study ref: 09-1093/2 / 09 - kreme.sistudy ref: 09-1093/2 / 09.0828 3 i. aim and principle of the...

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ASSESSMENT IN HUMAN OF THE ANTI-WRINKLE, MOISTURIZING AND WHITENING EFFECT AND THE EFFECT ON THE CELL RENEWAL OF THE EPIDERMIS OF A

COSMETIC PRODUCT

CHECKING OF ITS ACCEPTABILITY AFTER APPLICATION UNDER NORMAL CONDITIONS OF USE

SUBJECTIVE ASSESSMENT OF ITS COSMETIC QUALITIES AND EFFICACY

-Use test with clinical control by a dermatologist and with instrumental evaluation-

TABLE OF CONTENTS I AIM AND PRINCIPLE OF THE STUDY 3 II RELEVANCE OF THE STUDY 3 III TYPE OF THE STUDY 6 IV INVESTIGATOR CENTRE AND TECHNICAL STAFF 6 V DATES OF PERFORMANCE OF THE STUDY 6 VI TEST PRODUCT 7 VII VOLUNTEERS 7 VIII METHODOLOGY 9 IX RESULTS / DISCUSSION 20 X CONCLUSION 32

APPENDIX 1 34 APPENDICES 2 35-38 APPENDICES 3 39-40 APPENDICES 4 41-42 APPENDICES 5 43-46 APPENDIX 6 47 APPENDIX 7 48 APPENDIX 8 49 APPENDICES 9 50-51 APPENDICES 10 52-53

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I. AIM AND PRINCIPLE OF THE STUDY This study intended to assess the anti-wrinkle, moisturizing and whitening effect and the effect on the cell renewal of the epidermis, as well as to check the acceptability and to assess the cosmetic qualities and efficacy of a cosmetic product, after application under the normal conditions of use planned by the Sponsor. The efficacy of the product was assessed, objectively and quantitatively:

- anti-wrinkle effect: after taking of replicas, before and after treatment and image analysis with computer (Quantirides® software),

- moisturizing effect, by instrumental measurements of the capacitance of the skin, with the

Corneometer®, at different experimental times, before and after treatment,

- whitening effect, by instrumental measurements of the melanin index, with the Mexameter®, before and after treatment,

- cell Turn-over effect: by comparing the kinetics of decrease in skin colour obtained by

application of dihydroxyacetone (DHA) to a skin area previously treated with the product and to an untreated area.

The acceptability was:

- checked every day, by the volunteers themselves at home,

- controlled after visual examination of the experimental areas, by a dermatologist or the technician, under his authority, and after questioning of the volunteers.

The cosmetic qualities and efficacy were assessed, at the end of the study, using a target questionnaire. II. RELEVANCE OF THE STUDY Ethics The study aiming at a better knowledge of the cutaneous tolerance and efficacy of the test product and the foreseeable risk incurred by the volunteers who took part in the study being minor, there was a suitability between the aim of the study and its possible risks. Methodological approach The experimental conditions adopted (experimental areas, quantity of product applied, frequency and duration of the applications…) reproduced the normal conditions of use advocated and the test performed on a “small scale”, reflected the application by the future consumers. The observance of the experimental conditions by the volunteers who took part in the study was assessed by a questionnaire at the end of the study and by a control of the product consumption. The volunteers’ opinion was taken into account since it could reflect that of the potential consumers.

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* Assessment of the efficacy

The efficacy of the product was assessed by the Technical Department Manager of the investigator centre who has an appropriate experience or by a qualified and experienced technician under his authority. The method chosen for the assessment of the anti-wrinkle effect used the replica technique. Numerous publications support this methodology, notably:

- Lévêque J, EEMCO guidance for the assessment of skin topography, Jour. Of the European Academy of Dermatology and Venereology, 1999, 12, pp. 103-114,

- Corcuff P., Chatenay F., Brun A., Evaluation of antiwrinkle effects on humans, Int.J.

Cosmet. Sci., 1985, 7, pp. 117-126,

- Corcuff P., De Rigal J., Makki S., et al, Skin relief and aging, J. Soc. Cosmet. Chem., 1983, 34, pp. 177-190.

The experimental areas were the crow’s-feet which corresponded to the usual application site of the product. The method chosen for the assessment of the moisturizing effect used capacitance measurements. Numerous publications support this methodology, notably:

- Berardesca E , EEMCO guidance for the assessment of stratum corneum hydration: electrical methods, Skin Research and Technology, 1997, 3, pp. 126-132

- Clarys P , Barel A.O , In vitro calibration of the capacitance method (Corneometer CM

825 ®) and conductance method (Skicon 200 ®) for the evaluation of the hydration state of the skin, Skin Research and Technology, 1997, 3, pp. 107-113

- Clarys P , Barel A.O , Gabard B , Non-invasive electrical measurements for the evaluation

of the hydration state of the skin : comparison between three conventional instruments – the Corneometer ®, the Skicon ®, and the Nova DPM ®, Skin Research and Technology, 1999, 5, pp. 14-20

- Fluhr J.W , and col. , Comparative study of five instruments measuring stratum corneum

hydration (Corneometer CM 820 and CM 825 ®, Skicon 200 ®, Nova DPM 9003 ®, Dermalab ®) Part II In vivo , Skin Research and Technology, 1999, 5, pp. 171-178

The experimental areas were the forearms, less exposed than face to the intercurrent environmental factors.

The methodology chosen for the assessment of the whitening effect used the measurements of the melanin index on the skin. Numerous publications supported this methodology, notably:

- Chardon A., Crétois I., Housseau C.: Comparative colorimetric follow-up on humans of the tannings induced by cumulative exposure of UVB, UVA and UVB + A. 16th IFSCC congress, New York, Preprint 1, 1990, 51-70

- Piérard G.E., EEMCO guidance for the assessment of skin colour. Jour Europ Acad of

Dermatology and Venerology, 1998, 10, 1-11

- Chardon A., Crétois I., Hourseau C. Skin colour typology and suntanning pathways. International Journal of Cosmetic science, 1991, 13, 191-208

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- Muizzidin N., Marrenus K., Maes D., Smith W.: Use of chromameter in assessing the

efficacy of anti-irritants and tanning accelerator’s. J. Soc Cosmet. Chem. (Nov/Dec 1990), 41, 369-378

- Ok-Sub Lee, Eun-Joung Klm: Skin lightning, Cosmetics & Toiletries 1995, 110, pp. 51-56

- Zuidhoff H.W., Van Rijsbergen J.M.: Whitening efficacy of frequently used whitening

ingredients, Cosmetics & Toiletries 2001, 116, pp. 53-59 The experimental area was one hyperpigmented spot on face. The method chosen for the assessment of the effect on the cell renewal of the epidermis used colorimetric measurements. This methodology has been established on the basis of numerous publications, between these:

- Pierard G.E, Pierard-Franchimont C., Dihydroxyacetone Test as a Substitute for the Dansyl Chloride Test. Dermatology 1993; 186:133-1737.

- Meybeck A., Etude des mélanoïdines obtenues par la réaction de la dihydroxyacétone

avec les aminoacides. Parfums, Cosmétiques et arômes, 1977,14 pp. 63-71.

- Chardon A., Cretois I., Hourseau C. Skin colour topology and suntanning pathways. International Journal of Cosmetic science, 1991, 13, pp. 191-208.

The experimental areas were the forearms.

* Assessment of the acceptability

The acceptability of the product was controlled by the dermatologist, who has an appropriate experience or by a qualified and experienced technician under his authority. Numerous publications supported this methodology, notably:

- Jackson E.M. & Robillard N.F., The controlled use test in a cosmetic product safety substantation program, J.Toxicol.Cut. Ocular. Toxicol., 1982, 20, pp.117-132

- Jackwerth B., Krächter H.U., Matthies W., Dermatological test methods for optimising

mild tenside preparations, Parfümerie und Kosmetik, 1993, 74, pp.134-141

- Keswick B.H., Ertel K.D., Visscher M.O., Comparison of exaggerated and normal use techniques for assessing the mildness of personal cleansers, J. Soc. Cosm. Chem., 1992, 43, pp.187-193

Panel The panel corresponded to the population likely to use the product: the main inclusion criteria (dry skin, wrinkles on the crow’s feet area and hyperpigmented spots on face) corresponded to the target market of the product. The number of volunteers defined in the protocol was sufficient to check the acceptability and to assess the efficacy of the product.

