©

Operative Dentistry, 2023, 48-5, 564-574

Bleaching Gels Used After 1 Week

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of Mixing: Efficacy, Hydrogen
Peroxide Penetration, and
Physical–chemical Properties
H Forville • MW Favoreto • TS Carneiro • RMO Terra
LA Pinheiro • CPF Borges • AD Loguercio • A Reis

Clinical Relevance
The evaluation of in-office bleaching gels used after 1 week of mixing is important to
understand the behavior of efficacy and physical–chemical properties, being relevant to avoid
material waste.

SUMMARY and the following in-office bleaching (Opalescence

Objectives: This in vitro study aimed to evaluate
the bleaching efficacy (BE), hydrogen peroxide
penetration into the pulp chamber (HPP), and
physical–chemical properties (concentration,
pH, and viscosity) of in-office bleaching gels
immediately and after 1 week of mixing.
Methods and Materials: We randomly divided 49
premolars into seven groups: control (no bleaching)
Heloisa Forville, DDS, MSc, PhD student, School of Dentistry,
State University of Ponta Grossa, Parana, Brazil
Michael W. Favoreto, DDS, MSc, PhD student, School of
Dentistry, State University of Ponta Grossa, Parana, Brazil
Taynara S. Carneiro, DDS, MSc, PhD student, School of
Dentistry, State University of Ponta Grossa, Parana, Brazil
Renata M. O. Terra, DDS, MSc, PhD student, School of
Dentistry, State University of Ponta Grossa, Parana, Brazil
Luis A. Pinheiro, DDS, MSc, PhD, School of Materials
Engineering, State University of Ponta Grossa, Parana, Brazil
Boost 40%, Total Blanc One Step 35%, and
Whiteness HP Blue 35%) applied at two storage
times: immediately and after 1 week. We evaluated
the BE using a digital spectrophotometer and the
HPP through UV-Vis spectroscopy. We measured
the initial concentration, pH, and viscosity using
titration, a Digital pH meter and Rheometer,
respectively. For statistical analysis, we used a two-
way analysis of variance and Tukey and Dunnet
tests (α=0.05).
Christiane P. F. Borges, DDS, PhD, School of Chemistry, State
University of Ponta Grossa, Parana, Brazil
*Alessandro D. Loguercio, DDS, MSc, PhD, School of Dentistry,
State University of Ponta Grossa, Parana, Brazil
Alessandra Reis, DDS, PhD, School of Dentistry, State
University of Ponta Grossa, Parana, Brazil
*Corresponding author: Rua Carlos Cavalcanti, 4748, B
Uvaranas, Ponta Grossa, PR, Brazil, 84.030-900; email:
aloguercio@hotmail.com
http://doi.org/10.2341/23-010-L
Forville & Others: Bleaching Gels Used 1 Week After Mixing
Results: We observed higher BE and HPP for
Opalescence Boost and Total Blanc One Step after
1 week of mixing than for Whiteness HP Blue
(p<0.001). We observed a significantly lower initial
concentration for Whiteness HP Blue 1 week after
mixing compared to immediately (p=0.00001). All
bleaching gels showed a decrease in pH after 1
week of mixing (p=0.00003). However, Total Blanc
One Step had a lower pH at both evaluation times
(p<0.001). Only Opalescence Boost maintained
viscosity 1 week after mixing.
Conclusions: Opalescence Boost was the only
bleaching gel able to keep bleaching efficacy, with the
same characteristics of permeability and physical–
chemical properties after 1 week of mixing.
INTRODUCTION
Tooth bleaching is one of the most sought after and
well-established esthetic treatments in dental practice.1
Among the various bleaching techniques available, in-
office bleaching has become increasingly popular due
to it is ability to provide some degree of whitening after
only one clinical session.1-3 This protocol involves the
application of a high-concentration hydrogen peroxide
gel (20%-40%) by a dental professional during the
treatment period.4,5
In-office bleaching products are often marketed
in two bottles: one containing the active oxidizing
hydrogen peroxide and the other bottle containing
the thickening agent.6 The thickener serves to increase
the material’s viscosity, allowing it to adhere to dental
surfaces when applied7 and maintain the pH value
above the critical value of enamel demineralization.8
Once the two components are mixed together, the
hydrogen peroxide decomposes, releasing oxygen-free
radicals.9 These free radicals, which possess strong
oxidation activity, then react with the organic matter
