FIELD OF THE INVENTION
The present invention relates to a method for evaluating a dentifrice. More particularly, the present invention relates to an in-vitro method for evaluating a whitening dentifrice.
BACKGROUND OF THE INVENTION
Individuals increasingly seek to have an attractive smile, as it is considered to be synonymous with overall health and enhanced physical attractiveness. White shiny teeth are undoubtedly the most important component of an attractive smile. The aesthetic and social benefit associated with whiter teeth has led to an ever increasing demand for tooth whitening toothpaste. This growing demand has led to a flood of tooth whitening toothpastes in the market.
Tooth color is often a result of intrinsic and extrinsic staining. Extrinsic staining may be due to tobacco smoking or chewing, from the use of certain cationic agents, such as chlorhexidine, and from a diet rich in coloring foods. When beverages, such as coffee and tea, are frequently consumed, colors are adsorbed onto the dental pellicle present on the enamel surface, leading to tooth discoloration.
These extrinsic stains can be removed by chemical as well as mechanical means. A dentifrice which removes stains by mechanical means comprises of various abrasives. Although increased dentifrice abrasiveness leads to improved stain removal efficacy, it also increases tooth wear. Thus, dentifrices containing active ingredients specifically designed for tooth stain removal, known as whitening dentifrices, have been introduced in the market. Along with the widespread introduction of whitening toothpastes in the markets, various methods both in vivo and in vitro have been developed in the art to evaluate the whitening efficacy of dentifrice compositions.
WO 201519538 A1 discloses in vitro methods for demonstrating stain removal by oral care compositions. Also disclosed are staining solutions and their use in methods for demonstrating stain removal. The method comprises the following steps: immersing the substrate in the staining solution; removing the substrate from the staining solution; evaluating the color of the stained substrate; applying the treatment solution to the substrate; and evaluating the color of the treated substrate, wherein the staining solution comprises coffee. The substrate used may be calcium carbonate, the exoskeleton of a marine bivalve mollusk, or the exoskeleton of a marine bivalve mollusk in the family Arcidae.
Poster No. 835, presented by Venda et. al. at IADR/AADR/CADR 2015 Annual meeting Boston, Massachusetts on March 11-14, 2015, discloses the in-vitro evaluation of the instant whitening efficacy of commercially available dentifrices. The method disclosed therein involved the following steps: soaking teeth in artificial saliva; adding dentifrice in artificial saliva; brushing teeth followed by rinsing with deionized water; soaking teeth in artificial saliva; and measuring the colour intensity of teeth.
Poster No. 567, presented by Venda et. al. at IADR General Session, Cape Town, South Africa on June 25-28, 2014, discloses the in-vitro evaluation of the whitening efficacy of a new dentifrice containing 0.1% hydrogen peroxide as compared to a whitening tooth-paste containing silica abrasive technology. The method disclosed therein involved the following steps: brushing teeth with dentifrice, rinsing the teeth with deionized water and measuring the colour intensity of teeth; and repeating the first two steps for a predetermined number of times.
Poster No. 4046, presented by Baig et. al. at 80th IADR General Session, on March 6-9, 2002, discloses an in-vitro evaluation of the whitening efficacy of chemical agents and formulations using hydroxyapatite powder as substrate. The disclosed method comprises the following steps: vortexing a mixture of the supernatant of the dentifrice slurry with hydroxyapatite powder; centrifuging the above mixture and washing with water; vortexing a mixture of the hydroxyapatite powder and tea and centrifuging; washing with water and drying; and measuring the colour intensity of hydroxyapatite powder.

