Basic Research—Technology
Depletion Rate of Hydrogen Peroxide from
Sodium Perborate Bleaching Agent
Liliann Tran, BBiomed, Rebecca Orth, BSci (Hons), PhD, Peter Parashos, MDSc, PhD,
Ying Tao, BBiomed, Calvin W.J. Tee, BBiomed, Vineet Thenalil Thomas, BBiomedSc,
Georgina Towers, BApSci (MRS) DR, Diem Thuy Truong, BSc, Cynthia Vinen, BBiomed,
and Eric C. Reynolds, PhD
Abstract
Introduction: Internal bleaching of discolored teeth
uses sodium perborate reacting with water to form the
active agent, hydrogen peroxide (H2O2). Sodium perbo-
rate is replaced at varying time intervals depending on
clinician preference and until esthetically acceptable
results are achieved, but this is done without scientific
basis. This study measured the depletion rate of
hydrogen peroxide from sodium perborate as a bleaching
agent. Methods: Two sodium perborate bleaching
products (Odontobleach [Australian Dental Manufac-
turing, Kenmore Hills, Queensland, Australia] and Endo-
sure Perborate Micro [Dentalife, Ringwood, Victoria,
Australia]) and distilled deionized water mixtures at ratios
of 25 mg/mL, 50 mg/mL, and 100 mg/mL were placed into
sealed microtubes and incubated at 37 C. H2O2 concen-
trations were measured at 23 time points over 4 weeks.
Quantification of H2O2 concentrations was obtained
using a ferrothiocyanate oxidation reduction reaction
followed by spectrophotometry readings. Results: The
H2O2 concentration rapidly peaked within 27 hours and
reached a plateau by about 3 days (75 hours). Low levels
of H2O2 were evident beyond 3 days and for at least
28 days. No significant differences were found between
the 2 sodium perborate products. There was also no sig-
nificant difference in the depletion rate between the
different ratios. Conclusions: Based on the chemistry
of H2O2 depletion, the minimum replacement interval
for the bleaching agent is 3 days. Frequent replacements
of the perborate clinically may be unnecessary because of
the continued presence of low H2O2 levels for at least
28 days. Although these data cannot be extrapolated to
the clinical situation, they set a baseline for further
studies to address the many clinical variables influencing
internal bleaching. (J Endod 2016;-:1–5)
Key Words
Hydrogen peroxide depletion, internal bleaching, intra-
coronal bleaching, sodium perborate, walking bleach
From the Melbourne Dental School, Oral Health Cooperative Research Centre, University of Melbourne, Melbourne, Victoria, Australia.
Address requests for reprints to Prof Peter Parashos, Melbourne Dental School, University of Melbourne, 720 Swanston St, Melbourne, Victoria, Australia 3010.
E-mail address: parashos@unimelb.edu.au
0099-2399/$ - see front matter
Copyright ª 2016 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.10.043
JOE — Volume -, Number -, - 2016
W ith increasing con-
cerns for esthetics,
tooth discoloration is
Significance
This research is the first to investigate the chemis-
try of internal bleaching in order to begin to provide
often perceived as unde-
a baseline for further research to justify clinical pro-
sirable, especially in the
tocols, which to date are based mainly on clinical
anterior region. It is one
experience and opinions.
of the most frequent rea-
sons for seeking dental
treatment (1). Discoloration of nonvital teeth after endodontic treatment is relatively
common, occurring in 10% of all treated teeth (2) and can result from hemorrhage,
incomplete removal of pulp tissues, or the presence of endodontic materials such as
root canal irrigants, intracanal medicaments, and filling materials (3). Additional costs
related to bleaching can incur a significant financial strain and time burden because of
multiple dental appointments (4).
Sodium perborate is a safe, effective, and relatively inexpensive intracoronal
bleaching agent used in nonvital teeth in instances in which there is intrinsic discolor-
ation (5). It was first used as a mixture with water (6) and then a mixture with 30% H2O2
(7). Sodium perborate has been combined with many carrier agents over time to find
the most effective and safe combination. A combination of water and sodium perborate
has been found to be safe and equally effective in bleaching compared with mixtures of
sodium perborate with 2% chlorhexidine, 37% carbamide peroxide and 30% hydrogen
peroxide (H2O2), 10% carbamide peroxide, and 35% carbamide peroxide and 35%
H2O2 (4, 8–11).