present in the dental structure, thereby producing the
desired whitening effect.10
After bleaching, approximately 50% of the mixed
bleaching gel is typically applied in the bleaching
procedure (no data shown). Clinicians often discard
the mixed material because most manufacturers
recommend using the bleaching agents immediately
after mixing. This recommendation is given because
in-office bleaching gels can lose their physicochemical
properties, which may affect their bleaching efficacy
(BE).10 From a clinical perspective, the ability to reuse
the material in subsequent sessions can reduce material
waste and treatment costs.
Unfortunately, as far as we know, no in vitro studies
have been conducted to evaluate the BE and the
565
physical–chemical properties of the mixing products,
comparing those that are freshly mixed with those that
have been mixed for 1 week. Therefore, the aim in this
in vitro study was to evaluate the BE of various in-office
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bleaching gels when they are applied immediately
or after 1 week of mixing. The hydrogen peroxide
penetration into the pulp chamber (HPP) and some
physical–chemical properties (initial concentration,
pH, and gel viscosity) were also evaluated. The null
hypotheses tested were that 1) there is no difference in
the BE, 2) there is no difference in the HPP, and 3)
there is no difference in the initial concentration, pH,
and viscosity of various gels when they are applied after
1 week of mixing rather than immediately.
METHODS AND MATERIALS
Tooth Selection and Experimental Design
We obtained 49 healthy premolars of similar size from
the Human Teeth Local Bank at the State University
of Ponta Grossa (Parana, Brazil). We evaluated
the teeth under a microscope at 10× magnification
(Lambda LEB-3, ATTO instruments, Hong Kong,
China) to standardize the selection. We excluded
teeth with enamel cracks or morphological changes
and teeth lighter than A2, as measured with a digital
spectrophotometer (Vita Zahnfabrik, Bad Säckingen,
Germany). We also excluded teeth analyzed by
radiography (Timex 70C, Gnatus, Ribeirão Preto, SP,
Brazil) with a thickness (external point of the enamel
to the innermost point of dentin, corresponding to
the pulp horn) of less than 2.5 mm and greater than
4.0 mm.
We randomly divided the teeth into seven groups
(n=7). One group was not exposed to bleaching agents
(negative control). The other six groups were divided
according to bleaching gel (Opalescence Boost 40%,
Ultradent, South Jordan, UT, USA; Total Blanc
One Step 35%, DFL, Rio de Janeiro, RJ, Brazil; and
Whiteness HP Blue 35%, FGM, Joinville, SC, Brazil)
and storage time (bleaching procedure was performed
immediately after mixing and 1 week after mixing).
Calculation of the Sample Size
The primary outcome involved the BE assessed by
ΔEab. Based on a previous study,11 an average BE of
teeth subjected to in-office bleaching gels similar to
the bleaching gels evaluated in this study was 13.0 ± 1.1
units of ΔEab. Using an equivalence test with an alpha
of 5% and a study power of 90%, n = 7 teeth in each
group were needed to detect an equivalent difference
of 2.7 units of ΔEab so that it is acceptable to the
human eye.12
566
Specimen Preparation
With the help of a low-speed diamond disk (Isomet
1000, Buehler Ltd, Lake Bluff, IL, USA), we removed
the roots of the teeth approximately 3 mm from the
cementum-enamel junction, and the pulp tissue was
removed and rinsed with deionized water. With care
taken not to touch the internal occlusal region of the
pulp chamber, we expanded access to the pulp chamber
using a #1014 spherical bur (KG Sorensen, SP, Brazil),
so that 25 μL for solution could be introduced into the
pulp chamber using a micropipette (Labmate Soft,
HTL Lab Solutions, Warsaw, Poland). After preparing
a specimen, we took a new radiograph and initial color
evaluation. We took each radiograph with an exposure
time of 0.5 seconds and a 30-cm focus-object distance
(70 kVp-7 mA). We focused the central X-ray beam at a
90° angle to the tooth’s buccal surface. After exposure,
we digitally obtained the images and measured the
corresponding buccal tooth thickness with New IDA
software (Dabi Atlante, Ribeirão Preto, SP, Brazil).
