MOI)EL SYSTEM FOR DANDRUFF INVESTIGATION Table III Effcct of Cell Suspensions on the Initiation of Guinea Pig Sloughing in the Presence and Absence of Artificial Sebum 193 Sloughing Reaction Antibiotic s Treated No Antibiotic P. ovale q- saline P. ovale q- artificial sebum Staphylococcus JTM q- saline Staphylococcus JTM q- artificial sebum Diphtheroid q- saline Diphtheroid q- artificial sebum Control, artificial sebum 0 0 3 3 0 0 1 0 0 0 2 2 0 0 • O. 05% solution of tetracycline. tificial sebum before application to the guinea pig. The results (Table IIl) show that suppression of the natural flora of the guinea pig skin does not appear necessary for a positive test reaction and that concen- trated P. ovale cells produced the strongest reaction of the three groups of microorganisms tested. The most important conclusion from these data is that the artificial sebum must be present before the microorganisms tested can induce sloughing. The sloughing reaction appeared within 4 to 5 days and reached maximal severity within 6 to 7 days. Under normal circum- stances the reaction began to subside within an additional 10 days, with total clearing normally occurring within 2 to 3 weeks from the time of initial application. The scales observed were large, waxy segments of stratum corneum resembling human dandruff to the unaided eye. Microscopically, the guinea pig skin scales bore a remarkable resemblance to human dandruff scales. Thickened, horny, keratin layers were ob- served in both human dandruff and the sloughed guinea pig skin flakes. Attempts to accelerate and/or accentuate the sloughing reaction by preabrading the skin or applying the irritant mixture to other areas of the guinea pig were unsuccessful. Koch's postulates have been fulfilled repeatedly using either the P. ovale or diphtheroid cell suspensions in artificial sebum. There were no obvious differences in micro- or macro- scopic appearance traceable to the type of inoculum used. The relative ability of representative scalp microorganisms to produce sloughing is shown in Fig. 2. It is apparent that the staphylococcus was unable to produce appreciable sloughing with even very high cell con- centrations. The suspension of P. ovaIe was the most effective in this

194 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Figure 2. 41 0 .50 IO0 iS0 20o rr•g c½!s (wet weight) Effect of cell concentration (mg cells per gram of artificial sebum) on sloughing severity ß Diphtheroid ß Staphylococcus ß P. ova le respect, with a lnaximum sloughing reaction obtained with 100 mg o[ cells (wet weight) per gram of artificial sebum matrix. Other studies have shown that the addition of combinations of the three microbial types to the lipid matrix did not appear to produce more than an additive effect. The above data showed that a microbial inoctdum was required to produce a change in the artificial sebum which could induce sloughing. A possible source of this material was fatty acids released hy microbio- logical lipolysis of the artificial sebum triglycerides. Gas chroma- tography data pertaining to extracts of irritated areas treated with orga- nisms plus artificial sebum are shown in Table IV. These data show a Table IV Relative Composition of the Free Fatty Acid Component of Extracted Guinea Pig Irritation Sites Diphtheroid Carbon Chain Length Control (AS • only) P. ovale q- AS A q- AS _ 10 12 14 16 16 18 18 (1=) 18 •2=) 20 22 24 Irritation grade 1.41 2.23 14.39 19.52 1 63 9 92 21 98 3 01 3 44 0 83 8 69 0 0.31 0.44 0.98 4.34 7.56 9.52 21.15 24.75 2.45 2.29 12.79 10.30 38,47 36,06 4.32 3.49 1.66 1.73 0,45 0.17 7.18 5.66 3 2 • AS = artificial sebum.