WO2019219390A1

WO2019219390A1 - Method of evaluating efficacy of cosmetic cleansing compositions

Info

Publication number: WO2019219390A1
Application number: PCT/EP2019/061186
Authority: WO
Inventors: Guoqiang Chen; Nan Huang; Chengdong JI; Sheng MENG
Original assignee: Unilever N.V.; Unilever Plc; Conopco, Inc., D/B/A Unilever
Priority date: 2018-05-17
Filing date: 2019-05-02
Publication date: 2019-11-21

Abstract

Disclosed is an in vitro method to determine efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants adhering to the skin, comprising the steps of: (i) contacting a human skin-equivalent with a model fine particulate matter resembling a particulate atmospheric pollutant and measuring a first concentration of said model fine particulate matter on said human skin equivalent by an imaging technique; (ii) cleansing said human skin equivalent bearing said model fine particulate matter thereon with said cosmetic cleansing composition and measuring a second concentration of said model fine particulate matter on said human skin equivalent by the imaging technique; (iii) differentiating between said first and said second concentration; and, (iv) ascertaining said efficacy from the information of step (iii), wherein, said technique is a spectroscopic or microscopic chemical imaging technique, where said chemical imaging does not mean or include colorimetric or digital image analysis and wherein said human skin equivalent is artificial skin, donor skin, living skin equivalent or pig skin and wherein said model fine particulate matter is in the form of beads comprising polystyrene and a fluorescent material, further wherein said microscopic imaging technique is fluorescence microscopy and said human skin- equivalent is artificial skin.

Description

METHOD OF EVALUATING EFFICACY OF COSMETIC CLEANSING

COMPOSITIONS

Field of the invention

The present invention relates to a method of evaluating efficacy of cosmetic cleansing compositions. More particularly, the invention relates to a method of evaluating efficacy of such compositions to remove particulate atmospheric pollutants adhering to the skin.

Background of the invention

The World Health Organization (WHO) reports that outdoor air pollution originates from natural and anthropogenic sources. While natural sources contribute substantially to local air pollution in arid regions more prone to forest fires and dust storms, the contribution from human activities far exceeds natural sources.

Such human activities include fuel combustion, heat and power generation and industrial facilities (e.g. manufacturing factories, mines, and oil refineries). WHO classifies pollutants into particulate matter, black carbon, ground-level ozone and oxides of carbon, nitrogen and sulphur.

Particulate matter (PM) are inhalable particles composed of sulphate, nitrates, ammonia, sodium chloride, black carbon, mineral dust and water. Particles with a diameter of less than 10 microns (PM10), including fine particles less than 2.5 microns (PM2.5) pose the greatest risks to health, as they can enter the lungs and the bloodstream. Carbon black (soot) and dust (mineral oxides, such as iron oxides and the like) comprise much of the particulate matter in these size ranges.

WHO defines air pollution as contamination of indoor or outdoor environments by any chemical, physical, or biological agent that modifies the natural characteristics of the atmosphere. The U.S. Environmental Protection Agency (EPA) and the WHO have summarized the global extent of common atmospheric pollutants. In addition, there are innumerable reports and scientific publications pertaining to adverse effects of pollution on human skin. These adverse effects include premature ageing, development of fine lines and wrinkles, pigmented spots, hyperpigmentation, rash and inflammation.

A variety of cosmetic compositions claim to protect the skin from harmful effects of air pollution, e.g. WO2016/164216 A1 (ISP). Some compositions are not available commercially but are described in patent or non-patent literature. Some of these compositions are meant to remove or wash-off the atmospheric pollutants in contact with the skin while some others are meant for blocking or preventing the contact of pollutants with the skin. A variety of test methods are found in books, journals, periodicals and patents. The purpose of such methods is to ascertain the efficacy of a candidate cosmetic composition. At times the purpose also is to compare the efficacy of one or more compositions or active ingredients, e.g. polymers, and rank them accordingly. Some of these tests are conducted on human volunteers. Some others have been conducted on suitable skin-equivalents such as plastic membranes, living-skin equivalents, Vitro- Skin®, in vitro skin models, ex vivo skin and the like. While skin-equivalents is one component of such test methods, selection of an appropriate pollutant is equally important. However, it is not always possible to perform tests with real-life pollutants therefore model pollutants are often used.

