions on E. faecalis, a species associated
with failed endodontic therapy (13).
MATERIALS AND METHODS
Solutions
In this study 1, 2.62, and 5.25% sodium hypochlorite solutions (NaOCl in water,
wt/wt) were used. The aforementioned solutions were prepared by diluting a pure 14%
NaOCl solution (Thommen & Co. AG, Ru¨ti bei Bu¨ren, Switzerland). The available chlo-
rine (OCl
-
and HOCl) content of NaOCl solutions was measured using a standard iodine/
thiosulfate titration method (14). The solutions were protected from oxidation in tightly
covered amber glass bottles and stored in a refrigerator at 5°C between experiments.
Basic Research—Technology
JOE — Volume 31, Number 9, September 2005 Effects of Preheating NaOCl 669
Temperature Measurements
Four 10-ml syringes (Omnifix, B. Braun AG, Melsungen, Germany)
were filled with NaOCl solutions of 20°C and heated in a syringe warm-
ing device (Keydent, Vaterstetten, Germany) until they reached 45°C
and 60°C, respectively. A calibrated electronic thermometer with a mi-
cro-tip (Testo AG, Lenzkirch, Germany) was used to measure irrigant
temperature in the syringes. Times were recorded for the irrigants to
reach the temperatures indicated on the warming device.
Short-Term NaOCl Stability
Chemical stability of heated sodium hypochlorite solutions was
assessed by measuring the amount of available chlorine in solution over
time, using the aforementioned iodometric titration assay. Measure-
ments were done twice for each solution at 45°C and 60°C after a
heating period of 15, 30, and 60 min. Control solutions of 20°C were
cooled in a water bath to keep their temperature.
Tooth Collection
Twenty-two teeth (10 third molars and 12 premolars) were ex-
tracted for malposition or orthodontic reasons and collected at the
Departments of Orthodontics and Oral Surgery, University of Zu¨rich,
School of Dental Medicine. Patients volunteered to donate their teeth,
which were extracted according to their individual treatment plans, for
research purposes. Informed consent was obtained from all patients.
The current study protocol was approved by the institutional review
board. Teeth were immediately transferred to a freezer and kept at
⫺27°C in a tight container until further use. Because of possible inter-
ferences of disinfectants with the tissue dissolution assay (see below),
experimental teeth were not treated with any chemicals. Personnel han-
dling the specimens used universal precautions during the experiments.
Tissue Dissolution Assay
The teeth were thawed in sterile 0.9% saline. Subsequently, pilot
grooves were prepared in crowns, buccal, and oral aspects without
entering the pulp chamber using a diamond-coated fissure bur (Inten-
siv SA, Grancia, Switzerland). Teeth were then carefully split in two
fragments using a micro-chisel and the entire pulp tissue was removed
from the pulp chamber and the root canals using a spoon excavator.
Pulp specimens were immersed in a 0.9% saline solution at room tem-
perature for 30 min. Five human pulp specimens were randomly used
per irrigating solution: 1% NaOCl at 20°C, 1% NaOCl preheated to 45°C,
1% NaOCl preheated to 60°C and finally a 5.25% NaOCl solution at 20°C
(negative control). Two pulp specimens were irrigated with 0.9% saline
(positive controls).
The dissolution assay was performed as follows: a round polyeth-
ylene mesh with a pore size of 0.5 mm (PE-HD, VWR International,
Dietikon, Switzerland) was placed on a moistened filter paper with a
pore size ⱕ30
m (Schleicher & Schuell, Feldbach, Switzerland) in a
Buchner filter funnel mounted on a flask connected to a vacuum pump.
