journal of dentistry 39s (2011) e52e56

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

Effect of bleaching on tooth discoloration from food

colorant in vitro

Shereen S. Azer a,*, Anne L. Hague b, William M. Johnston c

a
Division of Restorative and Prosthetic Dentistry, The Ohio State University College of Dentistry, 305 West 12th Avenue, Columbus,
OH 43210-1267, USA
b
Division of Dental Hygiene, The Ohio State University College of Dentistry, Columbus, OH, USA
c
Division of Restorative and Prosthetic Dentistry, The Ohio State University College of Dentistry, Columbus, OH, USA

article info abstract

Article history: Objectives: The purpose of this study was to evaluate the effect of bleaching on tooth
Received 17 June 2011 discoloration from neutral food colorant media (NFCM).
Received in revised form Methods: Freshly extracted human molar teeth (n = 32) were divided into four groups (n = 8):
9 September 2011 non-bleached treatment-NBT, non-bleached control-NBC, bleached treatment-BT and
Accepted 12 September 2011 bleached control-BC. Bleached teeth specimens were subjected to 20% carbamide peroxide
bleaching agent for 10 h. Color measurements (pre-test) were done using a non-contact
spectroradiometer. The teeth specimens were immersed in a neutral buffer solution. NBT
Keywords: and BT groups had 0.025% red food color (Red 40) added to the buffer solution. The immersed
Teeth discoloration specimens were incubated for 4 h at 37 8C. Color was again measured after immersion/
Bleaching incubation (post-test) and after subsequent polishing using plain-pumice slurry (post-
Teeth whitening polish). Differences in CIE a* (redness parameter) were analysed by repeated measures
Food colorant media ANOVA with Bonferroni-corrected t-tests for the pairwise comparisons of interest.
Extrinsic stain Results: Tooth discoloration was measured as total stain (mean difference in a* between
Intrinsic stain post-test minus pre-test), extrinsic stain (mean difference in a* post-polish minus post-test)
and intrinsic stain (mean difference in a* post-polish minus pre-test) A statistically signifi-
cant difference in the mean Da* was observed (P = 0.009) between staining treatment for
bleached and non-bleached teeth. Also, a statistically significant difference (P < 0.001) was
noted between control and treatment groups of bleached teeth that were subjected to
staining.
Conclusions: Bleaching resulted in total and extrinsic tooth discoloration by the NFCM.
Clinical significance: It might be beneficial to avoid highly pigmented foods immediately
following bleaching in order to optimize the effects of tooth whitening.
Published by Elsevier B.V.

professional cleaning to remove stains, internal non-vital

1. Introduction
Bleaching of teeth, commonly referred to as teeth whiten-
ing, has become a well established method to improve dental
aesthetics.1,2 This can be achieved by a variety of techniques,
including whitening toothpastes and similar products,
tooth bleaching, and external vital tooth bleaching.36
The mechanism of bleaching of teeth is still not fully
understood or explained in the literature nor are the effects on
enamel or dentine.1,7 Oxidizing agents, such as hydrogen
peroxide and carbamide peroxide that are commonly used in
this process, diffuse and penetrate through the tooth enamel

* Corresponding author. Tel.: +1 614 292 7467; fax: +1 614 292 9422; mobile: +1 614 546 9468.
E-mail address: azer.1@osu.edu (S.S. Azer).
0300-5712/$ see front matter . Published by Elsevier B.V.
doi:10.1016/j.jdent.2011.09.006
 journal of dentistry 39s (2011) e52e56
into the dentine and break down to produce unstable free
radicals. These free radicals react with the organic colored
pigments between the inorganic enamel apatite crystals,
forming smaller, less heavily pigmented molecules that reflect
less color, which creates a whitening effect.812 Several
reports have been published on the side-effects of bleaching.
