CLINICAL RESEARCH

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Duarte Nuno da Silva Marques, DMD, PhD

Auxiliary Professor, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

João Miguel Lourenço Silveira, DMD, MSc

Lecturer, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

Joana Rita Oliveira Faria Marques, DMD, MSc

Lecturer, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

João Almeida Amaral, DMD, MSc

Lecturer, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

Nuno Marques Guilherme, DMD, MSc

Lecturer, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

António Duarte Sola Pereira da Mata, DMD, PhD, FICD

Associate Professor, University of Lisbon Faculty of Dentistry,
Oral Biology and Biochemistry Group, Lisbon, Portugal

Correspondence to: Professor Duarte Nuno da Silva Marques

Faculdade de Medicina Dentária da Universidade de Lisboa, Oral Biology and Biochemistry Research Group,

Biomedical and Oral Sciences Research Unit (FCT Unit 4062), Campus Universitário 1649-003 Lisboa, Portugal;

Tel: +351 217 922 600; Fax: +351 217 957 905; E-mail: duartemd@yahoo.co.uk

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Abstract
The objective of this in vitro study was
to evaluate the kinetics of hydrogen per-
oxide (HP) release from five different
bleaching products: VivaStyle® 10%
fitted tray gel, VivaStyle® 30% in-office
bleaching gel, VivaStyle® Paint-On Plus
paint-on bleaching varnish, Opales-
cence PF® 10% carbamide peroxide
gel and Trèswhite Supreme™ 10% HP
gel. Each product was firstly titrated for
its HP content by a described method.
HP release kinetics was assessed by a
modified spectrophotometric technique.
One sample t test was performed to test
for differences between the manufac-
turers’ claimed HP concentrations and
the titrated HP content in the whitening
products. Analysis of variance plus Tam-
hane’s post hoc tests and Pearson cor-
relation analysis were used as appropri-
ate. Values of P < 0.05 were taken as
significant.
MARQUES ET AL
Titrated HP revealed an increased con-
tent when compared to the manufac-
turer’s specifications for all the products
tested (P < 0.05), although only prod-
ucts from one manufacturer produced
significantly higher results. All products
presented a significant (P < 0.05) and
sustained release of HP. However, the
product with paint-on cellulose-based
matrix resulted in significantly (P < 0.05)
faster kinetics when compared to other
products tested. These results are con-
sistent with manufacturers’ reduced rec-
ommended application times.
The results of this study suggest that
modifying the matrix composition may
be a viable alternative to HP concentra-
tion increase, since this may result in
faster release kinetics without exposure
to high HP concentrations.
Key words: dental whitening, hydrogen
peroxide release, in vitro, kinetics
(Eur J Esthet Dent 2012;7:344–352)
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 CLINICAL RESEARCH
Introduction
Clinicians have performed teeth whiten-
ing procedures since the 19th century.1
However, during the last two decades
an increase in health awareness in our
society has led to a drop in dental de-
cay, an increase in life expectancy and
has given rise to a demand for a better
quality of life, including patients’ expec-
tations in oral esthetics.2 Therefore, cos-
metic dentistry has become widespread
and tooth whitening is now viewed as a
routine procedure, which has acquired
substantial popularity.
Hydrogen peroxide (HP) alone or
generated from other molecules like per-
oxide carbamide (CP) or percarbonate
has become the most often employed
whitening agent.3,4 As a response to the
demand for faster and easy-to-use whit-
ening treatments, most dental materials
manufacturers have started selling not
only different bleaching agents, but also
new types of whitening products. These
products, such as paint-on formulations
or fitted trays are clinician supervised,
but can be directly applied over dental
enamel by patients at home. Indeed, the
convenience of paint-on formulations
and fitted trays is driving the whitening
market. Moreover, in the most recently
marketed paint-on products, manufac-
turers are responding to a second trend
by suggesting extremely short applica-
tion times ranging from 30 to 10 minutes.
This study was designed with the aim
of analyzing different concentrations and
formulations used in different bleach-
ing techniques; one fitted tray gel (for
patients’ application under supervision
of a clinician), one clinician-supervised
bleaching gel, one paint-on formulation
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and two different 10% carbamide per-
oxide gels designed for custom fitted
tray application (one for day use and the
other for overnight use). Additionally, in
vitro HP release kinetics was compared
with the manufacturers’ recommended
application times. The primary hypoth-
esis considered in this study was that
recommended application times do in
fact correlate with different kinetics of
peroxide release among the products.
Studies on clear and precise HP re-
lease kinetics are lacking. Thus, the
purpose of this in vitro study was to de-
termine the initial HP contents and the
HP release kinetics. The null hypotheses
are:
"!There are no differences in titrated
HP content compared to the manu-
facturers’ claimed concentrations in
the whitening products tested in this
study.
"!The HP release does not correlate
with the manufacturers’ recommend-
ed application times of the whitening
products used in this study.
Materials and methods
Initial titration
The bleaching products (Table 1) were
firstly titrated for their HP content by a
described method using cerium as a ti-
trating agent.5,6
The samples were weighed on an
analytical balance with ± 0.1 mg sensi-
tivity (Denver TR204, Denver Instrument
Company, Denver, CO, USA) in a 600 ml
beaker and the weight recorded. A vol-
ume of 225 ml of deionized water was
added and the samples were stirred with
 MARQUES ET AL