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Results *Assessment of the efficacy The instrumental efficacy data were expressed in numbered data and were submitted to a suitable statistical treatment. The subjective efficacy data were submitted to a suitable statistical treatment (χ²). *Assessment of the acceptability The results of the acceptability were mainly expressed as descriptive data and did not require a statistical treatment. If the test product was well accepted by the volunteers, under these experimental conditions, by extrapolation they would be safe for human health when applied by a great panel of consumers. III. TYPE OF THE STUDY This monocentric study was performed in open. The subject was used as own control. It was performed according to the general conditions of Evic Hispania, established for the performance of Human test project. The test project was submitted to the previous agreement of the internal committee of Evic Hispania before its performance (opinion nº CI-S43/09 of October 16th, 2009). IV. INVESTIGATOR CENTRE AND TECHNICAL STAFF

IV.1. Investigator centre

EVIC HISPANIA Centro Experimental de Evaluación Cutánea, S.L. Travessera de Dalt n° 38, entlo. 4a

08024 Barcelona (Spain) tel : (+34) 93.285.14.46

IV.2. Technical staff Investigator: Dr. Isabel DEL BUSTO (Dermatologist) Technical Department Manager: Nuria SUSO Responsible technician: Mireia LLOBET V. DATES OF PERFORMANCE OF THE STUDY Beginning on: October 19th, 2009 End on: November 16th, 2009

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VI. TEST PRODUCT

VI.1. Identification of the test product

Denomination SNAKE ESSENCE

Reference 50709

Batch number 50709

Evic Hispania reference 09.0828

Galenic form and organoleptic characteristics Yellow emulsion

Number and type of samples 28 pots

Content of samples 50 g

VI.2. Information concerning the test product

The documents relating to the test product supplied with the samples were the qualitative and quantitative formula and the Sponsor’s letter of agreement particularly concerning the conformity of the formula to the regulations in force and its safety. VII. VOLUNTEERS

VII.1. Number The number of volunteers whose data had to be exploitable at the end of the study was 10. 13 volunteers were included in the study. No volunteer discontinued and no exclusion was decided by the investigator. The acceptability, cosmetic qualities and efficacy of the test product were therefore assessed in 13 volunteers. However, the anti-wrinkle and whitening effect of the test product were assessed on 12 volunteers because the results obtained for volunteer ref. 1 (anti-wrinkle effect) and volunteer ref. 11 (whitening effect) could not be exploitable.

VII.2. Specific inclusion criteria The specific inclusion criteria were the following ones:

- age: 40-70 years old, - sex: female, - phototype (Fitzpatrick): I to V, - all skin types,

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- having wrinkles and little wrinkles on the crow’s-feet area, which importance had to

correspond to a value of 2 or 3, evaluated following an ordinal scale in 4 points established by the investigator centre:

0 – absence of wrinkles and/or little wrinkles 1 – light wrinkle condition 2 – moderate wrinkle condition 3 – important wrinkle condition

- dry and hairless skin on the anterior side of the forearms (corneometric index at inclusion

< 55), - exhibiting at least one hyperpigmented spot on face (≥ 5mm).

All the volunteers corresponded to these specific inclusion criteria. Their typological characteristics are defined in Appendix 1.

VII.3. Specific non inclusion criteria The specific non inclusion criteria, defined in the protocol, were the following ones :

- cutaneous marks on the experimental areas which could interfere with the assessment of skin reactions (pigmentation troubles, scar elements, over-developed pilosity, ephelides and naevi in too great quantity, sunburn…..),

- allergy or reactivity to products of the same category than the tested one,

- allergy to latex,

- allergy or reactivity to the dihydroxyacetone (DHA),

- having a history of malignant melanomas (Dubreullh melanosis….),

- treatment with a whitening product or Vitamin A acid or its derivatives stopped less than

3 months before the beginning of the study,

- forecast of intensive sun or UVA exposures (U.V. lamps) during the test period,

- forecast of bath (sea or swimming-pool), of sauna or hammam sessions during the test period,

- anti-wrinkle treatment on the experimental area stopped less than 7 days before the

beginning of the study, - moisturizing treatment on the experimental area stopped less than 7 days before the

beginning of the study. All the volunteers corresponded to these specific non inclusion criteria.

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VIII. METHODOLOGY

VIII.1. Experimental conditions of use of the test product The experimental conditions, defined in the protocol, were the following ones:

Experimental areas Product directions for use Applications at the investigator centre

Applications at home Frequency/

duration

Face and neck

Apply on clean and dry skin, by gentle massage until complete

absorption

None

From D0 to D28

Application twice a day (morning and night), for 28

± 2 consecutive days

(4 weeks)

Anterior side of one forearm: one forearm

chosen at random receives the product

and the other forearm serves as control.

Apply by gentle massage until

complete absorption

On D0

Treated area: Application of the

product, by the technician, on the chosen forearm, to a delimited skin

surface of approximately 25 cm², at the rate of 2 mg/cm².

Control area: contra lateral forearm,

non treated

From D0 to D27

Application twice a day (morning and night), for 28

± 2 consecutive days

(4 weeks)

VIII.2. Constraints of the study

The constraints imposed on the volunteers were the following ones:

- no application of products similar to the tested one to the experimental areas, - full respect of the test product conditions of use,

- no change in hygiene and/or make-up habits, - on D0 no contact of the clothes with the experimental area (forearm), between the

application of the product and the end of the measurements,

- no intensive sun or UVA exposure (U.V. lamps) during the test period,

- no bath (bathtub, sea or swimming-pool), no sauna or hammam sessions during the test period,

- no application of make-up to the experimental area (face) the days of examination at the

investigator centre,

- neither anti-allergic, anti-inflammatory treatment nor treatment with patent medicines containing Vitamin A acid or its derivatives during the study (if therapeutic requirement : exclusion foreseen),

- no drugs or dietary supplements containing carotene, - no self-tanning products or tanning accelerator products on the experimental areas,

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- no clinical peeling or scrub or exfoliating products on the experimental areas,

- description of any treatment undertaken during the study and all eventual deviations.

VIII.3. Control of the observance of the modalities of the protocol The volunteers were questioned at the end of the study about the way they used the test product. The investigator assessed the importance of the possible deviations in comparison with the experimental conditions required at the beginning of the study. The investigator checked the respect of the constraints. The synthesis of the answers obtained is enclosed in Appendices 2/1 to 2/3. All the deviations from the protocol were analysed and the investigator assessed their effect on the validity of the results. Deviations from the protocol concerning the experimental conditions of use at home were noticed by the investigator and are enclosed in Appendix 2/1. The investigator considered these deviations as minor and without effect on the results. All the constraints of the study, defined in the protocol, were respected by the volunteers. A control of the product consumption was performed at the end of the study. The sample(s) given to each volunteer was (were) weighed before opening, at the beginning of the study and after the last application. The real average consumption of the panel was compared to the theoretical consumption defined by the investigator at the beginning of the study (and estimated around 1.0 gram per application) and the individual deviations from the theoretical consumption were analyzed. The results of the weighing of the samples and the individual consumptions worked out from these data are enclosed in Appendix 2/4. The mean consumption of the panel is 1.0 ± 0.3 g per application. Thus it is very close to the theoretical consumption defined at the beginning of the study.

VIII.4. Checking of the acceptability VIII.4.1. Frequency of the examinations The volunteers were requested to note every day any reaction observed and sensation of discomfort felt on the individual observation sheet they were given at the beginning of the study. A skin examination of the experimental areas had to be performed by the dermatologist or responsible technician. This examination had to be performed visually under standard “daylight” source, before then after 28 +/- 2 consecutive days of use. Concurrently with the clinical examination performed after the use of the product, each volunteer had to be questioned about the possible sensations of discomfort he felt. All the examinations were performed in accordance with the conditions defined in the protocol.

