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Volume 21
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e227878

Original Article

Braz J Oral Sci. 2022;21:e227878http://dx.doi.org/10.20396/bjos.v21i00.8667878

1 Department of Restorative 
Dentistry, Faculty of Dentistry, 
University of North Parana – 
UNOPAR, Londrina-PR.

2 Department of Restorative 
Dentistry, Faculty of Dentistry, 
Paranaense University– UNIPAR, 
Londrina-PR.

Corresponding author:  
Dr. Murilo Baena Lopes,  
University North of Parana - 
UNOPAR, Faculty of Dentistry,  
Rua Marselha, 183, Jd. Piza, 
Londrina-PR, 86041-140, Brazil,  
tel +554333717820,  
baenalopes@gmail.com 

Editor: Dr. Altair A. Del Bel Cury

Received: December 12, 2022

Accepted: April 2, 2022

Assessing the bleaching 
effect of an experimental 
stabilized chlorine dioxide 
agent used for internal 
bleaching purposes
Bruno Shindi Hirata1 , Sandrine Bittencourt Berger1 , 
Ricardo Danil Guiraldo1 , Klissia Romero Felizardo2 , 
Nádia Buzignani Pires Ramos1 , Murilo Baena Lopes1*

Aim: Assessing the intracoronal bleaching effectiveness 
of an experimental chlorine dioxide product, based on the 
walking bleach technique. Methods: Extracted bovine incisors 
were artificially stained with bovine blood and filled with zinc 
phosphate cement at cementoenamel junction level. Teeth were 
divided into 3 groups (n=10): (SP) sodium perborate added with 
distilled water, (CD) chlorine dioxide and (C) control – dry cotton 
inserted into the pulp chamber. Bleaching agents were used at 
0, 7 and 14 days. VITA Easyshadetm (∆Eab) was used to analyze 
tooth color at the 7th, 14th and 21st days, based on the CIE2000 
system. Data were analyzed through ANOVA and Tukey’s test. 
Results: There were no statistically significant differences in Δb, 
ΔE, ΔE00 and ΔWID between CD and the control group. These 
parameters have shown significant differences between CD 
and SP, which differed from the control. However, they did not 
show significant differences either in the control group or in CD 
at the 7th, 14th and 21st days. Values recorded for SP at the 7th 
day differed from those recorded at the 14th and 21st days. Δa 
has shown differences within the same group at the 7th, 14th and 
21st days. There was no difference between groups, when they 
were compared at the same day (7th and 14th). The control group 
differed from SP at the 21st day. CD did not differ from the other 
two groups. ΔL did not show differences between groups and 
times. Conclusion: Stabilized chlorine dioxide (0.07%, at pH 3.5) 
should not be used as intracoronal bleaching agent along with 
the walking bleach technique.

Keywords: Tooth bleaching. Borates. Chlorine compounds. 
Dental materials.

https://orcid.org/0000-0001-7043-8858
https://orcid.org/0000-0002-7915-3207
https://orcid.org/0000-0002-1625-3120
https://orcid.org/0000-0002-6296-4185
https://orcid.org/0000-0001-5845-8995
https://orcid.org/0000-0001-6198-7695


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1. Introduction

Tooth discoloration can result from intrinsic or extrinsic factors. Procedures such 
as polishing tooth surfaces, microabrasion, direct or indirect restoration and dental 
bleaching techniques have been developed to treat this cosmetic impairment. Dental 
bleaching treatments provide fast and expressive results without wearing the den-
tal structure away1. Consequently, they have become remarkably popular2. Internal 
bleaching is the technique most widely used in non-vital teeth due to its quite reason-
able cost, safety and pleasing aesthetic results3.

Root-filled teeth whitening was  firstly performed with hydrogen peroxide or sodium 
perborate, since the late nineteenth century4. Spasser5 (1961) has introduced a tech-
nique, according to which, sodium perborate and distillated water solution is injected 
into the pulp chamber for a few days; subsequently, this procedure is repeated until 
the enamel reaches the desired color. This technique is known as Walking Bleach. 
A simultaneous internal and external bleaching technique based on  peroxide carba-
mide was described in the literature in 19976. The adverse effect known as external 
cervical root resorption can result from certain internal bleaching techniques7. This 
issue appears to be directly linked to hydrogen peroxide diffusion in the periradicular 
area. Thus, hydrogen peroxide should not be used as treatment agent, either in asso-
ciation with sodium perborate, or not8,9.