H2O2 is released from sodium perborate when suspended in water. Its interaction
with metal ions, light irradiation, or laser irradiation produces hydroxyl radicals (OH)
(12). Many have attributed the shade lightening effect of OH to its ability to break
longer-chained chromophores into shorter-chained colorless compounds (13, 14).
It has been shown that OH levels increase in an H2O2-dependent manner,
confirming that the release of OH is from H2O2 (15). Furthermore, adding a chelating
agent decreased the release of OH, indicating that metal ions (especially Fe) aid in the
production of OH via the Fenton reaction (15, 16).
The most commonly used bleaching technique is the walking bleach method,
which involves filling the pulp chamber with a bleaching agent after root canal therapy
(17). The cavity is sealed off with a temporary restoration, and the bleaching agent is
left in the cavity and replaced regularly until a clinically satisfactory result is achieved
(18). However, the replacement time interval has been derived from clinical experi-
ence (19), and the optimal renewal time for efficient bleaching has yet to be evalu-
ated. Studies do not have a scientifically justified time period for the renewal of the
Hydrogen Peroxide Depletion Rate 1
Basic Research—Technology
sodium perborate bleaching agent. The intervals range from 3 to
4 days (20), 3 to 7 days (21), 2 to 6 weeks (22), 12 days (23), 4
to 7 days (24), and 21 days (25).
This study aimed to investigate the chemical reaction of sodium
perborate and water releasing H2O2, and its rate of depletion. Because
the trend in the rate of release of H2O2 from a sodium perborate bleach-
ing agent has yet to be investigated, the results will serve as a scientific
foundation for further and more clinically relevant research in the field
of intracoronal bleaching. The hypotheses tested were
1. That there were no significant differences in the depletion rate of
H2O2 between 2 sodium perborate products and
2. There was also no significant difference between the different
bleach/water ratios.
Materials and Methods
Preparation
Two brands of sodium perborate powders were used: Odonto-
bleach (ODB) (Australian Dental Manufacturing, Kenmore Hills,
Queensland, Australia) and Dentalife Endosure Perborate Micro
(END) (Endosure Perborate Micro; Dentalife, Ringwood, Victoria,
Australia). Initially, the intention was to include 2 g/mL as recom-
mended for clinical use of sodium perborate (26), and a pilot study
was developed to determine if this was feasible. Sodium perborate
samples (ODB and END) were used at 1, 2, and 3 g/mL Milli-Q
water (Millipore Corporation, Molshiem, France) for 16 time points
over 6 weeks: 1, 3, 6, 9, and 24 hours and 2, 3, 4, 5, 6, 7, 14, 21,
28, 35, and 42 days. At each time point, negative controls with
Milli-Q water and a standard curve consisting of serial dilutions
of H2O2 were prepared. However, sodium perborate was found to
be relatively insoluble and formed a cloudy suspension. Spectropho-
tometer results were skewed because of this opacity of samples.
Furthermore, there was insufficient supernatant available in the
3-g/mL samples for triplicates. The revised methodology reduced
the sodium perborate samples to 25, 50, and 100 mg/mL Milli-Q
water. This produced samples with minimal to no opacity and an
adequate volume of supernatant for analysis.
Figure 1. The layout of the 96-well plate for 1 time point.
2 Tran et al.
The revised method involved measurements of H2O2 at 23 time
points over 4 weeks: 0, 30, 60, 90, 120, 150, 180, 210, 240, 270,
300, 330, and 360 minutes; 24, 27, 48, 51, and 75 hours; and 9, 10,
14, 21, and 28 days. Sodium perborate powders (END and ODB)
were weighed into 2.2-mL microtubes (Greiner Bio-one GmbH, Frick-
enhausen, Germany), and each tube contained 50, 100, or 200 mg per
product. Milli-Q water (2 mL) was added to every reaction tube so that
for each time point, the concentration of the samples were as follows:
END 25 mg/mL (END25), END 50 mg/mL (END50), END 100 mg/mL
(END100), ODB 25 mg/mL (ODB25), ODB 50 mg/mL (ODB50), and
ODB 100 mg/mL (ODB100). All samples were incubated at 37 C.