Bleaching Efficacy Assessment
We measured the BE before the bleaching procedure
and 1 week after the treatment protocol was
performed in each experimental group, using a digital
spectrophotometer (VITA Easyshade Advance 4.0,
VITA Zahnfabrik). We immersed the specimens in
artificial saliva (Pharmacy Eficácia, Ponta Grossa, PR,
Brazil; pH=7.0, composed of carboxymethylcellulose,
sodium chloride, potassium chloride, magnesium
chloride, dibasic calcium phosphate, glycerin, xylitol,
and distilled water) between evaluation periods
and carried out daily changes of artificial saliva at
a controlled temperature of 37°C. To measure the
color of the specimens, we made guides with dense
condensation silicone to standardize the position of
the spectrophotometer (Coltoflax and Cub Kit Profile,
Vigodent, Rio de Janeiro, RJ, Brazil) through a 6-mm
diameter window with a metal device in the middle
third of the vestibular surface for each specimen, where
the tip of the spectrophotometer was inserted.13
We inserted the tip of the device in the silicone guide,
determining the color parameters (L*, a*, and b*). The
value L* represented the luminosity (values varying
from 0 [black] to 100 [white]), the value a* represented
the color along the red–green axis, and the value b*
represented the color along the yellow–blue axis. The
color change before (baseline) and after treatment
(immediately after bleaching on application days) was
given by the difference between the colors measured with
the spectrophotometer using the CIELab formula: ∆Eab
= [(∆L*) 2 + (∆a*) 2 + (∆b*) 2]1/2).14 In addition, the color
change was also calculated using the CIEDE formula:
Operative Dentistry
∆E00 = [(ΔL/kLSL)2 + (ΔC/kCSC)2 + (ΔH/kHSH)2 +
RT(ΔC*ΔH/SC*SH)]1/2.15 Perceptual changes were
accepted when the differences in the initial and after-
bleaching colors present ∆Eab > 2.7 and ∆E00 > 1.8.12
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Initial Concentrations of Hydrogen Peroxide in
the Bleaching Gels
We used potassium permanganate as an oxidizing
agent to describe the concentration of hydrogen
peroxide in each bleaching gel immediately and 1 week
after mixing. Hydrogen peroxide reacts with potassium
permanganate, reducing it to a colorless product based
on a reduction-oxidation reaction according to the
formula16,17: 2KMnO4 + 5H2O2 + 3H2SO4 → 1K2SO4 +
2MnSO4 + 5O2 + 8H2O.
For this, we mixed the bleaching gels according to the
manufacturer’s instructions (Table 1) and weighed them
analytically. We collected samples of each bleaching
gel (with approximately 0.2 g) and diluted them in 50
mL of distilled water and 10 mL of 1 mol.L-1 sulfuric
acid. We added the potassium permanganate solution
(0.02 mol.L-1) to this solution, with a violet solution
expected.16,17 The change in color was indicated when
the hydrogen peroxide was completely consumed. We
performed three titrations for each bleaching gel.
Obtaining the Analytical Curve
We used prior purification analytical products in the
study, and we prepared all solutions using deionized
water. At first, we drew a standard analytical curve
from a 5000 μg/mL stock solution prepared from
a concentrated solution (Eficácia Pharmacy, Ponta
Grossa, PR, Brazil) and diluted it in an acetate buffer
solution (pH=4), titrated using traditional methods and
with a potassium permanganate solution to determine
the actual concentration of the solution and the
analytical grade. Based on the primary concentration,
we performed serial volumetric dilutions of 0.000-0.416
μg/mL to draw the analytical curve. We obtained the
known concentrations of hydrogen peroxide using a
Cary UV-Vis 50 spectrophotometer (Varian, Palo Alto,
CA, USA). This procedure yielded a standard reference
line for the extrapolation of the study sample results
(R=0.996); these data are not shown.
Hydrogen Peroxide Penetration Into the
Pulp Chamber
For the application of the materials, a single calibrated
and experienced operator was responsible for all
treatment protocols. We placed the specimens with
the occlusal face in contact with a wax plate allowing
access to the pulp chamber. All teeth had their buccal
area isolated by the application of a light-cure resin
Forville & Others: Bleaching Gels Used 1 Week After Mixing 567