Table 1 Whitening products tested in this study

Label Corresponding
Whitening agent Manufacturer Batch no.
CP% HP%

Ultradent Products, B07NJ

Opalescence PF® 10% 3.62%
Utah, USA B08KG

VivaStyle® Paint Ivoclar Vivadent, G27174

6%
On Plus 6% Schaan, Liechtenstein NRG5051

HL1004
Ivoclar Vivadent, GL1020
VivaStyle® 10% 10% 3.62%
Schaan, Liechtenstein HL1015
HL1018

B2KHW
Trèswhite Supreme™ Ultradent Products,
B2K2D 10%
by Opalescence® Utah, USA
B2JRM

Ivoclar Vivadent,
VivaStyle® 30% HL1031 30% 10.86%
Schaan, Liechtenstein

a stir bar over a stir plate (200–300 rpm)
for approximately 30 minutes or until
the gel was completely dissolved. Af-
ter adding 25 ml of sulfuric acid 5.0 M
(Pancreac Química, Barcelona, Spain)
to all the samples, the beakers were
covered using a glass dish, then stirred
for approximately 5 minutes. Ten drops
of Ferroin indicator solution (Riedel-de-
Haën, Seelze, Germany) was added to
each sample by a dropper. The solution
changed from transparent to deep red.
Each sample was titrated with a 0.1 M
cerium (IV) sulfate tetrahydrate (CS)
(Pancreac Química, Barcelona, Spain)
solution. As the titrant was added, the
sample changed from deep red to pale
blue at the equivalence point. The re-
lationship between the volume of CS
used in the titration of the sample and
the sample weight (SW) was expressed
in a formula and used to determine the
percentage of HP (% by weight), which
was recovered in each sample:
% HP (% by weight) = CS volume (ml) x 0.17
SW (g)
In the bleaching products whose ac-
tive agent was carbamide peroxide, the
conversion was made using the follow-
ing formula, as previously described by
Matis et al7:
% CP (% by weight) = % H2O2
0.362
The initial concentration of each batch of
the whitening products was determined
until a minimum of three replicates with
results within an interval of 0.5% was ob-
tained.7
Sample preparation
A contact Petri dish (Sarstedt, Nümbre-
cht, Germany) of 6 cm diameter was
placed on an analytical balance (Den-
ver Instrument Company, Denver, CO,

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 CLINICAL RESEARCH

NH
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11 Manufacturer
10 Titrated
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9
8
7
6
5
Fig 1 Bar chart showing
4
the mean (± SEM) initial
3
hydrogen peroxide content
2
of the whitening products
1
0 used in this study. The man-
ufacturers’ claimed concen-
OPL

74


74


PO+

584
trations are also shown for
comparison. * = P < 0.05
when compared to claimed
manufacturer content.