CN106290176A (Shanghai Jahwa United Co Ltd, 2017) discloses a testing method for determining the efficacy of a cleansing composition against PM2.5. The method includes the steps of collecting pollutants in air to prepare into suspension liquid; determining chromaticity (aO) of a skin area of a human body needing to be tested, coating the skin area of the human body with the suspension liquid to form a coating area, and determining chromaticity (a1 ) of the coating area; cleaning the coating area by the cleaning product, and determining chromaticity (a2) of the cleaned coating area;

calculating the cleaning power by the aid of a formula which is eta: eta =(a1-a2)/(a1- aO).

Dow Coming’s website discloses a method in which a test material is coated on a synthetic substrate to form a thin film. Performance is assessed by quantifying the adhesion of particles of carbon black by image analysis. The method is also disclosed in US2016/0324757 A1.

Similarly, BASF’s website, while referring to its product PURISOFT®, also discloses a method in which particles of charcoal are used to mimic atmospheric particulate pollutants.

Schrader and Rohr have, in a scientific article published in Clinics in Dermatology; 1996;14:57-65, disclosed a skin washing machine to determine efficacy of cleansing compositions on human volunteers. Some standard model soils have also been disclosed which contain colouring ingredients and oily substances like mineral oil, lanolin and petrolatum. The measurement of the color reduction of“standard soil” (the actual detergency effect) is done with a Minolta® chromometer and reflection (L) is evaluated in percentage.

US20150362412 A1 (Shiseido) discloses a method to evaluate the condition of stratum corneum by tape-stripping the skin after cosmetic treatment of the skin. The sheet is contacted with a dye selected from the group consisting of fluorescein, rose bengal and their salts. The dye is then rinsed following which the staining intensity of the dye in the stratum corneum sheet is measured. Damaged stratum corneum is not as transparent and its barrier function is also adversely affected. The application also discloses a method of evaluating candidate cosmetics or cosmetic treatments and their ameliorating effect on the stratum corneum. US20150177221 A1 (L’Oreal) discloses a model-pollutant formulation configured to simulate contaminated human skin. The formulation includes triglycerides, fatty acids, waxes or wax esters, more hydrocarbons, cholesterol or a cholesterol ester and plurality of carbon particles of mean diameter of 10 pm or less. The formulation is applied to a test area of a subject's skin, followed by cleansing the area, measuring the color of the test area to provide a post-clean colour signal; and comparing the post- clean color signal to a baseline color signal obtained without the formulation. As the above methods relies on optical imaging to test the efficacy of the cleanser, a heavy dose of artificial soil needs to be applied on the test area for optimal contrast to permit image analysis for maximizing the difference(s) between candidate cleansing compositions. Therefore, although the test method provides an indication of efficacy of a cleansing composition, the method is not representative of a real-life situation in view of the heavy soil loading on skin.

US20180059007 A1 (L’Oreal) discloses an in vitro screening system which includes a friction assembly including at least one motorized friction structural component configured to engage and remove material on a substrate and an analytics assembly including at least one sensor and circuitry configured to generate cleanser-specific material removal information responsive to a detected response associated with removal of a material on a substrate by the friction structural component. Emily HENES et al,:“Evaluation of a novel sonic brush head to deeply cleanse pores”, JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY., vol.66, no. 41 , 1 January 2012, page AB26 discloses an In Vivo analysis method carried out on nose of some human volunteers in which a sonic care brush designed for deep pore cleansing was compared to manual cleansing for makeup removal. The techniques relies on photography/digital analysis of the noses of the volunteers before and after application of the makeup and cleansing but fluorescent microspheres are used.

Gregory PETERSON:“A novel fluorescent makeup methodology used to measure the cleansing efficacy of a sonic skin care brush” JOURNAL OF THE AMERICAN

ACADEMY OF DERMATOLOGY vol. 56, no. 2, 1 February 2007, page AB38 discloses an In Vivo analysis method carried out on face of some human volunteers in which a sonic care brush designed for facial cleansing was compared to manual cleansing for makeup removal. The techniques relies on photography/digital analysis of the faces of the volunteers before and after application of the makeup and cleansing.

