SAFETY TESTING OF HAIR DYE PREPARATIONS 755 tions of a product. To differentiate these effects, a more definitive modification of the Ascheim (2) method was used. At autopsy, the skin was immediately stripped, and plugs 4 mm. in diameter were taken, using a carbide steel punch or a uterine biopsy forceps. This punch biopsy material was then placed in IN NaOH for digestion overnight and examined for alkaline hematin content the next morning. The re- TABLE VIII Incidence of Histopathological Findings (• in Rabbits, 1-Hour Contact with P.P.D.-R. Dye q- H202 Organ and Findings None 5M 5F P.P.D..R. Dye q- H•oO2 Dose, ml./kg./day -2. •--4--• 5M 5F 5M 5F Incidence Lungs Interstitial inflammation 2 Focal chronic inflammation 2 Focal acute congestion Focal thickening of pleura Kidneys Focal chronic inflammation Testes Occasional abnormal germinal epithelial cell 1 Brain Perivascular cuffing Skin--Treated Chronic inflammation in dermis, focal 1 Epidermal thickening 1 Hyperkeratosis Epidermal inflammation Acute inflammation in dermis 1 Abscess in keratotic layer 1 Dermal fibrosis Edema in dermis Atrophy of adnexal structures in dermis Acute and chronic ulceration in dermis 1 1 1 1 1 1 1 I 2 1 1 1 1 1 Only positive findings are shown. suits could be expressed in terms of known erythroeyte counts (Fig. 1) or compared against standard curves for hemoglobin and expressed as mg. per 100 g. of skin. The latter alternative was preferred. Four graded concentrations of aqueous hydrogen peroxide solutions of 3, 6, 9, and 12% (equivalent to 10, 20, 30, and 40 volumes per cent) were applied to groups of 10 rabbits each. These materials were applied

756 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS TABLE IX Incidence and Severity of Histopathological Findings ø in Rabbits* Prolonged (6-hour) Daily Contact with P.P.D.-R. Dye q- H202 Organ and Findings P.P.D.-R. Dye q- H202 Dose, ml./kg./day-- - 1 3 10 Incidence Liver Focal acute necrosis Focal lymphocytic accumulation 1 Kidneys Focal chronic pyelonephritis Chronic interstitial nephritis Marrow Congestion Skin--Untreated Epidermal thickening Skin--Treated Chronic inflammation in dermis 2 • + Epidermal thickening Hyperkeratosis 1 • + Acute inflammatory dermis Abscess in keratis layer Dermal fibrosis Telangiectasia in dermis Abscess in hair follicle Atrophy of adnexal structures in dermis Foreign body material in dermis Diffuse necrosis in epidermis Early Miceration 22+, 10+ 12- 12+ 12+, 24+ 10+, 15+ 22+, 15+ lO% 14+ 24+, 15+ 12+, lO+, 14+ 14+ 15+ 22+ 34+ 3o+ 1•+, 14+ 1•+ 22+ 35+ 1 22t 25+ a Only positive findings are shown. * Groups of 10 rabbits received each treatment. once weekly for six weeks using a one-hour contact period. The skin was stripped at autopsy, a sample prepared for microscopic examina- tion and another prepared for hemoglobin analysis. In another series, groups of rabbits were placed on test to compare the results after 48-hours or two applications, each of one hour's dura- tion. A third comparison was based on a single acute continuous 24- hour application of four commercial oxidation dyes purchased on the open market and of a vehicle common to each of the bases. These tests were terminated immediately after the period of application, and the sites were treated in the manner described. RESULTS Primary irritation scores are shown in Table II for the daily six-hour contact. The maximum scores were 2 and indicative of slight ery-