Known methods for evaluating the efficacy of dentifrices utilize either human or animal teeth. Human teeth may be difficult to procure, whereas animal teeth may not be readily available in light of animal protection. Further, hydroxyapatite in the form of a powder may not be a suitable in-vitro model for evaluating the whitening efficacy of a dentifrice, as the surface properties of minute particles are different from that of human teeth.
The staining protocol disclosed in the prior art also differs significantly from the staining of a teeth under actual conditions. Most of the disclosed staining protocols include incubating animal teeth in staining slurry followed by application of the dentifrice composition. However, under actual conditions staining develops over time, involving repeated exposure of the teeth to the staining foodstuffs such as tea, coffee and so forth. Further, under actual conditions, staining is followed by exposure to saliva, leading to entrapment of the stain within the dental pellicle. The cycle of staining followed by entrapment within the pellicle leads to stubborn stains that are difficult to remove. Hence, protocol of staining adopted in prior art literature fails to simulate the staining of teeth under actual conditions.
For removal of stain from a substrate, prior art methods disclose the step of immediately brushing a substrate with the dentifrice composition after its staining. However, under actual conditions, brushing teeth is followed by exposure to saliva, which results in the formation of a protective dental pellicle over the enamel. Moreover, stain removal takes place over a period of many days over multiple cycles of brushing teeth followed by exposure to saliva. Hence, it is clear from the above discussion that prior art methods for evaluating the whitening efficacy of dentifrice compositions are found to be lacking in simulating the actual conditions for staining and whitening teeth. Hence, there exists a need in the art for a method for evaluating the whitening efficacy of a dentifrice which closely simulates staining and whitening of teeth under actual conditions.
The primary object of the present invention is to provide an in-vitro method for evaluating the teeth whitening efficacy of a dentifrice that mimics oral environment.
Another object of the present invention is to provide a novel staining solution, method of its preparation and use.
Yet another object of the present invention is to provide a novel in-vitro method for evaluating the whitening efficacy of a dentifrice using hydroxyapatite (HAP) test system.
SUMMARY OF THE INVENTION
A wide range of laboratory procedures to evaluate the extrinsic stain removal efficacy of teeth whitening dentifrices exist in the market. The present invention provides a novel in-vitro method for evaluating the whitening efficacy of a dentifrice under an environment that mimics actual oral conditions.
In the first aspect, the present invention relates to a method for evaluating the whitening efficacy of a dentifrice composition comprising the steps of:
(a) Soaking a hydroxyapatite disc in artificial saliva;
(b) Rinsing the hydroxyapatite disc with distilled water and measuring the colour intensity of the hydroxyapatite disc (T0);
(c) Soaking the hydroxyapatite disc in a staining solution followed by soaking in artificial saliva;
(d) Repeating step (c) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of the hydroxyapatite disc (T1);
(e) Treating the hydroxyapatite disc with a dentifrice and rinsing with distilled water followed by soaking in artificial saliva; and
(f) Repeating step (e) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of hydroxyapatite disc (T2).
In accordance with an embodiment of the invention, the staining solution comprises ingredients selected from the group consisting of tea, coffee, and combinations thereof.
In accordance with another embodiment of the invention, the staining solution is prepared by boiling tea and coffee in water.

In accordance with yet another embodiment of the invention, the amounts of tea and coffee in the staining solution range from 10% to 60%.

In accordance with yet another embodiment of the invention, the amount of tea and coffee in the staining solution ranges from 10% to 40%.

In accordance with yet another embodiment of the invention, the amount of tea and coffee in the staining solution ranges from 10% to 30%.

In accordance with a further embodiment of the invention, the amount of tea and coffee in the staining solution is 25%.
In accordance with still yet another embodiment of the invention, step (a) of the method comprises soaking the hydroxyapatite discs in artificial saliva at a temperature in the range of 35 to 40 °C for a period of 1 to 24 h.

In accordance with an embodiment of the invention, step (c) of the method comprises soaking the hydroxyapatite discs in the staining solution at a temperature in the range of 20 to 40 °C for a period of about 6 h.
In accordance with an embodiment of the invention, repetition of step (c) of the method is performed up to 20 times, the time interval between two consecutive repetitions being at least 24 h.

In accordance with an embodiment of the invention, repetition of step (e) of the method is performed up to 14 times, the time interval between two consecutive repetitions being at least 6 to 12 h.

In accordance with another embodiment of the invention, the dentifrice composition comprises abrasives, pyrophosphates, charcoal and excipients.
In accordance with yet another embodiment of the invention, the efficacy of the dentifrice composition is calculated by measuring the colour difference, ?E*.
DETAILED DESCRIPTION OF THE INVENTION
Discussed below are some representative embodiments of the present invention. The invention in its broader aspects is not limited to the specific details and representative methods. The illustrative examples are described in this section in connection with the embodiments and methods provided. The invention according to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification.
It is to be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The expression of various quantities in terms of “%” or “% w/w” means the percentage by weight of the total solution or composition unless otherwise specified.
The present invention, in its product and process aspects, is described in detail as follows:
The fast development of teeth whitening dentifrices and their wide use by the consumers led the present inventors to develop a novel in-vitro method for evaluating the whitening efficacy of dentifrice using hydroxyapatite (HAP) test system.