There is need for a robust and reliable in vitro method to test and compare the efficacy of cosmetic cleansing compositions treated in the same manner or the efficacy of a cleaning composition vis-a-vis water, which is the most common ingredient of skin cleansing compositions. Sometimes the methods are prone to erroneous conclusions in view of sample to sample variation even if the tests are performed by the same operator. In some other cases, the techniques are prone to erroneous conclusions in view of human error even if identical method is followed by two or more operators.

The present invention addresses the needs by overcoming at least one drawback, disadvantage or limitation of the state of the art.

Summary of the invention

In accordance with a first aspect is disclosed an in vitro method to determine efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants adhering to the skin, comprising the steps of:

(i) contacting a human skin-equivalent with a model fine particulate matter

resembling a particulate atmospheric pollutant and measuring a first concentration of said model fine particulate matter on said human skin equivalent by an imaging technique;

(ii) cleansing said human skin equivalent bearing said model fine particulate matter thereon with said cosmetic cleansing composition and measuring a second concentration of said model fine particulate matter on said human skin equivalent by the imaging technique;

(iii) differentiating between said first and said second concentration; and,

(iv) ascertaining said efficacy from the information of step (iii),

wherein, said technique is a spectroscopic or microscopic chemical imaging technique, where said chemical imaging does not mean or include colorimetric or digital image analysis and wherein said human skin equivalent is artificial skin, donor skin, living skin equivalent or pig skin and wherein said model fine particulate matter is in the form of beads comprising polystyrene and a fluorescent material, further wherein said

microscopic imaging technique is fluorescence microscopy and said human skin- equivalent is artificial skin.. In accordance with a second aspect is disclosed a process of demonstrating efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants adhering to the skin, said process comprising the steps of:

(i) contacting a human skin-equivalent with a model fine particulate matter

(iii) differentiating between said first and said second concentration; and,

(iv) ascertaining and thereby demonstrating said efficacy from the information of step (iii),

microscopic imaging technique is fluorescence microscopy and said human skin- equivalent is artificial skin..

Detailed description of the invention As used herein the term“comprising” encompasses the terms“consisting essentially of and“consisting of”. Where the term“comprising” is used, the listed steps or options need not be exhaustive. Unless otherwise specified, numerical ranges expressed in the format "from x to y" are understood to include x and y. In specifying any range of values or amounts, any particular upper value or amount can be associated with any particular lower value or amount. Except in the examples and comparative

experiments, or where otherwise explicitly indicated, all numbers are to be understood as modified by the word“about”. All percentages and ratios contained herein are calculated by weight unless otherwise indicated. As used herein, the indefinite article “a” or“an” and its corresponding definite article“the” means at least one, or one or more, unless specified otherwise. The various features of the present invention referred to in individual sections above apply, as appropriate, to other sections mutatis mutandis. Consequently, features specified in one section may be combined with features specified in other sections as appropriate. Any section headings are added for convenience only, and are not intended to limit the disclosure in any way. The examples are intended to illustrate the invention and are not intended to limit the invention to those examples per se. The term cosmetic cleansing composition means any cosmetic composition comprising at least one cleansing agent. Usually such cleansing agent is a surfactant. In one aspect the cleansing composition comprises a soap-based surfactant. Alternatively, the cleansing composition is a non-soap based surfactant. The terms soap-based and non- soap based surfactant are clear to persons skilled in the art of formulation of cosmetic cleansing compositions.

Examples of cosmetic cleansing compositions include soap bars, liquid soap, non-soap body wash compositions, foam-based cleansing compositions, serums, facewash creams, facewash gels, body wash gels, face pack, face mask and shampoo. The compositions may accordingly be in a variety of formats as described hereinbefore.

The term in vitro means that the method in accordance with the invention is not carried out on human volunteers, for example, on forearms of human volunteers. The term active ingredient means any ingredient, including surface-active agent, included in the cosmetic cleansing composition for any beneficial effect against particulate pollutants. Non-limiting examples thereof include polymers and extracts of natural products such as extract of roots or leaves of any particular plant. Human skin acts like a natural shield which protects our body from external influences. However, at times, and under certain conditions, the skin may no longer perform this function fully and efficiently. There is plethora of evidence to substantiate that atmospheric pollutants affect the normal functioning of human skin. Particulate pollutants tend to top the list at least in some countries or some regions of the world.