Tooth sensitivity, gingival irritation, and aesthetic restora-
tions compromise were amongst the most recorded effects13,14
in addition to tooth demineralization and decreased enamel
hardness.1522
Unaesthetic tooth discoloration is generally caused by
intrinsic stains (e.g. heredity, fluorosis, tetracycline staining,
and ageing) and/or extrinsic stains (e.g. food pigments and
mouth rinses).2328 Intrinsic tooth discoloration due to
tetracycline staining derive their color from photo-oxidation
of the tetracycline molecules within the tooth structures.29
Bleaching of such teeth is sometimes possible if the peroxide
agent is successful in causing chemical degradation of the
unsaturated quinone-type compounds found in tetracycline
leading to less colored molecules.30
Although researchers have examined intrinsic tooth
discoloration, the mechanism of how extrinsic stains form
on the tooth surface is still not fully understood. Some
investigators have shown that anionic polyphenols, found in
highly pigmented foods/beverages such as red wine and black
tea, react with cationic salivary pellicles, forming thickened
layers of stained material that adheres to the tooth surface.31
33
Others have shown that physical and chemical forces (e.g.
van der Waals electrostatic forces, hydrophobic interactions,
and hydrogen forces) allow chromogens to adhere to the tooth
surface.26,27 Investigators have also shown that biofilm, food
debris, and metallic compounds influence the formation of
extrinsic tooth discoloration.23,26 The degree and type of tooth
discoloration have been shown to be influenced not only by
the dietary pigments or food colorant, but also by the lowered
pH medium.34
The effects of food pigments on the tooth discoloration of
bleached teeth remain unclear. The current in vitro investi-
gation was conducted to evaluate the effects of a neutral
solution with red food color on tooth discoloration of
bleached teeth, and to then determine if extrinsic tooth
discoloration could be removed by polishing, leaving behind
any intrinsic discoloration. Since bleaching has been shown
to increase enamel surface roughness,3540 it was hypothe-
sized that food colorants would increase the discoloration
of teeth that have been bleached. The null hypothesis in
this study was that a neutral food colorant medium (NFCM)
would not influence tooth discoloration of bleached teeth
in vitro.
2. Materials and methods
2.1. Specimen preparation and testing
Freshly extracted intact human third molar teeth (n = 32) were
stored in a 10% formalin solution, rinsed with distilled water,
and autoclaved. The roots of each tooth specimen were
embedded and mounted into dental stone and sealed with
clear room temperature vulcanization (RTV) silicone seal
e53
(Permatex, Inc. Salon, Ohio) in accordance with a previously
published technique.34 All teeth specimens were immersed
and stored in distilled water for 96 h at 37 8C before testing to
ensure 100% relative humidity and hydration.
Reflectance measurements were obtained with a noncon-
tact spectroradiometer (PR705 spectroradiometer) with Spec-
trawin 2.0 software (Photo Research Inc., Chatsworth, CA,
USA) and a fibre optic light cable (Model 70050; Newport
Stratford Inc., Stratford, CT, USA) connected to a Xenon Arc
Lamp (300W; Newport Stratford Inc.). An optical table (Mecom
Inc., Rising Sun, OH, USA) was used to position the spectro-
radiometer and cable to provide a 458/08 configuration. The
measuring diameter was 1 mm and the distance from the lens
to the specimen surface was 80 mm. The calibration of
reflectance was obtained via a reflectance standard (Lab-
Sphere Spectralon SRT-99-010-#3796A, Labsphere Inc., North
Sutton, NH, USA). Color calculations were executed for CIE
Illuminant D65 and CIE Standard (28) Observer using wave-
lengths of 380 nm through 780 nm at every 2 nm. For each
specimen, reflectance measurements were taken and color
calculations performed three times at pre-test, post-test, and
post-polish using the CIE a* as the chromacity parameter,
since it describes redness. The test specimens were carefully
handled with clean forceps and were kept hydrated in distilled
water between measurements.
