Study 103/97/00 Page 1/12 Milan, 8/10/1997 EVALUATION OF THE HYDRATION, ELASTIClTY AND IMAGE ANALYSIS AFTER USE METHOD: Ref. E15006H1/E15003H1/E16001H1 STUDY: 103/97/00 CUSTOMER: SINERGA srl via Pitagora, 11 20016 Pero (Mi) PRODUCT: Customer Ref. AZELAOILDIGLICINATO MONOPOTASSICO (in 3% aqueous solution) Ref. ISPE 103/97/01 STARTING DATE OF THE STUDY: 15/09/97 COMPLETION DATE: 07/10/97 The data given in the present report are exclusively related to the tested sample. This report can be only in full reproduced.
Study 103/97/00 Page 2/12 1. METHOD OF PERFORMANCE Aim of the test is to evaluate the product efficacy in improving elasticity, moisture smoothness of the face skin after long-term use. For these purposes, 5 subjects aged between 30 and 50 years were selected. They applied the tested product twice a days for 3 weeks. The test was carried out in a bioclimatic room (24 C; 50% rh) after the subjects had been climatized for 30 minutes. At the beginning and at the end of the treatment, instrument measurements of moisture elasticity were taken. The micro-relief of skin stratum corneum was assessed in-vivo with the image analysis plastic replica of the skin surface. Measurements were taken in two different areas of the face: forehead: in the middle area, 2 cms away from the arch of the eyebrows; cheek (either right or left cheek, randomly chosen) in the middle area; 4 cms away from earlobe. The data obtained were then analyzed and statistically compared.
Study 103/97/00 Page 3/12 2.a. CORNEOMETER CM 820 Pc 2. DEVICES AND MATERIALS The Corneometer (CORNEOMETER CM 820 Pc Courage & Khazaka) consists of a square-shaped sensor with a special glass covering the front, on a mobile axis (area: 49 mm 2 ), connected to the base unit by a spiral cable. When the front surface of the sensor is pressed against the skin a number (max. 3 figures) appears on the liquid crystal display. The measurement must be made on a flat area, taking care to use constant pressure for not too long a time, to avoid provoking occlusion phenomena with the probe. In cases of occlusion, the number that will appear on the sensor display will be much higher (more than 3.0 corneometrical units) than the measurements made on neighboring areas. This value should not be considered acceptable when calculating the mean value. 2.h. CUTOMETER SEM 474 The Cutometer (CUTO METER SEM 474 Courage & Khazaka) is a device that measures the stretch capacity and residual deformation of the skin, when sucked into the opening of a measuring probe. The Cutometer is connected to a computer; the data obtained by the device are expressed in millimeters. The skin surface is sucked into the opening of a measuring probe, in which a constant level of depression is created (350 mbar) for an established time (5 seconds). The air depression is then annulled and the released skin can return to its original position Three measurement cycles (1 cycle: suction/release) are performed on the same point. The computer monitor displays, on a Cartesian axes system, 3 successive curves that indicate the heights (in mm) reached by the skin during the suction operations and the different levels of skin return during the release times. A cursor on the computer screen follows the progression of the curves on the graph and gives, for each curve, the values correspondent to the following points: the maximum skin stretch point (stretch capacity) indicated as RO, R3 or R6 depending on the considered curve (first, second or third); the minimum value (skin return) indicated as RI, R4 or R7 depending on the considered curve; skin elasticity (indicated as R2, R5 or R8 depending on the considered curve) which correspondent to the ratio: R1-RO/RO (or R4-R3/R3 or R7-R6/R6).
