55 MULTIDISCIPLINARY PROCESS OF HAND SANITIZER is used in skin-care products as a nonvolatile emollient, which leaves the skin soft and smooth (43). Several formulations were tested (data not shown), but the one with 0.05% (w/w) of polyacrylate crosspolymer-6 was the one capable of incorporating 0.4% (w/w) of hemisqualane without the oil phase splitting out. Improvement of TEWL by addition of an occlusive agent. Since a reduction in the TEWL decrease was observed with formulation F#7 (Figure 1. C.2), an occlusive ingredient was added to the formulation. This compound, cetyl alcohol, is a fatty alcohol obtained from the reduction of coconut, palm, or other vegetable oil. This is because it slows down evaporation and water loss by forming a hydrophobic film on the skin’s surface (44,45). These alterations led to formulation F#8 (Table II). Formulation F#8 has a low viscosity and a short drying time (Table IV). Compared with the benchmark, F#8 has the capability to increase skin hydration, and it has a similar effect in reducing the TEWL (Table III and Figure 1). Appropriate skin hydration and intact skin barrier function are essential for maintaining healthy skin (25,46). The frequent use of ABHS can cause adverse effects such as skin dryness, pruritus, erythema, and bleeding, in severe cases. For health-care workers, these adverse effects are more common, because they must frequently cleanse their hands (2). Therefore, it is important to select an ABHS with a good balance between antimicrobial effectiveness and skin protection. As formulation F#8 has the desired sensorial characteristics (Table I), a moisturizing effect, and 80% (v/v) ethanol (therefore antimicrobial activity is expected), it was selected for final validation. ABHS PERFORMANCE TESTS Antimicrobial efficacy. The results of bactericidal and yeasticidal activity are shown in Table V, using colony-forming units (CFU)/mL. When diluted at 80% (v/v), the ABHS F#8 had a log reduction of at least 5 logs for bacteria and 4 logs for C albicans, using an interfering substance (dirty conditions) after a contact time of 15 seconds. According to the European standards EN 13727 and EN 13624, formulations displaying 5 logs of reduction for bacteria and 4 logs of reduction for yeast/fungi are considered effective (11,12). Thus, the conclusion was that the developed formulation has bactericidal and yeasticidal activity. When diluted at 8% (v/v) and 0.08% (v/v), the formulation was not active. The test for virucidal activity was done following the European standard EN 14476. ABHS F#8 was evaluated at 50% (v/v) of its concentration for 30 seconds, but it was cytotoxic against the cell line used. Therefore, following the standard indications (13), the residual virus titer was determined by the large volume plating, at a dilution of 1:10,000. The result was no residual virus out of 528 cell cultures. The number of infectious virus particles was determined by the Poisson distribution (13) and resulted in 2.75 log 10 TCID 50 (tissue culture infectious dose 50%). As the control virus titer was 9.5 log 10 TCID 50 ,the reduction factor of ABHS F#8 was 6.75 log 10 TCID 50 (9.5−2.75). According to EN 14476 (13), a disinfectant is effective in inactivating a virus if the titer is reduced by at least 4 log 10 steps within the exposure time. Thus, ABHS F#8 is considered to possess virucidal activity against enveloped viruses after an exposure time of 30 seconds. Finally, antimicrobial activity was accessed in vivo following EN 1500, and the results are presented in Table VI. This standard establishes whether a product for hand hygiene can, by friction, reduce the release of transitory microbial flora (14). For a product to be validated, the reduction of the release of the test organism (E coli) achieved with the test

56 JOURNAL OF COSMETIC SCIENCE product must at least not be inferior to that achieved by the reference product: 60% (v/v) propan-2-ol. Both the reference product and ABHS F#8 had a reduction factor of 2.88 logs (Table VI). To evaluate the noninferiority of the test product, the Hodges–Lehmann upper one-sided 97.5% confidence limit for the difference in log reductions between the reference product and test product must be under 0.6 log units (14). As the value obtained was 0.31 log units, one can conclude that ABHS F#8 is noninferior to the reference product. ABHS F#8 has been proven to have antimicrobial efficacy, as it complies with the European standards EN 13727, EN 13624, EN 14476, and EN 1500. Ethanol antimicrobial mechanism of action consists of the denaturation of proteins, leading to damages in the microorganism’s membranes and in virus envelopes (47,48). This mechanism is supported by the observation that mixtures of ethanol and water are more bactericidal than absolute ethanol, because proteins are denatured more quickly in the presence of water (47). The efficacy of ethanol in ABHS can be reduced by the other ingredients (emollients, emulsifiers, and others) incorporated in the ABHS formulation (4). This was not the case with ABHS F#8, which can provide dermoprotection for the user’s hands and still have antimicrobial efficacy. FORMULATION SAFETY In vitro irritation. It is very important to note the potential of ABHS to induce skin irritation (hazards), because they are directly applied to the skin several times per day (49). According to the United Nations, “skin irritation” refers to reversible damage to the skin following the application of a substance for up to 4 hours (50). In the European Union, skin irritation Table V CFU/mL of the Initial Microorganisms’ Suspensions and the Suspensions After 15 Seconds of Contact with ABHS F#8, at Different Concentrations, and the Respective Logarithmic Reductions Test microorganisms Initial suspension (CFU/mL) ABHS F#8 at 80% (v/v) ABHS F#8 at 8% (v/v) ABHS F#8 at 0.08% (v/v) CFU/mL Log reduction CFU/mL Log reduction CFU/mL Log reduction S aureus 3.21 × 107 1.40 × 102 5 3.30 × 104 3 3.30 × 104 3 P aeruginosa 2.99 × 107 1.40 × 102 5 3.30 × 104 3 3.30 × 104 3 E hirae 3.10 × 107 1.40 × 102 5 3.30 × 104 4 3.30 × 104 4 E coli 2.85 × 07 1.40 × 102 5 3.30 × 104 3 3.30 × 104 3 C albicans 3.21 × 106 1.40 × 102 4 3.30 × 104 2 3.30 × 104 2 Table VI Cellular Log Cycles (Mean ± SD) of Initial and Final Values and Their Difference for the Reference and Test Products 60% (v/v) propan-2-ol (reference product) ABHS F#8 (test product) Hodges–Lehmann upper one-sided 97.5% confidence limit Log prevalues 6.11 ± 0.49 6.12 ± 0.35 N/Aa Log postvalues 3.23 ± 0.62 3.25 ± 0.75 N/A Log difference 2.88 ± 0.69 2.88 ± 0.74 0.31 a Different letters (a, b) under the column drying time indicate statistically significant differences (p 0.05) determined by the post hoc Duncan’s Multiple Range test.