2.1.1. Pre-test
The tooth specimens were randomly assigned to four groups
(eight specimens per group): non-bleached treatment (NBT),
non-bleached control (NBC), bleached treatment (BT), and
bleached control (BC). Carbamide peroxide bleaching agent
(Opalescence 20%, Ultradent Products Inc., South Jordan, Utah)
was applied to the exposed surfaces of the teeth for 10 h,
followed by rinsing under running distilled water. Pre-test
measurements of the enamel were then taken at a given
location on the enamel surface as previously described.34
Following initial measurement (pre-test), the specimens were
immersed into 100 mL of neutral (pH 7) buffer solution. Both
staining treatment groups (n = 16) were further subjected to
25 mL (0.025%) of red food color (Red 40). The specimens were
then incubated for 4 h at 37 8C.
2.1.2. Post-test
After the incubation period, the tooth specimens were then
gently rinsed with running distilled water, and the color
measurements were again taken at the same point on the
enamel surface as the pretest measurements.
2.1.3. Post-polish
The enamel surfaces were subsequently polished for a period
of 2 min with plain pumice slurry and a slow-speed handpiece
and rinsed with distilled water. Post-polish color measure-
ments were taken again at the same point on the enamel
surface as the pre-test and post-test measurements.
2.2. Statistical analyses
CIE a* differences were statistically analysed with repeated
measures ANOVA with Bonferroni-corrected t-tests for the
pairwise comparisons of interest.
e54 journal of dentistry 39s (2011) e52e56
3. Results
Tooth discoloration was measured as total stain (mean
difference in a* between post-test minus pre-test values),
extrinsic stain (mean difference in a* post-polish minus post-
test values), and intrinsic stain (mean difference in a* post-
polish minus pre-test values).
3.1. Total stain (post-test minus pre-test)
A statistically significant difference was found in the mean Da*
color parameter between staining treatment application for
bleached and non-bleached teeth (P = 0.009). Also, a statisti-
cally significant difference was noted between the control and
treatment groups of bleached teeth that were subjected to
staining treatment (P < 0.001) (Fig. 1).
3.2. Extrinsic stain (post-polish minus post-test)
A statistically significant difference was found in the mean Da*
color parameter between staining treatment application for
bleached and non-bleached teeth (P < 0.006). Additionally, a
Fig. 1 Mean Da* total stain (post-test minus pre-test).
Fig. 2 Mean Da* extrinsic stain (post-polish minus
post-test).
Fig. 3 Mean Da* intrinsic stain (post-polish minus
pre-test).
statistically significant difference was found between the
control and treatment groups of bleached teeth that were
subjected to staining treatment (P < 0.001) (Fig. 2).
3.3. Intrinsic stain (post-polish minus pretest)
No significant difference was found in the mean Da* color
parameter between staining treatment application for
bleached and non-bleached teeth. Similarly, no significant
difference was found in the mean Da* between the control and
treatment groups of bleached teeth (Fig. 3).
4. Discussion
The null hypothesis in this study was rejected since the mean
Da* for bleached teeth treated with a neutral food colorant
agent was significantly different than for non-bleached teeth
that were subjected to the same coloring media for both total
and extrinsic staining.
These findings were consistent with previous investigators,
who have shown that highly pigmented beverages such as
black tea and red wine caused extrinsic tooth discolor-
ation.32,33 Other similar investigations pointed out that highly
pigmented, acidic beverages not only caused tooth discolor-
ation but also dissolution of enamel.4043 Interestingly, the
results of the current investigation, which used only neutral
FCM, showed extrinsic tooth discoloration effects on bleached
teeth rather than intrinsic effects (Fig. 2). These findings
suggest that bleached teeth not only are more susceptible to
tooth discoloration in general, but more particularly to
extrinsic discoloration.
There was no significant difference in the mean Da* between
control and treatment non-bleached teeth, treated by neutral
FCM at post-test minus pre-test (total stain), post-polish minus
post-test (extrinsic), and post-polish minus pre-test (intrinsic).
