j. Cosmet. Sci., 54, 263-270 (May/June 2003) The hair strand testA new method for testing antifungal effects of antidandruff preparations PETER MAYSER, HORST ARGEMBEAUX, and FRANK RIPPKE, Zentrum fiir Dermatologie und Andrologie der Justus-Liebig-Universit•it Giefien, Gaffkystr. 14, 35385 Giefien (P.M.), and Beiersdorf AG, Hamburg (H.A., F.R.), Germany. Accepted for publication August 19, 2002. Parts of this manuscript were presented at the 35th Annual Meeting of the Deutschsprachige Mykologische GesellschaJg, Marburg, Germany, September 13-15, 2001. Synopsis Seborrheic dermatitis and its minimal variant, dandruff (pityriasis simplex capillitii), are among the most frequent diseases caused by Malassezia (M.) yeasts. Treatment studies have shown that antimycotics achieve clinical improvement, while recolonization leads to recurrent symptoms. Among the antimycotics used are azoles, hydroxypyridones, and various agents such as zinc pyrithione, tar, and selenium disulfide. However, comparative efficacy studies in vitro should not only consider the minimal inhibitory concentrations against Malassezia yeasts but also the bioavailability of the individual substances with regard to hair and scalp. By means of a new method, the hair strand test, hairs from ten volunteers were subjected to standardized 5-min incubation with different shampoo formulations. Thereafter they were rinsed with running water for 1 min and dried. Two hundred each of these hairs (length 1 cm) were given into a medium (olive oi! on selective agar for pathogenic fungi) inoculated with M. sympodialis or M. globosa (5 x 103 CFU/lal), and the influence on growth was semiquantitatively determined over a period of up to 18 days. According to preliminary results, 1% climbazole proved to be particularly effective. The hair strand test, which can also be performed ex vivo, is a new method to find out whether antimycotic agents bind differently to the hair substance and, via a depot effect, may influence the growth of Malassezia yeasts and thus affect dandruff. This allows conclusions about the efficacy of antidandruff formulations. INTRODUCTION Yeasts of the genus Malassezia (M.) are part of the human microflora and that of several homeotherms (1,2). As their growth depends on an exogenous lipid source, they are primarily found in seborrheic areas that are rich in sebaceous glands. Seven species are currently differentiated: M. furfur, M. pachydermatis, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae (3,4). Initial epidemiological studies have shown that M. sympodialis and M. globosa are among the most frequent species on human skin (1-4). Address all correspondence to Peter Mayser. 263

264 JOURNAL OF COSMETIC SCIENCE Apart from their significance as saprophytes, Malassezia spp. can cause diseases. In addition to pityriasis versicolor, seborrheic dermatitis and its minimal variant, dandruff (pityriasis simplex capillitii), are the most common (1,5). Although some environmental factors should not be overlooked, Malassezia spp. are the main protagonists of dandruff as a reactive response of the scalp epidermis (6). The relationship between Malassezia and dandruff was demonstrated ex juvantibus in a series of treatment studies that showed that antimycotics achieved clinical improvement while recolonization resulted in reoccurrence of symptoms (5-10). However, it is still unknown in which way these yeasts cause skin diseases (5). Increased growth cannot be the only reason. Some studies denied a constantly and significantly increased yeast carriage in patients compared with healthy individuals (14-16), while others did not (8,11-13). Therefore, qualitative rather than quantitative changes in the resident scalp flora (Malassezia spp., aerobic cocci, corynebacteria) appear to be significant. McGinley et al. (17) found a 100% incidence of Malassezia in dandruff patients compared to 98% in individuals with healthy skin. However, there was a decisive difference in the portion of Pityrosporum/Malassezia on the scalp flora: 46% in healthy persons vs 74% in dandruff patients and 82% in those with seborrheic dermatitis. The geometric mean value for microorganisms per cm 2 was 5.05 x 105 in healthy individuals, 9.22 x 105 in dandruff patients, and 6.45 x 105 in those with seborrheic dermatitis (p 0.05). Among the antimycotics, azoles (climbazole, clotrimazole, ketoconazole, oxiconazole), polyenes (amphotericin B), hydroxypyridones (ciclopirox, octopirox), and various other preparations, such as zinc pyrithione, coal tar, selenium disulfide and polidocanol, are used (5,6,10,18). Comparative studies in vitro should not only consider the minimal inhibitory concentrations of the individual agents against Malassezia yeasts, but also their bioavailability in the target compartment (hair and scalp). By means of a simple method using the presented hair strand test it was investigated whether antimycotic agents bind differently to the hair substance and via a depot effect influence the growth of Malassezia yeasts. This would allow further conclusions about the efficacy of antidan- druff preparations. EXPERIMENTAL METHODS MALASSEZIA SPECIES As it is still unknown which Malassezia spp. are involved in the development of dan- druff, reference strains of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands, were used for M. sympodialis (CBS 7222) and M. globosa (CBS 7966). These two species are most commonly found on the scalp (2). Hair specimens were taken from ten volunteers of different hair color (six female, four male mean 28.2 years, 5-53 years), who did not use antidandruff preparations or hair dyes. By means of scissors the strands were cut near the scalp surface (hair roots were not included in the sample). Five different shampoo formulations were used, blinded by the manufacturer (Beiersdorf AG, Hamburg, FRG): (A)anti-dandruff shampoo (2% poli- docanol + 0.5% octopirox + 1% climbazole) (B) shampoo base + 2% polidocanol (C) shampoo base + 0.5% octopirox (D) shampoo base + 1% climbazole and (E) shampoo base (INCI: aqua, sodium laureth sulfate, undecylenamidopropyl betaine, laureth-9,