Measuring the Concentration of H2O2 Released
At each time point, 6 samples were removed from the incubator
and vortexed for 5 seconds. Triplicates of 100-mL samples were pipet-
ted into a microplate as labeled in Figure 1. At each time point, a fresh
set of H2O2 standards was prepared with serial dilution to establish the
standard curve, and the concentration varied from 10 1 mol/L to
10 9 mol/L. Triplicates of each concentration were pipetted into the
wells on the same plate. Milli-Q water was used as the negative control.
The measurement of H2O2 was performed according to the method
described by Camps et al (27), and 0.2 mL 10 mmol/L FeSO4 and 0.1 mL
2.5 mmol/L potassium thiocyanate were added to experimental wells.
The microplate was read with a spectrophotometry machine (Wallac
VICTOR3 1420 Multilabel Counter; PerkinElmer, Waltham, MA) at a
wavelength 480 nm. Results were exported into Microsoft Excel for
Mac 2011 Version 12.4.1 (Microsoft Corporation, Redmond, WA).
Analysis consisted of creating a standard curve using the absor-
bance of the standards, and concentrations of H2O2 were determined
using the following linear equation: y = mx + c, where y equals the
average absorbance of each sample type minus the negative (28).
The results were graphed and statistically evaluated with 2-way analysis
of variance (SPSS version 11.5.0; SPSS Inc, Chicago, IL) with P < .05.
Results
The first 27 hours showed a rapid increase in the concentration of
H2O2, produced, which peaked at 27 hours before beginning to decline
JOE — Volume -, Number -, - 2016

Figure 2. The concentration of H2O2 over time for 0 to 51 hours with each of the samples (END25, END50, END 100, ODB25, ODB50, and ODB100).
(Fig. 2). This rapid depletion continued until the concentration pla-
teaued at about 3 days (75 hours); at this point, an equilibrium was
reached in which the net production and depletion were equal
(Fig. 3). Beyond 3 days, there remained a low level of H2O2 up to the
last time point of 28 days. At 336 hours, ODB200 was not included
in the analysis because it was a negative value (Fig. 3); this inaccurate
value was because the sample was not pipetted into the well.
The results indicated that there were no significant differences
between END25 and ODB25, END50 and ODB50, and END100 and
ODB100. Within the ODB and END samples, there was no significant dif-
ference in H2O2 concentration between the different ratios. All samples
(END25, END50, END100, ODB25, ODB50, and ODB100) followed a
similar pattern of H2O2 depletion (Fig. 2).
Discussion
Despite the wide use of sodium perborate and water as an intra-
coronal bleaching agent, little is known about the dynamics of the
Figure 3. The concentration of H2O2 over time 1 to 28 days with each of the samples (END25, END50, END 100, ODB25, ODB50, and ODB100).
JOE — Volume -, Number -, - 2016
Basic Research—Technology
reaction itself. Previous literature indicates that a pH change occurs
over time after mixing sodium perborate with water (29). It is known
that the addition of water to sodium perborate results in the release
of H2O2, sodium metaborate (NaBO3), and nascent oxygen (20). The
H2O2 decomposes into radicals and ions (20), and these radicals are
responsible for oxidation, reduction, and, hence, the bleaching proper-
ties of H2O2. They are able to break unsaturated double bonds of long
chromatic molecules into simpler molecules or reduce colored metallic
oxides to colorless metallic oxides (20). However, the trend in the pro-
duction of H2O2 is unknown; hence, the length of time the bleaching
agent should be left in the pulp chamber remains clinically derived.
The results suggest that the greatest effect of the bleaching agent
would have ceased by the 75-hour (3 days) time point, with the op-
timum bleaching activity reached by 27 hours. However, the persistence
of H2O2 levels beyond the initial peak indicates the continual bleaching
activity of sodium perborate for at least 28 days. This indicates that as a
bleaching agent sodium perborate does not need to be replaced
frequently and is supportive of clinical experience in which there are
Hydrogen Peroxide Depletion Rate 3
Basic Research—Technology
considerable variations between the replacement time periods of so-
dium perborate depending on clinician preference. However, it is
important to note that clinically many factors need to be evaluated
when considering the bleaching of a tooth including the etiology and
type of the discoloration, the severity and intensity of discoloration,
the duration of discoloration, the age of the patient, and the size of
the dentinal tubules (17, 20, 21, 27, 30, 31). Also, the absence of a
significant difference between the different ratios suggests that
perhaps the water to powder ratio does not significantly influence the
outcome.