Formulation scientists have explored and continue to explore newer and more effective cosmetic compositions to protect the skin from particulate pollutants, including the compositions which can wash-off such pollutants. However, as discussed at length under the section of background and prior art, there is need for a more robust and reliable method for demonstrating the efficacy of such compositions. The present invention addresses such a need, at least in part.

The term particulate pollutant, also called particulate matter or PM, means a mixture of solid and liquid droplets floating in the air. Some particles are released directly from a specific source, while others form in complicated chemical reactions in the atmosphere. Suitable examples include dust, dirt, soot, or smoke. Particulate pollutants are described in terms of particle size: PM2.5 and PM10 having an aerodynamic diameter less than 2.5 pm and 10 pm, respectively. It is preferred that in the method in accordance with the invention, the model fine particulate matter resembles PM2.5 or PM10 at least in size. The method in accordance with the invention is carried out on a human skin-equivalent (HSE), i.e. a material that resembles the skin of human beings. Preferably the human skin-equivalent is artificial skin, donor skin, living skin equivalent or pig skin. Further preferably the artificial skin is a polymer-based substrate mimicking human skin.

Currently, several HSEs are commercially available for such applications. Examples include Apligraf^®, Epicel^®, Dermagraft^®, Alloderm^®, Transcyte^®, Orcel^®, Integra^® DRT, Epistem^® and StrataGraft^®. These HSEs can be divided into three major categories: epidermal, dermal, and full-thickness models. A particularly preferred human skin- equivalent is Vitro-Skin® which is a type of artificial skin. To simulate real-life situation to the extent possible, it is preferred that in the method in accordance with the invention, the model fine particulate matter is dispersed in an artificial human sebum composition comprising one or more fatty acids, one or more fatty acid esters and cholesterol to thereby form a dispersion, and said dispersion is contacted with the human skin-equivalent. A preferred composition of artificial human sebum is disclosed in US20080254074 A1 (Unilever) but any other suitable artificial human sebum may also be used instead. Further, in the method in accordance with this invention, the human skin-equivalent is contacted with 0.1 to 0.5 pg of the model fine particulate matter per cm².

Chemical imaging refers to the analytical method to create a visual image of components distribution from simultaneous measurement of spectra and spatial, time information. Imaging instrumentation has three components: a radiation source to illuminate the sample, a spectrally selective element, and usually a detector array (the camera) to collect the images. The data format is called a hypercube. The data set may be visualized as a data cube, a three-dimensional block of data spanning two spatial dimensions (x and y), with a series of wavelengths (l) making up the third (spectral) axis. The hypercube can be visually and mathematically treated as a series of spectrally resolved images (each image plane corresponding to the image at one wavelength) or a series of spatially resolved spectra.

The chemical imaging technique is a spectroscopic chemical imaging technique.

Preferably such a technique is IR, Raman or XRF and said model fine particulate matter is correspondingly responsive to said technique.

Alternatively, the chemical imaging technique is a microscopic chemical imaging technique. Preferably such a technique is fluorescence microscopy, MS Imaging, SEM or light microscopy and said model fine particulate matter is correspondingly responsive to said technique.

Chemical imaging does not mean or include colorimetric or digital image analysis.

It is preferred that the model fine particulate matter comprises a synthetic polymeric material, a natural polymeric material, a water- insoluble salt, a mineral, a metal, an alloy, glass or a mixture thereof. Further preferably the synthetic polymeric material is a polyamide, polyacetate, polyester, polyacrylate, polystyrene, polyethylene, polypropylene, rayon, polyvinyl chloride or a mixture thereof. Further preferably the natural polymeric material is cellulose, regenerated cellulose, starch, microcrystalline cellulose or a mixture thereof.

In accordance with the method in accordance with the invention, in step (i), a human skin-equivalent is contacted with a model fine particulate matter resembling a particulate atmospheric pollutant and a first concentration of the model fine particulate matter on the human skin equivalent is measured by an imaging technique, where said chemical imaging does not mean or include colorimetric or digital image analysis. The first concentration could be, and preferably is, measured as a function of a signal emitted by the particulate atmospheric pollutant. Preferably the concentration is directly proportional to the intensity of the signal. The signal would vary depending on the nature of the analysis and the technique used. For example, when the technique is fluorescence microscopy, the first concentration is measured as a function of the fluorescence intensity (fluorescence signal).

