SAFETY TESTING OF HAIR DYE PREPARATIONS 757 thema. In the Draize system, effects scored as 2 or less are considered only mildly irritating, products eliciting scores of 2 to 5 are moderate irritants, and those with scores above 6 are considered to be severe irritants. There was no evidence of irritation in this modified pro- cedure. Primary irritation scores for the graded peroxide concentrations are shown in Table III. For this study, samples were applied on each animal in a randomized fashion so that in every instance each concen- tration used was placed adjacent to a different concentration. Comparison of the data in Tables II and III indicates that the in- tensity of response to oxidative irritants of equal peroxide concentra- tion is not a function of contact time. This may be due to the rapid decomposition of the hydrogen peroxide in contact with organic matter and air. TABLE X Incidence--Peroxide Studies, i-Hour Contact Histopathological Findings in Skin" --Per cent H202-- Findiugs 3 6 9 Chronic inflammation in dermis Epidermis thickening Epidermis inflammation Hyperkeratosis Epidermis abscess Dermal abscess Dermal fibrosis 1+ 4 2+ 3+ 1 lq- 1 1+ 1 1+ 1+ lq- 1 1+ 3 2+ 2 3 3 1 Two groups of 6 rabbits, each treated with each concentration. Examination of a variety of physiological factors failed to demon- strate any evidence of systemic toxicity, following subacute derreal applications. The data shown in Tables IV and V indicate no adverse responses in body weights. No deleterious changes were seen (Tables VI and VII) in hemoglobin concentration, hematocrit, or total erythrocyte counts • ith either treatment groups. The variance in poly: lymphocyte ratio seen at all levels in the one-hour group (Table VI) indicates a complete reversal. This is commonly noted in rabbits and occurs spontaneously in nontreatment groups. Literature values for the ratio of these two leukocytes confirm the spontaneity of this shift. Critical examination of the differential slides revealed no change in the

758 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS incidence of juvenile or adult forms, indicating no treatment-related shift either to the left or right. The histopathological assessments of the vital organs and of the skin of rabbits in which the two test methods were employed are shown in Tables VIII to X. The increase in number of skin lesions characterized by dermal fibrosis and ulceration in the 4 mi. per kg. group underscores the usefulness of the one-hour contact procedure for safety evaluation of this type of formulation. In general, these findings were observed in one or two rabbits in each group. Therefore, there were no differ- ences between the treated and control groups or between the two treat- ment levels. The lack of significant findings in the viscera in both control and treated groups is indicative of the generally innocuous nature of the daily shampooing in this species. TABLE XI Hemoglobin Concentration in Skin Peroxide Studies, 1-Hour Contact • H202 Concentration, %, , Untreated 3 6 9 12 mg./100 g. Males 60 230 550 520 330 20 180 230 640 840 10 270 20 220 350 60 230 210 20 330 30 270 270 250 710 10 240 240 470 290 20 230 290 350 290 60 460 340 310 360 Females 50 370 230 180 250 20 510 580 350 430 90 270 320 220 170 10 230 230 250 340 20 220 300 230 170 10 240 210 380 270 10 200 200 200 270 20 220 500 320 400 The observations (Table IX) in both the abraded and intact skin treated for six hours each day appeared to have no adverse significance. Telangiectasia, atrophy of adnexal structures in the dermis, epidermal thickening, focal or diffuse chronic inflammation of dermis, hyperkera- tosis, and occasional ulceration have been frequently seen in control