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79]

Original Research

The effect of McInnes solution on enamel and the

effect of Tooth mousse on bleached enamel: An in
vitro study
H E Darshan, N D Shashikiran1
Department of Pedodontics, JSS Dental College and Hospital, Mysore-570 015, 1College of Dental Sciences, Davangere-577 004, India.

Abstract
Aims: To evaluate the effect of McInnes bleaching agent on the micro hardness of enamel before and after bleaching and to
evaluate the effect of G C Tooth Mousse on the bleached enamel surface for its microhardness.
Materials and Methods: McInnes bleaching solution, Casein phosphopeptide-amorphous calcium phosphate CCP-ACP
(G C Tooth mousse) artificial saliva (Dept of Oral Pathology, College of Dental Sciences, Davengere), deionized water, Vickers Micro Hardness tester (Zwick/ZHV, Germany), freshly extracted teeth, cold cure acrylic, Diamond disc (Horico - PFINGST
New jersey USA, KAVO- Germany), straight handpiece (kavo peca reta) and plastic moulds (6.5 x 2 mm). The purpose of this
study was to evaluate and compare microhardness of the sound enamel surface by Vickers Hardness Number before and after
bleaching with McInnes solution, and to evaluate the effect of casein phosphopeptide amorphous calcium phosphate (G C
Tooth Mousse) on the bleached enamel surface for its microhardness.
Statistical analysis: The data obtained from the test were subjected for statistical analysis and are presented as range, mean
and standard deviation. P value of 0.05 or less was considered for statistical significance. The changes in microhardness at
different times of assessment were analyzed using the paired t test
Results: All the samples showed decrease in the microhardness after two cycles of bleaching, though immediately after bleaching the decrease in the microhardness was not significant (P = 0.34). However, after the second cycles, it showed a significant
decrease (P<0.01) in the microhardness. After application of remineralization solution (GC Tooth mousse), the samples showed
a marginal increase in the microhardness (P<0.05) after seven days and a marked increase after fourteen days (P<0.001).
Conclusion: McInnes bleaching agent does decrease the microhardness of enamel by causing enamel demineralization and
GC Tooth mousse used in the study causes an increase in the microhardness of bleached enamel by maintaining a high gradient of calcium and phosphate ions at the enamel subsurface.
Keywords: Amorphous calcium phosphate; bleaching; casein phosphopeptides; enamel; hardness; remineralization; Vickers
hardness number.

INTRODUCTION
Tooth enamel is the most mineralized tissue of the
human body. Its composition is 96 wt% inorganic
material, 4 wt% organic material and water. Human
teeth are exposed to a different point to point pressure
during mastication. Therefore, an understanding of the
masticatory strain distribution throughout the tooth
and an understanding of the fact that the prediction
of stresses and strains are altered by dental restorative
procedures, age and disease are important.[1]
Correspondence:

Dr. H. E. Darshan, Assistant Professor, Department of

Pedodontics, JSS Dental College and Hospital,
Mysore-570 015, India. E-mail: dashi_us@yahoo.com
Date of submission: 25.12.2008
Review completed: 20.01.2008
Date of acceptance: 08.02.2008

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The technique of bleaching or whitening teeth was

first described in 1877.[2] It was in the year 1916 that
Dr. Walter Kane used hydrochloric acid to successfully
remove the fluorosis stains. In the year 1937 Ames
reported an alternative for removing fluorosis using
hydrogen peroxide instead of hydrochloric acid. Later
it was McInnes who reported a technique where
hydrogen peroxide, hydrochloric acid and ethyl ether
were used. This technique has been found to be
successful for bleaching the teeth of patients with
endemic fluorosis.[3]
Increased frequency of acid exposure in bleaching tends
to alter the total demineralization/ remineralization
amounts, resulting in significantly greater amounts
of mineral loss.[4] The presence of fluoride acts as a
remineralizing agent, by forming a calcium fluoride
layer.[5]

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Darshan and Shashikiran: Effect of McInnes solution

Casein phosphopeptide-amorphous calcium phosphate

(CPP-ACP) nanocomplexes, which have been shown to
prevent demineralization and promote remineralization
of enamel subsurface lesions in in situ caries models.[6]
The CPP, by stabilizing calcium phosphate in a solution,
maintains high-concentration of gradients of calcium
and phosphate ions into the subsurface lesion and thus
affects high rates of enamel remineralization.[7]
Various methods have been used to analyze tooth
demineralization and remineralization when acidic
bleaching is used, including techniques ranging
from direct measures of mineral gain/ loss as in
microradiography to indirect measures like iodide
permeability and surface microhardness.[8]
Based on these observations the present study was
undertaken at College Of Dental Sciences, Davengere,
to investigate the effect of McInnes bleaching on enamel
microhardness and subsequent remineralization by
CCP-ACP on bleached enamel.