Thereafter, step (ii) involves cleansing the human skin equivalent bearing the model fine particulate matter thereon with the cosmetic cleansing composition and measuring a second concentration of said model fine particulate matter on said human skin equivalent by the imaging technique. For example, when the technique is fluorescence microscopy, the second concentration is also measured as a function of the intensity of fluorescence.

While cleansing the human skin equivalent with the personal cleansing composition, it is preferred that an aqueous dispersion of the composition which comprises an amount thereof equivalent to the amount under normal in-use conditions of said composition. This is useful for accurate analysis because it simulates in-use conditions.

Yet further preferably, to further reduce the chances of human error, it is preferred that an automated cleansing procedure is used. A suitable example includes a procedure according to SDL ATLAS M235 Martindale.

Step (iii) of the method involves differentiating between the first and said second concentration. Thus, in the case where the technique is fluorescence microscopy, differentiation between the first and the second response is based on the respective fluorescence signals measured as intensity of fluorescence in each case.

Finally, in step (iv) of the method of the invention, the efficacy of the candidate cosmetic composition is ascertained based on the information of step (iii).

To reduce errors or incorrect analysis, it is preferred that the first and said second response is measured under identical conditions, i.e., by following identical steps of measurement.

It is preferred that the efficacy is determined and recorded in terms of % cleaning efficacy which preferably is measured in accordance with the following equation: Efficacy

The background reading is the concentration measured on the human skin-equivalent comprising said artificial human sebum composition thereon but devoid of said model fine particulate matter. To further lend credibility to the data, it is preferred that the data pertaining to cleansing efficacy is further subjected to statistical analysis and represented as mean efficacy ± standard deviation. These terms have their usual standard meaning as known from standard textbooks of statistical analysis. It is further particularly preferred that the data after statistical analysis is considered significant if the confidence level is 95% (p < 0.05).

In accordance with the present invention, the model fine particulate matter is in the form of beads comprising polystyrene and a fluorescent material, the microscopic imaging technique is fluorescence microscopy and the human skin-equivalent is artificial skin. A particularly preferred material is Fluorescent Probes which are polystyrene-based particles (about 1 pm diameter) with fluorescent tagging Ex. Polysciences Inc. Further it is preferred that the fluorescent material absorbs and emits radiation in the range of wavelength 400 to 800 nm, more preferably 400 to 600 nm. Accordingly, the

measurements are made in the abovementioned range.

Accordingly, in such case, each of the first and second concentration is measured by measuring intensity of fluorescence of the beads comprising polystyrene and a fluorescent material. Further preferably, in such case, in said step (i) the human skin- equivalent is mounted on a glass plate to facilitate measurement. Yet further preferably in such case, in the step (ii) the human skin-equivalent is mounted on non-woven cloth to facilitate measurement. A particularly preferred automated cleansing procedure is according to SDL ATLAS M235 Martindale which is followed for cleansing the human skin-equivalent bearing the model fine particulate matter thereon using an aqueous dispersion of the composition which comprises an amount of the composition equivalent to the amount under normal in-use conditions of the composition. In this case, further, the difference in intensity of fluorescence is used for differentiating between the first and the second concentration and the difference is measured by an image analysis software such as Image J.

In the above case, it is preferred that efficacy is determined by the following equation:

Fluoresence Intensity after cleansing Background reading

Efficacy = 1 1— x 100%

Fluoresence Intensity before cleansing Background reading where said background reading is the intensity of fluorescence measured on the human skin-equivalent comprising the artificial human sebum composition thereon but devoid of the model fine particulate matter.

Demonstration of efficacy

In a second aspect of the invention is disclosed a process of demonstrating efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants in contact with the skin. Such a demonstration may be useful for any consumer promotion event, or a consumer demonstration such as in a mall or supermarket or a consumer fair. The demonstration may also be useful for claim support and advertising. The method provides reliable and results at much lower pollutant dosage of

approximately 0.25 pg/cm² of skin which better represents a real-life situation of skin exposed to pollutants, especially particulate pollutants.