Table 1: Description of the Materials (Manufacturers), Batch Number, Composition of the Products That Were Used
in This Study, Protocols, and Manufacturers’ Mixing and Application Instructions
Bleaching Composition Protocol Manufacturers’ Mixing and

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Product Application Instructions
and Batch
Number
40% hydrogen Bleaching Bleaching 1 1. Mix the two phases with the syringes
peroxide, 20% water, immediately week after mix connected, pushing the plungers alternately
and desensitizing after mix up to 50 times (25 times in each direction/
agents (3% side). Press all mixed gel into the red
Opalescence
potassium nitrate and syringe.
Boost 40%
1.1% fluoride) 2.. Twist to separate the two syringes and
(Ultradent)
securely attach the recommended tip (black
D09XR
mini tip) onto the red syringe and apply
a 0.5- to 1.0-mm thick layer of gel to the
clean labial surface of the tooth.
3. Apply for 40 min.a
35% hydrogen Bleaching Bleaching 1 1. Remove the caps of the syringes with
peroxide, immediately week after mix peroxide and thickener and attach the
thickener, water, after mix (manufacturer connector at both syringes. Assure that
vegetable extracts, did not both syringes are well connected to avoid
sequestrating agents, recommend material leakage.
Total Blanc amide, colorants, and the use of 2. Push the plungers alternately, 6-7 times,
One Step glycol this product until the product reaches a homogeneous
35% (DFL) 1 week after yellow color, indicating that it is active. Then,
21030273 mix) move all mixture into one of the syringes.
3. Couple the applicator tip in the syringe
and apply the bleaching gel over the teeth
labial surface.
4. Follow the application time (40 min)
described for the manufacturer.
35% hydrogen Bleaching Bleaching 1 1. Mix the two phases connected to the
peroxide, deionized immediately week after mix syringe by pushing the plungers alternately
water, thickener, after mix (manufacturer 8 times (4 times for each side). Move all
Whiteness
violet colorant, glycol, did not mixture into one of the syringes.
HP Blue
neutralized agents, recommend 2. Twist an application tip in the syringe with
35% (FGM)
and desensitizing the use of the bleaching gel and apply Whiteness HP
051120
agents (3% calcium this product Blue 0.5- to 1-mm layer on the buccal.
gluconate) 1 week 3. Apply for 40 min.b
after mix)
a
The manufacturer recommends the application time for only 20 minutes.
b
The manufacturer recommends the application time for only 30 minutes.

barrier (Top Dam, FGM) to standardize an area of 6
mm². The delimitation of 6 mm² was guided by a dry-
ended compass with the standardized measurement on
the instrument. A 25 μL aliquot of the acetate buffer
(pH=4) was inserted into the pulp chamber of each
tooth to absorb and preserve any hydrogen peroxide
that may have penetrated the pulp chamber during the
bleaching procedure.18,19
In the groups that received the bleaching treatment,
we applied the bleaching agents, after mixing as
recommended by the manufacturer (Table 1), in
the buccal enamel area according to the different
experimental groups. We applied the bleaching gel
until it completely covered the area of the teeth to
be whitened. For all bleaching gels, the application
period was 40 minutes in each session, being carried
568
out immediately or 1 week after mixing the gel. We
performed the removal of the bleaching gel with gauze
and carefully washed it with deionized water only on
the buccal surface. We maintained the control group
without contact with bleaching agents. During the
interval between applications, we kept the bleaching
gels under refrigeration.20
After the end of the bleaching procedure, we removed
the acetate buffer solution in the pulp chamber of each
sample. We transferred the removed solutions to a glass
tube and repeated this procedure with the cleaning of
the pulp chamber of each tooth four times with 25 μL
of the acetate buffer. We transferred this solution to the
same glass tube previously used. After that, in the same
glass tube, we added 100 μL of 0.5 mg/mL (Leucocrystal
Violet, Sigma Chemical Co, St Louis, MO, USA) and
50 μL of 1 mg / mL of horseradish peroxidase enzyme
(Peroxidase Type VI-A, Sigma Chemical Co) to the
glass tube, along with deionized water (2.725 μL). We
repeated this sequence separately for each of the teeth
at different times. We measured the resulting solution
using a Cary 50 UV-Vis spectrophotometer (Varian).
According to Beer law, the concentration is directly
proportional to the absorbance. Thus, we determined
the concentration of hydrogen peroxide (μg/mL) by
comparison with the calibration curve already obtained.
pH Measurements of Bleaching Gels
We measured the two phases of the bleaching agents
before mixing to check the pH of the different phases
separately (thickener and hydrogen peroxide).
Following this, we applied the bleaching gels on the
buccal surface of the teeth and repeated the same
procedure for each bleaching gel and by each application
time (immediately and after 1 week of mixing). For this
purpose, a 6-mm circular pH meter and a flat surface
pH electrode (Extech pH100 ExStik pH Meter, Extech
Instruments, Nashua, NH, USA) were positioned in
the areas delimited for the application of the bleaching
gel and held in position until they were stable. Three
measurements were carried out on each tooth.11
Viscosity of the Bleaching Gel
We measured the viscosity of the bleaching gels
according to their shear rate, using a controlled-strain
rheometer (DHR-2, TA Industries, Houston, TX, USA)
and a 2º geometry in a conical plate 40 mm in diameter.
The rheometer was equipped with a Peltier accessory,
integrated with a heating/cooling system, ensuring the
uniform temperature of the samples. We performed all
tests at a constant temperature of 37°C, equivalent to
mouth temperature. We evaluated bleaching agents
according to the manufacturer’s recommendations
Operative Dentistry
regarding application time, immediately and 1 week
after mixing. The thixotropic behavior of the gels was
evaluated using a permanent flow for 40 minutes at a
constant shear rate of 5 s-1.
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Statistical Analysis
We analyzed the data using the Kolmogorov–Smirnov
test to assess whether the data were normally distributed
and via the Bartlett test for equality of variance to verify
the assumption of the equality of variances (no data
shown). As the data showed normality, the data of
the BE (ΔEab and ΔE00), the initial hydrogen peroxide
concentration, HPP (µg/mL), and pH were subjected
to two statistical evaluations: 1) a two-way analysis of
variance (bleaching gels vs time) and Tukey post hoc
test to compare different bleaching techniques and 2)
a two-way analysis of variance (bleaching vs time) and
Dunnet post hoc test to compare the values obtained in
different bleaching techniques with those of the control
group (α=0.05). The results of viscosity were subjected
to descriptive analysis.
RESULTS
Bleaching Efficacy Assessment
A lower color change (ΔEab and ΔE00) was found in
the control group when compared to all experimental
groups (Table 2; p<0.00002). In the immediate time,
we observed no significant difference in the BE when
different in-office bleaching gels were evaluated (Table
2; p>0.05). However, after 1 week, we observed a
significantly lower color change for Whiteness HP Blue
(FGM) when compared to other bleaching materials,
as well as when compared to the same product at
the immediate time (Table 2; p<0.001). Regarding
Opalescence Boost (Ultradent) and Total Blanc One
Step (DFL), when both times for each bleaching gel
was compared, no significant difference was observed
in terms of BE (Table 2; p>0.05). It is worth mentioning
that all the changes in the color for ΔEab and ΔE00 were
primarily due to delta L changes (data not shown).
Initial Concentrations of Hydrogen Peroxide in
the Bleaching Gels
In the immediate time, the Opalescence Boost
(Ultradent) showed a significant and higher
concentration of hydrogen peroxide when compared
to the other bleaching gels (Table 3; p<0.001). We
observed no significant difference between Total Blanc
One Step (DFL) and Whiteness HP Blue (FGM; Table
3; p>0.05). After 1 week, we observed a significantly
higher concentration of hydrogen peroxide for
Forville & Others: Bleaching Gels Used 1 Week After Mixing 569

