36 JOURNAL OF COSMETIC SCIENCE Table I 24-Hour Patch Tests: Grading 24 Hours After Removal of Patches Dry Water 0.005% SLS 0.025% SLS 0.1% SLS Subject Clin Fluo Clin Fluo Clin Fluo Clin Fluo Clin Fluo P.B. 0 0 0 2 0 2 0 2 1 4 M.S. 0 0 0 0 0 1 1 0 3 2 D.R. 0 0 0 2 0 2 0 3 1 4 M.H. 0 0 0 1 0 1 0 3 0 4 D.F. 0 0 0 0 0 2 0 4 1 3 N.L. 0 1 0 2 0 3 0 3 0 4 T.B. 0 2 0 0 0 0 0 0 0 3 L.C. 0 0 0 1 0 2 0 3 I 3 E.L. 0 0 0 0 0 1 3 1 3 4 B.C. 0 0 0 0 0 0 2 2 2 3 L.P. 0 0 0 0 0 0 0 0 0 3 12345 Figure 1. 24 hours post removal of 24-hour SLS applications. Fluorescent evaluation: 0.1% SLS (#3) shows large patches of bright fluorescence (score = 4). 0.025% SLS (#4) is scored 3.0.005% SLS (#2) and water (#1) are scored 1. The empty chamber (#5) is scored 0. Note the halo of fluorescence at the sites in contact with the tape, suggesting that a slight skin damage has occurred. Fluorescence grades and TEWL values of 24-hour exposure chambers correlated very well at one and 24 hours after removal (r = 0.86 and r = 0.94). Clearly, even the slightest damage is detected by both methods. DISCUSSION Until now, identification of dubious, subclinical reactions to anionic surfactants required the use of complex, expensive, bioengineering instruments such as 20 MHz ultrasound, conductance, and evaporimetry (2,3,5). By contrast, the pyranine dye technique is the ultimate in simplicity, requiring only a Wood's lamp to visualize fluorescence. This method has also great sensitivity since we regularly observed strong fluorescence (grades 3 and 4) at 24-hour exposure sites that were clinically negative. TEWL mea-

PYRANINE DETECTS INJURY TO SLS 37 350 300 250 200 150 lOO 5O -5O TEWL 24-HOUR EXPOSURE - [] 1 hr post removal _ []24 hrs post removal * ! IIIIIII1.._., .... , , , , DRY WATER 0.005% SLS 0.025% SLS 0.1% SLS Figure 2. At 24-hour exposure sites TEWL for 0.025% and 0.1% SLS increased significantly (*p 0.1) compared to water. surements correlated very well with the intensity of fluorescence, validating the reli- ability of the method. Fluorescence remained positive for days, enabling serial follow-up. Interestingly, strong inflammatory reactions provoked by higher concentrations of SLS (for example, 0.5 % SLS) do not fluoresce after application of the pyranine dye (unpub- lished results). At first glance this seems paradoxical, but it is explainable by the knowledge that higher concentrations result in total loss of the severely damaged horny layer. Preliminary studies showed that as little as a 30-minute application of the dye to normal volar skin enabled most of the stratum corneum to become fluorescent. It took 30 cellophane tape strippings to completely remove the dye, short of the strippings nec- essary to reach the glistening layer. Moreover, overnight dye exposure on normal skin stained corneocytes, veilus hairs, and the follicular infundibula for over one week (un- published observations). The intensity of fluorescence and the time for extinction of fluorescence was less than after a 24-hour exposure to 10% dansyl chloride. We speculate that pyranine binds to keratin filaments in corneocytes, but this has not been properly studied. It is important to realize that 24-hour exposure to water alone increased TEWL and resulted in a slightly enhanced fluorescence. Therefore, a water control must be included as a reference control in all tests of water-soluble materials. It is well known that water alone can overhydrate the horny layer and make it more permeable (11). We have chosen SLS as a paradigm for this study because its primary effect is to disrupt the barrier. We predict that the pyranine test will be helpful in evaluating the mildness