The Cosmetic Cleansing Composition

Suitably, the composition comprises 10 to 90 wt%, preferably 20 to 85 wt% and more preferably 60 to 85 wt% surfactant, which usually is a combination of 2 or more surfactants. Typical surfactants include those surface active agents which contain an organic hydrophobic group with from 8 to 14 carbon atoms, preferably from 10 to 14 carbon atoms and at least one water-solubilising group which is preferably selected from sulphate, sulphonate, sarcosinate and isethionate. Specific examples of such anionic cleansing surfactants include ammonium lauryl sulphate, ammonium laureth sulphate, trimethylamine lauryl sulphate, trimethylamine laureth sulphate, triethanolamine lauryl sulphate, trimethylethanolamine laureth sulphate, monoethanolamine lauryl sulphate, monoethanolamine laureth sulphate, diethanolamine lauryl sulphate, diethanolamine laureth sulphate, lauric monoglyceride sodium sulphate, sodium lauryl sulphate, sodium laureth sulphate, potassium lauryl sulphate, potassium laureth sulphate, sodium lauryl sarcosinate, sodium lauroyl sarcosinate, lauryl sarcosine, ammonium cocoyl sulphate, ammonium lauroyl sulphate, sodium cocoyl sulphate, sodium lauryl sulphate, potassium cocoyl sulphate, potassium lauryl sulphate, monoethanolamine cocoyi sulphate, monoethanolamine lauryl sulphate, sodium tridecyl benzene sulphonate, sodium dodecyl benzene sulphonate, sodium cocoyl isethionate and mixtures thereof. A preferred class of

anionic cleansing surfactants for use in the invention are alkyl ether sulphates. Specific examples of such preferred anionic surfactants include the sodium, potassium, ammonium or ethanolamine salts of C10 to C12 alkyl sulphates and C10 to C12 alkyl ether sulphates (for example sodium lauryl ether sulphate), Mixtures of any of the above described materials may also be used.

Suitable silicones for use in the cosmetic cleansing compositions include

polydiorganosiloxanes, in particular polydimethylsiloxanes (dimethicones), polydimethyl siloxanes having hydroxyl end groups (dimethiconols), and amino-functional polydimethylsiloxanes (amodimethicones). Such silicones are preferably non-volatile (with vapour pressure of less than 1000 Pa at 25 °C), and preferably have a molecular weight of greater than 100,000 D, more preferably greater than 250,000 D.

In a typical composition the amount of silicone (per se as active ingredient) will generally range from 1 to 20 wt%, preferably 2 to 8 wt%.

The compositions suitably include at least one inorganic electrolyte. The inorganic electrolyte may be used to help provide viscosity to the composition. Suitable inorganic electrolytes include metal chlorides (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, ferric chloride and aluminium chloride) and metal sulphates (such as sodium sulphate and magnesium sulphate).

The term "benefit agent" in the context of this invention includes materials which can provide a benefit to the hair and/or the scalp and/or the skin (preferably the hair and/or the scalp) as well as those materials which are beneficially incorporated into

cosmetic cleansing compositions, such as aesthetic agents. The benefit agent may suitably be selected from perfumes, cosmetic active ingredients such as antimicrobial agents, antidandruff agents, moisturisers, conditioning agents, sunscreen agents, physiological coolants and emollient oils; and mixtures thereof. A composition of the invention may contain further optional ingredients to enhance performance and/or consumer acceptability. Examples of such ingredients include fragrance, dyes and pigments, pH adjusting agents and preservatives or antimicrobials. Each of these ingredients will be present in an amount effective to accomplish its purpose. Generally, these optional ingredients are included individually at a level of up to 5 percent by weight based on the total weight of the composition. The pH of the composition preferably ranges from 4 to 7, more preferably from 5.5 to 6.5. Mode of Use

The composition of the invention is primarily intended for topical application to the body, preferably the hair and scalp.

Most preferably the composition of the invention is topically applied to the hair and then massaged into the hair and scalp. The composition is then rinsed off the hair and scalp with water prior to drying the hair.

The invention will now be described in detail with the following non-limiting examples. Examples

Example 1 : In vitro method to determine cleansing efficacy of cosmetic cleansing compositions using fluorescence microscopy In this experiment, Vitro-Skin® was used as the human skin equivalent and

Fluorescent Probes® was used the model fine particulate matter. Artificial human sebum composition was prepared in accordance with the formulation disclosed in US20080254074 A1 (Unilever). Step 1 of the method involved contacting the Vitro-Skin® with Fluorescent Probes®.