Table 2: Means (± Standard Deviations) of the Bleaching Efficacy in Different

Objective Assessments (ΔEab and ΔE00) in Different Experimental Groupsa
Experimental Bleaching Time ΔEab ΔE00

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Groups
Control Control 2.4 ± 0.6b 1.9 ± 0.6b
Immediate 7.0 ± 0.7 A 4.5 ± 0.4 a
Opalescence Boost
1 week 7.0 ± 0.9 A 4.4 ± 0.4 a
Immediate 6.2 ± 1.0 A 4.1 ± 0.6 a
Total Blanc One Step
1 week 6.6 ± 0.9 A 4.6 ± 0.7 a
Immediate 6.9 ± 0.5 A 4.6 ± 0.4 a
Whiteness HP Blue
1 week 3.7 ± 1.0 B 2.6 ± 0.8 b
a
Identical uppercase or lowercase letters in each column indicate statistically similar means
(two-way analysis of variance and Tukey test, α=0.05).
b
Control group was statistically different when compared to all groups (two-way analysis of
variance and Dunnet test, α=0.05).

Opalescence Boost (Ultradent) when compared to
Whiteness HP Blue (FGM) and Total Blanc One
Step (DFL; Table 3; p<0.001). A significantly lower
concentration of hydrogen peroxide was also observed
for Whiteness HP Blue (FGM) when compared to
Total Blanc One Step (DFL; Table 3; p=0.00001).
When we compared each bleaching gel at different
times, we found a significantly lower concentration
only for Whiteness HP Blue (FGM; Table 3; p<0.001).
Hydrogen Peroxide Penetration Into the
Pulp Chamber
The mean and standard deviation buccal thickness
of the specimens, according to the radiographs taken,
were 3.2 ± 0.1 mm, with no statistical significance
among groups (p=0.37; data not shown). We found a
lower HPP in the pulp chamber in the control group
when compared to all experimental groups (Table
3; p<0.00001). In the immediate time, we observed
no significant difference of HPP, regardless of how
different in-office bleaching gels were evaluated (Table
3; p>0.05). However, after 1 week, a significantly lower
HPP was observed for Whiteness HP Blue (FGM)
when compared to other bleaching materials (Table 3;
p=0.0007). A significantly higher HPP was also observed
for Total Blanc One Step (DFL) when compared to
other bleaching materials (Table 3; p=0.0007). When
we compared the bleaching gels at different times, we