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VIII.4.2. Expression of the results of the questioning and skin examination The information gathered during the questioning was compared to that noted every day by the volunteer on his individual observation sheet. The expression of the results of the skin examination and questioning was that defined for this type of study in accordance with the corresponding procedure. Expression of the data gathered in the individual observation sheet The volunteer had to note any reaction or sensation of discomfort on the sheet reserved for that effect, using their own words to express what they felt. Expression of the results of the questioning and skin examination In case of reactivity :

− the main sensations of discomfort were described, i.e. : Heating, Stinging, Pruritus (itching), Pulling, Burning, Watering. The intensity of the sensations of discomfort was assessed according to an ordinal scale : slight, moderate, severe.

− the main visible signs were noted, i.e. :

Erythema, Oedema, Vesicle, Bulla, Papule, Scab, Dryness, Coloration, Macula. The intensity of the erythema and oedema was assessed according to an ordinal scale : slight, moderate, severe. The appearance of the erythema was specified : diffuse, punctuated. The importance of the number of vesicles was assessed according to an ordinal scale : 1 to 2 vesicles, more than 2 vesicles. The papules, bullae and maculae were counted. The scabs, dryness and coloration were described. The importance of the dryness and coloration was assessed according to an ordinal scale : slight, moderate, severe. The dermatologist or responsible technician noted for any clinical sign or sensation of discomfort described its location, duration, date of occurrence after application of the product, frequency, intensity, evolution, medical treatment possibly undertaken, ascribability of the product : none, doubtful, possible, obvious. He also noted the usual or unusual characteristic of the clinical sign or sensation of discomfort described, by questioning the volunteers about the effects observed when applying similar product. The results were expressed in a descriptive manner for the visible signs or the sensations of discomfort and the percentage of “reactive” volunteers was calculated. VIII.4.3. Interpretation of the results of the questioning and skin examination All the volunteers included in the study were taken into account to check the acceptability of the test product as long as they were submitted at least to one post application examination at the defined time or else.

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The interpretation of the results of the questioning and skin examination and of the data gathered in the individual observation sheet was that defined for this type of study in accordance with the corresponding procedure. This interpretation, performed by the dermatologist was absolute, referring to the following grading scale, which takes into account the sensations of discomfort described by the volunteers and the visible reactions of irritation (clinical signs). The test product could therefore have a very good, good, moderate or bad acceptability.

Acceptability % of volunteers exhibiting clinical signs ascribable to the test product

% of volunteers exhibiting sensations of discomfort ascribable

to the test product

Very good 0 % 0 %

Good 0 % < 25 %

< 10 % +/- Moderate

0 % 25 to 50 %

≥ 10% +/- Bad

0 % > 50 %

According to the nature and intensity of the clinical signs or sensations of discomfort, the dermatologist could have to under-class or over-class the product in relation to the grading scale.

VIII.5. Assessment of the efficacy

VIII.5.1. Instrumental assessment of the anti-wrinkle effect VIII.5.1.1. Principle The skin relief was appreciated quantitatively, after taking of replicas and image analysis with computer, before and after 28 days of use of the test product. The anti-wrinkle effect of the test product was evaluated by comparison of the results obtained before and after treatment.

VIII.5.1.2. Silicone replicas and equipment The taking of replicas was performed according to the standard procedure defined by the investigator centre. After cleansing of the skin, an adhesive ring was fixed on the defined skin site. The silicone material (Silflo®) was applied in the middle of the ring. After a defined time, necessary for the silicone to harden, the silicone was removed. A negative replica of the skin was obtained. The capture of images (shadows process) was performed from the replica by a CDD camera connected to a computer, the replica being illuminated by a boring light source of 35° incidence. The image analysis was performed with the Quantirides® software (Monaderm).

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VIII.5.1.3. Environmental conditions

The environmental conditions imposed on the volunteers, 15 minutes before performing the replica and during the process were the following ones:

Controlled temperature: T = 20°C ± 2°C Controlled relative humidity: RH = 45% ± 15%

The environmental conditions were respected.

VIII.5.1.4. Measurement site

The measurements were performed on a perfectly defined and documented site on the crow’s-foot chosen at random.

VIII.5.1.5. Frequency of measurements

The measurements were performed on D0 and on D28.

VIII.5.1.6. Expression and interpretation of the results The Quantirides® software allowed to calculate the following parameters: − N : total number of wrinkles − LT : total length of wrinkles, expressed in mm − PM : mean depth of wrinkles, expressed in µm − ST : total surface of wrinkles, expressed in mm² The individual results were expressed: − in absolute values for each experimental time,

− in percentages of variation in comparison with the D0 values. The means and standard deviations were calculated for each parameter and for all the experimental times. All the volunteers included in the study were taken into account to assess the efficacy of the test product as long as they were subjected to the examinations on D0 and D28.

The interpretation of the results was absolute, referring to:

− the percentage of “reactive” volunteers:

o volunteers for whom two (at least) parameters of the three parameters N, LT, ST decreased with a percentage of variation in comparison with the value on D0 superior or equal to 10% : effect on the little wrinkles (effect 1),

o volunteers for whom the parameter PM decreased with a percentage of variation in comparison with the value on D0 superior or equal to 10% : effect on the deep wrinkles (effect 2),

− the mean percentage of improvement of the skin relief after treatment, for the

“reactive” volunteers and for each parameter related to the effect 1 or 2,

− the results of the statistical analysis of the data (comparison of the values obtained on the treated site before and after treatment with a suitable statistical test).

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VIII.5.2. Instrumental assessment of the moisturizing effect

VIII.5.2.1. Principle

The hydration level of the upper layers of the skin was quantitatively assessed by capacitance measurements at different experimental times. The effect of the product on the skin hydration level was assessed by comparing the measurements taken on the 2 experimental areas (treated and control). VIII.5.2.2. Equipment The measurements were performed with the Corneometer CM 825 PC ® (Courage & Khazaka) which works at a mean frequency of 1 MHz and is equipped with a 0.64 cm² probe made up of 75 µm electrodes. The capacitance of the system, represented by the electrodes and the upper layers of the skin, being influenced by the changes of the dielectric constant of the skin surface, when the hydration level of the stratum corneum varies, the dielectric properties of this medium are modified and consequently the capacitance of the system is modified.

VIII.5.2.3. Environmental conditions The environmental conditions imposed on the volunteers during the 15 minutes before each measurement and over the duration of the measurements were the following ones: Controlled temperature: T = 20°C ± 2°C Controlled relative humidity: RH = 45 % ± 15% The environmental conditions were respected.

VIII.5.2.4. Measurement sites The instrumental measurements were performed on a perfectly defined and documented site, in the centre of the control and treated area.

VIII.5.2.5. Rate of the measurements The measurements were performed on the control and treated areas before and after 2 and 4 hours (D0/T0, D0/T2h and D0/T4h) after the application performed on the treated area of the forearm on D0 and on D28, after the last application performed on D27. VIII.5.2.6. Assessment criteria The capacitance values of the system were expressed in arbitrary units ranging from approximately 0 to 120 (corneometric indexes).

VIII.5.2.7. Expression and interpretation of the results All the volunteers included in the study were taken into account to assess the efficacy of the test product as long as they were submitted to all the examinations at the defined time.

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The individual results were expressed:

− in absolute values for each experimental time,

− in percentages of variation in comparison with the D0/T0 values,

− in differences between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area.

The means and standard deviations were calculated. The interpretation of the results was absolute, referring to:

. the percentage of “reactive” volunteers at each experimental time : volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area is superior or equal to 10%,

. the mean percentage of the skin hydration level improvement due to the product at each experimental time (difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area), . the results of the statistical analysis of data (comparison of the values obtained on the control area with those obtained on the treated area using a suitable statistical test).

VIII.5.3. Instrumental assessment of the whitening effect VIII.5.3.1. Principle The whitening effect was assessed by comparing the skin colour on the dark spot chosen, before and after treatment (D0 and D28).

VIII.5.3.2. Experimental area The experimental area was the spot selected on the face, by the responsible technician. This spot (≥ 5 mm) was perfectly documented in the investigator’s brochure as the measurements before and after treatment had to be performed exactly on the same spot.