Study 103/97/00 Page 4/12 In the final calculation, the following values were used: the values R0 and R1 correspondent to the first curve the value R2 correspondent to the elasticity value of the first curve (ratio between the skin return and the maximum skin stretch point) the R9 value correspondent to the mean between the elasticity values of the first and third curve (R2 + R8)/2. 2.c. IMAGE ANALYSIS A fast hardening synthetic polymer (SILFLO - Flexico Ltd, United Kingdom) and bi-adhesive discs (HEWLETT PACKARD) were used for preparing duplicates of the skin surface. The adhesive disc was applied onto the subject s thigh to delimit the considered area (1,8 cm 2 ) and to avoid skin surface stretching during the polymer affixing. A little amount of polymer was then spread in the internal circular area of the disc. A few minutes after the application of the hardened polymer the disc was removed, so obtaining a duplicate of the skin surface. All duplicates were analyzed by means of a computerized morphological reading. The following equipment was used for the readings: halogen lamp for lateral illumination of the replica (26 ); macro cinecamera connected with a personal computer; image processor with a densitometric measuring program based on 286 gray degrees. The duplicate was illuminated laterally and filmed by the cinecamera. Its image was transmitted to the processor, which is able to identify the wrinkles (related as negative on the duplicate) and to measure their depth by the color difference and the intensity created by the shadows. The computer supplies for each duplicate information related to the following parameters: Mean of grays: corresponds to the mean value of the shadows obtained by the wrinkles stamped on the silicon rubber duplicate. This parameter gives information about two different aspects of the skin: the micro-furrows and the large furrows. The information given by the analysis arc to be interpreted differently, according to the type of foreseen cosmetic action and to the time interval of the study. Sigma: corresponds to the uniformity of the white and the dark zones given by the shadows. This parameter gives information about the directionality and uniformity of the skin roughness. High values of Sigma correspond to a higher unidirectionality/anisotropy of the furrows. The normal skin (young skin) is characterized by a higher micro-roughness with a multidirectional network (isotropy); the deep furrows are absent or very low in number and depth.
Study 103/97/00 Page 5/12 3. MATHEMATICAL ELABORATION The averages and the standard deviations were calculated for all initial and final instrumental values (hydration, elasticity and image analysis). Furthermore, it was calculated: T f - T 0 = VARIATION OF THE PARAMETER T f -T 0 x 100 =VAR1ATION% T 0 Where: T f = mean value at the end of the treatment T 0 = mean value at the beginning of the treatment The initial and final values of the three considered areas were compared with the PAIRED SAMPLES T-TEST. The difference between the initial and final values was considered significative when the probability p was = < 0,05.
Study 103/97/00 Page 6/12 4a. CORNEOMETRY 4. RESULTS The product use led to an increase in the basal value of the skin moisture in both skin areas considered (forehead = 12.7%; cheek=8.2 %; TAB.1). The statistical comparison between the series of initial and final data by the t-test for paired data shows that these increases are significant (p < 0,05). 4.b. ELASTICITY Forehead The product led to a significant increase in skin elasticity (parameter R9) by 0,022 equivalent to 2,5% (p =0,02). This parameter is the most directly correlated with the improvement of the skin elasticity property. Moreover it was found an increasing tendency in the skin elasticity of the first curve (parameter R2) by 0,057 mms equivalent to 6,7%. This parameter represents the ratio of skin return to maximum skin stretch point of the first curve. After product use, maximum skin stretch point (parameter R0) showed an increase by 0,016 mms equivalent to 9,7%. This variation is only to be regarded as tendential, since it is not statistically significant. As for parameter R1, no significant increase in the value of skin return after extension was found. This value was of 0,01 mms, equivalent to a variation of 38,5% (TAB.2-3). In this case, a decrease in the final value compared to the basal value shows the product efficacy. In fact, this value refers to the skin property of regaining its initial condition after being pulled, which was an extension of 0 mms. This is reported in the graph enclosed in the appendix. Cheek It was not found any significant variation concerning the skin elasticity parameters recorded in this area. 4.c. IMAGE ANALYSIS The analysis of the replicas showed a decrease in the mean of grays parameter, concerning skin wrinkledness. This decrease was not significant in either area considered (forehead = 6,7%; cheek = 9,4%; TAB.4-5). As for the wrinkle direction (sigma parameter), a decrease by 11,7% was found in the forehead area. This value was not significant.
Study 103/97/00 Page 7/12 5. CONCLUSIONS The AZELAOILDIGLICINATO MONOPOTASSICO (in 3% aqueous solution) was used twice a day for 3 weeks on 5 subjects in order to evaluate the hydration, elasticity and smoothness improvement efficacy of the product. At the beginning and at the end of the test instrument measurements were taken. The obtained data showed: a significative increase in the skin hydration values (forehead = 12,7% cheek = 8,2%); a significative increase in the skin elasticity (R9 parameter) concerning the forehead area (2,5%).