Table 3: Means (± Standard Deviations) of the Hydrogen Peroxide Concentration (µg/mL)

Detected Inside the Pulp Chamber and Initial Concentration of Hydrogen Peroxide (%) in
Different Experimental Groupsa
Experimental Bleaching Time Hydrogen Peroxide Initial Concentration
Groups Concentration (μg/mL) of Hydrogen
Peroxide (%)
Control Control 0.0004 ± 0.0004b —
Immediate 0.2360 ± 0.1888 B 37.4 ± 1.1 a
Opalescence Boost
1 week 0.2400 ± 0.1180 B 37.4 ± 0.5 a
Immediate 0.2083 ± 0.1763 B 31.5 ± 1.6 b
Total Blanc One Step
1 week 0.3930 ± 0.1790 C 31.4 ± 3.4 b
Immediate 0.2499 ± 0.1298 B 32.5 ± 1.9 b
Whiteness HP Blue
1 week 0.0497 ± 0.0378 A 13.0 ± 0.5 c
a
Identical uppercase and lowercase letters in each column indicate statistically similar means (two-way analysis
of variance and Tukey test, α=0.05).
b
Control group was statistically different when compared to all groups (two-way analysis of variance and Dunnet
test, α=0.05).
570 Operative Dentistry

Table 4: Means (± Standard Deviations) pH Values of the Thickener, Hydrogen Peroxide and Initial
in Different Experimental Groupsa
Experimental Groups Bleaching Time Thickener Hydrogen Peroxide Mixture

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Immediate 13.5 ± 0.1 2.2 ± 0.0 7.5 ± 0.3 A
Opalescence Boost
1 week — — 6.7 ± 0.4 B
Immediate 9.6 ± 0.0 3.8 ± 0.0 6.7 ± 0.3 B
Total Blanc One Step
1 week — — 4.5 ± 0.2 C
Immediate 11.6 ± 0.0 1.6 ± 0.0 8.1 ± 0.2 A
Whiteness HP Blue
1 week — — 6.6 ± 0.2 B
a
Identical uppercase letters indicate statistically similar means (two-way analysis of variance and Tukey test, α=0.05).

observed a significant difference between Total Blanc
One Step (DFL) and Whiteness HP Blue (FGM).
However, for Whiteness HP Blue (FGM), a significant
decrease in HPP occurred, whereas for Total Blanc One
Step (DFL), a significant increase in HPP was observed
when 1-week results were compared to immediate
results (Table 3; p<0.001).
pH Measurement of Bleaching Gels
Usually, all thickener bottles showed an alkaline pH,
but a hydrogen peroxide bottle showed an acidic pH
(Table 3). In the immediate time, Total Blanc One
Step (DFL) showed a significant and lower pH in
comparison with other bleaching gels (Table 4; Figure
1; p<0.001). After 1 week, all bleaching gels showed a
significant decrease in their pH (Figure 1; p=0.00003).
When all bleaching gels were compared at different
times, a significant decrease in pH was observed only
for Total Blanc One Step (DFL; Figure 1; p<0.001).
Viscosity of the Bleaching Gel
The thixotropic nature of the bleaching gels are
shown in Figure 2. Regarding Whiteness HP Blue
(FGM), we observed a higher oscillation during the
immediate time, a fact that could be clinically observed
due to the large number of bubbles generated by this
product. One week after mixing, a significant decrease
occurred in the number of bubbles formed, which
could be confirmed by the decrease in the oscillation
for Whiteness HP Blue (FGM) in Figure 2. When
we compared the two evaluation times, Whiteness
HP Blue (FGM) showed a decrease in thixotropy 1
week after mixing. For Total Blanc One Step (DFL),
an increase in thixotropy occurred in the first few
minutes, followed by a decrease after 10 minutes when
compared to the immediate application time (Figure
2). After 1 week, Total Blanc One Step (DFL) showed
an increase in thixotropic behavior. On the other
hand, Opalescence Boost (Ultradent) showed similar