Before the experiments described below, the mesh had been pre-
weighed in an air-tight container using a precision balance (AT 261,
Mettler-Toledo AG, Greifensee, Switzerland). The moist pulp specimens
were laid on this carrier; the subjacent filter paper was used to avoid
small pulp remnants from being sucked into the flask. To remove excess
fluid from the pulps, the vacuum pump was turned on for 60 s. Pulp
specimens were then irrigated with 5 ml of test or control solutions for
60 s, using a 10-ml syringe. To stop the tissue dissolution process, pulp
remnants were subsequently rinsed for 120 s with distilled water and
finally the vacuum pump was left on for 60 s to remove excess fluid. The
mesh containing remaining pulpal tissue was then removed from the
flask and weighed. Pilot studies using the mesh only without pulp spec-
imens showed that it did not alter its weight during the experimental
process. Results are reported as per cent of initial pulp tissue weight
remaining after irrigation.
Antimicrobial Test
Stationary phase E. faecalis ATCC 29212 cells were cultured in
Tryptic Soy Broth (TSB, Difco, Detroit, MI) for 48 h at 37°C from stocks
(TSB ⫹10% glycerol vol/vol) stored at ⫺70°C. The cells were washed
once with phosphate-buffered saline (PBS) and harvested by centrifu-
gation at 10,000 ⫻gfor 10 min at ⫹4°C. Subsequently, bacteria were
suspended in PBS to an optical density A
660
⫽0.1, corresponding to a
cell concentration of approximately 4 ⫻10
6
colony forming units
(CFU) per milliliter. Test solutions were: NaOCl 0.001, 0.0001, and
0.00001% (wt/vol); control solutions were: 0.1 M sodium thiosulfate,
0.1 M sodium thiosulfate ⫹0.001% NaOCl (1:10, vol/vol), and PBS.
Solutions were either preheated to 45°C or cooled to 20°C in a water
bath. Ten microliters of bacteria in PBS was added to 890
l of these
solutions, and incubated for 10 min at the respective temperatures.
Subsequently, 100
l of a 0.1 M sodium thiosulfate solution was
added to stop antimicrobial NaOCl activity (15). A 10-fold dilution se-
ries was made in PBS. Droplets of 20
l from these series were cultured
on tryptic soy agar (Difco) for 48 h at 37°C, and colonies were counted.
Data Analysis
Data obtained in the pulp dissolution experiments were compared
using one-way ANOVA. Post hoc comparisons were performed using t
tests followed by Bonferroni’s correction. The level of the alpha-type
error was set at ⬍0.05.
RESULTS
Using the syringe irrigant warming device under investigation, the
hypochlorite solutions reached 45°C and 60°C in 7 min and 20 min,
respectively. The temperature scale on the device was found to be ac-
curate. Aqueous hypochlorite solutions of 1, 2.62, and 5.25% kept
100% of their available chlorine at 20, 45, and 60°C during the whole
experimental period (60 min).
A comparison of original tissue weights (mean ⫽41.1 ⫾20.8
mg) showed no significant difference between groups (p ⬎0.25). The
two control specimens irrigated with saline only kept their weight
throughout the experimental process. When percent values of dissolved
tissue after hypochlorite irrigation were compared, significant differ-
ences (p ⬍0.05) were recorded between all experimental groups, i.e.
a 1% NaOCl solution at 60°C was significantly more efficient than a
corresponding solution at 45°C, which, in turn, was more efficient than
a 1% solution at 20°C (Fig. 1). A 1% solution at 45°C was equally
effective as the negative control solution, 5.25% NaOCl, at 20°C. A 1%
solution at 60°C dissolved 96.0 ⫾3.7% of the original pulp weight in
60 s, thus, was significantly (p ⬍0.05) more effective than the 5.25%
solution at 20°C.
Microbiological experiments using E. faecalis showed that incu-
bation at 60°C in an inert PBS solution killed all bacteria, while they
tolerated a 10-min incubation at 45°C in PBS without reduction in
viability counts (Table 1). Equal results were obtained when bacteria
were incubated in 0.1 M thiosulfate or 0.001% NaOCl blocked with the
latter solution. With the pure 0.001% and 0.0001% NaOCl solutions, the
antimicrobial efficacy was roughly two orders of magnitude higher at
45°C as compared to 20°C. A 0.00001% NaOCl solution was ineffective
at both 20°C and 45°C.