In the first aspect, the present invention relates to a method for evaluating the whitening efficacy of dentifrice composition comprising the steps of:
(a) Soaking a hydroxyapatite disc in artificial saliva;
(b) Rinsing the hydroxyapatite disc with distilled water and measuring the colour intensity of the hydroxyapatite disc (T0);
(c) Soaking the hydroxyapatite disc in a staining solution followed by soaking in artificial saliva;
(d) Repeating step (c) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of the hydroxyapatite disc (T1);
(e) Treating the hydroxyapatite disc with a dentifrice and rinsing with distilled water followed by soaking in artificial saliva; and
(f) Repeating step (e) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of hydroxyapatite disc (T2).
Hydroxyapatite (HAP) discs are more chemically related to human teeth, hence, serve as useful synthetic analogs for studying the adsorption and desorption chemistries involved in chromogen attachment, retention and curing during stain formation. Dense HAP discs (9.5 mm diameter x 2 mm thick; 95% purity; Clarkson Chromatography products, INC, USA) were used as substrates in the present invention to screen various dentifrice formulations for their teeth whitening efficacy. The Hydroxyapatite (HAP) discs are soaked in artificial saliva followed by incubating it under definite conditions to facilitate pellicle formation in order to mimic natural teeth environment. In a preferred embodiment, the HAP disc is soaked in artificial saliva at a temperature in the range of 35 to 40 °C for a period of 1 to 24 h.
Pellicle-coated enamel is known to possess a net negative charge. The ions from food and drinks are rich in tannins as well as chromogens, for example- copper, nickel, and iron. Such molecules attach themselves to the negative charge on the pellicle-coated enamel, thus causing extrinsic dental stains.
In an embodiment of the invention, the specimens (HAP discs) were soaked in staining solution under definite conditions, followed by soaking it in artificial saliva. The staining solution comprises tea, coffee, any staining food composition or combinations thereof. In a preferred embodiment, the HAP disc is soaked in staining solution at a temperature in the range of 20 to 40 °C for a period of about 6 h. In another embodiment the sequence of soaking in a staining solution followed by soaking in artificial saliva is repeated up to 20 times, wherein the time interval between two consecutive repetitions is at least 24 h.
In accordance with another aspect of the present invention, there is provided a staining solution for substrate comprising ingredients selected from the group consisting of tea, coffee and combinations thereof. Examples of tea that can be used in the present invention, include but not limited to green tea, black tea, for example Tata Tea, and combinations thereof. Examples of coffee that can be used in the present invention, include but not limited to green coffee extract, roasted coffee extract, instant coffee, for example Nescafe, and combinations thereof. It is postulated that tea might be even more likely to stain teeth than coffee due to its higher tannin content. Hence, in an embodiment, the staining solution is prepared by boiling tea and coffee in water, wherein tea and coffee are present in the range from 10% to 60%, preferably from 10% to 40%, more preferably from 10% to 30% and most preferably at 25%. Thereafter, the solution is filtered and centrifuged to obtain the staining solution used in the present invention.
After that, the stained HAP discs are treated with a dentifrice formulation. This is followed by rinsing with distilled water and soaking in artificial saliva under controlled conditions. The above sequence comprising treating with a dentifrice, rinsing with water and soaking in artificial saliva may be repeated up to 14 times, the time interval between two consecutive repetitions being at least 6 to 12 h. In a preferred embodiment, the dentifrice used in the treatment comprises abrasives, pyrophosphates, charcoal and excipients.
In an embodiment of the invention, the evaluation of change in colour (improvement in whiteness) of the stained substrate after dentifrice treatment is quantified by measurement of mean total colour difference, ?E*, using L*a*b*colour space (L*a*b* refers to stain score in accordance with the Commission International de L’Eclairage Laboratory (CIELAB) colour scale). The mean total colour difference, ?E* is calculated by using the formula:
?E* = v (L2-L1)2 + (a2-a1)2 + (b2-b1)2
wherein L* represents lightness-darkness scale, L1 represents pre-treatment reading, L2 represents post- treatment reading; a* represents red/green chroma, a1 represents pre- treatment reading, a2 represents post- treatment reading; b* represents yellow/blue chroma, b1 represents pre- treatment reading, b2 represents post- treatment reading.
The measurement of L*a*b* values is performed using colour spectrophotometer at different points of the experiment, i.e. before staining or baseline reading (T0), after staining or pre-treatment reading (T1) and after treatment with dentifrice or post-treatment reading (T2). Greater the ?E* values, higher is the teeth whitening efficacy of the dentifrice.
The present invention is more particularly described in the following non-limiting examples that are intended as illustrations only since numerous modifications and variations within the scope of the present invention will be apparent to a skilled artisan. Unless otherwise noted, all parts, percentages, and ratios reported in the following examples are on a weight basis, and all reagents used in the examples were obtained or made available from the chemical suppliers.
Examples
Example 1
Preparation of test system
Hydroxyapatite (HAP) discs bear a greater resemblance chemically to human teeth than do calcium carbonate substrates or powders or exoskeletons of other marine animals. Hence, the present test system closely mimics the environment of the oral cavity. The HAP discs (substrate) were soaked in 5 mL of artificial saliva. After that, the discs were incubated in a CO2 incubator at a temperature of about 37±1 °C for 24 h to aid in pellicle formation to mimic teeth inside the oral cavity. Finally, the HAP discs were rinsed with distilled water thrice and L*a*b* values were recorded, in triplicates, to obtain baseline reading (T0) for shade scoring using spectrophotometer. Finally, a total of 42 discs with similar CIELAB values were selected for further experimental studies.
Example 2
Staining procedure and preparation of staining solution
30 grams of tea and 30 grams of coffee powder were dissolved in 150 mL of distilled water and boiled for about 20 to 21 min. Thereafter, the staining solution was filtered and centrifuged at 800 rpm for about 3 min to obtain uniform slurry of coffee-tea cocktail. Each HAP disc was placed in a well plate immersed in 800 µL of coffee-tea cocktail slurry for about 6 h at 25 °C. After that, the substrates (HAP discs) were rinsed, thrice, with distilled water and brushed with a tooth brush (2 strokes with de-ionised water) to remove any loose deposits. Thereafter, the stained HAP discs were washed with distilled water and immersed in 800 µL of fresh saliva solution for further incubation at 37±1°C for 18 h. Further, the aforesaid sequence comprising, staining, rinsing and incubating was repeated ten times, and L*a*b* values were recorded for the HAP discs, in triplicates, to obtain pre-treatment reading (T1) for shade scoring using spectrophotometer.
Example 3
Experimental study was carried out to highlight the significance of the method and the staining solution of the present invention. Some of the prepared test systems (HAP discs with pellicle) were dipped in artificial saliva (control), while others were kept in staining solution, i.e. coffee-tea cocktail for seven days. L, a, b values were recorded for the sample HAP discs, in triplicates, to obtain baseline or pre-staining reading (T0) and post-staining reading (T1) for shade scoring using spectrophotometer as shown in table-1 below:

