EFFECTS OF ADJUVANTS ON DIHYDROXYACETONE TANNING 267 These experimental conditions enabled the screening of the best amino acid but they were not convenient for later in vivo trials (except for temperature which is that of the skin) because of too weak a concentration for sufficient tanning. RESULTS The results obtained are shown in Table I. Among the amino acids tested, the following were eliminated: Cystine and cysteine do not react sufficiently with D.H.A. Lysine gives good coloration but the molecule does not contain a functional group able to fix the product on the stratum corneum. Methionine is only slightly soluble and has a bad odor. The derivatives of methionine seemed interesting since they had better solubility and substantivity than the starting material. The sulfonium compounds were, however, eliminated because they had a bad odor and because iodides and bromides are not innocuous and therefore unsuitable for cutaneous applications. Chlorides were not tested since the odor due to the sulfonium group would preclude their use. Table I Evaluation of the Color Obtained After Mixing the D.H.A. and Amino-Acid Solutions* Optical Density at 420 nm (32 ø C) After 24 Hours 48 Hours 72 Hours Cysteine 0.05 + 0.01 0.08 + 0.01 0.10 + 0.01 Cystine 0.06 -+ 0.01 0.09 -+ 0.01 0.15 -+ 0.01 Methionine 0.10 _+ 0.01 0.20 q- 0.01 0.35 q- 0.01 Methionine methylsulfonium iodide 0.15 _+ 0.01 0.28 _+ 0.01 0.35 -+ 0.01 Mixture of amino acids found in the stratum corneum** 0.17 + 0.01 0.30 + 0.01 0.36 + 0.01 Methionine methylsulfonium bromide 0.18 q- 0.01 0.29 -+ 0.01 0.39 -+ 0.01 Lysine 0.19 -+ 0.01 0.33 -+ 0.01 0.45 -+ 0.01 Methionine sulfoxide 0.17 -+ 0.01 0.32 -+ 0.01 0.47 -+ 0.01 * Values are averages from three determinations. ** L-Serine 24.7%, L-Citrulline 16.4%, L-Glycine 9.3%, L-Alanine 8.9%, L-Threonine 7.753, L-Aspartic Ac. 5.5%, L-Arginine 4%, L-Histidine 3.6%, L-Lysine 3.4%, L-Tyrosine 3.2%, L-Leucine 3%, L-Valine 2.5%, L-Glutamic Ac. 2.3%, L-Phenylalanine 2.3%, L-Proline 1.9%, L-Ornithine 1.353. Table I shows that the intensity of the color obtained with M.S.O. is greater than with the other amino acids or with a mixture of amino acids found in the stratum cor- neum (15). Moreover, we observed that after 48 hours at a pH of 7 and a temperature of 32 ø C, the M.S.O with a 0.5 M phosphate buffer containing 10% NaC1 produced the max- imum color (Table II). The methionine sulfoxide was therefore selected because it has good reactivity with D.H.A., good water solubility, good organoleptic character, and innocuity. Indeed, in a Japanese patent (16) it is used instead of methionine for enrichment of soybean

268 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Influence of Ionic Strength on Color Intensity* Optical Density at 420 nm, 32 ø C, pH 7 After 24 Hours 48 Hours 72 Hours Without Without Without With NaC1 NaC1 With NaC1 NaC1 With NaC1 NaCI M.O.S. Methionine Methionine Methyl- sulfonium iodide 0.19 _-_ 0.01 0.17 _-_ 0.01 0.46 ___ 0.01 0.32 --- 0.01 0.51 --- 0.01 0.46 + 0.01 0.18 ___ 0.01 0.10 ___ 0.01 0.28 + 0.01 0.20 + 0.01 0.49 + 0.01 0.35 + 0.01 0.20 +_ 0.01 0.15 +_ 0.01 0.29 +- 0.01 0.28 ñ 0.01 0.45 +_ 0.01 0.35 +- 0.01 * Values are averages from three determinations. Sodium chloride concentration: 10%. milk and synthetic meat. Moreover, some studies of toxicity, particularly an Ame's test and cutaneous tolerance tests (17,18) on rabbits, were done. Lastly, due to its sulfoxide function the M.S.O. develops some affinity for keratin and permits the retention of the colored complex as shown by in vivo trials (19). IN VIVO STUDIES The in vivo studies were extensions of the in vitro studies but only the M.S.O. was used. It was necessary to select two different vehicles, the first well absorbed by the skin and able to fix the amino acid on the stratum corneum, and the second a film former able to regulate and facilitate easy spreading of the D.H.A. It was also necessary to define a method of application and to perfect an objective method of color evaluation. FORMULATION Some trials were performed with aqueous solutions of M.S.O. directly spread on the skin or with M.S.O. at a concentration of 0.1 M in a Carbopol © gel, but they were unsatisfactory because of irregular spreading or unsatisfactory release of M.S.O. from the vehicle. It seemed that only emulsions would be convenient. Selection of emulsion bases. We chose to formulate three emulsions differing in their oil phase percentage, the nature of the oils, and the type of emulsion (Table III). The A and B emulsions of the O/W type containing autoemulsive base are fluid emulsions. They contain either substances permitting cutaneous permeation (isopropyl myristate-PEG-4 isostearyl glyceride) or a film-forming component (mineral oil). The C emulsion of the W/O type was produced in order to test the reactivity of D.H.A. in such a medium and the water resistant properties of the color produced. Incorporation of specific substances. The direct mixture of amino acid with D.H.A. is not stable. Therefore we used separate formulations for D.H.A. and for the amino acid. Former trials showed that the best formulation for M.S.O. was emulsion A and for