USA). Subsequently, about 500 mg of Spectrophotometric

the whitening product was placed in the measurements
Petri dish with the use of a microbrush
or a spatula. The method for measuring peroxide con-
The exact net weight of the whitening tent used in this study is a modification
product was recorded within 1 minute to of the original technique described pre-
prevent any significant solvent evapo- viously by Childs and Bardsley.8 Brief-
ration. Immediately, 5.0 ml of deionized ly, a phosphate buffer solution (PBS)
water was poured onto the edge of the was made by mixing 87.9% vol/vol of
Petri dish, and the samples were shaken 0.05 M potassium dihydrogenophos-
on the laboratory shaker at 50 rpm. At phate (KH2PO4) solution (Merk, Darm-
different periods of time, after the addi- stadt, Germany) to 12.1% vol/vol of
tion of water, samples of 100 µl were tak- 0.05 M disodium hydrogen phosphate
en, using a micro-pipette (Gilson, Villiers (Na2HPO4) solution (Chemische Fab-
le Bel, France), and analyzed in a He- rik Budenheim, Budenheim, Germany)
lios alpha spectrophotometer (Geneq, and then pH was set at 6.0. A solution of
Montreal, Canada) for HP content by a 2.08 mM of 2,2’-azino-bis (3-ethylbenzo-
peroxidase-based colometeric method. thiazoline-6-sulfonic acid) diammonium
An initial measurement with deionized salt reagent (ABTS) (Fluka Chemie, Bu-
water was performed to set up the base- chs, Switzerland) in PBS and a horse-
line. Release of the HP was recorded up radish peroxidase solution (HRP) (Fluka
to twice the recommended application Chemie, Buchs, Switzerland) of 400 U/
time for each product tested. ml in PBS were also made. After prepar-

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ing the solutions, several 4 ml cuvettes
(Nuova Aptaca, Canelli, Italy) were filled
with 3.84 ml of ABTS solution and 80 µl
of HRP solution, one for each collection
time. With the use of a micropipette,
80 µl of the water samples were collect-
ed and placed in the corresponding cu-
vette. In order for the components to mix
properly, the cuvette was covered with
parafilm (Pechiney Plastic Packaging
Company, Chicago, IL, USA) and hand
stirred. Finally, the cuvette was placed
in a 30ºC thermostat cell holder of the
spectrophotometer, and after 2 minutes,
an absorption reading was recorded at
420 nm.
Calibration curve
At least 7 solutions of different HP con-
centrations were made out of a HP
standard solution (hydrogen peroxide
solution 3% by weight in H2O, Sigma-
Aldrich, Buchs, Switzerland) in order to
get an absorption in the range of 0.1 to
1.5. Only confidence coefficients > 0.99
were accepted as valid.
Statistical analysis
Results are expressed as mean ± stand-
ard error of mean (SEM) of mg of HP
(100 mg of total product)–1, or percent
of total active product delivered as ap-
propriate.
One sample t test was performed to
test for differences in titrated HP content
compared to the manufacturers’ claimed
concentrations in whitening products.
The analysis of variance (ANOVA) plus
Tamhane’s post hoc tests were used to
test for differences in HP release among
different products and Pearson corre-
MARQUES ET AL
lation analysis was used to test mean
differences and correlation between the
variables HP percentage and time of re-
lease. Values of P < 0.05 were taken as
significant.
Results
Initial titration
The HP concentrations claimed by the
manufacturer and the values obtained
from cerium-derived titration are shown
in Figure 1. Titration values presented a
higher content for all the products tested
compared to the the claimed values, but
were found to be only significantly dif-
ferent from the claimed values for the
Ivoclar Vivadent systems (VivaStyle®
Paint on Plus 6% (PO+), Vivastyle® 10%
(VS10%), and VivaStyle® 30% (VS30%)
(one sample t test against claimed value).
The Ultradent systems (Opalescence
PF® (OPL) and Trèswhite Supreme™ by
Opalescencec® (TWS)) presented no
significant differences between titrated
values and the claimed values.
In Figure 2, the kinetics of HP release
are shown, displayed with the percent-
age of HP released to the aqueous me-
dium per 100 mg of whitening agent over
time, for a total application time that was
double the manufacturers’ recommend-
ed application time. There was a sus-
tained increase in the release of HP over
time for all products tested. A Pearson
correlation analysis between the varia-
bles HP percentage and time was found
to be extremely significant (P < 0.01) for
all products tested. The paint-on varnish
(Fig 2a) produced the fastest HP kinet-
ics release with PO+ releasing 100% of
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 CLINICAL RESEARCH