These findings suggest that food colorants may not influence
intrinsic tooth discoloration of bleached teeth.
Unlike acidic pigmented solutions, highly pigmented
neutral or alkaline solutions were not found to cause
 journal of dentistry 39s (2011) e52e56
significant intrinsic tooth discoloration.34 A possible explana-
tion might be the lack of the chelating effect on hard tooth
structures by alkaline food media, resulting in lesser demin-
eralization effect.40,44 Extrinsic staining appears to be effected
by the increased surface roughness due to bleaching, which
allows subsurface penetration of colorant into the body of the
tooth.23,27 On the other hand, intrinsic tooth discoloration
appears to be caused by the dissolution of calcified tooth
structure, accompanied by an increase in enamel porosity,
with a resultant decrease in enamel hardness. This effect has
been shown especially following immersion in acidic food
media.4043 In the current study, the bleaching agent used
(Opalescence 20%) had a pH of 6.5 which is considered a fairly
neutral agent compared to other bleaching agents. This
variable could have possibly helped to control the bleaching
effect, and thus might have resulted in reduction of intrinsic
tooth staining as was reported by previous investigators.
In the current study, the use of noncontacting spectro-
radiometry to measure color change was based on previous
investigators who have shown such measurements to yield
acceptable testretest validity and reliability.4551
The scope of this in vitro study did not include investigating
the role of saliva or salivary pellicles in tooth discoloration,
although this has been shown by other investigators to play a
minor role in reducing the activity of peroxide compounds in
both tooth bleaching and staining.12,52
5. Conclusions
Bleaching resulted in the increased total and extrinsic staining
of teeth, but not intrinsic tooth discoloration. The degree and
type of extrinsic tooth stain from food pigments appear to be
greater in bleached versus non-bleached teeth. Although more
research is needed, these results suggest that it might be
beneficial to avoid highly pigmented foods immediately
following bleaching in order to optimize the effects of tooth
whitening.
Conflict of interest statement
None.
Acknowledgements
The authors wish to thank Joel Richards (dental student) for
his assistance with specimen preparation and measurements.
references
1. Kihn PW. Vital tooth whitening. Dental Clinics of North
America 2007;51:31931.
2. Luo W, Westland S, Ellwood R, Pretty I, Cheung V.
Development of a whiteness index for dentistry. Journal of
Dentistry 2009;37s:e216.
3. Joiner A. Whitening toothpastes: a review of the literature.
Journal of Dentistry 2010;38s:e1724.
e55
4. Joiner A, Pickles MJ, Matheson JR, Weader E, Noblet L,
Huntingdon E. Whitening toothpastes: effects on tooth stain
and enamel. International Journal of Dentistry 2002;52:42430.
5. Sarrett DC. Tooth whitening today. Journal of the American
Dental Association 2002;133:15358.
6. Berman LH. Intrinsic staining and hypoplastic enamel:
etiology and treatment alternatives. General Dentistry
1982;30:4848.
7. Hannig C, Zech R, Henze E, Dorr-Tolui R, Attin T.
Determination of peroxides in saliva-kinetics of peroxide
release into saliva during home-bleaching with Whitestrips
and Vivastyle. Archives of Oral Biology 2003;48:55966.
8. Bowles WH, Ugwuneri Z. Pulp chamber penetration by
hydrogen peroxide following vital bleaching procedures.
Journal of Endodontics 1987;13:3757.
9. Bowles WH, Thompson LR. Vital bleaching: the effect of heat
and hydrogen peroxide on pulpal enzymes. Journal of
Endodontics 1986;12:10812.
10. Fuss Z, Szajkis S, Tagger M. Tubular permeability to calcium
hydroxide and to bleaching agents. Journal of Endodontics
1989;15:3624.
11. McCaslin AJ, Haywood VB, Potter BJ, Dickinson GL, Russell
CM. Assessing dentin color changes from nightguard vital
bleaching. Journal of the American Dental Association
1999;130:148590.
