BOTANICALLY DERIVED SKIN SURFACE LIPID MIMETIC 61 MATERIALS AND METHODS SUBJECTS AND SAMPLE COLLECTION The study, which was performed in Chandler, AZ, was approved by the Argus Indepen- dent Review Board (Tucson, AZ) prior to beginning any study procedures. Written in- formed consent was obtained from all subjects. Fifty-nine healthy, 22-year-old females were selected according to the following criteria: no active skin diseases on the face (e.g., acne, psoriasis, atopic dermatitis, eczema, rosacea, and skin cancer), no immunological disorders, and not pregnant or nursing. The subjects consisted of fi fty Caucasian, three African American, two Asian, and four mixed race persons. Solvent-washed cigarette rice papers (Rizla UK, Ltd, Pontypridd, United Kingdom) were used as lipid-free absorbent papers. Among the papers tested, it was found that adhesive- free rice paper contained the least amount of contaminants, particularly lipids. This is often an issue when using adhesive-containing collection methods such as Sebutape (CuDerm Corporation, Dallas, TX). Approximately 50 papers were washed at a time with 250 ml HPLC-grade diethyl ether in an ultrasonic water bath for 15 min at room temperature. After extraction, the cigarette rice papers were removed from the solvent and dried in a rotary evaporator. The papers were stored in polyethylene jars until further use on subjects. Subjects were instructed to wash their faces approximately 12 h before sampling using Cetaphil® Gentle Skin Cleanser (Galderma, Fort Worth, TX), supplied by Floratech (Chandler, AZ), to remove dirt and oil from the facial skin. After this 12-h period, sub- jects reported to the testing facility where they acclimated in a controlled environment [20°–22°C, 50% relative humidity (RH)]. Noninvasive sampling of SSLs from the fore- head of each subject was then conducted over the course of 2 h in the following manner. Two sheets of lipid-free absorbent paper were placed on top of one another in the center of the forehead and held in position for 30 min. The paper was then removed, and placed in a sealed container for extraction. The lipid absorption step was then repeated, consecu- tively, three additional times. EXTRACTION AND SAMPLE ANALYSIS Each subject’s collection papers were extracted twice with 25 ml HPLC-grade diethyl ether (Honeywell Burdick and Jackson, Muskegon, MI) in an ultrasonic water bath for 10 min. The collection papers were removed from the extraction fl ask, and the diethyl ether was evaporated under a gentle stream of nitrogen at 70°C on a hot plate until dry. After drying, the samples were weighed and diluted with an appropriate amount of isooctane (EMD Millipore, Billerica, MA) to obtain a uniform concentration range for the series of samples, approximately 0.5–0.7 μg/μl. The instrument consisted of an Agilent 6890 GC with a programmable cool on-column injector coupled to an Agilent 5973N with turbo pump (Santa Clara, CA). The in- strumental analysis methods were developed using strategies referenced from Michael- Jubeli et al. (19). Software control and data analysis were accomplished with Agilent MSD Chemstation D.02.00.237, NIST 11 (Gaithersburg, MD), and AMDIS v2.70 (Gaithersburg, MD).

JOURNAL OF COSMETIC SCIENCE 62 SKIN SURFACE LIPID MIMETIC FORMULATION Refi ned jojoba oil, macadamia oil, ethyl macadamiate (Floratech), squalene (Ekiz, Izmir, Turkey), and phytosterols (ADM Nutrition, Decatur, IL) were used to formulate the mi- metic. The jojoba and macadamia oil underwent a transesterifi cation process under typical conditions in order to distribute the palmitoleic acid among both the wax-ester and triglyc- eride portions of the material. Ethyl macadamiate and phytosterols were transesterifi ed in a similar process to create a phytosteryl ester of macadamia FAs thereby incorporating palmi- toleic acid in the phytosteryl ester portion of the mimetic. The transesterifi ed products of wax esters, triglycerides, and phytosteryl esters along with squalene and phytosterols were mixed in specifi c quantities resembling SSLs in the fi nal formulation. EFFICACY ANALYSIS OF TOPICAL APPLICATION OF SSL MIMETIC Four effi cacy studies were conducted in Chandler. Independent review board approval and a written informed consent from each subject were obtained before any protocol-related procedures were undertaken. All study participants were healthy females. Upon arriving at the testing facility, subjects acclimated for 30 min in a controlled environment (20°– 22°C, 50% RH). All studies were carried out in a double-blind, vehicle-controlled, randomized manner according to the testing matrix (see Table I). The vehicle contained the following: water (q.s.), methylisothiazolinone and caprylyl glycol (0.9%), ammonium acryloyldimethyltaurate/VP copolymer (0.6%), sorbitan and sucrose cocoate (0.5%), hy- droxyethylcellulose (0.3%), and disodium ethylenediaminetetraacetic acid (0.1%). The SSL mimetic was compared to olive oil (OO) and caprylic/capric triglyceride oil (CCT) because these also contain skin-lipid-like components. RESULTS SKIN SURFACE LIPID COMPOSITION AND COMPARISON The initial portion of this study was to evaluate variation in the SSL composition within a population where age and sex were controlled. The sensitivity of GC-MS analysis allows Table I Effi cacy Testing Matrix Function Washout (days) Anatomical location n Sex Instrumenta Insult/condition Short-term barrier recovery 2 Volar forearms 14 M/F Tewameter TM 300 Acetone exposure Short-term hydration 2 Lower legs 12 F Corneometer® CM 825 Dry skin Long-term skin hydration and barrier function 3 Lower legs 18 F Corneometer CM 825/Tewameter TM 300 Dry skin Viscoelasticity and hydration 2 Forearms 13 M/F MPA Cutometer/ Corneometer CM 825 Aged/sun-damaged skin (60–80 years of age) aAll instruments are products of Courage + Khazaka (Köln, Germany).