j. Soc. Cosmet. Chem., 36, 355-362 (September/October 1985) In vitro and in vivo study of the substantivity of p-amino benzoic acid and two of its esters M. I. MORASSO, A. M. THIELEMANN, C. PINTO, M. FIGUEROA, and A. ARANCIBIA, Departamento de Ciencias Farmacologicas, Universidad de Chile, Casilla 233, Santiago, Chile. Received November 26, 1984. Synopsis The substantivity of p-amino benzoic acid (PABA) and two of its esters (Escalol 507 © and Amerscreen "P"©), in an alcoholic vehicle, has been studied both in vitro and in vivo in order to establish the possible correlation between the two methods. Eight healthy volunteers participated in the in vivo method. Solutions of the sunscreen substances were prepared in isopropanol (IPA): PABA at 5%, and Escalol 507 © and Amerscreen "P"©, both at 2%. In order to study the substantivity of each of the sunscreen substances to the skin, the subjects placed their hands in the solutions. The hands were then rinsed with water and extracted with IPA at 50øC to quantitate the amount of substance retained by the skin. A crossover design was used all the subjects participated in experiments with each of the substances at one week intervals. In the in vitro experiment, keratin powder obtained from hard, insensible tissues (human calluses of the feet) was used. The keratin was kept in an ethanol solution of the sunscreen for 72 hr, then treated with water for 48 hr, and finally extracted with ethanol at 50øC for 24 hr in order to determine the amount of sunscreen retained by the keratin. The sunscreen concentrations in the different solutions in both in vitro and in vivo methods were determined by spectrophotometry. The in vivo percent substantivities (i.e. percentage of sunscreen deposited which was not rinsed off ,by water) obtained in the study were: PABA = 0.30 _+ 0.05 Escalol 507 © = 57.96 --- 0.63 Amerscreen "P"© = 0.46 + 0.08. In the in vitro method the following values were obtained: PABA = 0.30 +-- 0.02 Escalol 507 © = 50.84 + 0.66 Amerscreen "P"© = 0.47 + 0.03. The differences are statistically significant (p 0.01) and Escalol 507 © is the substance that shows this property in the highest degree. The results obtained by the in vitro and in vivo methods were linearly correlated. INTRODUCTION The function of a sunscreen preparation is to absorb as completely as possible the erythemic ultraviolet radiation of sunlight while transmitting a maximum of the other wavelengths. During the last few years, several researchers have focused their attention on the loss of efficacy of sunscreens due to their removal from the skin by perspiration and/or swimming (1,14). The term substantivity is currently used to express the ca- 355

356 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS pacity of a compound to adhere or combine with a keratinized substrate (2). In general it is accepted that a sunscreen preparation must have some degree of substantivity. Our literature search has failed to reveal studies oriented toward establishing standard methods of evaluating in the laboratory the ability of a U.V. absorber to resist being removed by water. Some studies of affinities of sunscreens for human skin in vivo and in vitro (3,4) or animal skin in vitro (5) have been reported. Nevertheless, very little is known about the significance of the in vitro methods and their capability to predict the behaviors of the compounds in human skin. An in vitro method that correlates with actual in vivo application on human skin is desirable to avoid the inconveniences and limitations of human experimentation. In our work we have studied the substantivities of p-amino benzoic acid (PABA) and two of its esters (Escalol 507 © and Amerscreen "P"©), using both an in vivo and in vitro method in order to find a correlation between the two methods. Healthy volunteers participated in the in vivo method, thus studying the substantivity of the sunscreen compounds under conditions which were near to actual use conditions. Human keratin in the form of excised human callus tissue was employed in the in vitro method. The results in terms of percentage of substantivity obtained by the two methods were compared and a very strong linear correlation was found. EXPERIMENTAL DESIGN MATERIALS The test materials were: p-amino benzoic acid, analytical reagent grade (USP) octyl dimethyl (Escalol 507 ©, Van Dyk & Co., Inc.) and ethyl dihydroxypropyl (Amerscreen "P"©, Amerchol Corporation) p-aminobenzoate and ethanol 95% and 2-propanol (E. Merck, Darmstadt, W. Germany). The human keratin was obtained from human foot callus tissue which had not been treated with chemical agents prior to removal. The callus tissue was pulvarized while suspended in acetone refrigerated below 0øC with solid CO2 using a Waring Blender ©. After the comminution procedure, the material was washed with ether and allowed to dry (6). After pulverization the keratin was sieved before use, utilizing the fraction that passed through a 70-mesh sieve but which was retained by a 100-mesh sieve. The mean particle size of the powder was 150 DETERMINATION OF SUBSTANTIVITY IN VIVO Eight healthy volunteers, one male and seven females, ranging in age from 20 to 30 yr (mean 24 yr) and in weight from 45 to 70 kg (mean = 60 kg), with no external signs of allergy or other skin disease, participated in this study. The method previously described by Cumpelik (4) was employed, utilizing 2% w/v, solutions of Escalol 507 © and Amerscreen "P"© in isopropanol (IPA). A 5% PABA solution in IPA was used for comparison. Hands and arms of all subjects were washed up to the elbows with soap and tapwater, dried, and then washed in IPA at 50øC repeatedly until 100% transmittance of the washings was obtained between 240 and 400 nm, using a Carl Zeiss spectrophotometer. Hands were air dried. Following the cleaning procedure, all the subjects had their right hand dipped into the PABA solution for 2 min. For comparison the left hand of each subject was dipped