Table 1
Group Sample No. Pre staining (T0) Post staining (T1) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E= Square root of ((?L)2+ (?a)2+ (?b)2)
Lab value Reading 1 Mean-1 Reading 1 Reading 2 Mean 2
No staining solution (only dipped in artificial saliva) 1 L 93.8 93.80 93.84 93.84 93.84 -0.04 0.00 0.02 0.15
a -0.71 -0.71 -0.8 -0.8 -0.80 0.09 0.01
B 1.9 1.90 2 2.03 2.02 -0.12 0.01
Staining solution with artificial saliva 2 L 94.89 94.89 85.97 85.97 85.97 8.92 79.57 156.01 12.49
a -0.69 -0.69 1.25 1.25 1.25 -1.94 3.76
B 1.33 1.33 9.86 9.85 9.86 -8.53 72.68

From table 1, it is evident that significant amount of staining is achieved in the HAP discs placed in the staining solution, which proves that the use of a staining solution comprising coffee and tea is much better for evaluating a dentifrice composition than staining with saliva alone.
Example 4
Teeth whitening efficacy studies
After staining of HAP discs in staining solution as mentioned in example 2, the stained HAP discs were randomly allocated into seven treatment groups with each group having six stained HAP discs. For the application of treatment (test item/control), a cast was prepared using plaster of paris (POP), which was used to hold the HAP discs. The cast was prepared for each group and was labelled. Each group was treated with the respective dentifrice/control (test item 1 to 5/positive control/negative control), twice a day, the time interval between two consecutive treatments being 12 h, by brushing with tooth brush for about 2-3 min (approx. 120 strokes of tooth brush) for a period of 14 consecutive days (two weeks), wherein distilled water is used as negative control; a commercially available dentifrice functions as positive control and Test items 1 to 5 are dentifrice compositions as described in table-2. The twice daily application of treatment (test item/control) was performed in the morning and evening with a gap of 12 h and the amount of treatment (test item/control) for application was about 25 mg/HAP disc. After the application of treatment (test item/control), the HAP discs were rinsed, thrice, with distilled water and were brushed (2 strokes) in order to remove any traces of treatment (test item/control) on HAP discs. Later, the rinsed HAP discs were transferred to 24 well plates containing fresh artificial saliva and were kept in the incubator at 37±1°C until the next application of treatment (test item/control). At the end of treatment duration, i.e. on day 14, L*a*b* values were recorded for the HAP discs, in triplicates, to obtain post treatment reading (T2) for shade scoring using spectrophotometer (Tables 3-9). The teeth whitening efficacy (improvement in whiteness) of dentifrice/control was quantified using measurements of mean total colour difference, ?E* as shown below in tables 2 to 9:
Table 2: Absolute mean difference in ?E* on day 14 with respect to day 1
Treatment groups Treatment ?E* on day-14
Negative control Distilled water 2.93
Positive control A commercially available dentifrice 4.34
Test item-1 A dentifrice composition comprising abrasives, pyrophosphates, charcoal and excipients 5.68
Test item-2 A dentifrice composition comprising abrasives, charcoal and excipients 3.52
Test item-3 A commercially available dentifrice composition comprising abrasives, pyrophosphates, charcoal and excipients 4.09
Test item-4 A dentifrice composition comprising abrasives and excipients 4.34
Test item-5 A dentifrice composition comprising abrasives and excipients 3.31