Study 103/97/00 Page 8/12 6. TABLES TABLE 1: SKIN CORNEOMETRY AREA BASAL FINAL VARIATION VARIATION T-TEST FOREHEAD mean 76,4 st.dev. 2,7 mean 86,1 st.dev. 6,5 9,7 12,7 % p = 0,01 CHEEK mean 77,7 st.dev. 4,4 mean 84,1 st.dev. 3,6 6,4 8,2 % p = 0,005
Study 103/97/00 Page 9/12 TABLE 2: SKIN ELASTICITY: FOREHEAD BASAL FINAL VARIATION VARIATION % T-TEST R0 mean 0,164 st.dev. 0,08 mean 0,180 st.dev. 0,05 0,016 9,7 % p> 0,05 R1` mean 0,026 st.dev. 0,03 mean 0,016 st.dev. 0,01-0,01-38,5 % p > 0,05 R2 mean 0,854 st.dev. 0,1 mean 0,911 st.dev. 0,05 0,057 6,7 % p > 0,05 R9 mean 0,868 st.dev. 0,03 mean 0,890 st.dev. 0,04 0,022 2,5 % p = 0,02 TABLE 3: SKIN ELASTICITY: CHEEK R0 BASAL FINAL VARIATION VARIATION % mean 0,270 st.dcv. 0,05 T-TEST mean 0,264 st.dev. 0,07-0,006-2,2 % p > 0,05 R1 R2 R9 mean 0,022 st.dev. 0,01 mean 0,922 st.dev. 0,04 mean 0,892 st.dev. 0,04 mean 0,022 st.dev. 0,01 0 p > 0,05 mean 0,915 st.dev. 0,03-0,007-0,8% p >0,05 mean 0,906 st.dev. 0,03 0,014 1,6 % p> 0,05
Study 103/97/00 Page 10/12 TABLE 4: IMAGE ANALYSIS: MEAN OF GRAYS AREA BASAL FINAL VARIATION VARIATION % T-TEST FOREHEAD CHEEK mean 93 st.dev. 3,9 mean 92,9 st.dev. 12,6 mean 86,8 st.dev. 5,6-6,2-6,7 % p > 0,05 mean 84,2 st.dev. 3,8-8,7-9,4 % p > 0,05 TABLE 5: IMAGE ANALYSIS: SIGMA AREA BASAL FINAL VARIATION VARIATION % T-TEST FOREHEAD mean 18 st.dev. 14 mean 15,9 st.dev. 10,6-2,1-11,7 % p > 0,05 CHEEK mean 9,9 st.dev. 2,5 mean 11,3 st.dev. 2 1,4 14 % p > 0,05
Study 103/97/00 Page 11/12 7.BIBLIOGRAPHY 1) G. Borroni, P. Gabba, Berardesca, G.Rabbiosi: VALUTAZIONE DELL EFFETTO IDRATANTE Dl ALCUNE EMULSIONI A/0 E 0/A DEL COMMERCIO - Cosmesi Dermatologica n 17, 34-40. 2) P.Morganti, S.D.Randazzo: L UTILIZZAZIONE DEGLI INDICI DI CORREZIONE PER IL TRATTAMENTO COSMETICO DELLA CUTE SECCA E DISIDRATATA - Il Prodotto Chimico, 17-21, (Aprile 1989) 3) A. Giardina: IDRATAZIONE E DERMOCOSMESI - Corso di aggiomamento - XXVI Congresso Nazionale A.D.0.I. - Siracusa, 22-26 Settembre 1987 4) F.Deniker: L IDRATAZIONE DELL EPIDERMIDE - LA SUA REGOLAZIONE LE SUE MODIFICAZIONI - IL RUOLO DEL PIRROLIDON CARBOSSILATO SODICO -Cosmetics & Toiletries Ed.It. nøl, 45-49, (1987) 5) C.G. Baker: MOISTURIZATION: NEW METHODS TO SUPPORT TIME PROVEN INGREDIENTS - Cosmetics & Toiletries Vol.102, 99-102 (April 1987) 6) E.K. Boisits, G.E.Nole, M.C.Cheney: THE REFINED REGRESSION METHODS - J.of Cutaneous Aging & Cosm.Dermatology, Vol.], N03, 155--163, (1989). 