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100 80
80 60
60
40
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PO+ 20 TWS
20
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20
30
40
50
60
70
80
90
100
110
120
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10

20

30
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80 80

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180

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450

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Fig 2 Original chart recording of mean ± SEM for percentage of hydrogen peroxide release times (initial
content times min)-1 for the whitening products used in this study.

its HP content at both 10 and 20 min- and assisted bleaching respectively.

utes, which corresponds to the recom- Only the paint-on formulation (Fig 2a)
mended and double the recommended and the custom fitted tray formulation
application times respectively. intended for overnight use (Figure 2e)
Both VS10% and VS30% (Figs 2c and presented 100% HP release for the
2d) presented similar kinetics in vitro but manufacturers’ recommended applica-
are intended for different whitening tech- tion time. The fitted tray system (Fig 2b),
niques – custom fitted tray application the dentist supervised bleaching gel

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(Fig 2c) and the 10% CP intended for
one-hour use (Fig 2d) failed to release
the entirety of HP quantity in the applied
product. Student t test for testing mean
differences between the amount of HP
applied and the amount of HP released
during recommended application time
was found to be extremely significant
(P < 0.01) for these three products.
Discussion
Since 1989, with the introduction of the
carbamide peroxide nightguard vital
bleaching technique,9 at-home bleach-
ing has become increasingly popular.
This technique, which was initially de-
signed for overnight use, has been pro-
gressively shortened in its application
time during the last few years. This is
mainly due to an increased appeal for
dental procedures and demands from
the patients for faster and less time-con-
suming bleaching protocols.
The current concept is that HP con-
centration of the bleaching materials
correlates with the time period in which
clinical dental whitening is achieved.10
However, several studies have found
that similar products yield different
rates to accomplish dental whitening
results.11-14 One of the factors, which
could also be linked with treatment dura-
tion and efficacy, is the intrinsic variability
of the HP content derived from manufac-
turing and storing conditions. Therefore,
one of the aims of this study was to as-
sess the real HP concentration of differ-
ent whitening products and determine
the existence of any variability among
different batches. This was achieved by
analyzing deviations from manufactur-
MARQUES ET AL
ers claimed HP concentration with the
described titration method. The titration
results show that all the products used
in this study presented mean titrated
values above claimed manufacturers
concentration values. The use of higher
concentrations of HP than claimed in the
whitening products may be a possible
way used by the manufacturers to coun-
teract potential degradation of the HP
during shipping and storage. This fact,
among others, can contribute to a differ-
ence in clinical outcomes.
On the other hand, recommended
application times in different products
range from 10 minutes to 8 hours with-
out supporting kinetics studies. The re-
sults of the present study suggest that
the recommendation for overnight use
of carbamide peroxide nightguard vital
bleaching techniques is artificial and
meant for patients’ comfort only.
The results of the HP release kinet-
ics agree with the initial hypothesis that
the recommended application times do
in fact correlate with different kinetics
of peroxide release. The study results
suggest that the HP release patterns
are not only related to initial product HP
content, but also to its individual com-
position since products with similar HP
concentrations, but with different ma-
trixes present different kinetics. The
customized tray of daily use or the as-
sisted whitening product were found to
produce HP kinetics ranging from 64%
to 74% release of the initial HP content
at manufacturers application time. At
120 minutes the product intended for
overnight use (OPL) releases 99.62%
of the HP content, which correlates
with the manufacturer’s recommended
application time of 2 to 8 hours.10 The
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 CLINICAL RESEARCH