12. Joiner A. The bleaching of teeth: a review of the literature.
Journal of Dentistry 2006;34:4129.
13. Pohjola R, Browning WD, Hackman ST, Meyers ML, Downey
MC. Sensitivity and tooth whitening agents. Journal of
Esthetic and Restorative Dentistry 2002;14:8591.
14. Anagnostou M, Chelioti G, Chioti S, Kakaboura A. Effect of
tooth-bleaching methods on gloss and color of resin
composites. Journal of Dentistry 2010;38s:e12936.
15. Akal N, Over H, Olmez A, Bodur H. Effects of carbamide
peroxide containing bleaching agents on the morphology
and subsurface hardness of enamel. Journal of Clinical
Pediatric Dentistry 2001;25:2936.
16. Basting RT, Rodrigues AL, Serra MP. The effects of seven
carbamide peroxide bleaching agents on enamel
microhardness over time. Journal of the American Dental
Association 2003;134:133542.
17. Lewinstein I, Fuhrer N, Churaru N, Cardash H. Effect of
different peroxide bleaching regimens and subsequent
fluoridation on the hardness of human enamel and dentin.
Journal of Prosthetic Dentistry 2004;92:33742.
18. Attin T, Muller T, Patyk A, Lennon A.M.. Influence of
different bleaching systems on fracture toughness and
hardness of enamel. Operative Dentistry 2004;29:18895.
19. Rodrigues JA, Marchi GM, Ambrosano GMB, Heymann HO,
Pimenta LA. Microhardness evaluation of in situ vital
bleaching on human dental enamel using a novel study
design. Dental Materials 2005;21:105967.
20. Attin T, Vollmer D, Wiegand A, Attin R, Betke H. Subsurface
microhardness of enamel and dentin after different
external bleaching procedures. American Journal of Dentistry
2005;18:812.
21. Ulukapi H. Effect of different bleaching techniques on
enamel surface microhardness. Quintessence International
2007;38:2015.
22. Azer SS, Machado C, Sanchez E, Rashid R. Effect of home
bleaching systems on enamel nanohardness and elastic
modulus. Journal of Dentistry 2009;37:18590.
23. Chan KC, Hormati AA, Kerber PE. Staining calcified dental
tissues with food. Journal of Prosthetic Dentistry 1981;46:
1758.
24. Watts A, Addy M. Tooth discoloration and staining. A review
of the literature. British Dental Journal 2001;190:30916.
25. Eriksen HM, Nordb H, Kantanen H, Ellingsen JE. Chemical
plaque control and extrinsic tooth discoloration. A review of
e56 journal of dentistry 39s (2011) e52e56
possible mechanisms. Journal of Clinical Periodontology
1985;12:34550.
26. Nathoo SA. The chemistry and mechanisms of extrinsic and
intrinsic discoloration. Journal of the American Dental
Association 1997;128(Suppl.):6S10S.
27. Hattab FN, Qudeimat MA, Al-Rimawi HS. Dental
discoloration: an overview. Journal of Esthetic Dentistry
1999;11:291310.
28. Addy M, Mahdavi SA, Loyn T. Dietary staining in vitro by
mouthrinses as a comparative measure of antiseptic
activity and predictor of staining in vivo. Journal of Dentistry
1995;23:959.
29. Mello HS. The mechanism of tetracycline staining in
primary and permanent teeth. Journal of Dentistry for Children
1967;34:47887.
30. Feinman RA, Madray G, Yarborough D. Chemical, optical, and
physiologic mechanisms of bleaching products: a review.
Practical Periodontics and Aesthetic Dentistry 1991;3:326.
31. Sheen S, Banfield N, Addy M. The effect of unstimulated and
stimulated whole saliva on extrinsic staining in vitro a
developmental method. Journal of Dentistry 2002;30:3659.
32. Joiner A, Muller I, Elofsson UM, Malmsten M, Arnebrant T.
Adsorption from black tea and red wine onto in vitro
salivary pellicles studied by ellipsometry. European Journal of
Oral Sciences 2003;111:41722.
