34 JOURNAL OF COSMETIC SCIENCE laser-doppler velocimetry, ultrasound imaging, and conductance (1-3). No chemical irritant has been more intensively and comprehensively studied than sodium lauryl sulfate (SLS), an anionic surfactant. A huge literature has been built up dealing with multifarious ways of characterizing the response of human skin to SLS. SLS is now the classic prototype of an irritant whose major impact is on the horny layer barrier. The most complete description and review of the numerous methods available to study SLS reactions, entitled "Guidelines on sodium lauryl sulfate exposure tests," has recently been published by the European Society of Contact Dermatitis (4). The consensus is that TEWL is the most sensitive procedure for detecting tissue damage by SLS. Invisible damage to the horny layer can be reliably, quickly, and easily estimated by the evapo- rimeter (2,5), which measures vapor tension at two fixed positions above the surface. International standards for proper use of the evaporimeter have also been published (6). Interest is growing in methods that will detect early, subclinical damage by anionic surfactants. Dansyl chloride is a fluorescent dye that stains the entire thickness of the horny layer. Disappearance of the dye under Wood's light is a measure of the turnover of the stratum corneum (7). More rapid extinction of fluorescence has been shown to correlate with subclinical injury to SLS (8). However, there are technical limitations to the technique, which is also time-consuming (9,10). Dermatologists, including us, frequently mark patch test sites with yellow fluorescent pens. The indicator dye in these pens is pyranine. We now present a method using pyranine dye to detect early, invisible damage to the horny layer barrier by SLS. MATERIALS AND METHODS The subjects were 11 healthy Caucasian women, ages 30 to 50. One hundred fifty microliters of the test solutions was pipetted into the cotton pad of 11-mm Hill Top chambers (Hill Top Research Inc., Cincinnati, OH). These were fixed to the volar forearm skin. On one forearm, chambers containing distilled water and 1%, 5%, and 10% SLS (Sigma Chemical Co.) were applied for one hour. On the opposite forearm, chambers containing distilled water and 0.005%, 0.025%, and 0.1% SLS and one empty chamber were applied for 24 hours. The sites were briefly washed with water after removal and left open. The sites were evaluated clinically one and 24 hours later and scored as follows: 0 = no reaction 1 = patchy mild erythema 2 = uniform mild erythema 3 = moderate ery- thema, slight infiltration, few vescicles 4 -- strong erythema, infiltration, vescicles 5 = strong erythema, infiltration, bullae with exudation. The source of pyranine (solvent green no. 7) was a yellow Hi-Liter © pen (Avery- Dennison, Diamond Bar, CA). At first, the dye was squeezed out of the pen into a jar. One hour after removing the chambers, each patched site was evenly covered with 0.15 ml of the dye. The dye was applied with a cotton-tipped applicator to each area of 6.5 cm 2 that included both patched and surrounding untreated skin. The solution was left to air dry for 30 minutes, and the site was briefly washed with Dove © soap and patted dry. The sites were visualized under Wood's light immediately and 24 hours later. Fluores- cence was scored as follows: 0 = no fluorescence or less than surrounding area 1 -- mild fluorescence comparable to surrounding area 2 = moderate fluorescence 3 -- strong

PYRANINE DETECTS INJURY TO SLS 35 fluorescence, with punctuate accentuation 4 = strong, bright fluorescence in large patches. Fluorescent photographs were also taken under two oppositely placed Wood's lamps (Spectroline ©, MB-100, 365 nm, Spectronics Corp., Westbury, NY) positioned 15 cm from the surface at a 45 ø angle. A Haze 2A Tiffen filter was placed in front of the camera lens. The film was 1600 ASA Kodak Ektachrome, processed at P2. TRANSEPIDERMAL WATER LOSS (TEWL) TEWL was measured 30 minutes after removal of the chambers, with the evaporimeter (EP2, Servo Med, Stockholm, Sweden) connected to a dedicated software. Measurements were conducted inside an environmental chamber at 21øC degrees and 41% relative humidity, after equilibration, following international guidelines for the use of this instrument (6). STATISTICS Significance was estimated by the Wilcoxon signed rank two-tailed test. RESULTS CORRELATION OF VISIBLE IRRITATION WITH FLUORESCENCE None of the one-hour patch tests, including 10% SLS, induced any visible reaction. However, with 10% SLS there was a non-significant increase in fluorescence when compared to water. With 24-hour exposures, only four of the 11 subjects showed a visible reaction to 0.025% SLS (grade 2) and 0.1% SLS (grades 2 and 3) one hour after removal. At 24 hours after removal of the chambers, two subjects with 0.025 % SLS and three with 0.1% SLS still had a mild erythema (Table I). The irritation was non-significant at any time compared with the water chamber. By contrast, strong fluorescence was observed (grades 3 and 4) in 100% of the sites exposed to 0.1% SLS (p 0.005, compared with all the other test sites), and in 63% of those exposed to 0.025% SLS (p 0.005, compared with water), one hour after removal. Fluorescence was still strong 24 hours after removing the chambers, although reaching significance only for 0.1% SLS (p = 0.001), in com- parison with water (Table I). No increase in fluorescence was observed with 0.005 % SLS (Figure 1). TEWL After the one-hour exposures, the TEWL values did not increase significantly. By contrast, with 24-hour patches TEWL values became significantly elevated (p 0.1), with all concentrations at one and 24 hours post removal (Figure 2). These increases were significant compared to water for 0.025% and 0.1% SLS (p 0.1). It should be noted that water also caused barrier damage compared to the empty chamber.