Step 1

Vitro-Skin® was cut into pieces of approximately 2 cm ^c 7 cm area and the pieces were mounted on glass slides (exposure size ~10 cm²) by using a sticky tape.

Thereafter, in the absence of light, Fluorescent Probes was mixed with model sebum by using a vortex mixer. Thereafter, the mixture was doped on Vitro-Skin®. The slide was tiled to allow the model pollutant to distribute evenly. The final concentration of Fluorescent Probes on the Vitro-Skin® was 0.25 pg/cm². In Step 2, Vitro-Skin® with Fluorescent Probes thereon, was placed in a fluorescence microscope (Leica® SP5) under the following parameters/settings. In Step 2, fluorescence imaging was done before using any candidate cosmetic cleansing composition.

Table 1

In Step 3, the Vitro-Skin® with Fluorescent Probes thereon was subjected to automated cleansing, as follows:

The candidate cosmetic cleansing composition was mixed thoroughly with deionized water (2 g composition, 10 g water)) to generate enough lather to simulated in-use conditions. A piece of non-woven cloth was first mounted on a Teflon® stand and then immersed (cloth and the stand) in the lather for 30 seconds. Thereafter, an automated cleansing procedure was followed according to SDL ATLAS M235 Martindale, [(33 g force, 23.8 rpm for 20 circles (~ 50 seconds)].

After the cleansing procedure, deionized water was used to rinse the cloth. The cloth was air dried thoroughly before being subjected to fluorescence imaging again, following the procedure of Step 2.

In Step 4, all the images were analysed by ImageJ (1.47v, NIH) software without any processing. The overall intensity was captured by‘measure’ function. The efficacy was calculated by following the equation:

Fluoresence Intensity after cleansing Background reading

Efficacy = 1 1— x 100%

Fluoresence Intensity before cleansing Background reading The background reading was the intensity of fluorescence measured on Vitro-Skin® supplemented with model sebum but without Fluorescent Probes. The background reading remained constant under the same fluorescent imaging parameters, thereby permitting a robust and reliable comparison of the data generated through two or more sets of experiments.

Thereafter, the data was subjected to statistical analysis and the values were represented as mean cleansing efficacy ± standard deviation. Statistical significance of the data was determined by using an independent one-tailed Student’s t- test for two sets of data using EXCEL software. If the Confidence level was 95% (p < 0.05), the data was considered statistically significant.

Cleansing efficacy was measured and represented as the percentage removal of fluorescence particles (intensity).

The data, after statistical analysis, is shown in Table 2.

Table 2

The data in Table 2 clearly indicates that the method can clearly and unambiguously distinguish between the efficacy of two closely related, yet distinct products, with a reasonable degree of certainty. The cleansing efficacy of the soap-based cleanser was significantly greater than the non-soap surfactant based cleanser (95 ± 2% vs 86 ± 3%, p < 0.05) as well as plain water (95 ± 2% vs 56 ± 3%, p<0.0001 ). The cleansing efficacy of the non-soap surfactant-based cleanser was significantly greater than that of water (86 ± 3% vs 56 ± 3%, p < 0.0001 ). Statistical significance of the data reinforces the inferences. While it is generally believed that a soap-based cleanser is more as compared to its counterpart non-soap surfactant based cleanser, the extent to which the efficacy differs can very clearly be proven and demonstrated by the method in accordance with the invention.

Further, the test method was repeated in another set of experiments which were designed to find out the variation in results that often occur when an analyst repeats the tests with multiple samples of the same product, or when the same test is repeated was repeated by the same analyst or when two or more analysts perform the same test with one and the same product following identical procedure.

In such cases it is often seen that the coefficient of variance of the data (ratio of standard deviation to the average values) is high therefore the results are not reliable enough.

The data pertaining to these two sets of experiments done as per the method in accordance with the invention is shown in Table 3.

Table 3

The data in Table 3 clearly indicates that the co-efficient of variance is well within acceptable range. This further confirms that the method in accordance with this invention is robust and reliable.