MATERIALS AND METHODS

The present study was conducted in the Department
of Pedodontics and Preventive Dentistry, Collage of
Dental Sciences, Davangere, in collaboration with the
Department of Metallurgy, Indian Institute Of Science,
Bangalore.
Materials used
1. McInnes Bleaching solution
2. CCP-ACP(G C Tooth mousse)
3. Artificial saliva (Dept of Oral Pathology, College of
Dental Sciences, Davengere)
4. Deionized water
Equipment used for testing
1. Vickers Micro Hardness tester (Zwick/ZHV,
Germany)
Materials used for preparation of samples
Freshly extracted teeth
Diamond disc (Horico horico- PFINGST New jersey
USA)
Straight handpiece (kavo peca reta)
Plastic moulds(6.5 2mm)
Cold cure acrylic ( DPI)
Materials used for preparing artificial saliva
Calcium chloride 0.22g/l
Sodium phosphate 1.07g/l

Sodium bicarbonate 1.68/l

Sodium azide 2g/l
Distilled water one liter

Materials used for remineralization

GC tooth mousse (Recaldent) which contains: CPP-ACP,
Glycerol, Silicon dioxide, Zinc oxide, cmc-na,propylene
glycol, titanium dioxide, phosphoric acid, guargum,
sodium saccarien, ethyl p-hydroxybenzoate, butyl
p-hydroxybezoate, propyl p-hydroxybenzoate,
Magnesium oxide,D Sorbitol, Xylitol and Pure water
Inclusion criteria
Only permanent non carious teeth that were indicated
for extraction were included in this study.
Preparation of specimens
Freshly extracted teeth were selected. The teeth were
cut sagittally, using diamond disc (Horico horicoPFINGST New jersey USA) and the buccal surface
were impregnated in the cold cure acrylic resin facing
upwards. Using the plastic moulds the resins were
made into pellets (6.5mm x 2mm thickness). A total
of ten specimens were made, which were then kept in
artificial saliva to prevent dehydration.[8] The samples
were rinsed in water and dabbed dry with absorbent
paper before subjecting them for baseline hardness
test.[9]
Procedure for microhardness test
For microhardness testing the Zwick Roell Indentec
(Zwick, Inc, Germany) was used for measuring Vickers
hardness test. The tests were carried out according to
the manufacturers instructions.[10] The test specimens
were placed on the stage of the tester and stabilized.
Then area to indent was selected by focusing with 10x
objective lens. After this, the test was carried out where
the indentations were made with a rate of 100g load for
30 seconds, never close to any edge of the specimen.
The indentation formed was viewed and measured on
the display monitor with 10x objective lens. The average
microhardness of the specimen was determined from
two indentations to avoid any discrepancy, since the
enamel surface has a curvature. The procedure was
repeated for all the ten specimens.
Preparation of bleaching agent
McInnes bleaching solution consists of a mixture of
1ml of 36 % hydrochloric acid, 1ml of 30 % hydrogen
peroxide and 0.2 ml of anesthetic ether which is mixed
in the ratio of 5:5:1. The mixture is prepared freshly in
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Darshan and Shashikiran: Effect of McInnes solution

a dappen dish before each application.[11]

The bleaching agent thus prepared bleaching agent was
applied to the enamel surface using a cotton applicator
for five minutes (first cycle of bleaching). It was then
washed under deionized water, damped dry with
absorbent paper and then subjected for microhardness
test. After this, the samples were stored in artificial saliva
for 24 hours to prevent dehydration. Again after 24 hours
the second application of bleaching agent was carried
out as described earlier and the microhardness values
were recorded. Then the samples were applied with
remineralizing agent (GC Tooth mousse) for fourteen
days (second cycle of remineralization).[12,13,14]
Application of GC tooth mousse
GC- Tokyo Tooth mousse (Recaldent) was applied with
cotton applicator tips on the post bleached samples,
everyday for seven days with minimum application time
of three minutes. The samples were then washed under
deionized water, stored in artificial saliva for seven days
(first cycle of remineralization) after which the samples
were tested for microhardness and the values were
recorded as described earlier. Following this, GC Tooth
mousse was applied for seven more days and at the
end of fourteen days (second cycle of remineralization)
the samples were subjected for microhardness testing
using the same procedure as described earlier.
The recorded values are subjected to statistical
analysis.
Results
The data obtained from the following test were
subjected for statistical analysis and are presented
as range, mean and standard deviation [Table 1]. A