According to the material safety data sheets, the proportion of so-
dium perborate in each of the products (ie, ODB and END) is 99% and
96%–98%, respectively. The lack of a significant difference in the con-
centration of H2O2 produced by the 2 bleaching agents indicates that
there is unlikely to be a difference clinically when evaluating shade
change.
Teeth were not used in this study in order to investigate the reac-
tion mechanics in a scenario in which most confounding factors could
be controlled. However, a limitation of this study was that the results are
unable to be correlated to clinical shade changes during bleaching.
Furthermore, the sodium perborate to water ratios could not include
the 2 g/mL recommended for clinical use of sodium perborate. Diluting
the sodium perborate samples allowed an adequate amount of super-
natant for analysis, with minimal to no clouding of these samples.
This was required to determine the chemical basis as a foundation to
future clinical studies (32). Also, because of the intrinsic nature of
H2O2 to rapidly decay, interoperator variability in the time interval be-
tween the preparation of samples and the measurement of samples may
have led to variations in absorbance readings and hence variations in
concentration correlations.
Future studies could consider using these laboratory data to
design studies using tooth samples to correlate directly to clinical sce-
narios. It may be valuable to consider and investigate factors other
than H2O2 influencing the tooth bleaching process. For instance, prod-
ucts causing discoloration may affect the pharmacodynamics of H2O2
diffusion (30). In the case of pulpal hemorrhage causing discolor-
ation, evaluating the action of H2O2 in the presence of heme proteins
would better simulate the clinical situation. This is because the pres-
ence of Fe ions can interact with H2O2 via the Fenton reaction (15).
Also, any chemical reactions of H2O2 with dentin should be consid-
ered. However, the exact mechanism in which H2O2 interacts with
dentin has not been fully elucidated. Kawamoto and Tsujimoto (15)
showed evidence for organic dissolution of dentin with the application
of H2O2, whereas Eimar et al (33) suggested there are no changes to
the inorganic and organic components and instead bleaching is
through oxidation.
Micromorphologic differences in the dentin and cementum may
also influence bleaching. Diffusion of H2O2 has been shown to be
greater in areas of cemental root defects (34). Furthermore, the age
of the patient requiring tooth bleaching will influence the efficacy of
the bleaching agent. Diffusion of H2O2 through young dentin was re-
ported to be twice as high as through old dentin (27). Previous studies
have also determined that discoloration in younger teeth responded bet-
ter to chemical bleaching than old teeth (35). Camps et al (27) attrib-
uted the reduced diffusion through old dentin to intratubular deposits
and a small dentin tubule diameter, which decrease the interaction be-
tween H2O2 and the dentin tubule contents. Consequently, to establish
firm clinical guidelines may be impractical because of the many clinical
variables and may rely on individually tailoring bleaching protocols for
specific clinical scenarios. However, these experimental findings have
provided a basis for clinical extrapolation, and further research using
an ex vivo model is indicated.
4 Tran et al.
In conclusion, there was a trend in the release of H2O2 from mix-
tures of sodium perborate and water, with a rapid increase peaking at
27 hours followed by a rapid decline and finally a plateau by 75 hours
with no relation to the powder to water ratio or the commercial product
used. Based on this chemistry, frequent replacements of the bleaching
agent are probably unnecessary because of the continued presence of
low levels of H2O2. These data cannot be directly extrapolated clinically
but provide a scientific basis on which clinical factors must also be
considered and will likely indicate a longer time period to allow
adequate clinical outcomes.
Acknowledgments
The authors would like to thank Dr Samantha Byrne,
Prof Stuart Daspher, Dr Harry Mohan, and Assoc Prof Menaka
Abuzar for their support.
Supported by the Melbourne Dental School, The University of
Melbourne.
The authors deny any conflicts of interest related to this study.
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