From the above results, it is evident that Test item -1 showed maximum teeth whitening efficacy in comparison with other test items and commercial market samples. Test item-1 is a stable dentifrice formulation with optimised charcoal and pyrophosphate concentration that reduces the adverse salt effects and dental enamel abrasion. (Test items 1, 2, 3, 4 and 5 differ in the % w/w of the various component ingredients)
Example 5
The test items 1 to 5 and controls were evaluated for the teeth whitening efficacy (improvement in whiteness) using measurement of mean total colour difference, ?E*, calculated using L*a*b*colour space. The L*a*b* values were recorded for the HAP discs, in triplicate, to obtain pre-treatment reading (T1) and post-treatment reading (T2) for shade scoring using spectrophotometer as shown in tables 3-9. Test item 1 scored the highest in terms of ?E* value, and hence, the most efficient at teeth whitening.

Table 3: CALCULATION OF ?E VALUE FOR CONTROL GROUP
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b)2)
L* a* b* Values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Control (Distilled water) 1 L 90.43 90.43 90.43 90.4 91.15 91.15 91.14 91.1 0.7 0.5 3.7 1.94
a 0.27 0.27 0.28 0.3 -0.04 -0.04 -0.04 0.0 -0.3 0.1
b 6.33 6.33 6.33 6.3 4.56 4.56 4.56 4.6 -1.8 3.1
2 L 86.72 86.72 86.71 86.7 88.05 88.05 88.05 88.1 1.3 1.8 23.2 4.82
a 1.37 1.37 1.37 1.4 0.7 0.69 0.68 0.7 -0.7 0.5
b 11.25 11.26 11.25 11.3 6.67 6.68 6.68 6.7 -4.6 20.9
3 L 85.49 85.49 85.49 85.5 84.52 84.52 84.52 84.5 -1.0 0.9 1.3 1.14
a 1.48 1.47 1.49 1.5 1.85 1.85 1.84 1.8 0.4 0.1
b 10.27 10.28 10.26 10.3 9.81 9.79 9.8 9.8 -0.5 0.2
4 L 82.76 82.76 82.75 82.8 83.85 83.85 83.86 83.9 1.1 1.2 9.3 3.05
a 2.82 2.82 2.81 2.8 2.31 2.32 2.31 2.3 -0.5 0.3
b 12.3 12.3 12.29 12.3 9.5 9.49 9.49 9.5 -2.8 7.9
5 L 82.59 82.58 82.58 82.6 79.98 79.98 79.98 80.0 -2.6 6.8 18.4 4.29
a 2.89 2.89 2.9 2.9 3.2 3.21 3.19 3.2 0.3 0.1
b 11.88 11.88 11.88 11.9 8.49 8.48 8.49 8.5 -3.4 11.5
6 L 73.42 73.42 73.41 73.4 74.91 74.9 74.91 74.9 1.5 2.2 5.6 2.36
a 4.67 4.66 4.66 4.7 3.84 3.85 3.85 3.8 -0.8 0.7
b 13.32 13.33 13.32 13.3 11.7 11.68 11.67 11.7 -1.6 2.7
?E*(Mean ?E) 2.93

Table 4: CALCULATION OF ?E VALUE FOR POSITIVE CONTROL
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Positive Control (A commercially available dentifrice) 1 L 89.97 89.97 89.96 90.0 90.56 90.56 90.55 90.6 0.6 0.3 3.2 1.80
a 0.19 0.19 0.20 0.2 -0.09 -0.08 -0.09 -0.1 -0.3 0.1
b 6.33 6.35 6.33 6.3 4.65 4.65 4.67 4.7 -1.7 2.8
2 L 86.64 86.64 86.63 86.6 87.88 87.87 87.87 87.9 1.2 1.5 7.3 2.71
a 1.1 1.1 1.1 1.1 0.89 0.91 0.89 0.9 -0.2 0.0
b 11.31 11.31 11.31 11.3 8.9 8.9 8.9 8.9 -2.4 5.8
3 L 85.7 85.69 85.69 85.7 84.99 84.98 84.98 85.0 -0.7 0.5 1.1 1.07
a 2.13 2.14 2.13 2.1 2.08 2.07 2.07 2.1 -0.1 0.0
b 9.95 9.94 9.94 9.9 9.14 9.15 9.14 9.1 -0.8 0.6
4 L 82.79 82.79 82.79 82.8 81.29 81.28 81.28 81.3 -1.5 2.3 6.3 2.51
a 2.62 2.62 2.62 2.6 2.89 2.90 2.90 2.90 0.3 0.1
b 11.87 11.89 11.88 11.9 9.9 9.89 9.9 9.9 -2.0 3.9
5 L 86.29 86.28 86.28 86.3 88.02 88.02 88.01 88.0 1.7 3.0 43.0 6.56
a 1.26 1.27 1.27 1.3 0.80 0.81 0.82 0.80 -0.5 0.2
b 14.63 14.62 14.61 14.6 8.32 8.31 8.31 8.3 -6.3 39.8
6 L 83.21 83.2 83.2 83.2 88.98 88.98 88.98 89.0 5.8 33.4 129.2 11.37
a 2.98 2.98 2.99 3.0 0.84 0.84 0.84 0.8 -2.1 4.6
b 17.39 17.39 17.38 17.4 7.84 7.83 7.83 7.8 -9.6 91.3
?E*(Mean ?E) 4.34