VIII.5.3.3. Equipment The measurements were perfomed with a Mexameter® MX 18 (Courage & Khazaka) which has a measuring probe with a diameter of measuring surface of 5mm. The melanin and hemoglobin (erythema) are the main responsible for the skin’s colour. The special probe emits light of two defined wavelengths (red and near infrared) in which the melanin absorbs and the absorption of the hemoglobin pigment is minimal, avoiding therefore the interference of the vascular state of the skin in the evaluation of the melanin content. The amount of light absorbed by the skin is calculated from the amount of light emitted by the device and the amount of light reflected by the skin, obtaining then the melanin index (M parameter):

M = ( 500/Log 5 ).{ Log (Infrared - Reflection / Red - Reflection) + Log 5 }

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The melanin content is expressed in arbitrary units which range from 0 to 1000 (melanin index). The higher the M parameter was, the more quantity of melanin was detected.

VIII.5.3.4. Environmental conditions The environmental conditions imposed on the volunteers during the 15 minutes before each measurement and over the duration of the measurements were the following ones: Controlled temperature: T = 20°C ± 2°C Controlled relative humidity: RH = 45 % ± 15% The environmental conditions were respected.

VIII.5.3.5. Measurement sites At each experimental time, the triplicate measurements of the melanin index were performed on the spot (see paragraph VIII.5.3.2), i.e. M1, M2, M3 and on the non pigmented skin around the spot i.e. M1’, M2’, M3’ according to the following schema: M1’ M2’ M3’

VIII.5.3.6. Frequency of measurements The measurements of the melanin index were performed on D0 and D28.

VIII.5.3.7. Expression and interpretation of the results All the volunteers included in the study were taken into account to assess the efficacy of the test product as long as they were submitted to all the examinations at the defined time. The results were expressed in mean values of the parameter M or melanin index. The difference between the value of the M parameter obtained on the pigmented spot and that obtained on the adjacent area (ΔM) was calculated. The individual results were expressed:

- in absolute values for the parameter M for each experimental time,

- in ΔM for each experimental time,

- in % of variation of ΔM on D28 in comparison with D0. The means and standard deviations were calculated.

M1 M2 M3

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The interpretation of the results was based on:

o the percentage of “reactive” volunteers: volunteers for whom the percentage of variation of ΔM on D28 in comparison with D0 is superior or equal to 10%,

o the mean percentage of skin whitening due to the effect of the product, for

the “reactive” volunteers (percentage of variation of ΔM on D28 in comparison with D0),

o the results of the statistical anaylisis of data: comparison of the values obtained on D0 and D28 using a suitable statistical test. VIII.5.4. Instrumental assessment of the effect on the cell renewal of

the epidermis VIII.5.4.1 Principle

The effect of the test product on the cell “Turn-over” was assessed quantitatively by colorimetric measurements performed at different experimental times. After 14 days of treatment with the test product, one test with DHA was performed on the two forearms (treated and control). The DHA is usually used in self-tanning products thanks to its properties to colour the skin. The effect of each product was assessed by comparing the kinetics of decrease in skin colour obtained by application of DHA to a skin area previously treated with the product and to an untreated area.

VIII.5.4.2 Equipment

The measurements were performed with the Colorimeter® CL400 (Courage & Khazaka), which has a console and a measuring probe connected to the console with a flexible cable.

The probe has a plastic and cylindrical part at the end of which we can find the measurement opening (diameter: 0.8 cm). This probe sends a white LED light, aligned in a circular way in order to illuminate the skin uniformly. The light emitted is dispersed in all directions, some parts go through the skin layers and some others are dispersed outside the skin. The light reflected by the skin is measured by the probe and is expressed in a corresponding way. The colour analyzed is converted in numeric values according to the C.I.E, obtaining thus the 3 coordinates in the colour space: L*, a* and b*.

VIII.5.4.3 Environmental conditions

The environmental conditions imposed on the volunteers during the 15 minutes before each measurement and over the duration of the measurements were the following ones:

Controlled temperature: T = 20°C ± 2°C Controlled relative humidity: RH = 45% ± 15%

The environmental conditions were respected.

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VIII.5.4.4 Measurement sites

At each experimental time, the colorimetric measurements were performed on a perfectly defined and documented site, in the centre of the control and treated area. These areas had to be different from the ones chosen to assess the moisturizing effect.

VIII.5.4.5 Frequency of measurements

On D14, after 14 days of treatment with the test product, the technician applied, on each forearm (treated and control), a product containing 6% of DHA under semi-occlusive patch (TruMed®: absorbent support in Webril® on which the product was put down (160 μl or around 160 mg) and kept in position by a non woven medical adhesive (surface: 400 mm²)). The semi-occlusive patch was kept in contact with the skin for 24 hours.

The first colorimetric measurement was performed by the technician on D16, 48 hours after the application of the DHA. The rest of the colorimetric measurements were performed by the technician from D18 to D28, every 48 hours, performing a total of 6 colorimetric measurements.

VIII.5.4.6 Expression and interpretation of the results The effect on the cell renewal of the epidermis of the treated and control areas was appreciated with the help of a kinetic curve. The ITA° angle was taken into account for the evaluation of the skin colour according to time, where:

ITA° = [tangent arc ( (L*-50)/b*] 180/3.1416 The results were expressed in mean values of the ITA° parameter. All the volunteers included in the study were taken into account to assess the efficacy on the test product as long as they were subjected to the examinations and measurements at the defined times. The individual results were expressed:

- in absolute values for each experimental time, - in gradient of the curves obtained for the values of the ITAº parameter, according to

time. The means and standard deviations were calculated. The interpretation of the results was absolute and was based on:

.the percentage of “reactive” volunteers: volunteers for whom the gradient of the curve obtained for the ITA° parameter according to time (from D16 to D28) on the treated area was higher than the gradient obtained for the untreated area, .the results of the statistical analysis of the data (comparison of the gradient of the curves obtained for the treated and untreated areas using a suitable statistical test).

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VIII.6. Assessment of the cosmetic qualities and efficacy (self-assessment)

The volunteers had to answer a questionnaire at the end of the study which gathered the items concerning the cosmetic qualities and efficacy of the product, defined with the Sponsor according to the category and target market of the test product, i.e. : * for the cosmetic qualities : - (Easy) Application - (Pleasant) Texture - (Quick) Absorption - (Pleasant) Fragrance - (Pleasant) Colour - Non oily * for the cosmetic efficacy : - Moisturizes skin - Nourishes skin - Anti-wrinkle effect - Leaves skin softer and smoother - Leaves skin more supple and elastic - Whitening effect - Reduces the size of the hyperpigmented spots - Lightens the hyperpigmented spots - Eliminates the hyperpigmented spots All the volunteers included in the study were taken into account to assess the cosmetic qualities and efficacy of the test product as long as they were submitted at least to one post application examination at the defined time or else. For each item relating to the cosmetic efficacy (except for items 11 and 17), the results were submitted to a suitable statistical analysis (2-rank χ² test: comparison between 2 percentages – satisfied volunteers and non satisfied volunteers). For each item, the volunteers had to express their satisfaction in an ordinal scale in 4 points: 0 = disagree; 1 = quite disagree; 2 = quite agree; 3 = agree; and the results were expressed in percentage of satisfied volunteers (scores 2 and 3).