Figure 1. Means of pH during the time of application of the bleaching agents in different experimental groups (*).
Forville & Others: Bleaching Gels Used 1 Week After Mixing
Figure 2. Average viscosity (thixotropy in Pa s) according to the two application times in different experimental groups (*). Abbreviations:
IM, immediate after mixing; 1S, 1 week after mixing.
and uniform thixotropic behavior, even after 1 week of
mixing (Figure 2).
DISCUSSION
The results of this study showed that immediately after
treatment, all bleaching gels had BE, with values much
higher than acceptability thresholds (∆Eab > 2.7 and
∆E00 > 1.8).12 These results aligned with the expected
outcomes for bleaching gels with high concentrations
of hydrogen peroxide.21 The acceptability parameter
allowed us to evaluate the perceptibility and acceptability
of the color change by evaluators and patients.12
Despite some differences in the application time of the
present study when compared to the manufacturer’s
instructions (Opalescence Boost [Ultradent] is
indicated to be applied for 20 minutes, Total Blanc One
Step [DFL] is indicated to be applied for 30 minutes,
and Whiteness HP Blue [FGM] is indicated for 40
minutes), no significant difference, in terms of BE, was
observed among them, in agreement with the previous
literature.11 However, after 1 week, different results were
observed. Whereas Opalescence Boost (Ultradent) and
Total Blanc One Step (DFL) resulted in larger whitening
effects, Whiteness HP Blue (FGM) produced a smaller
whitening effect, thus partially rejecting the first
null hypothesis.
The difference in BE results observed for
Whiteness HP Blue (FGM) was expected because
the manufacturer recommends immediate use after
571
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mixing. This reduction in the concentration of HPP in
the bleaching gel (by 40%) may explain the variation
in the results compared to the immediate time. Studies
have demonstrated that the concentration of hydrogen
peroxide plays a crucial role in the whitening results,
with bleaching gels containing low concentrations,
requiring more time and sessions to achieve the same
color change as those with higher concentrations but
same efficacy.22 It is worth noting that Opalescence
Boost (Ultradent) and Total Blanc One Step (DFL) did
not exhibit a significant loss in the initial concentration
of hydrogen peroxide despite some variation in
concentration levels between the two materials.
BE is based on the theory that HP penetrates into
the dental structure, which is highly permeable to
certain fluids. These fluids interact with the organic
chromophores and promote a visible change in color.23
However, it is well known that whitening is not the only
outcome of this process.23 HP can penetrate the enamel
and dentin, and if it does not react with substances
inside the structure, it can reach the pulp chamber. This
may explain adverse effects, such as tooth sensitivity.24,25
Therefore, understanding the appropriate mixing time
and the effects of the bleaching gel’s properties is crucial
to avoiding these issues.
The results of hydrogen peroxide’s penetration
into the pulp chamber showed similar values for all
bleaching gels, which could be attributed to their
similar initial concentrations of peroxide.17 However,
572
the evaluation of HPP after 1 week of mixing revealed
different results. Whiteness HP Blue (FGM) exhibited
less hydrogen peroxide inside the pulp chamber. As
previously mentioned, this material had less hydrogen
peroxide available after 1 week of mixing. Because the
concentration of available hydrogen peroxide is the
most significant factor affecting HPP,26,27 we expected
Whiteness HP Blue (FGM) to exhibit less hydrogen
peroxide inside the pulp chamber.
Interestingly, despite some differences in the available
concentration of hydrogen peroxide, Total Blanc One
Step (DFL) and Opalescence Boost (Ultradent) exhibited
different behavior in terms of HPP. The differences in
the physical–chemical properties, particularly pH and
viscosity,28,29 may help explain these results. Although
Opalescence Boost (Ultradent) and Total Blanc One
Step (DFL) had an alkaline/neutral pH in immediate
time, after 1 week of mixing, only Opalescence Boost
(Ultradent) maintained an alkaline/neutral pH,
whereas Total Blanc One Step (DFL) exhibited a
significantly lower pH of approximately pH = 5.
Recent studies have demonstrated that the presence of
residual peroxide in the pulp chamber may be attributed
to other factors, such as the bleaching gel’s pH and the
substances of which it is composed.30,31 The bleaching
gel’s pH is associated with the dissociation of hydrogen
peroxide (pKa=11.5) in the dental tissues. An alkaline
pH is favorable for the formation of free radicals and
increase of ions available for the bleaching process, thus
avoiding the presence of excess peroxides in the pulp
chamber.10,29,30,32 However, the bleaching gel’s acidic
pH can lead to greater demineralization of the dental
surface when applied, which can produce changes
in the enamel’s surface morphology.33 In addition,
undissociated hydrogen peroxide may reach the pulp
chamber in an acidic pH because less decomposition
of hydrogen peroxide occurs in free radicals.11 These
factors can influence the passage of hydrogen peroxide
to the pulp chamber and may be responsible for the
higher pain observed with more acidic bleaching gels.34
Another factor that can interfere with HPP is viscosity,
which is a physical property that can be related to the
amount of thickener and the strength of the molecular
interaction inside the product.28 In the case of in-office