Reports on external bleaching carried out with chlorine dioxide precursors were 
published, mainly in the UK, in the first decade of the current century. This technique 
applies sodium chloride, in association with anhydrous citric acid, to whiten tooth 
surface; its effects result from chlorine dioxide release on tooth surface10. Several 
studies have shown that this technique can harm the dental tissue due to the solu-
tion’s low pH; thus, its application should be discouraged10-14. However, chlorine 
dioxide has oxidative antimicrobial activity and it may remain used as alternative to 
other internal bleaching agents15. Nevertheless, the literature lacks studies focused 
on investigating its use in a lesser acidic stabilized formula. Therefore, the aim 
of the current study was to assess the effects of stabilized chlorine dioxide used 
as internal bleaching agent in comparison to those of sodium perborate and the  
control group.

2. Materials and Methods
Sixty (60) permanent bovine mandibular incisors with intact crowns were used in 
the current study. All organic and inorganic debris were removed from extracted 
teeth with the aid of scalers; dental elements were stored in chloramine-T solution, 
at 37°C/pH 7, for one week, for disinfection purposes. Teeth were selected and only 
the ones presenting A2 color, in compliance with the Vittapan Classical scale, were 
used. Root canals were sectioned right below the amelocemental junction, with the 
aid of diamond disk attached to a handpiece, whereas crowns were opened with 
spherical diamond burs (KG Sorensen, Barueri, SP, Brazil, model 1018HL). Tooth pulp 
was removed with the aid of scalers and the first third of the root canal was expanded 
with spherical diamond burs (KG Sorensen model 1016HL).



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All teeth were stained by using a variation of the technique described by Freccia and 
Peters16 (1982). Crowns were centrifuged together with fresh bovine blood, at high 
speed (5,000 rpm), three times a day. The aforementioned blood was replaced on 
a daily basis; the pigmentation procedure was repeated for 15 days. Then, dental 
elements were rinsed with running water for 2 minutes, and stored in saline solution.

Thirty (30) stained crowns were selected and covered with 1-mm thick protective 
base (ZnPO4, SS White, Juiz de Fora, Brazil), which was fixed 1 mm below the buc-
cal cementoenamel junction. Each crown was randomly assigned to one of the  
three groups.

The SP group was treated with sodium perborate (Odontofarma, Londrina, Brazil) and 
distilled water solution at 2:1 mg/ml. The bleaching agent was applied to the tooth 
until it filled the buccal surface inside the pulp chamber; then, a provisional resto-
ration (Coltosol, Coltrane, France) was used to seal the cavity. The bleaching agent 
was applied at treatment days 0, 7 and 14. Specimens were rinsed with distilled water 
and gently dried with triple syringe, before each bleaching exchange.

The CD group was treated with 0.07% chlorine dioxide (from the TescaClor product, 
which has 5% chlorine dioxide), in association with carbopol, and it generated a prod-
uct with pH 3.5. The gel was applied to the tooth until it filled the buccal surface inside 
the pulp cavity; then, a provisional restoration (Coltosol, Coltrane, France) was used to 
seal the cavity. The bleaching agent was applied at treatment days 0, 7 and 14. Spec-
imens were rinsed with distilled water and gently dried with triple syringe, before each 
bleaching exchange. Dry cotton was inserted into the pulp chamber of teeth in the 
control group at 0, 7 and 14 days after the procedures described above. All dental ele-
ments were stored in gauze (soaked in water), at 37°C and relative humidity of 100%. 

Easyshade intraoral spectrophotometer (Vita, Zahnfabrik H. Rauter GmbH & Co. KG, 
Bad Sackingen, Germany) was used for tooth color assessment under controlled 
conditions (artificial light and temperature of 16°C). A mold, whose opening had the 
same diameter as the tip of the spectrophotometer, was prepared with hot glue to 
ensure that the same point was assessed on each specimen. Color change (T1 and 
T2) was herein obtained through CIEDE2000 formula, which uses coordinates h (hue) 
and C (chroma): ΔE00 = [(ΔL 0 /KLSL)2 + (ΔC 0 / KCSC)2 + (ΔH 0 /KHSH)2 + RT * (ΔC 
0 /KCSC) * (ΔH 0 /KHSH)]1/2. (23) In addition, the perceptibility threshold was set at 
ΔE00 ≤ 0.8, whereas the clinical acceptability threshold was set at ΔE00 ≤ 1.8.17 Data 
were also obtained through a formula recommended by CIELAB, according to which, 
coordinates L (indicating brightness), a and b (red-green and yellow-blue, respec-
tively) interact as follows: ΔEab = [(ΔL) 2 + (ΔA) 2 + (ΔB) 2] 0.5. Color variations Δa, Δb and 
ΔL, as well as the Whiteness index ΔWID, were calculated

18.

