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,

HAIR STRAND TEST 265 sodium chloride, trideceth-2 carboxamide MEA, citric acid, sodium benzoate, PEG-200 hydrogenated glyceryl palmate, sodium salicylate, and polyquaternium-10). STRUCTURE OF THE TRIAL Sterile glass Petri dishes (3 cm in diameter) were filled with 4 ml of selective agar for pathogenic fungi (SPF Merck, Darmstadt, FRG). Cold-sterilized olive oil was inocu- lated with the different Malassezia strains, which were cultured for four days on SPF overlaid earlier with olive oil, and adjusted to an inoculation density of 5 x 103 CFU/!al according to McGinley et al. (17) using a Neubauer chamber (18). Two-hundred mi- croliters of this suspension were piperted into the prepared Petri dishes so as to cover an area of about one square centimeter (-106 CFU/cm2). A model to imitate hair washing procedure was developed as follows: From each vol- unteer, hair strands approximately 5 cm in length were incubated with one of the five test substances at 30øC for 5 min in sterile Petri dishes. The hairs were then transferred to a sieve with filter paper, rinsed for 1 min in running water (30øC), and dried at room temperature. By means of sterile scissors, 1-cm pieces were cut from the dried hair and distributed in the center of the different test dishes. To approximate natural scalp conditions, 200 hairs/cm 2 were inoculated. Growth of Malassezia yeasts as compared with a positive control (200-1al inoculation suspension without addition of hair) was evaluated as follows: + = growth, (+) = weak growth, and 0 = no growth after incubation at 30øC. Two hundred microliters of pure olive oil on SPF with addition of hair was used as a negative control. The trials were performed two times. STATISTICAL ANALYSIS The trial was a single-blind, vehicle-controlled in vitro study. The individual test for- mulations were compared in pairs by means of the McNemar test at a local level of 5 %. RESULTS Different results were obtained with regard to test formulations, test hairs, and test strains. M. sympodialis showed faster growth in the control, so that a first evaluation was already possible after four days. As growth of M. globosa is known to be significantly slower (3), the dishes could not be evaluated until the fifteenth day. The results are shown in Table I. Repeated testing revealed no differences. Hair strands incubated in pure olive oil on SPF showed no growth at any time. Individual evaluations for M. sympodialis after four-day incubation are shown in Table II. They were nearly identical for test substances B, C, and E. All ten hair specimens that had been treated with these preparations showed growth ofM. sympodialis after four days. In some of them, growth was only observed in the marginal region, i.e., in an area where there was no direct contact with the inoculated hairs. With increasing incubation time, however, homogenous growth was observed. The results of paired comparisons of test formulations A/D and B/C/E are summarized in Table III. The difference between formulations A and D was found to be insignificant. All other paired comparisons of formulations A and D showed statistically relevant differences--after four days, prepa-