Table 5: CALCULATION OF ?E VALUE FOR TEST ITEM-1
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Test item-1 1 L 89.95 89.95 89.95 90.0 83.84 83.85 83.85 83.8 -6.1 37.3 38.8 6.23
a 0.37 0.37 0.37 0.4 1.51 1.49 1.51 1.5 1.1 1.3
b 6.91 6.93 6.92 6.9 7.43 7.44 7.43 7.4 0.5 0.3
2 L 86.36 86.36 86.36 86.4 86.77 86.76 86.76 86.8 0.4 0.2 5.5 2.35
a 1.64 1.63 1.65 1.6 1.19 1.19 1.2 1.2 -0.4 0.2
b 10.40 10.41 10.40 10.4 8.13 8.14 8.13 8.1 -2.3 5.2
3 L 83.76 83.76 83.76 83.8 81.73 81.73 81.73 81.7 -2.0 4.1 7.9 2.81
a 2.44 2.44 2.43 2.4 2.54 2.55 2.56 2.6 0.1 0.0
b 8.5 8.49 8.5 8.5 6.55 6.56 6.54 6.6 -1.9 3.8
4 L 84.63 84.63 84.62 84.6 86.73 86.72 86.72 86.7 2.1 4.4 24.0 4.90
a 1.57 1.58 1.59 1.6 0.98 0.96 0.97 1.0 -0.6 0.4
b 13.7 13.69 13.67 13.7 9.3 9.3 9.31 9.3 -4.4 19.2
5 L 83.09 83.09 83.09 83.1 83.59 83.6 83.59 83.6 0.5 0.3 25.8 5.07
a 1.86 1.86 1.87 1.9 1.36 1.34 1.35 1.4 -0.5 0.3
b 12.87 12.88 12.87 12.9 7.84 7.86 7.85 7.9 -5.0 25.2
6 L 79.82 79.82 79.82 79.8 88.78 88.78 88.77 88.8 9.0 80.2 161.0 12.69
a 3.51 3.51 3.53 3.5 0.22 0.19 0.21 0.2 -3.3 11.0
b 13.47 13.48 13.46 13.5 5.11 5.11 5.13 5.1 -8.4 69.8
?E*(Mean ?E) 5.68

Table 6: CALCULATION OF ?E VALUE FOR TEST ITEM-2
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Test item-2 1 L 88.67 88.67 88.67 88.7 88.36 88.36 88.35 88.4 0.3 0.1 4.1 2.03
a 0.86 0.86 0.87 0.9 0.47 0.48 0.47 0.5 0.4 0.2
b 8.24 8.24 8.22 8.2 6.27 6.27 6.25 6.3 2.0 3.9
2 L 86.76 86.75 86.75 86.8 83.54 83.53 83.53 83.5 3.2 10.4 18.4 4.29
a 1.47 1.47 1.46 1.5 0.8 0.79 0.8 0.8 0.7 0.4
b 9.07 9.08 9.09 9.1 6.34 6.33 6.33 6.3 2.7 7.5
3 L 84.95 84.95 84.94 84.9 84.56 84.56 84.56 84.6 0.4 0.1 2.3 1.53
a 1.76 1.77 1.77 1.8 1.88 1.9 1.9 1.9 -0.1 0.0
b 11.28 11.28 11.27 11.3 9.81 9.81 9.8 9.8 1.5 2.2
4 L 81.39 81.39 81.38 81.4 81.13 81.13 81.13 81.1 0.3 0.1 13.5 3.67
a 2.88 2.86 2.87 2.9 2.44 2.44 2.43 2.4 0.4 0.2
b 9.39 9.38 9.38 9.4 5.76 5.74 5.74 5.7 3.6 13.2
5 L 84.08 84.08 84.07 84.1 84.82 84.82 84.83 84.8 -0.7 0.6 26.6 5.16
a 2.16 2.15 2.17 2.2 1.52 1.53 1.52 1.5 0.6 0.4
b 13.43 13.44 13.42 13.4 8.37 8.35 8.37 8.4 5.1 25.7
6 L 79.68 79.67 79.67 79.7 83.86 83.87 83.86 83.9 -4.2 17.6 20.0 4.47
a 3.01 3.0 3.01 3.0 1.81 1.8 1.81 1.8 1.2 1.4
b 10.58 10.58 10.59 10.6 9.59 9.6 9.58 9.6 1.0 1.0
?E*(Mean ?E) 3.52