P value of 0.05 or less was considered for statistical

significance. The changes in microhardness at different
times of assessment were analyzed using the paired
t test
[Table 1] shows the mean, P value and percentage
changes of microhardness from the baseline. The first
comparison was between the baseline and the first
cycle of bleaching. The mean and standard deviation
at baseline was 297.2 17.6 and for the first cycle of
bleaching was 296.2 17.3. When these values were
compared it showed a decrease by a small margin of
0.3%, which was not significant (P = 0.034). The next
comparison was between the baseline and the second
cycles of bleaching carried out 24 hours later. The
mean and standard deviation after the second cycle
of bleaching was M=290 SD= 16.6, which showed
decrease by 2.3% and was highly significant (P<0.001).
The third comparison was between the baseline and
the first cycle of remineralization (M=304.3, SD=18.5),
which showed increase by 2.2% and was significant (P
value <0.05). The fourth comparison was between
the baseline and the second cycle of remineralization
(M=307.0 17.4) which showed 3% increase from the
baseline values which was significant (P < 0.05).
[Table 2] shows the mean, P value and percentage
changes of microhardness from the first cycle of
bleaching. The values obtained after first cycle of
bleaching (M=296.2, SD= 17.3) were compared
with the values of the second cycle of bleaching
(M= 290.0, SD=16.6) which showed 2% decrease
and was significant (P< 0.05). The next step was to
compare the values of first cycle of bleaching with first
cycle remineralization (M=304.3, SD= 18.5) which
showed 2.5% increase in Vickerss hardness number

Table 1: Mean, P value and percentage changes of microhardness from baseline

Mean SD
P value
Percentage changes from
baseline 297.2 17.6

1st Cycle of bleaching

2nd Cycle of bleaching

1st Cycle of remineralization

2nd Cycle of remineralization

296.2 17.3
= 0.034 NS
0.3% Decrease

290 16.6
<0.001 HS
2..3% Decrease

304.3 18.5
<0.05 S
2.2% Increase

307 17.4
<0.05 S
3% Increase

SD = Standard deviation; S= Significant; HS= Highly significant

Table 2: Mean, P value and percentage changes of microhardness from the 1st cycle of bleaching

Mean SD
P value
Percentage change from first cycle of
bleaching 296.2 17.3

2nd cycle of bleaching

1st cycle of remineralization

2nd cycle of
remineralization

290 16.6
P<0.05 S
2% decrease

304 .3 18.5
P<0.01 S
2.5% increase

307 17.4
P<0.01 S
3.24% increase

S= Significant; SD= Standard deviation

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Darshan and Shashikiran: Effect of McInnes solution

Table 3: Mean, P value and percentage changes of microhardness from the 1st cycle of remineralization
2nd cycle of remineralization
Mean SD
P value
Percentage change from first cycle of remineralization 304.3 18.5

307 17.4
<0.05 S
1%

S= Significant; SD= Standard deviation.

Figure 1: Application of bleaching agent and application of

GC tooth mousse

Figure 3: Storage and Zweck indentor

Figure 2: Micro hardness Indentations

(VHN), and was significant (P <0.01). When the first

cycle of bleaching was compared with 2nd cycle of
remineralization (M= 307, SD = 17.4), it showed an
increase by 3.24% and was significant (P < 0.01).

DISCUSSION

[Table 3] shows the mean, P value and percentage

changes of microhardness changes between first
cycle of remineralization and the second cycle of
remineralization. The comparison between the first
cycle of remineralization (M=304.3 SD= 18.5)
and the second cycle of remineralization (M=307
SD 17.4) showed increase in the VHN by 1% and was
significant (P<0.05)

Many patients are keeping their teeth for longer time

but dislike the physiological darkening process that
follows the laying down of secondary dentine.[2,15] The
use of chemical agents to remove certain intrinsic
stains from the enamel or the dentin of vital teeth is
not new. Hydrochloric acid and hydrogen peroxide
have been used alone and in combination to remove
stains associated with fluorosis, tetracycline and more
recently trauma related injuries.[16]
It was in the year 1966 that McInnes reported a
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Darshan and Shashikiran: Effect of McInnes solution

