192 JOURNAL OF COSMETIC SCIENCE THE ISOLATED PERFUSED BOVINE UDDER SKIN (BUS) MODEL: SURFACTANTS/SKIN INTERACTIONS AFTER SHORT AND LONG TERM EXPOSURE W. Pitterman •, J. Kahre •, H. - U. Kriichter 2, Th. Fi•rster 1, L. Kintrup • and M. Kietzmann 3 •Henkel KGaA, D-40191 DiisseldorjS, Germany :Henkel Corp., Ambler, PA 19002 31nstitute for Pharmacology, Pharmacy and Toxicology, Vet. School Hannover, D-30559, Hannover, Germany INTRODUCTION: The isolated perfused bovine udder skin model (1) was originally introduced as an in vitro model to study the percutaneous absorption. Under in vitro conditions barrier properties and metabolization comparable to living skin are maintained in perfused skin models. Due to this viable status additional information concerning time- dependent skin irritation can be obtained in punched skin biopsies representing epidermal and derreal layers. The model gives the opportunity to distinguish cytotoxicity (modified methyl tetrazolium assay) (2) from irritancy (prostaglandin E2-tissue concentration). The period of exposure (0.5 hour up to 5.0 hours) as well as the type of application (open / occluded Finn Chamber R) is variable within the perfusion time of approx. 8 hours. EXPERIMENTAL: Surfactants: Alkyl polyglycoside [APG] (pH.: 5.5 3 % and 10 % active substance) Sodium Lauryl Sulfate [SLS] (pH.: 5.5 3 % and l0 % active substance) The unpreserved surfactants were handled as frozen samples during the logistic procedures before the application. The surfactants were applied occlusively using a chamber (D=18 mm 500pl / Finn Chamber ©) under adhesive tape. Model: The test set-up is extensively described (1). The viability of the perfused udder was demonstrated by a nearly unchanged glucose consumption, an initially decreasing and thereafter unchanged lactate production and an unchanged dehydrogenase activity in the perfusate after it has flowed through the vascular system. It may be assumed that no significant edema developed within 6 hours since the skin fold thickness was constant. Morphology: Samples of the treated and untreated skin underwent a routine preparation for H&E histology and transmission electron microscopy. Biochemistry: Cytotoxicity: The MTT-assay indicates the level of impaired mitochondria in the cells of the epidermal layers and derreal tissue. The modified MTT (methyl tetrazolium salt dye conversion, pg Formazan / pg DNA) is used (Maag, 1993). Irritancy: The results of the assessment of the prostaglandin E2-concentration (ng/pg DNA) in the epidermal and derreal layers presents the level of a mediator substance responsible for erythema and edema formation in the case of clinically evident skin irritation (MaaB, 1993). For both assays the whole skin biopsy (D=6mm, approx. 4 mm depth) is used. Due to the different cell concentrations in the epidermal and derreal layers, the tissue preparations were adjusted to the comparable DNA content before being analyzed. For the statistical analysis the t-test was used. CONCLUSION: Cytotoxicity (MTE, Figure/):After an occlusive exposure period of 1 hour the application of APG 3 % and 10 % AS does not induce a statistically significant cytotoxic potential compared to the untreated skin area whereas the application of SLS 3 % and 10 % resulted in a statistically significant difference (p 0.05). After 1 hour of exposure the cytotoxicity may be predominantely influenced by physicochemical processes which in the case of SLS was much more pronounced than when applying APG. After an exposure period of five hours the cytotoxic potentia, l shows a comparable level (p 0.05). This demonstrates that the exaggerated test conditions allow a skin penetrating effects of the surfactants which is much less pronounced in the case of APG than when using SLS. APG 10 % AS has about the same potential as APG 3 %or SLS 3 % AS. SLS 10 % AS after a period of exposure of 1 hour and 5 hours impairs about 30 % and 50 % resp. of the epidermal and dermal cells.

PREPRINTS OF THE 1998 ANNUAL SCIENTIFIC SEMINAR 193 Irritancy (PGoe2-synthesis, Figure 2): After one and five hours exposure period there was a statistically significant difference (p 0.05) for APG 3 % AS and APG 10 % AS compared to the untreated control. Again no real difference could be observed between APG 3 % and APG 10 % AS. Morphology: The routine histology did not reveal major alterations in the structure and morphology of the stratum corneum induced by SLS after an exposure period of one hour. The transmission electron microscopy evidences after the prolonged occlusive exposure period of five hours swollen stratum corneum lamellae and small focal clefts between the lamellae. The pathogenesis of cytotoxicity in the skin is a quite different process from irritancy and is not linked up closely. The MTT- and PGE2-1evels of SLS 10 % and 3 % AS at I hour and 5 hours of exposure differ markedly, wheras with APG, the values are rather close, thereby disclosing a higher skin compatibility of the latter up to 10 % AS. The difference between both surfactants regarding skin compatibility may also be explained with a remarkable low cytotoxic potential of APG after I hour of exposure which means almost no physicochemical effects, combined with a low influence on the synthesis of mediator substances. FIGURE 1 1,20 1,1o 1 ,oo 0,90 0,80 0,70 0,60 0,50 0,40 0,30 MTT mean values (cytotoxicity) ß '--• u•lreeted (n-20) ß --O•APG, 3% (n,,a) •APG, 10% (n-a) ß •O•SOS. 3% (n-4) •SDS, 1o% (fire4) 1,0h 5,0h FIGURE 2 1,35 Z• 1,25 Q 1,15 • 1,05 • 0,95 • 0,85 0,75 • 0,65 0,55 PGE2 mean values (Irrltancy) T • '--4'•u•eated (n-20) ••.•,• --cz.• A,.oG, 3% (•) •• •,•o%(•) • • •, 3% (•) •, 1• (•) 1,o h 5,0 h (1) Kietzmann, M. et. al. J. Pharmacol. Toxicol. Meth. 30. (1993) pp 75-84 (2) Pittermann, W. et al. In Vitro Toxicology 10 (1997) pp 17-21 (3) MaaB, P. Das isoliert perfundierte Rindereuter-Ein Modell zur Prtlfung der Hautirritation ? (Thesis) Tiertlrztliche Hochschule Hannover (1993)