Table 7: CALCULATION OF ?E VALUE FOR TEST ITEM-3
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Test item-3 1 L 87.55 87.55 87.55 87.6 87.39 87.39 87.39 87.4 0.2 0.0 3.7 1.93
a 0.82 0.83 0.83 0.8 0.59 0.59 0.59 0.6 0.2 0.1
b 8.41 8.4 8.41 8.4 6.49 6.5 6.51 6.5 1.9 3.6
2 L 83.89 83.89 83.9 83.9 81.57 81.57 81.57 81.6 2.3 5.4 6.6 2.57
a 1.68 1.69 1.67 1.7 2.03 2.04 2.04 2.0 -0.4 0.1
b 5.7 5.71 5.72 5.7 4.68 4.68 4.66 4.7 1.0 1.1
3 L 85.62 85.62 85.61 85.6 86.31 86.3 86.31 86.3 -0.7 0.5 15.0 3.87
a 1.47 1.46 1.46 1.5 1.21 1.21 1.2 1.2 0.3 0.1
b 11.75 11.76 11.75 11.8 7.95 7.95 7.95 8.0 3.8 14.5
4 L 85.38 85.37 85.38 85.4 84.69 84.69 84.69 84.7 0.7 0.5 31.6 5.62
a 1.55 1.55 1.55 1.6 1 1 1.01 1.0 0.5 0.3
b 12.49 12.48 12.49 12.5 6.94 6.93 6.94 6.9 5.6 30.8
5 L 84.17 84.17 84.16 84.2 82.6 82.6 82.59 82.6 1.6 2.5 34.5 5.87
a 2.38 2.38 2.38 2.4 1.84 1.81 1.82 1.8 0.6 0.3
b 13.58 13.59 13.59 13.6 7.97 7.95 7.95 8.0 5.6 31.7
6 L 80.72 80.72 80.72 80.7 79.87 79.86 79.87 79.9 0.9 0.7 22.1 4.70
a 3.43 3.44 3.43 3.4 2.83 2.84 2.83 2.8 0.6 0.4
b 12.72 12.71 12.71 12.7 8.14 8.13 8.13 8.1 4.6 21.0
?E*(Mean ?E) 4.09

Table 8: CALCULATION OF ?E VALUE FOR TEST ITEM-4
Group Sample No. Pre treatment (T1) Post Treatment(T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Test item-4 1 L 85.82 85.86 85.88 85.9 85.08 85.08 85.08 85.1 0.8 0.6 3.8 1.9
a 1.65 1.65 1.64 1.6 1.64 1.64 1.65 1.6 0.0 0.0
b 6.18 6.18 6.16 6.2 4.39 4.4 4.4 4.4 1.8 3.2
2 L 86.77 86.76 86.76 86.8 83.37 83.38 83.38 83.4 3.4 11.5 12.9 3.6
a 1.2 1.2 1.19 1.2 2.38 2.38 2.4 2.4 -1.2 1.4
b 9.88 9.89 9.89 9.9 9.81 9.81 9.81 9.8 0.1 0.0
3 L 82.59 82.59 82.58 82.6 82.42 82.42 82.42 82.4 0.2 0.0 2.5 1.6
a 2.55 2.55 2.57 2.6 2.54 2.53 2.53 2.5 0.0 0.0
b 8.02 8.04 8.02 8.0 6.46 6.46 6.46 6.5 1.6 2.5
4 L 84.58 84.58 84.58 84.6 85.22 85.21 85.22 85.2 -0.6 0.4 23.5 4.8
a 1.32 1.3 1.31 1.3 1.95 1.96 1.94 2.0 -0.6 0.4
b 13.17 13.17 13.18 13.2 8.42 8.4 8.41 8.4 4.8 22.7
5 L 80.02 80.02 80.03 80.0 78.83 78.83 78.83 78.8 1.2 1.4 8.4 2.9
a 2.97 2.96 2.97 3.0 2.15 2.13 2.14 2.1 0.8 0.7
b 7.51 7.53 7.53 7.5 5.02 5.02 5.03 5.0 2.5 6.3
6 L 81.9 81.89 81.89 81.9 90.1 90.1 90.11 90.1 -8.2 67.4 125.3 11.2
a 2.73 2.72 2.72 2.7 0.05 0.05 0.05 0.1 2.7 7.1
b 12.44 12.45 12.44 12.4 5.31 5.32 5.32 5.3 7.1 50.8
?E*(Mean ?E) 4.34