DISCUSSION
Using endodontically relevant tissues and microbiota, this in vitro
study corroborated three desirable effects of preheated NaOCl solu-
Basic Research—Technology
670 Sirtes et al. JOE — Volume 31, Number 9, September 2005
tions, namely their stability for a clinically relevant short period of time,
improved tissue dissolving capacity and antimicrobial efficacy.
It should be noted that the current investigation was performed in
an ideal in vitro environment. Results may therefore not be directly
extrapolated to the clinical situation. Furthermore, it remains unclear
whether preheated solutions would be less toxic than more concen-
trated solutions of similar efficacy administered at room temperature.
Moreover, heat transfer may occur during rinsing of the root canal
system with preheated solutions through dentin and jeopardize adjacent
periodontal tissues (16). However, it may deduced from existing liter-
ature that irrigating solutions used clinically would reach a temperature
equilibrium relatively quick (11). Consequently, more concentrated
solutions most probably will, if administered to the periodontal tissues,
be more toxic than preheated counterparts of lower concentrations (8).
Furthermore, human dentin demonstrates low thermal conductivity
(17). An intact vasculature in the area and the thermal conductivity of
the periodontal membrane and the alveolar bone may also help dissi-
pate the heat rise on the root surface. Nevertheless, further studies are
necessary to find the ideal temperature/concentration combination for
hypochlorite solutions to be used in vivo.
The current results pertaining to heat stability of NaOCl solutions
are in agreement with earlier reports on the topic (18). The found an
increased amount of tissue dissolution by increasing the concentration,
contact time, and volume of sodium hypochlorite and by maintaining
the temperature of the solution at 36°C (19). Results pertaining to an
increased tissue dissolution capacity of heated NaOCl solutions were
later confirmed by other authors (11, 12), and were corroborated in
the current study using human pulp specimens. Furthermore, heated
hypochlorite solutions remove organic debris from dentin shavings
more efficiently than unheated counterparts (20). The antimicrobial
properties of heated NaOCl solutions have also been discussed in the
past. As early as 1936, the effect of NaOCl temperature on Mycobacte-
rium tuberculosis survival was demonstrated (21). A 50 ppm
(0.005%) NaOCl solution, obtained complete kill in 30 s at 60°C, in 60 s
at 55°C, and in 150 s at 50°C. With the taxa tested so far, bactericidal
rates for sodium hypochlorite solutions are more than doubled for each
5°C rise in temperature in the range of 5 to 60°C (22). This was cor-
roborated in the current study using E. faecalis cells; a temperature
raise of 25°C increased NaOCl efficacy by a factor 100. An increased
killing effect of heated solutions against endodontic microbiota appears
to be present not only with NaOCl but also with other antiseptic irrigants
such as chlorhexidine gluconate (23).
In conclusion it may be stated that preheating sodium hypochlorite
solutions appears to improve their necrotic pulp tissue dissolution ca-
pacity and efficacy against stationary phase E. faecalis cells. Heating
NaOCl solutions chair-side using a heating device bears the advantage
that the desired irrigant temperatures can be reached within a relatively
short period of time from stock solutions stored at low temperatures.
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Figure 1. Bar chart depicting relative amounts (percent of original weight) of
remaining human dental pulp tissue (n⫽5 per group) after a 60-s irrigation
with 5 ml of aqueous NaOCl. Differences between all groups were statistically
significant at the 0.05 level (ANOVA/Bonferroni), except for the 1% NaOCl
solution at 45°C and the 5.25% solution at 20°C.
TABLE 1. Mean log
10
CFUs (48 h) of surviving stationary-phase E. faecalis
cells exposed for 10 min to test and control solutions
NaOCl concentration (%, wt/vol) 20°C 45°C
0.001 2.0 (no CFUs)
0.0001 4.9 3.1
0.00001 5.4 5.2
PBS (positive control) 5.3 5.3
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