33. Proctor GB, Pramanik R, Carpenter GH, Rees GD. Salivary
proteins interact with dietary constituents to modulate
tooth staining. Journal of Dental Research 2005;84:738.
34. Azer SS, Hague AL, Johnston WM. Effect of pH on tooth
discoloration from food colorant in vitro. Journal of Dentistry
2010;38s:e1069.
35. Bitter NC, Sanders JL. The effect of four bleaching agents on
the enamel surface: a scanning electron microscopic study.
Quintessence International 1993;24:81724.
36. Potocnik I, Kosec L, Gaspersic D. Effect of 10% carbamide
peroxide bleaching gel on enamel microhardness,
microstructure and mineral content. Journal of Endodontics
2000;26:2036.
37. McGuckin RS, Babin JF, Meyer BJ. Alterations in human
enamel surface morphology following vital bleaching.
Journal of Prosthetic Dentistry 1992;68:75460.
38. Yeh S-T, Su Y, Lu Y-C, Lee S-Y. Surface changes and acid
dissolution of enamel after carbamide peroxide bleach
treatment. Operative Dentistry 2005;30:50715.
39. Cavalli V, Arrais CAG, Giannini M, Ambrosano GMB. High-
concentrated carbamide peroxide bleaching agents effects
on enamel surface. Journal of Oral Rehabilitation 2004;31:
1559.
40. Ren Y-F, Amin A, Malmstrom H. Effects of tooth whitening
and orange juice on surface properties of dental enamel.
Journal of Dentistry 2009;37:42431.
41. Al-Dlaigan YH, Shaw L, Smith A. Dental erosion in a group
of British 14-year-old school children. Part II: influence of
dietary intake. British Dental Journal 2001;190:25861.
42. Prati C, Montebugnoli L, Suppa P, Valdre G, Mongiorgi R.
Permeability and morphology of dentin after erosion
induced by acidic drinks. Journal of Periodontology
2003;74:42836.
43. Owens BM, Kitchens M. The erosive potential of soft drinks
on enamel surface substrate: an in vitro scanning electron
microscopy investigation. Journal of Contemporary Dental
Practice 2007;8:111.
44. Reis C, De-Deus G, Leal F, Azevedo E., Coutinho-Filho T,
Paciornik S. Strong effect on dentin after the use of high
concentrations of citric acid: an assessment with co-site
optical microscopy and ESEM. Dental Materials 2008;24:1608
15.
45. Gozalo-Diaz DJ, Lindsey DT, Johnston WM, Wee AG.
Measurement of color for craniofacial structures using 45/0-
degree optical configuration. Journal of Prosthetic Dentistry
2007;97:4553.
46. Brook AH, Smith RN, Lath DJ. The clinical measurement of
tooth color and stain. International Dental Journal 2007;57:324
30.
47. Lath DL, Smith RN, Guan YH, Karmo M, Brook AH.
Measurement of stain on extracted teeth using
spectrophotometry and digital image analysis. International
Journal of Dental Hygiene 2007;5:1749.
48. Wee AG, Lindsey DT, Kuo S, Johnston WM. Color accuracy of
commercial digital cameras for use in dentistry. Dental
Materials 2006;22:5539.
49. Johnston WM. Color measurements in dentistry. Journal of
Dentistry 2009;37s:e26.
50. Chu SJ, Trushkowsky RD, Paravina RD. Dental color
matching instruments and systems. Review of clinical and
research aspects. Journal of Dentistry 2010;38s:e216.
51. Hu X, Johnston WM, Seghi RR. Measuring the color of
maxillofacial prosthetic material. Journal of Dental Research
2010;89:15227.
52. Zahradnik RT, Moreno EC, Burke EJ. Effect of salivary pellicle
on enamel subsurface demineralization in vitro. Journal of
Dental Research 1976;55:66470.