Table 9: CALCULATION OF ?E VALUE FOR TEST ITEM-5
Group Sample No. Pre treatment (T1) Post Treatment (T2) ?L, ?a, ?b (?L)2, (?a)2, (?b)2 (?L)2+ (?a)2+ (?b)2 ?E=Square root of ((?L)2+ (?a)2+ (?b2))
L* a* b* values R1 R2 R3 Mean- 1 R1 R2 R3 Mean-2
Test item-5 1 L 88.33 88.33 88.32 88.3 87.71 87.71 87.72 87.7 0.6 0.4 1.2 1.09
a 0.83 0.81 0.81 0.8 0.98 0.97 0.97 1.0 -0.2 0.0
b 8.49 8.5 8.5 8.5 7.6 7.61 7.6 7.6 0.9 0.8
2 L 87.72 87.72 87.72 87.7 87.06 87.06 87.06 87.1 0.7 0.4 0.6 0.78
a 1.08 1.07 1.08 1.1 1.44 1.44 1.46 1.4 -0.4 0.1
b 8.14 8.18 8.16 8.2 7.96 7.97 7.96 8.0 0.2 0.0
3 L 86.22 86.22 86.21 86.2 87.79 87.78 87.78 87.8 -1.6 2.5 79.5 8.92
a 2.06 2.06 2.06 2.1 1.23 1.22 1.22 1.2 0.8 0.7
b 13.76 13.77 13.76 13.8 5.01 5.02 5.04 5.0 8.7 76.4
4 L 84.0 84.0 84.0 84.0 83.48 83.48 83.5 82.3 0.5 0.3 0.9 0.93
a 1.98 1.98 1.98 2.0 2.29 2.29 2.28 2.3 -0.3 0.1
b 11.27 11.28 11.28 11.3 10.57 10.57 10.55 10.6 0.7 0.5
5 L 82.86 82.86 82.86 82.9 86.55 86.55 86.56 86.6 -3.7 13.6 29.7 5.45
a 2.73 2.73 2.74 2.7 1.41 1.41 1.4 1.4 1.3 1.8
b 13.36 13.39 13.37 13.4 9.58 9.6 9.58 9.6 3.8 14.3
6 L 81.23 81.23 81.23 81.2 80.44 80.44 80.43 80.4 0.8 0.6 7.1 2.66
a 3.41 3.4 3.41 3.4 3.43 3.43 3.43 3.4 0.0 0.0
b 10.76 10.75 10.77 10.8 8.21 8.23 8.22 8.2 2.5 6.5
?E*(Mean ?E) 3.31

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is thereof intended to cover in the appended claims such changes and modifications that are within the scope of the invention.

We claim:
1. A method for evaluating the whitening efficacy of a dentifrice composition comprising the steps of:
a) Soaking a hydroxyapatite disc in artificial saliva;
b) Rinsing the hydroxyapatite disc with distilled water and measuring the colour intensity of the hydroxyapatite disc;
c) Soaking the hydroxyapatite disc in a staining solution followed by soaking in artificial saliva;
d) Repeating step (c) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of the hydroxyapatite disc;
e) Treating the hydroxyapatite disc with the dentifrice composition and rinsing with distilled water followed by soaking in artificial saliva; and
f) Repeating step (e) for a predetermined number of times, rinsing with distilled water and measuring the colour intensity of the hydroxyapatite disc.
2. The method for evaluating the whitening efficacy of a dentifrice composition as claimed in claim 1, wherein the staining solution comprises ingredients selected from the group consisting of tea, coffee, and combinations thereof.
3. The method for evaluating the whitening efficacy of a dentifrice composition as claimed in claims 1 and 2, wherein the staining solution is prepared by boiling tea and coffee in water.
4. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 2 and 3, wherein the amounts of tea and coffee in the staining solution range from 10% to 60%.
5. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 2 to 4, wherein the amount of tea and coffee in the staining solution ranges from 10% to 40%.
6. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 2 to 5, wherein the amount of tea and coffee in the staining solution ranges from 10% to 30%.
7. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 2 to 6, wherein the amount of tea and coffee in the staining solution is 25%.
8. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 7, wherein step (a) of the method comprises soaking the hydroxyapatite discs in artificial saliva at a temperature in the range of 35 to 40 °C for a period of 1 to 24 h.
9. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 8, wherein step (c) of the method comprises soaking the hydroxyapatite discs in the staining solution at a temperature in the range of 20 to 40 °C for a period of about 6 h.
10. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 9, wherein repetition of step (c) of the method is performed up to 20 times, the time interval between two consecutive repetitions being at least 24 h.
11. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 10, wherein repetition of step (e) of the method is performed up to 14 times, the time interval between two consecutive repetitions being at least 6 to 12 h.
12. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 11, wherein the dentifrice composition comprises abrasives, pyrophosphates, charcoal and excipients.
13. The method for evaluating the whitening efficacy of a dentifrice composition, as claimed in claims 1 to 12, wherein the whitening efficacy of the dentifrice composition is calculated by measuring the colour difference, AE*.