The illustrated example clearly indicates that the method in accordance with the invention is a robust and reliable tool for measuring and comparing the efficacy of cosmetic cleansing compositions to remove particulate pollutants which are in contact with skin. The method could be used to measure and demonstrate cleansing efficacy of cosmetic cleansing compositions against atmospheric pollutants, especially particulate pollutants such as PM2.5 and PM10. The testing could be monadic, alternatively paired- testing or further alternatively discrete-choice testing. Outcome of the method could potentially be useful for relative ranking of various cleansing compositions belonging to the same category of products, such as a soap-based cleanser v/s a non-soap surfactant-based cleanser, or even, where necessary, between two or more products belonging to different categories of products for example, a shampoo against a soap bar. The method could also be useful to determine the efficacy of one or more active ingredients such as surfactants and polymers by suitably formulating the candidate compositions to be tested. Further, the method of the invention could also be used as a demonstration tool for consumer promotion or activation of new or existing cosmetic cleansing compositions. Outcome of the method could also be used for claim-support.

Claims

1 . An in vitro method to determine efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants adhering to the skin, comprising the steps of:

(i) contacting a human skin-equivalent with a model fine particulate matter resembling a particulate atmospheric pollutant and measuring a first concentration of said model fine particulate matter on said human skin equivalent by an imaging technique;

(iii) differentiating between said first and said second concentration; and,

(iv) ascertaining said efficacy from the information of step (iii),

wherein, said technique is a spectroscopic or microscopic chemical imaging technique, where said chemical imaging does not mean or include colorimetric or digital image analysis and wherein said human skin equivalent is artificial skin, donor skin, living skin equivalent or pig skin and wherein said model fine particulate matter is in the form of beads comprising polystyrene and a fluorescent material, further wherein said microscopic imaging technique is fluorescence microscopy and said human skin-equivalent is artificial skin.

2. A method as claimed in claim 1 wherein said model fine particulate matter

resembles PM2.5 or PM10 at least in size.

3. A method as claimed in claim 1 or 2 wherein said artificial skin is a polymer-based substrate mimicking human skin.

4. A method as claimed in any of claims 1 to 3 wherein said model fine particulate matter is dispersed in an artificial human sebum composition comprising one or more fatty acids, one or more fatty acid esters and cholesterol to thereby form a dispersion, and said dispersion is contacted with said human skin-equivalent.

5. A method as claimed in any of claims 1 to 4 wherein said spectroscopic chemical imaging technique is IR, Raman or XRF and said model fine particulate matter is correspondingly responsive to said technique.

6. A method as claimed in any of claims 1 to 4 wherein said microscopic chemical imaging technique is fluorescence microscopy, MS Imaging, SEM or light microscopy and said model fine particulate matter is correspondingly responsive to said technique.

7. A method as claimed in any of claims 1 to 6 wherein said model fine particulate matter comprises a synthetic polymeric material, a natural polymeric material, a water- insoluble salt, a mineral, a metal, an alloy, glass or a mixture thereof.

8. A method as claimed in claim 7 wherein said synthetic polymeric material is a polyamide, polyacetate, polyester, polyacrylate, polystyrene, polyethylene, polypropylene, rayon, polyvinyl chloride or a mixture thereof.

9. A method as claimed in any of claims 5 to 8 wherein said efficacy is determined by the following equation:

Efficacy

where said background reading is the concentration measured on said human skin-equivalent comprising said artificial human sebum composition thereon but devoid of said model fine particulate matter.

10. A method as claimed in claim 10 wherein data pertaining to efficacy is further subjected to statistical analysis and represented as mean efficacy ± standard deviation.

1 1. A method as claimed in claim 1 wherein each of said first and second

concentration is measured as a function of the intensity of fluorescence of said beads.

12. A method as claimed in claim 11 wherein said efficacy is determined by the

following equation:

Fluoresence Intensity after cleansing Background reading

Efficacy = 1 1— x 100%

Fluoresence Intensity before cleansing Background reading where said background reading is the intensity of fluorescence measured on said human skin-equivalent comprising said artificial human sebum composition thereon but devoid of said model fine particulate matter.

13. A process of demonstrating efficacy of a cosmetic cleansing composition or an active ingredient comprised therein to remove particulate pollutants adhering to the skin, said process comprising the steps of:

(iii) differentiating between said first and said second concentration; and,