χ² = Σ(|oi - ci|²) ci Oi = strength observed Ci = strength calculated α = 5 % risk

Significant difference if χ² > 3,84 (for α = 5 %) Non significant difference if χ² ≤ 3,84

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IX. RESULTS / DISCUSSION

IX.1. Checking of the acceptability The individual data of the questioning of the volunteers and skin examination are enclosed in Appendices 3/1 and 3/2. In brief:

Types of skin reactions ascribable to the test

product

% of volunteers exhibiting clinical signs ascribable to the test

product

Types of sensations of discomfort ascribable to

the test product

% of volunteers exhibiting sensations of discomfort ascribable to

the test product

None 0% None 0%

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21

IX.2. Assessment of the efficacy IX.2.1. Instrumental assessment of the anti-wrinkle effect

The individual data (absolute data for each experimental times and the percentage of variation in comparison with D0 for each parameter), are enclosed in Appendix 4. The analysis of the replicas taken from volunteer ref. 1 was impossible to perform due to an experimental error occurred during the replica sampling. Therefore, the results of the anti-wrinkle efficacy of the test product have been calculated on 12 volunteers. The percentage of “reactive” volunteers and the type of effect for these volunteers are reported on the table below:

Difference D28/D0 in %

Volunteer reference ST N LT PM

Effect type

1 NON EXPLOITABLE

2 36 -21 -16 -12 1+2

3 28 21 13 -5 /

4 -14 -11 -21 14 1

5 -40 9 -39 8 1

6 4 -21 -2 1 /

7 -22 -16 -29 14 1

8 -30 -7 -26 -1 1

9 -54 -56 -54 16 1

10 5 -53 -33 20 1

11 -46 -48 -51 22 1

12 -90 -81 -88 -13 1+2

13 -42 9 -26 -5 1

% of “reactive” volunteers (effect 1 and/or 2) 83%

% of “reactive” volunteers: effect 1 83%

% of “reactive” volunteers: effect 2 17%

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The percentages of “reactive” volunteers who showed an effect 1 and/or 2, after 28 days of treatment are reported on the graphics below:

83% 83%

17%

0%

20%

40%

60%

80%

100%%

of

" re

acti

ve "

vo

lun

teer

s

effect 1 and/or 2 effect 1 effect 2 Effecttype

The percentage of improvement for each of the parameters and for the “reactive” volunteers are reported on the table below:

Parameters % of improvement

ST 42%

N 38%

LT 38%

PM 12%

42% 38% 38%

12%

0%

20%

40%

60%

80%

100%

% o

f im

prov

emen

t fo

r th

e "r

eact

ive"

vol

un

teer

s

parameters

The iconography of the “reactive” volunteer ref. 13 is included in Appendix 10.

ST N LT PM

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* statistical analysis of the results

Comparison between D28 and D0 values Non parametric Mann-Whitney (Wilcoxon) W

test W p Significance

of the difference

ST 54.0 0.31232 NS

Student’s t test for paired series t p Significance

of the difference

N 2.68018 0.0214015 S

LT 4.23475 0.00140117 S

PM -1.41823 0.183826 NS

S = significant NS = Non significant

The statistical analysis of the results shows a significant decrease for two of the parameters defined, indicating an effect mainly on little wrinkles.

IX.2.2. Instrumental assessment of the moisturizing effect

The individual data (absolute values for each experimental time, percentages of variation in comparison with the D0/T0 values and the differences between the percentage of variation in comparison with D0/T0 treated area and the percentage of variation in comparison with D0/T0 control area) and the statistical analysis are enclosed in Appendix 5.

* Mean results on D0

Experimental times

Variation of the corneometric

indexes in comparison with

D0/T0 T2h T4h

Treated area 18.3% 20.4%

Control area 2.5% 3.4%

Difference between treated area

and control area 15.8% 17.0%

% of “reactive” volunteers* 69% 69%

% of improvement for “reactive” volunteers*

20.0% 23.5%

*volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area is superior or equal to 10%.

Maximal moisturizing effect 4 hours after application % of increase in the corneometric index = 17%

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For each experimental time, the percentage of “reactive” volunteers and the improvement for these volunteers is reported in the graphics below:

69%

20%

69%

24%

0%

20%

40%

60%

80%

100%

T2H T4H

% of "reactive" volunteers* % of improvement for "reactive" volunteers

*volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area is superior or equal to 10%.

* Statistical analysis of the results

(ANOVA test) F P Significance of the difference

Comparison Tr/C 2.20 > 0.05 NS

Comparison time 26.29 < 0.05 S

Comparison groups and time 13.73 < 0.05 S

S= significant NS= non significant The analysis of the results, using the statistical Anova test, shows a statistically significant increase in the corneometric indexes on the treated area, at both experimental times and therefore a statistically significant moisturizing effect.

Student’s t test for paired series t value p value Significance of the

difference

T0 and T2h -6.58526 0.0000259271 S

T0 and T4h -4.68447 0.000528193 S

S= significant NS= non significant

This test was performed on the variation treated-control. These results show a statistically significant moisturizing effect 2 hours after application of the product with a remaining effect 4 hours after application.

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* Mean results on D28

Variation of the corneometric indexes in comparison with D0/T0 D28

Treated area 19.9%

Control area 3.6%

Difference between treated area and control area 16.3%

% of “reactive” volunteers* 54%

% of improvement for “reactive” volunteers* 31.1%

*volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area is superior or equal to 10%. The results show that in comparison with D0/T0, the skin hydration level on D28 in the treated area is superior (+16.3%), which indicates that the repeated application at home had an accumulative moisturizing effect.

* Statistical analysis of the results

The statistical analysis (Student’s t test for paired series) allowed to compare the variation on the corneometric index before and after the treatment: The results of this analysis are shown in the table below:

Student’s t test for paired series t value p value Significance of the

difference

D0/T0 and D28 -2.99188 0.0112349 S

S= significant NS= non significant

This test was performed on the variation treated-control. The results of the statistical analysis show a statistically significant accumulative moisturizing effect, for the test product, after 28 days of use.

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IX.2.3. Instrumental assessment of the whitening effect

The measurements of the melanin index were performed on D0 and D28 and expressed in ΔM. The whitening effect was appreciated in percentage of “reactive” volunteers which showed a reduction on the measurements higher or equal to 10% on D28 in comparison with the values obtained on D0. The individual data (absolute values for each experimental time, in ΔM for each experimental time as well as the percentages of variation on D28 in comparison with D0) are included in Appendix 6. The measurements performed for volunteer ref. 11 were impossible to analyse due to an error occurred during the performing of the measurements. Therefore, the results of the whitening effect of the test product have been calculated on 12 volunteers. The mean values and standard deviations obtained for the M parameter at each experimental time for the treated and control areas are showed in the tables below and also the ΔM values for each experimental time and the % of variation on D28 in comparison with D0.

Melanin index (M)

D0 D28 Control

area Treated

area ΔM D0 (Tr-Co)

Control area

Treated area

ΔM D28 (Tr-Co)

% variation

ΔM (D28-D0)

Nº and % of “reactive”

volunteers*

% variation

ΔM (D28-D0)*

Mean 189.0 285.0 96.0 171.9 251.9 79.9 -20% -26%

Std. Dev. 46.7 86.2 49.8 44.6 88.9 54.3 17% 9 / 75%

14%

*volunteers for whom it is observed a diminution higher or equal to 10% on the pigmented spots (ΔM parameter) between D28 and D0. After28 days of treatment, it is observed a mean reduction of the melanin index (-20%) for the total of volunteers and a mean relative reduction of 26% on the "reactive" volunteers (75% of the panel).

* Statistical analysis of the results The statistical analysis (non parametric Mann-Whitney (Wilcoxon) W test) allowed to compare the variations on the melanin index before and after treatment. The results of this analysis are gathered below:

S=significant NS=non significant The statistical analysis does not show a statistically significant variation of the melanin index and therefore a statistically significant reduction of the skin pigmentation after 28 days of use.

ΔM Parameter W value p value Significance

D0 and D28 54.0 0.312215 NS

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IX.2.4. Instrumental assessment of the effect on the cell renewal of the

epidermis *Descriptive analysis

The individual data (values of ITA° angle for each volunteer, for each experimental time and for the treated and control area) are enclosed in Appendix 7. In brief:

The kinetic curves obtained for the treated and control areas are shown on the graphic below:

0,05,0

10,015,020,025,030,035,040,045,050,0

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Days of measurement

ITA

º val

ues

PRODUCT CONTROL

ITA° angle Treated area Control area

Day of measurement

Mean Standard deviation Mean Standard

deviation

D16 8.8 8.3 10.7 11.4

D18 12.7 8.4 15.8 9.2

D21 16.4 11.0 28.2 10.9

D23 21.8 11.8 33.5 10.0

D25 26.4 11.4 35.5 10.1

D28 34.0 14.1 39.1 10.2

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28

Moreover, for each volunteer and for each area the gradient of the curves obtained was calculated. The results obtained are represented on the following table:

GRADIENTS Volunteers references

Treated area Control area

“reactive”

volunteers*

1 2.0 1.9 +

2 3.7 3.4 +

3 2.5 3.6 -

4 1.3 1.7 -

5 2.8 3.3 -

6 2.5 2.3 +

7 2.6 1.5 +

8 1.4 2.1 -

9 1.2 1.9 -

10 2.8 3.6 -

11 1.4 2.7 -

12 0.8 2.0 -

13 2.0 2.2 -

Mean 2.1 2.5

Standard deviation 0.8 0.8 31%

*volunteers for whom the gradient of the curve obtained for the treated area is higher than the gradient of the curve obtained for the control area

These results show that there are 31% of “reactive” volunteers, for whom it can be observed an acceleration of the cell renewal epidermis, on the treated area, due to the test product, in comparison with the control area. On the other hand, the comparison of the kinetic curves obtained for the treated and control areas show that the skin colour produced by DHA does not decrease faster on the treated area than on the control area, which means that the product does not accelerate the cell renewal of the epidermis.