Data were subjected to Kolmogorov-Smirnov test, which has shown that they were 
within the normality range; then, they were subjected to analysis of variance (ANOVA) 
and to Tukey’s test, at 5% significance level.

3. Results
There were no statistically significant differences in color measurement (Δb, ΔE and 
ΔE00) and whiteness index (ΔWID) between CD and the control group. There were sig-



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nificant differences in these parameters between the CD and SP groups, which also 
differed from the control group (p<0.05). Moreover, no significant differences were 
observed either in the control group or in the CD group at the 7th, 14th and 21st days. 
However, values recorded for the SP group at the 7th day differed from those recorded 
at the 14th and 21st days. Data are shown in Table 1.

Δa did not show  differences inside the same group at the 7th, 14th and 21st days 
(Table 1). There was also no difference between groups, when they were compared 
at the same day (7th and 14th). The control group differed from SP in the analysis con-
ducted at the 21st day. CD did not differ from any of the two other groups.

The analysis applied to ΔL (Table 1) did not show differences between groups  
and times.

4. Discussion
Sodium perborate efficiency, either in association with distilled water or with hydro-
gen peroxide, has been known for years4,5,19-23. In addition, it is widely used for inter-
nal bleaching due to its reasonable cost and reliability. Sodium perborate was herein 
associated with distilled water at 2:1 g/ml, rather than with hydrogen peroxide, which 
may be linked to external root resorption3,8. However, sodium perborate, either in its 
mono-, tri- or tetrahydrate form, releases hydrogen peroxide. H2O2 release can gen-
erate different radicals and reactions in the body. Besides being related to external 
root resorption, free radicals can act on lipids, proteins and nucleic acids8. Thus, the 
discovery of a bleaching agent that does not release hydrogen peroxide would be a 
remarkable breakthrough.

Chlorine dioxide has been randomly used in beauty salons in the UK10,11. However, 
precursor elements such as sodium chlorite and citric acid are used to obtain chlorine 
dioxide, since they cause a chemical reaction that releases it right away10-12. However, 

Table 1. Effect of bleaching agents on the color change of materials subjected to bleaching treatment

Groups
Period 
(days)

Δa* Δb* ΔL* ΔE Δ00 ΔWID

Control

7 -0.15±1.14 a 0.72±5.82 a 1.22±3.02 a 5.6±3.4 c 2.87±1.84 c 0.18±8.16 c

14 -0.33±1.43 ab 0.62±5.96 a 1.72±3.20 a 5.6±3.8 c 3.14±1.93 c 0.96±8.14 c

21 -0.30±1.46 ab 1.12±6.37 a 2.32±2.80 a 6.4±3.6 c 3.40±1.77 c 0.65±8.48 c

Sodium 
Perborate

7 -1.66±1.32 abc -12.34±8.92 b 3.41±2.77 a 14.5±6.3 b 7.98±3.36 b 19.17±10.36 b

14 -2.02±0.77 bc -25.44±2.82 c 4.01±2.33 a 26.1±2.8 a 14.12±1.30 a 34.73±3.90 a

21 -2.22±1.09 c -28.84±3.40 c 6.01±2.99 a 29.7±3.8 a 16.04±1.71 a 39.95±5.37 a

Chlorine 
Dioxide

7 -0.03±1.55 a -0.09±3.28 a 1.38±4.52 a 5.3±2.3 c 3.4±1.28 c 0.87±8.22 c

14 -0.42±1.31 abc 1.21±3.75 a 2.28±5.11 a 6.2±2.3 c 3.92±1.44 c 0.81±7.45 c

21 -0.56±1.43 abc -0.59±5.17 a 3.28±4.24 a 6.9±2.6 c 4.02±1.41 c 3.63±8.17 c

Different letters indicate statistically significant difference based on Tukey’s test, at 5% significance level.



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this reaction has severe impact on hard dental tissue, since it leads to increased den-
tinal sensitivity and enables faster future staining11. Studies have shown that topo-
graphic changes take place when dental tissues get in  contact with solutions at pH 2, 
or lower10. This factor leads to enamel tissue deterioration, tooth surface roughening, 
minerals’ erosion and to changes at atomic level, including calcium leaching12.

Several studies have shown that chlorine dioxide works as bleaching agent in dis-
colored teeth10-13 and composites24. Chlorine dioxide attacks stains, be them organic 
or inorganic, at subatomic level, such as ClO2 + e- = ClO212. However, the chlorine 
dioxide used in these studies derived from anhydrous citric acid and sodium chlo-
ride. Because of the acidic nature of this association, apart from its other deleterious 
effects, the aforementioned studies have emphasized that sodium chloride should be 
recommended to be used in tooth bleaching procedures.

