266 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS acid was made substantive without altering the amino function which is necessary for the Maillard reaction. To achieve this a sulfur atom was used to form a sulfonium or a sulfoxide functional group. The selected product was methionine sulfoxide (M.S.O.) which has good solubility in water, can be easily prepared, and meets the previously established requirements re- garding coloration. IN VITRO STUDIES The in vitro studies permit the selection of a substance producing the optimum intensity and speed of color development without changing the formulation. This product should have appropriate organoleptic characteristics and should be technologically easy to ob- tain. AMINO ACIDS SELECTION We studied the color produced by the combination of D.H.A. and (a) sulfur-containing amino acids: cystine, cysteine, and methionine* (b) modified sulfur-containing amino acids derived from methionine: methionine methylsulfonium iodide, methionine meth- ylsulfonium bromide, and methionine sulfoxide (M.S.O.)** and (c) lysine*: a sulfur- free amino acid which reacts particularly well with D.H.A. and was therefore used as a reference product. PROCEDURE The reaction of amino acids with D.H.A. (the Maillard reaction) produces complex polymers or "melanoidins" (4, 9-13). This reaction is influenced by the temperature, the amount of water, the proportion of D.H.A. to amino acid, the nature of the amino acid, the pH, and, if the medium is buffered, the nature of the buffer and its ionic strength. The quantitative evaluation of the color obtained was difficult. The spectrum did not show any maximum of absorption (3,4) between 360 and 600 nm, for all the amino acids chosen. Because there is no maximum absorption, the intensity of the color development was measured by spectrophotometry (Beckman © 25 spectrophotometer) at 420 rim. This wavelength seems characteristic of the mixture's color. The different parameters affecting the Maillard reaction were successively studied to obtain the best development of color (14). After various experiments, the following parameters were selected: (a) a phosphate buffer, 0.5 M pH 7, with Na H2 PO4 ß I H20 (138 g/l) (39 ml) and Na2H PO4 ' 12 H•O (358.5 g/l) (61 ml) to obtain a solution of amino acid at 0.01 M and a solution of DHA at 2 M (b) a mixture of these two solutions such that D.H.A. was 20 times the concentration of the amino acid (c) reaction temperature: 32 ø C and (d) evaluation of the color development at 420 nm, 24, 48, and 72 hours after mixture of the two solutions. * Riedel, de Ha•n Seelze, Hannover: purity 99%. ** Elf Aquitaine, Centre de Recherche de Lacq, FRANCE.

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