7) P.Creidi, B.Faivre, P.Agache: ASSESSMENT OF SKIN HYDRATION AND SOFTENING EFFECTS OF COLLOIDAL OAT FRACTION CONTAINING CREAM -J.Appl.Cosmetol. 10, 1-6, (January-March 1992) 8) M.Batt, E. Fairhurst: COSMETIC AND PHYSIOLOGICAL ASPECTS OF SKIN MOISTURIZATION - Society of Cosmetic Scientists, 1-16 (1985) 9) B.Reece,D.Deeds: IMAGE ANALYSIS USED TO DETERMINE MOISTURIZER EFFICACY - Cosmetics & Toiletries Vol.107,81-82 (July 1992) 10) V.Rogiers, M.P.Derde, G.Verleye, D.Roseeuw: STANDARDIZED CONDITIONS NEEDED FOR SKIN SURFACE HYDRATION MEASUREMENTS Cosmetics & Toiletries Vol.105, 73-82 (October 1990)
Study 103/97/00 Page 12/12 11) A.Di Nardo, P.Pepe, E.Torti, A.Giannetti: QUANTIFICAZIONE DEL POTERE IDRATANTE DI SOSTANZE DI BASE - Chron. Derm., Anno II, N 1, 83-86. 12) R.O.Potts: STRATUM CORNEUM HYDRATION: EXPERIMENTAL TECHNIQUES AND INTERPRETATIONS OF RESULTS - JSoc.Cosmet. Chem. 37, 9-33 (January-February 1986) 13) M.Monti, M.Barbareschi, S. Motta: STUDIO DEL CALCO CUTANEO MEDIANTE MICROSCOPIA ELETRONICA, ANALISI DELL IMMAGINE E PROFILOMETRIA - Incontri di Cosmetologia N 3, 62-80, (Giugno 1989). 14) J.Kim, J.H.Lee, Y.Y. Lee, C.K.Kim: NEW EVALUATION SYSTEM OF COSMETIC EFFECTS ON MORPHOLOGY OF SKIN SURFACE USING TSRLM WITH IMAGE ANALYZER - Vol.1, 156-171, I.F.S.C.C. Congress, New York (1990) 15) D.G.Groh, J.Ayres, P.Maher: MODIFIED REPLICATION TECHNIQUE FOR PRELIMINARY LABORATORY EVALUATION OF HUMAN SKIN SURFACES - Vol.2, 223-230, I.F.S.C.C. Congress, New York (1990) 16) J. Ayres, P. Maher, D. Groh: DEMONSTRATION OF SIMPLE AND INEXPENSIVE TECHNIQUES FOR EVALUATING MODIFIED SKIN REPLICAS - Vol.2, 231-238, I.F.S.C.C. Congress, New York (1990) 17) G.Durupt Ch. Montastier: METHODES D APPRECIATION DE L ACTIVITE DES PRODUITS COSMETIQUES ANTIRIDES - J. Med. Esth. et Chir. Derm., Vol. XII - 47, 181-185, (Sept.1985).
Page 13 EVALUATION OF THE HYDRATION, ELASTICITY AND IMAGE ANAYSIS AFTER USE ANNEX R0 = e (a) R1 = e (a + b) R2 = (e (a) - e (a±b)) / e (a) R3 = e ((r*a) + ((r-1) *b)) R4 = e ((a±b) *r R5= (e (a) - e (a ± 0.l) R6= (e (a) - e (0.1))/e (0.1) R7= (e (a)-e (a + 0.1))/e (a) R8= (R7-R6)/R6 R9 = (R2+R8/2) maximum skin stretch at time a minimum skin stretch at time (a+b) maximum skin stretch of the last curve, r repetition number minimum skin stretch of the last curve, r repetition number