Chlorine dioxide pH level was selected due to its proximity to the agents10,12,  
as well as to its comparability to that of hydrogen peroxide bleaching solutions, which 
range from 2.113 to 3.720. The adopted chlorine dioxide concentration was based on 
Ablal et al.13 (2013), although the aforementioned authors used an agent produced by 
mixing sodium chloride with anhydrous citric acid, rather than a stabilized agent. The 
bleaching observed in their study was associated with  teeth dehydration caused by 
the temperature resulting from light activation; this factor could explain the imme-
diate bleaching effect observed in their study13. Thus, prolonged exposures did not 
improve bleaching effect14.

If one takes into consideration the non-difference in luminosity (ΔL) among all assessed 
groups, the significant difference in color measurement (Δb, ΔE and ΔE00) and white-
ness index (ΔWID) recorded for SP, in comparison to the CD and Control groups, can be 
associated with the potential of SP bleaching to remove stains and, consequently, to 
decrease Δa and Δb. This factor provided the analyzed specimens with stronger green-
ish and bluish color, and it has changed the perceptibility threshold.

Although the perceptibility threshold observed for the control and CD groups was 
slightly higher than 1.8 (Δ00), which clinically indicated discoloration, it happened due 
to elimination of easily-removed extrinsic pigments from bovine teeth. It is possi-
ble saying that immersing the specimens into water, similar to the control group, did 
not have effective bleaching effect like the one observed for Chlorine dioxide. The 
citric acid and sodium chlorite combination enabled fast chlorine dioxide release, in 
its gaseous form, which acted almost immediately on nearby surfaces11. However, 
the chlorine dioxide-based agent was not capable of bleaching teeth discolored with 
bovine blood in the current study. The negative result observed for the chlorine diox-
ide-based experiment was likely associated with the use of stabilized chlorine diox-
ide liquid, rather than with the active destabilized form deriving from the association 
between citric acid and sodium chloride, which, however, is unsafe for such a use10,11. 

The use of bovine dental elements in the current study was justified by common 
human teeth standardization difficulties associated with both dentin thickness and 
the incidence of reactionary dentin. Bovine incisors are similar to human teeth; thus, 
they have been used as substitutes in assays focused on assess the effectiveness of 
dental products23. Other studies have used cattle incisors in mandibular tooth whiten-



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ing tests, which included both external25 and internal bleaching26,27. According to these 
tests, specimens were pigmented with bovine blood in order to simulate discolor-
ation types often found in clinical practice16. The herein used teeth were standardized 
in A2 and randomly distributed among the analyzed groups, after staining protocol 
application. Although teeth with slight difference in color were used in the current 
study, the visually different ones were removed from it. This factor can be considered 
a limitation of the present study, since spectrophotometry-based analysis was not 
performed at that time.

Although shade evaluation could have been performed by calibrated observers, 
based on the Vita scale, in controlled environment20, the use of the spectrophotom-
eter provides lower margin of error, enables more accurate results and presents 
higher reproducibility28.

Chlorine dioxide is categorized as primary oxidizing agent. However, its stabilized 
form was not capable of bleaching bovine incisors in the current study, unlike sodium 
perborate, which is also a well-known oxidizing product. Using chlorine dioxide at 
other concentrations or pH levels, or adding certain adjuvant agents to it, may help 
achieving the desired effect.

In conclusion, keeping in mind the limitations of the current study, it was possible 
concluding that stabilized chlorine dioxide (0.07% at pH 3.5) cannot perform as inter-
nal bleaching agent.

Data availability
Datasets linked to the current article will be available upon request to the corre-
sponding author.

Disclosure statement
The authors declare no conflict of interest.

Author contribution
Conceptualization: Lopes MB, Berger SB. Data curation: Felizardo KR. 

Formal analysis: Lopes MB, Berger SB Funding acquisition: Lopes MB Investigation: 
Hirata BS, Felizardo KR

Methodology: Hirata BS, Guiraldo RD Project administration: Lopes MB Resources: 
Ramos NBP, Guiraldo RD. 

Supervision: Lopes MB 

Validation: Guiraldo RD Visualization: Berger SB

Writing - original draft: Lopes MB, Guiraldo RD, Ramos NBP, Hirata BS 

Writing - review &editing: Felizardo KR, Berger SB, Lopes MB

All authors actively participated in the discussion of the manuscript’s findings and 
have revised and approved the final version of the manuscript.



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