paint-on formulation yielded a very Conclusions

high release kinetics, which also cor-
relates with its recommended applica-
tion time.
If these in vitro results correlate with a
swift in vivo efficacy, this could be of clin-
ical importance since it could generate
new clinical protocols that equate with
considerably less time-consuming treat-
ments. The results of this study suggest
that modifying the matrix composition
may be a viable alternative to HP con-
centration increase, since it may lead to
faster release kinetics without increas-
ing the exposure to elevated hydrogen
concentrations.
Despite these results demonstrating
in vitro high release kinetics, in vivo ef-
ficacy studies are needed to assure the
real efficacy of these formulations.
Within the limitations of this study, we can
conclude that bleaching products matrix
modifications influence HP release kinet-
ics. Reducing the time of application in
overnight products could be an advan-
tage for patients who fail to comply with
the use of products requiring extended
time-based protocols. Moreover, the intro-
duction of different matrixes in HP-based
products with faster release kinetics
would result in reduced time-application
protocols. This could be of major clinical
importance provided that further in vivo
studies confirm their clinical efficacy.
Acknowledgment
The bleaching products used in this study were
provided by Ivoclar-Vivadent (Liechtenstein) and
Ultradent Products (USA).

References
1. Freedman G, McLaughlin G.
Tooth whitening as a treat-
ment modality. In: Hacke G
(ed). Color Atlas of Tooth
Whitening. St. Louis: Ishiyaku
EuroAmerica Inc, 1991:1–9.
2. Theobald AH, Wong BK,
Quick AN, Thomson WM.
The impact of the popular
media on cosmetic dentistry.
N Z Dent J 2006;3:58–63.
3. O’Neil MJ, Heckelman
PE, Koch CB, Roman KJ.
Antiseptics: peroxides/
permanganates. In: O’Neil
MJ (ed). The Merck index.
An encyclopedia of chemi-
cals, drugs, and biologicals.
Whitehouse Station: MERK &
Co Inc, 2006:288, 831.
4. Dahl JE, Pallesen U. Tooth
bleaching: a critical review
of the biological aspects.
Crit Rev Oral Biol Med
2003;4:292–304.
5. Mendham J, Denney RC,
Barnes JD, Thomas MK. Titri-
metric analysis: oxidations
with cerium (IV) sulphate
solution. In: Mendham J (ed).
Vogel’s textbook of quantita-
tive chemical analysis. New
Jersey: Prentice Hall, 2000:
425–427.
6. Skoog DA, West DM, Hol-
ler FJ Electrogravimetric
and colourimetric methods:
colourimetric methods of
analysis. In: West DM, Holler
FJ (eds). Fundamentals of
Analytical Chemistry. Phila-
delphia: Saunders College
Publishing, 1996:446–457.
7. Matis BA, Gaiao U, Black-
man D, Schultz FA, Eckert
GJ. In vivo degradation
of bleaching gel used in
whitening teeth. J Am Dent
Assoc 1999;2:227-–235.
8. Childs RE, Bardsley WG.
The steady-state kinetics of
peroxidase with 2,2’-azino-
di-(3-ethyl-benzthiazoline-
6-sulphonic acid) as
chromogen. Biochem J
1975;1:93–103.
9. Haywood VB, Heymann HO.
Nightguard vital bleach-
ing. Quintessence Int
1989;3:173–176.
10. Joiner A. The bleaching of
teeth: a review of the litera-
ture. J Dent 2006;7:412–419.
11. Braun A, Jepsen S, Krause
F. Spectrophotometric and
visual evaluation of vital tooth
bleaching employing dif-
ferent carbamide peroxide
concentrations. Dent Mater
2007;2:165–169.
12. Niederman R, Tantraphol
MC, Slinin P, Hayes C,
Conway S. Effectiveness of
dentist-prescribed, home-
applied tooth whitening. A
meta analysis. J Contemp
Dent Pract 2000;4:20–36.
13. Sulieman M. An overview
of bleaching techniques:
2. Night guard vital bleach-
ing and non-vital bleaching.
SADJ 2006;8:352, 354, 356.
14. Sulieman M, MacDonald E,
Rees JS, Newcombe RG,
Addy M. Tooth bleaching by
different concentrations of
carbamide peroxide and HP
whitening strips: an in vitro
study. J Esthet Restor Dent
2006;2:93–100.

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