* Statistical analysis of the results The statistical analysis (Student’s t test for paired series) allowed to compare the gradient of the curves obtained for the treated and control areas after treatment. The results of this analysis are shown following:

t value p value Significance of the difference

Student’s t test for paired series

-2.09627 0.0579268 NS

S= significant NS= non significant The results show that after 28 days of treatment it is not observed a statistically significant effect on the cell renewal of the epidermis for the test product.

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IX.3. Assessment of the cosmetic qualities and efficacy (self-assessment)

Cosmetic qualities For each item, the number of volunteers according to the score and the percentage of satisfied volunteers is mentioned below:

Items 1. (Easy) Application 2. (Pleasant) Texture 3. (Quick) Absorption

Score 0 1 2 3 0 1 2 3 0 1 2 3

Number of volunteers 0 3 2 8 0 2 1 10 1 1 4 7

% of satisfied

volunteers 77% 85% 85%

Items 4. (Pleasant) Fragrance 5. (Pleasant) Colour 6. Non oily

Score 0 1 2 3 0 1 2 3 0 1 2 3


% of satisfied


For each item, the percentage of satisfied volunteers is enclosed in Appendix 8 and is reported in the graphics below:

77%

85% 85% 85% 85%92%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% o

f sa

tis f

ied

volu

n tee

rs

1 2 3 4 5 6 Items

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Cosmetic efficacy For each item, the number of volunteers according to the score and the percentage of satisfied volunteers is mentioned below:

Items 1. Moisturizes skin 2. Nourishes skin 3. Anti-wrinkle effect

Score 0 1 2 3 0 1 2 3 0 1 2 3


% of satisfied


Items 4. Leaves skin softer and

smoother 5. Leaves skin more supple and

elastic 6. Whitening effect

Score 0 1 2 3 0 1 2 3 0 1 2 3


% of satisfied


Items 7. Reduces the size of the

hyperpigmented spots 8. Lightens the

hyperpigmented spots 9. Eliminates the hyperpigmented spots

Score 0 1 2 3 0 1 2 3 0 1 2 3


% of satisfied


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31

For each item, the percentage of satisfied volunteers is enclosed in Appendix 9 and is reported in the graphics below:

85%92%

77%

85%

69%77% 77% 77%

54%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%%

of

sati

sfie

d vo

lun

teer

s

1 2 3 4 5 6 7 8 9 Items

For a significant percentage of volunteers, the product was judged that it:

- moisturizes and nourishes skin, - leaves skin softer and smoother.

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APPENDICES

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Appendix 1/1

TYPOLOGICAL CHARACTERISTICS OF THE VOLUNTEERS

Volunteers

Ref. Surname/ Name

Age (years)

Sex F=female

Phototype*

With expression wrinkles on the crow’s-feet area

Dry skin on the anterior side of

the forearms (corneometric

index < 55)

Exhibiting at least one

hyperpigmented spot on face

(≥ 5mm)

1 LEIV/LI 65 F IV x x x

2 AGUT/JO 70 F II x x x

3 FERN/FL 68 F III x x x

4 PATA/MN 49 F IV x x x

5 BOIX/CA 61 F III x x x

6 GARC/MJ 61 F III x x x

7 MANZ/MI 49 F II x x x

8 OTAL/MV 62 F II x x x

9 PERM/MM 65 F IV x x x

10 BALL/IS 63 F III x x x

11 DEPE/RO 55 F IV x x x

12 REUS/MA 55 F IV x x x

13 MATE/FE 62 F II x x x

Legends: / = no x = yes *phototype according to Fitzpatrick, established on the principle of a first 30 to 40-minute sun exposure after the winter or a period without exposure of an equivalent duration :

Type I : Always burns easily, never tans Type II : Always burns easily, tans minimally Type III : Burns moderately, tans gradually Type IV : Burns slightly, always tans easily Type V : Burns rarely, tans intensely Type VI : Never burns, strongly pigmented

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Appendix 2/1

CONTROL OF THE OBSERVANCE Modalities of use

Modalities of use (13 exploitable results)

Number of volunteers who respected the

modalities

Percentage of volunteers who respected the

modalities

Experimental areas: Face and neck and one forearm (chosen at random) Deviation : none

13 100%

Product mode of use: Application on clean and dry skin, by gentle massage until complete absorption Deviation : none

13 100%

Frequency of application at home : Twice a day (morning and night) Deviations : 4 volunteers

- Volunteer ref. 1: applied the product once a day on D1 and D2. - Volunteer ref. 6: applied the product 3 times a day on D16. - Volunteer ref. 8: applied the product once a day on D17, D19 and

D21. - Volunteer ref. 12: applied the product once a day on D26.

9 69%

Duration of application at home : 28 +/- 2 consecutive days Deviation : none

13 100%

Non concurrent use by another person Deviation : none

13 100%

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Appendix 2/2

CONTROL OF THE OBSERVANCE Constraints

Constraints (13 exploitable results)

Number of volunteers who respected the constraints

Percentage of volunteers who respected the constraints

No application of products similar to the tested one to the experimental areas Deviation : none

13 100%

No change in hygiene and/or make-up habits Deviation : none

13 100%

On D0 no contact of the clothes with the experimental area (forearm), between the application of the product and the end of the measurements Deviation : none

13 100%

No intensive sun or UVA exposure (U.V. lamps) during the test period Deviation : none

13 100 %

No bath (bathtub, sea or swimming-pool), no sauna or hammam sessions during the test period Deviation : none

13 100%

No application of make-up to the experimental area (face) the days of examination at the investigator centre Deviation : none

13 100%

Neither anti-allergic, anti-inflammatory treatment nor treatment with patent medicines containing Vitamin A acid or its derivatives during the study – no medical treatment which could interfere with the study Deviation : none

13 100%

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Appendix 2/3

CONTROL OF THE OBSERVANCE Constraints

Constraints (13 exploitable results)

Number of volunteers who respected the constraints

Percentage of volunteers who respected the constraints

No drugs or dietary supplements containing carotene Deviation : none

13 100%

No self-tanning products or tanning accelerator products on the experimental areas Deviation : none

13 100%

No clinical peeling or scrub or exfoliating products on the experimental areas Deviation : none

13 100%

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Appendix 2/4

CONTROL OF THE OBSERVANCE Product consumption

Sample Sample Consumption Number of Consumption weighing D0 weighing D28 pdt. P1-P2 = applications Pdt./application

Ref. Surname/Name P1 in (g) P2 in (g) C in (g) N C/N in (g)

1 LEIV/LI 156 126 30 54 0.6

2 AGUT/JO 306 260 46 57 0.8

3 FERN/FL 316 240 76 56 1.4

4 PATA/MN 312 261 51 57 0.9

5 BOIX/CA 159 120 39 54 0.7

6 GARC/MJ 153 100 53 56 0.9

7 MANZ/MI 157 108 49 53 0.9

8 OTAL/MV 315 241 74 52 1.4

9 PERM/MM 310 218 92 55 1.7

10 BALL/IS 311 228 83 54 1.5

11 DEPE/RO 157 114 43 53 0.8

12 REUS/MA 164 118 46 51 0.9

13 MATE/FE 158 117 41 54 0.8

1.0 +/- 0.3 gMean consumption of

the panel +/- standard deviation

Volunteers

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Appendix 3/1

ACCEPTABILITY Sensations of discomfort

Volunteers

Ref. Surname/ Name

Synthesis of the results of the questioning at the end of the study and of the data written by the volunteers in their individual observation sheet