bleaching gels, a viscosity is necessary that allows
for the application of the gel onto the dental surface
to avoid leakage and burns in the soft tissues.28,35 We
observed that all in-office bleaching gels showed
an adequate viscosity to be applied clinically when
mixed immediately. However, the viscosity evaluated
1 week after mixing, in association with a lower pH,
supports the recommendation to not use the gel after
1 week, as provided in the manufacturer’s instruction.
Operative Dentistry
Some studies have shown that a lower viscosity in the
bleaching gel may result in a higher penetration of
peroxide into the tooth structure.28,35
Among all in-office bleaching gels evaluated,
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Opalescence Boost (Ultradent) was the only one
capable of maintaining all properties evaluated,
whether measured immediately or after 1 week. To the
best of the authors’ knowledge, this is the first study to
show that Opalescence Boost (Ultradent) can be used
after 1 week, in accordance with the manufacturer’s
recommendation. It is worth mentioning that the
manufacturers of Whiteness HP Blue (FGM) and Total
Blanc One Step (DFL) did not recommend saving and
using their products 1 week after mixing. Therefore, it is
important to reinforce that these materials should not be
used outside of the manufacturers’ recommendations.
As previously mentioned, the alkaline pH of
Opalescence Boost (Ultradent) was likely responsible
for the low pH amount of hydrogen peroxide inside
the pulp chamber. Additionally, the presence of
remineralizers in the gel may have helped protect
the dental tissues and reduce HPP. However, it is
noteworthy that Opalescence Boost (Ultradent)
maintains not only the pH and viscosity but also the
amount of hydrogen peroxide available to react with
the dental structure after 1 week. This behavior is not
fully understood, but differences in the thickener and
hydrogen peroxide compared to other gels may provide
insight. Further studies are necessary to investigate
these hypotheses more thoroughly.
When measuring the pH separately for the thickener
and hydrogen peroxide, we observed that different
commercial brands have a different pH for each
component. More acidic gels were introduced on the
market to prolong the product’s shelf life29 because the
product remains more stable in an acidic medium.
Manufacturers are able to maintain this characteristic
before mixing, allowing commercial products to have
the same shelf life as the older generation of in-office
bleaching gels.10 Additionally, according to a previous
study, the type of thickener significantly affects the
chemical changes of the gel during application.28
In most cases, the gel component that contains
hydrogen peroxide is acidic because peroxide
decomposition is reduced in an acidic environment,
making this component stable for storage.29 Among
the evaluated products, Opalescence Boost (Ultradent)
has the most alkaline thickener gel and the most acidic
peroxide, which may have contributed to better gel
properties. Meanwhile, Total Blanc One Step (DFL)
and Whiteness HP Blue (FGM) showed a smaller
difference between the pH of the thickener and the
peroxide. The process that occurs in the change of
Forville & Others: Bleaching Gels Used 1 Week After Mixing
viscosity in the bleaching gels is still unclear because
multiple possibilities could influence it. Researchers
should investigate this matter.
It is important to mention that all bleaching gels were
stored in a refrigerator because low temperatures help
maintain their properties.30 However, the results of
the present study cannot be extended to situations in
which storage is carried out under various temperature
conditions. Future studies should be conducted to
evaluate the behavior of the properties at various
temperatures. Although Opalescence Boost (Ultradent)
had good results, it is important to note that this is an in
vitro study; therefore, future clinical studies are needed
to understand this bleaching gel’s behavior with respect
to clinical parameters and side effects.
CONCLUSIONS
Although all bleaching gels showed similar efficacy
and physical–chemical proprieties immediately after
mixing, only Opalescence Boost (Ultradent) was able
to maintain its pH and viscosity 1 week after mixing,
as the manufacturer recommended. This ensures that
its bleaching potential is retained even after a certain
amount of time has passed provided it is kept under
ideal conditions.
Acknowledgments
The authors would like to thank DFL (Brazil), FGM Dental
Products (Brazil), and Ultradent (Brazil) for the donation
of the bleaching gel used in this investigation. This study
was partially supported by the National Council for
Scientific and Technological Development (CNPq) under
grants 304817/2021-0 and 308286/2019-7 and Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior—
Brasil (CAPES)—Finance Code 001. The authors are
grateful for the technical support of the characterization
laboratory physicochemical properties of State University of
Ponta Grossa.
Regulatory Statement
This study was conducted in accordance with all the
provisions of the human subjects oversight committee
guidelines and policies of ethical committees of State
University of Ponta Grossa, PR, Brazil. The approval code
issued for this study is 453.323.
Conflict of Interest
The authors of this article certify that they have no proprietary,
financial, or other personal interest of any nature or kind in
any product, service, and/or company that is presented in
this article.
573
(Accepted 20 May 2023)
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