technique that combined hydrochloric acid and

hydrogen peroxide to remove fluorosis stains. He
used a solution of five parts 30% hydrogen peroxide,
five parts 36% hydrochloric acid and one part ethyl
ether and applied the solution with a cotton wrapped
toothpick to the areas of the teeth affected by the
stain. After 10 to 15 minutes the teeth were washed
with water and neutralized with a sodium bicarbonate
paste.[17]
In the present study McInnes bleaching agent was
selected, as it was most commonly used in clinical setup
for treating dental fluorosis. McCoy SE[3] has reported
that McInnes bleaching technique were specifically
recommended for the treatment of teeth exhibiting
endemic dental fluorosis because of its superficial nature,
easy manipulation and its quality of being less expensive
when compared to other commercially available agents
like carbamide peroxide.[18] All the samples used in
the study showed no change in the microhardness
immediately after first cycle of bleaching. Around 0.3%
reduction in the microhardness was seen which was
not significant (P=0.034).
When the bleaching procedure was repeated after
24 hours (the second cycle of bleaching) there was
significant reduction in the VHN, which shows 2.3%
reduction in the microhardness, which was significant
(P<0.001).
In many studies[19,20] artificial saliva was used for
storing the specimens in between the bleaching cycles,
because it is believed that artificial saliva contributed
to a slight increase in the microhardness, after
demineralization.
The amount of demineralization of enamel by
bleaching was assessed using a microhardness tester.[8]
Microhardness test was selected mainly because it was
economical compared to the other tests and was easily
available (Indian Institute of Science, Bangalore).
Microhardness measurement of tooth material can be
done in three different ways like; Knoops hardness
number (KHN), Vickerss hardness number (VHN) and
Brennels hardness number (BHN). In the present study
Vicker hardness number was chosen over Knoops
because a square shape of indent obtained in VHN was
easy and more accurate to measure. Even the minute
changes in the square shape indent obtained after
the test can be easily detected, where as the Knoop

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hardness test gave rhomboid shape indentation with

opposing surfaces parallel to each other and detecting
the error was difficult.
According to Chow LC, Takagi S,[21] both KHN and VHN
have reported approximately the same value. The
average hardness value for enamel is in the range from
270 to 350 KHN range or from 250 to 360 VHN range. In
this study the average values for enamel microhardness
were in the range from 266.5 VHN to 318.5 VHN range,
which was within the standard range.
One of the factors that affects the hardness measurement
was the specimen preparation, because any tilt or not
flat surface would yield a too large an indentation and
thus a smaller Vickerss hardness number. Therefore
to produce a flat surface in the specimens was crucial
in this study, but the cut enamel surface tested for
microhardness did not have a flat surface. The convex
surface gave variations in the VHN.[10] Hence two
indentations were made to avoid any operational
bias, then average of two indentations was taken for
statistical analysis.
Various techniques have been followed to neutralize
the effect of bleaching; the use of baking soda,
prophylactic paste containing fluoride, APF gel and use
of copious amount of water.[22] In the present study GC
Tooth mousse has been used, which is a commercially
available Casein phosphopeptides (CPP), stabilized
amorphous calcium phosphate (ACP) product.[23]
The values obtained after the application of
remineralizing solution for seven days (first cycle
of remineralization) showed marginal recovery
in the microhardness by 2.2% from the baseline.
Although most of the remineralizing solutions were
supersaturated with respect to the amorphous and
crystalline calcium phosphate phases, the solutions
which were stabilized by the CPP-ACP, such as in
GC Tooth mousse, the spontaneous precipitation of
calcium phosphate did not occur and thus the longer
the duration of the solution in contact with the teeth
the better was the remineralization.[9,24,] The second
cycle of remineralization for seven more days showed
a 3% increase from the baseline value which was
significant (P<0.05). According to the manufactures
instructions for the maximum benefit the solutions of
GC Tooth mousse solution should be left on the tooth
surface as long as possible. Hence in the present study
the excess GC Tooth mousse was wiped off after four

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Darshan and Shashikiran: Effect of McInnes solution

minutes leaving the residues on the samples and then

stored in artificial saliva.
In summary, McInnes bleaching agent used in this
study caused significant decrease in the enamel
microhardness when it was compared with the baseline
values. The more frequency of application, the lesser
was the VHN. However subsequent remineralizing
by GC Tooth mousse for fourteen days does cause
recovery in the microhardness, which was almost equal
or slightly more than the baseline value.

7.
8.
9.
10.
11.
12.

CONCLUSIONS
13.

From the above study it can be concluded that

McInnes bleaching agent does decrease the
microhardness of enamel by causing enamel
demineralization.
GC Tooth mousse used in the study causes an
increase in the microhardness of bleached enamel
by maintaining high gradient of calcium and
phosphate ions at the enamel subsurface.
This study was conducted with a small number of
samples utilizing in vitro conditions. Application to
general population requires further research and
analysis.

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Source of Support: Nil, Conflict of Interest: None declared.

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