The sensations non ascribable to the test product are not reported in this table

1 LEIV/LI /

2 AGUT/JO /

3 FERN/FL /

4 PATA/MN /

5 BOIX/CA /

6 GARC/MJ /

7 MANZ/MI /

8 OTAL/MV /

9 PERM/MM /

10 BALL/IS /

11 DEPE/RO /

12 REUS/MA /

13 MATE/FE /

Legends : / : nothing to report

Hea : Heating 1 : Slight intensity Sti : Stinging 2 : Moderate intensity Pr : Pruritus (itching) 3 : Severe intensity Bur : Burning

Pu : Pulling W. Watering

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Appendix 3/2

ACCEPTABILITY Skin examination: clinical signs

Volunteers

Ref. Surname/ Name

D0

The clinical signs compatible with the inclusion criteria are not reported in this table

D28

1 LEIV/LI / /

2 AGUT/JO / /

3 FERN/FL / /

4 PATA/MN / /

5 BOIX/CA / /

6 GARC/MJ / /

7 MANZ/MI / /

8 OTAL/MV / /

9 PERM/MM / /

10 BALL/IS / /

11 DEPE/RO / /

12 REUS/MA / /

13 MATE/FE / /

Legends : / : nothing to report

E : Erythema Bu : Bulla 1 : Slight intensity Oe : Oedema Pa : Papule 2 : Moderate intensity V : Vesicle Sc : Scab 3 : Severe intensity D : Dryness C : Coloration d : diffuse M : Macula p : punctuated

Vesicles 1 : nb = 1 or 2 2 : nb > 2

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Appendix 4/1

INSTRUMENTAL ASSESSMENT OF THE ANTI-WRINKLE EFFECT

ST N Volunteer reference

D0 D28 % of variation

“Reactive” volunteers

effect type 1D0 D28 % of

variation


effect type 1

1 NON EXPLOITABLE

2 6.346 8.599 35.5% / 117 93 -20.5% +

3 8.797 11.221 27.5% / 73 88 20.5% /

4 12.344 10.651 -13.7% + 185 164 -11.4% +

5 20.554 12.264 -40.3% + 173 188 8.7% /

6 5.031 5.243 4.2% / 136 108 -20.6% +

7 12.141 9.447 -22.2% + 207 174 -15.9% +

8 9.392 6.616 -29.6% + 167 156 -6.6% /

9 7.954 3.653 -54.1% + 186 82 -55.9% +

10 8.157 8.567 5.0% / 158 75 -52.5% +

11 6.446 3.470 -46.2% + 147 77 -47.6% +

12 5.245 0.534 -89.8% + 188 35 -81.4% +

13 10.860 6.294 -42.0% + 139 151 8.6% /

Mean 9.44 7.21 -22.13% 156.33 115.92 -22.88%

Standard deviation 4.28 3.55 36.00% 36.88 48.59 30.65%

% of improvement for the “reactive” volunteers corresponding to an effect type 1 - 42%

% of improvement for the “reactive” volunteers corresponding to an effect

type 1 - 38%

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42

Appendix 4/2

INSTRUMENTAL ASSESSMENT OF THE ANTI-WRINKLE EFFECT

LT PM Volunteer reference

D0 D28 % of variation


effect type 1D0 D28 % of

variation


effect type 2

1 NON EXPLOITABLE

2 43.590 36.653 -15.9% + 52.984 46.653 -11.9% +

3 38,797 43.897 13.1% / 56.371 53.622 -4.9% /

4 97.614 77.441 -20.7% + 53.872 61.132 13.5% /

5 138.583 85.063 -38.6% + 53.188 57.610 8.3% /

6 41.365 40.536 -2.0% / 49.868 50.307 0.9% /

7 91.920 65.584 -28.7% + 55.681 63.404 13.9% /

8 75.990 56.430 -25.7% + 53.467 53.062 -0.8% /

9 59.835 27.679 -53.7% + 51.984 60.463 16.3% /

10 43.735 29.138 -33.4% + 42.564 51.119 20.1% /

11 64.638 31.976 -50.5% + 39.103 47.783 22.2% /

12 63.674 7.956 -87.5% + 40.397 35.259 -12.7% +

13 67.512 50.330 -25.5% + 52.439 49.858 -4.9% /

Mean 68.94 46.06 -30.75% 50.16 52.52 4.99%

Standard deviation 29.12 22.18 25.84% 5.99 7.70 12.29%

% of improvement for the “reactive” volunteers corresponding to an effect type 1 - 38%

% of improvement for the “reactive” volunteers corresponding to an effect

type 2 - 12%

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43

Appendix 5/1

INSTRUMENTAL ASSESSMENT OF THE MOISTURIZING EFFECT

Corneometric indexes

Control area

Absolute values V co %

Percentages of variation in comparison with D0/T0

Volunteer Ref.

D0/T0 D0/T2h D0/T4h T2h T4h

1 33.8 34.6 34.8 2.4% 3.0%

2 24.3 23.9 23.2 -1.6% -4.5%

3 25.2 26.8 29.8 6.3% 18.3%

4 24.3 25.2 24.1 3.7% -0.8%

5 35.8 34.0 36.3 -5.0% 1.4%

6 29.4 32.5 30.4 10.5% 3.4%

7 36.5 40.5 36.6 11.0% 0.3%

8 35.1 36.0 33.1 2.6% -5.7%

9 43.7 40.1 41.8 -8.2% -4.3%

10 32.5 35.8 33.7 10.2% 3.7%

11 39.1 38.9 41.1 -0.5% 5.1%

12 33.1 31.8 34.7 -3.9% 4.8%

13 45.5 47.7 54.4 4.8% 19.6%

Mean 33.7 34.4 34.9 2.5% 3.4%

Standard deviation 6.8 6.7 8.0 6.2% 7.8%

Study ref: 09-1093/2 / 09.0828

44

Appendix 5/2



Treated area

Absolute values V tr %

Percentages of variation in comparison with D0/T0

Volunteer Ref.

D0/T0 D0/T2h D0/T4h T2h T4h

1 34.2 38.7 37.8 13.2% 10.5%

2 24.8 33.4 35.2 34.7% 41.9%

3 24.4 30.5 31.2 25.0% 27.9%

4 25.6 28.5 31.3 11.3% 22.3%

5 35.3 35.0 37.7 -0.8% 6.8%

6 32.3 44.5 39.8 37.8% 23.2%

7 38.1 47.1 46.2 23.6% 21.3%

8 36.1 45.0 44.8 24.7% 24.1%

9 44.1 44.3 46.7 0.5% 5.9%

10 30.5 36.7 40.3 20.3% 32.1%

11 42.6 44.6 43.5 4.7% 2.1%

12 32.1 39.7 41.1 23.7% 28.0%

13 44.2 52.8 52.6 19.5% 19.0%

Mean 34.2 40.1 40.6 18.3% 20.4%

Standard deviation 6.9 7.1 6.2 12.0% 11.4%

Study ref: 09-1093/2 / 09.0828

45

Appendix 5/3



Difference V tr – V co in % Volunteer reference T2h T4h

1 10.8% 7.6%

2 36.3% 46.5%

3 18.7% 9.6%

4 7.6% 23.1%

5 4.2% 5.4%

6 27.2% 19.8%

7 12.7% 21.0%

8 22.1% 29.8%

9 8.7% 10.2%

10 10.2% 28.4%

11 5.2% -3.0%

12 27.6% 23.2%

13 14.6% -0.6%

% of improvement for “reactive” volunteers*

20.0% 23.5%

% Standard deviation* 9.0% 11.1%

% of “reactive” volunteers* 69% 69%

*volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area is superior or equal to 10%

Statistical analysis: Anova test for repeated measurements

Parameters taken into account in the

analysis Liberty degree

LD Sum of the

squares Mean square Variance F value

Threshold of significance

p value

Groups 1 300.901 300.901 2.20 > 0.05

Nb of vol. and groups 24 3288.583 137.024

Experimental times 2 224.373 112.187 26.29 < 0.05

Experimental times and groups 2 117.201 58.6005 13.73 < 0.05

Experimental times Nb of vol. 48 204.852 4.26776

Study ref: 09-1093/2 / 09.0828

46

Appendix 5/4



Control area Treated area

Absolute values Absolute values Volunteer Ref.

