47 MULTIDISCIPLINARY PROCESS OF HAND SANITIZER For each volunteer, and for each probe and each time point (before and 30 minutes after application), measurements were made in triplicate, and only the average value was considered for future data analysis. Hydration and TEWL fold change were calculated according to the following formula: fold change =(average of three applications − baseline average/baseline average) *100. VISCOSITY ANALYSIS Viscosity was measured using a Vibro Viscometer SV-10 (A&D Company, Tokyo, Japan) following the manufacturer’s instructions. Briefly, 10 mL of ABHS was placed into a polycarbonate cup that was lifted until the center of the narrow part of the sensor plates was aligned with the liquid surface. Viscosity was measured until a stable value was obtained. DRYING TIME MEASUREMENTS To measure the formulations’ drying time, six volunteers performed hand rubbing according to the WHO’s “How to Hand Rub” technique using 1.5 mL of formulation (10). The drying time was recorded with a stopwatch from the moment the volunteer began to rub to when the person indicated that her/his hands felt dry. This was performed twice for each formulation on different days, and only the average of both measurements was considered for statistical analysis. All formulations were tested by the same six volunteers. SENSORY CHARACTERISTICS EVALUATION Small-scale consumer evaluations were performed with the volunteers that participated in the skin parameter measurements. After the first application, volunteers evaluated the following attributes: formulation texture (how pleasant the product felt between the fingers), spreadability (ease of distributing the product over the skin), drying time (how fast the product dried in the skin), afterfeel (how soft and smooth the skin felt), and overall opinion of the product. These attributes were classified on a scale of 1 to 9 (1 was extremely dislike 5 was indifferent and 9 was extremely like). The formulations were evaluated on different days and kept in an uncharacterized container. For the final formulation, a consumer evaluation was performed in a hospital in the Oporto region. For 3 days, the benchmark was replaced by the formulation developed in this study and the workers were given access to an online questionnaire, via a QR code, to evaluate the new ABHS. The questionnaire asked the hospital workers to evaluate the same attributes previously described (same scale) and to compare the new ABHS to the benchmark that is usually used in the hospital, on a scale of 1 to 5 (1 was much worse 3 was equal and 5 was much better). Answering the questionnaire was done anonymously and was optional. PRODUCT ANTIMICROBIAL EFFICACY The ABHS bactericidal and yeasticidal efficacy were evaluated, respectively, according to EN 13727 (11) and EN 13624 (12), using the dilution–neutralization method. In short, ABHS at concentrations of 80% (v/v), 8% (v/v), and 0.08% (v/v), after dilution in distilled water, was added to microbial suspensions in the presence of a solution with an interfering

48 JOURNAL OF COSMETIC SCIENCE substance. The mixtures were conducted at 20°C ± 1°C, and the contact time was 15 seconds. After this contact time, an aliquot was removed and immediately neutralized. The number of cells recovered was determined by plate count, and the reduction was calculated considering the initial suspension log. The bactericidal tests were performed with Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Enterococcus hirae (ATCC 10541), and Escherichia coli K12 (NCTC 10538). The yeasticidal test was performed with Candida albicans (ATCC 10231). Virucidal activity was verified following EN 14476 (13). In summary, the ABHS at 50% (v/v), after dilution in distilled water, was added to a suspension of Modified vaccinia virus Ankara (ATCC 1508), at 20°C ± 1°C for 30 seconds. At the end of this contact time, an aliquot was taken and immediately suppressed by diluting the samples in an ice-cold cell maintenance medium. The dilutions were transferred to wells in microtiter plates with a monolayer of BHK-21 cells (ATCC® CCL10™, American Type Culture Collection, Manassas, United States). Infectivity tests were done by quantal tests. Reduction of virus infectivity was calculated from differences of log 10 virus titers before (virus control) and after treatment with the ABHS. Finally, the ABHS hand hygiene performance was assessed in vivo according to EN 1500 (14) in 18 people. Briefly, an evaluation was performed on the number of microorganisms (E coli K12, NCTC 10538) released from the fingertips of volunteers’ hands, which were artificially contaminated, before and after hand sanitizing with the product under testing (ABHS formulation) and with a reference product (60% v/v propan-2-ol). All materials and reagents (including culture mediums, interfering substances, neutralizers, reference substances, etc.), detailed methods, and experimental conditions are described in the referenced European standards (EN 13727, EN 13624, EN 14476, and EN 1500). Additionally, the validation assay criteria and controls, as defined in each European standard, were found to be effective in all experiments, indicating validity of the presented data. IN VITRO SKIN IRRITATION TEST To test potential skin irritation, the ABHS was topically applied to a reconstructed human epidermis (RhE) model followed by a cell viability test. The authors used the commercial RhE model EpiDerm™ (EPI-200-SIT, MatTek Corporation, Ashland, United States) following the standard operating procedures of the test system provider (15). Cell viability was measured by mitochondrial dehydrogenase conversion of (3-4,5-dimethyl thiazole 2-yl)2,5- diphenyltetrazoliumbromide (MTT) into a blue formazan salt, whose OD was measured at 570 nm after extraction from tissues. According to the OECD TG 439, the test substance can be classified as nonirritant if the measured viability of epidermal cells is 50%, or it can be classified as an irritant of category 2 if the measured viability of epidermal cells is ≤50% (16). Three RhE tissues were used for each condition: test substance (ABHS F#8), negative control (phosphate buffered saline without Ca2+ and Mg2+), and positive control (5% sodium dodecyl sulphate). Data are presented as relative viability (%)calculated following OECD TG 439 (16). EX VIVO SKIN MODEL AND HISTOLOGICAL ANALYSIS An ex vivo study was performed by Genoskin (Toulouse, France) that followed all legal authorizations necessary from the French government and the appropriate ethics committee.