SAFETY TESTING OF HAIR DYE PREPARATIONS 749 As a consequence, serious doubts have arisen concerning the validity of such exaggerated exposure conditions. The problem is compounded in the case of formulations which contain active ingredients where the dosages recommended are based on multiples of the human dosage. These are often scaled to 1, 3, and 10 times the human dose on a mg. per kg. body weight basis. The higher dosages often require volumes of test material considerably greater than can be applied to the trunk of the rabbit in single applications. When the total dose is placed under the plastic sleeve, the quantity of test material in direct contact with the skin is considerably less than if the same total quantity were applied as a thin layer covering the entire trunk. To avoid this problem of dosage, many workers administer the total dosage as a series of divided doses. These volumes are without doubt unrealistic in relation to the total body surface of the animal. Recently, the Food and Drug Administration reviewed a protocol prepared by a group representing manufacturers of hair dyes. This group submitted a realistic procedure for evaluating oxidation hair dyes which bore a closer relation to the conditions of use. The results of several studies employing the original and modified procedures are covered by this report. Characterization of the irritation response is one of the single most important criteria in these studies. Up to this time, only the Draize scoring procedure and subsequent histomorphological evaluation of tissue pathology have been applicable. The Draize system is an at- tempt to reduce subjective evaluations to numerical terms. Micro- scopic examination, a critical aspect of the total assessment, has serious limitations in terms of differentiating subtle differences in response. A new procedure was employed which quantitates the inflammatory changes associated with irritation in terms of the fluids and cellular elements present at the application sites. It reflects the increased numbers of erythrocytes present due to the increased leakage through the capillaries and possible impaired integrity of these vessels. Frank hemorrhage is not required for definition. This procedure has re- vealed differences in the tissue fluids where no discrete visual evidence of erythrocytes had been noted. METHODS These modified procedures were designed to approximate normal usage patterns. Adult albino rabbits weighing between 2 and 3 kg. were distributed into groups of 10 animals equally divided as to sex.

750 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS On the day prior to the initiation of applications, the dorsal hair was clipped to a length of 1/• to •/• in. with an electric clipper, care being taken to avoid nicking or abrading the skin. One group served as the control, receiving all preliminary preparations without treatment. The p-phenylenediamine-resorcinol (P.P.D.-R.) type dye-peroxide mix- ture was prepared in accordance with label directions, all dilutions being discarded after a single use. Two graded concentrations of the mixture were applied within 20 minutes of preparation. The contact •c l i i, ,• , i-i-, •t• • • • • ............ ====================== :::: Figure 1. Standard values of rabbit blood calls-calibration curve for alkaline digestion procedure time was fixed at 60 minutes. At the end of this exposure period, the dye mixture was rinsed off with lukewarm water. Four milliliters of a commercial shampoo intended for use after dye application was spread over the dyed area, worked into a rich lather, and then rinsed with luke- warm water until all traces of lather were gone. The rabbit was then