D0/T0 D28

V co % Percentages of

variation in comparison with

D0/T0 D0/T0 D28

V tr % Percentages of

variation in comparison with

D0/T0

Difference V tr – V co in %

1 33.8 30.9 -8.6% 34.2 37.9 10.8% 19.4%

2 24.3 22.6 -7.0% 24.8 31.8 28.2% 35.2%

3 25.2 25.3 0.4% 24.4 30.1 23.4% 23.0%

4 24.3 30.3 24.7% 25.6 32.5 27.0% 2.3%

5 35.8 25.3 -29.3% 35.3 37.3 5.7% 35.0%

6 29.4 34.4 17.0% 32.3 39.1 21.1% 4.0%

7 36.5 49.6 35.9% 38.1 46.7 22.6% -13.3%

8 35.1 38.7 10.3% 36.1 42.0 16.3% 6.1%

9 43.7 39.5 -9.6% 44.1 48.4 9.8% 19.4%

10 32.5 35.4 8.9% 30.5 41.9 37.4% 28.5%

11 39.1 44.8 14.6% 42.6 46.5 9.2% -5.4%

12 33.1 32.5 -1.8% 32.1 49.9 55.5% 57.3%

13 45.5 41.7 -8.4% 44.2 40.5 -8.4% 0.0%

Media 33.7 34.7 3.6% 34.2 40.4 19.9% 16.3%

Desv. est. 6.8 8.0 17.2% 6.9 6.4 15.9% 19.7%

% of “reactive” volunteers* 54%

% of improvement for “reactive” volunteers* 31.1%

% Standard deviation* 13.3%

*volunteers for whom the difference between the percentage of variation in comparison with D0/T0 / treated area and the percentage of variation in comparison with D0/T0 / control area was superior or equal to 10%

Study ref: 09-1093/2 / 09.0828

47

Appendix 6

INSTRUMENTAL ASSESSMENT OF THE WHITENING EFFECT

D0 D28 Volunteer reference Control

area Treated

area ΔM D0 (Tr-Co)

Control area

Treated area

ΔM D28 (Tr-Co)

% variation ΔM (D28-D0)

“Reactive” volunteers*

1 245 303 58 184 244 60 3% /

2 169 293 124 142 248 106 -14% +

3 191 332 141 187 319 132 -6% /

4 269 476 207 246 464 218 5% /

5 155 249 94 131 209 78 -17% +

6 172 207 35 157 184 27 -23% +

7 133 165 32 111 138 27 -14% +

8 148 213 65 133 173 40 -38% +

9 243 369 126 238 313 75 -40% +

10 218 331 113 213 312 99 -13% +

11 NON EXPLOITABLE

12 199 276 77 189 228 39 -50% +

13 125 205 80 133 190 57 -28% +

Mean 189,0 285,0 96,0 171,9 251,9 79,9 -20%

Standard deviation 46,7 86,2 49,8 44,6 88,9 54,3 17%

Mean -26% “Reactive” volunteers*

Standard deviation 14%

9 / 75%

*volunteers for whom it is observed a diminution higher or equal to 10% on the pigmented spots (ΔM parameter) between D28 and D0.

Study ref: 09-1093/2 / 09.0828

48

Appendix 7

INSTRUMENTAL ASSESSMENT OF THE EFFECT ON THE CELL RENEWAL OF THE EPIDERDIS

ITA° Parameters

CONTROL AREA

ITA° VALUES

DAYS OF MEASUREMENT Volunteer reference D16 D18 D21 D23 D25 D28 Gradient

1 7 8 14 24 25 27 1.9 2 20 18 47 49 51 56 3.4 3 -8 17 30 33 35 40 3.6 4 1 7 16 19 19 22 1.7 5 5 15 35 40 40 44 3.3 6 9 14 19 27 29 36 2.3 7 33 33 39 42 45 50 1.5 8 20 27 36 41 43 45 2.1 9 9 5 13 20 24 28 1.9

10 3 8 22 35 40 42 3.6 11 3 8 28 28 31 33 2.7 12 10 15 27 29 30 34 2.0 13 27 30 40 48 49 51 2.2

Mean 10.7 15.8 28.2 33.5 35.5 39.1 2.5

Std. Dev. 11.4 9.2 10.9 10.0 10.1 10.2 0.8

TREATED AREA

ITA° VALUES

DAYS OF MEASUREMENT Volunteer reference D16 D18 D21 D23 D25 D28 Gradient

1 10 10 11 13 26 33 2.0 2 5 14 14 26 34 52 3.7 3 0 25 32 33 33 37 2.5 4 2 4 8 14 15 16 1.3 5 7 8 10 19 29 39 2.8 6 12 18 22 28 33 43 2.5 7 30 30 40 48 48 61 2.6 8 12 13 13 19 20 30 1.4 9 7 8 8 10 12 24 1.2

10 5 7 10 18 29 37 2.8 11 1 4 9 10 14 18 1.4 12 4 4 6 9 11 12 0.8 13 19 20 30 36 39 40 2.0

Mean 8.8 12.7 16.4 21.8 26.4 34.0 2.1

Std. Dev. 8.3 8.4 11.0 11.8 11.4 14.1 0.8

Study ref: 09-1093/2 / 09.0828

49

Appendix 8

COSMETIC QUALITIES

Items (13 exploitable results)

Number of satisfied

volunteers

Percentage of satisfied

volunteers

(Easy) Application 10 77%

(Pleasant) Texture 11 85%

(Quick) Absorption 11 85%

(Pleasant) Fragrance 11 85%

(Pleasant) Colour 11 85%

Non oily 12 92%

PREFERENCE AND PURCHASE INTENTION

Items

Number of answers


volunteers

Preference for the test product compared with the usual product (13 volunteers concerned by the item) 7 54%

Purchase intention if the test product was launched (13 exploitable results) 8 62%

Study ref: 09-1093/2 / 09.0828

50

Appendix 9/1

COSMETIC EFFICACY

Items (13 exploitable results)

Number of satisfied volunteers


volunteers

Moisturizes skin 11 85%

Nourishes skin 12 92%

Anti-wrinkle effect 10 77%

Leaves skin softer and smoother 11 85%

Leaves skin more supple and elastic 9 69%

Whitening effect 10 77%

Reduces the size of the hyperpigmented spots 10 77%

Lightens the hyperpigmented spots 10 77%

Eliminates the hyperpigmented spots 7 54%

STATISTICAL ANALYSIS

Test of χ² Item 1: Moisturizes skin

Repartition yes no Total Value χ² Significance of the difference

Theoretical proportion 50% 50% 100% Strength calculated (ci) 6.5 6.5 13 Strength observed (oi) 11 2 13

6.2 S

χ² = Σ(|oi - ci|²) ci Oi = strength observed Ci = strength calculated α = 5% risk

Significant difference (S) si χ² > 3,84 (for α = 5 %) Non significant difference (NS) if χ² ≤ 3,84

Study ref: 09-1093/2 / 09.0828

51

Appendix 9/2

Item 2: Nourishes skin Repartition yes no Total Value χ² Significance of

the difference Theoretical proportion 50% 50% 100% Strength calculated (ci) 6.5 6.5 13 Strength observed (oi) 12 1 13

9.3 S

Item 3: Anti-wrinkle effect



3.77 NS

Item 4: Leaves skin softer and smoother



6.2 S

Item 5: Leaves skin more supple and elastic



1.9 NS

Item 6: Whitening effect



3.77 NS

Item 7: Reduces the size of the hyperpigmented spots



3.77 NS

Item 8: Lightens the hyperpigmented spots



3.77 NS

Item 9: Eliminates the hyperpigmented spots



0.08 NS

Study ref: 09-1093/2 / 09.0828

52

Appendix 10/1

ICONOGRAPHY OF THE REPLICAS IMAGE

D0 (VOLUNTEER REF.13)


Study ref: 09-1093/2 / 09.0828

53

Appendix 10/2

ICONOGRAPHY OF THE REPLICAS IMAGE IN 3D



study ref: 09-1093/2 / 09 - kreme.sistudy ref: 09-1093/2 / 09.0828 3 i. aim and principle of the...

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