292 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS able to have tools which have the high level of sensi- tivity needed to determine the presence of condi- tioning agents which may be present only in mono- layer or even submonolayer quantities on the surface of these substrates. The search for such tools is on- going. Here we report an investigation of electron spectroscopy for chemical analysis (ESCA), also known as X-ray photoelectron spectroscopy, for this purpose. In our study, emphasis was given to water soluble conditioning agents which were either poly- meric or cationic, or both. Substantivity was also emphasized, i.e., the ability of the agent to absorb from aqueous solution and resist rinse-off by water. The substrates were neonatal rat stratum corneum membranes and virgin brown human hair. ESCA, which probes the outermost 25-50 A of the substrate, is shown clearly to have the sensitivity to detect adsorbed layers of cationic polymers, al- lowing quantitative estimates of surface coverage from the increase in concentration of elements con- stituting the adsorbing species (or, indeed, from a reduction in measured concentration of elements constituting the original substrate surface). High resolution spectra, which are sensitive to the state of chemical bonding of the constituent elements, pro- vide characterizing information on the adsorbed species even when present in submonolayer amounts. Using the technique, we show that deposition can vary considerably along the surface of hair fibers but not with all conditioning polymers. The ability of anionic surfactants to strip deposited material is also demonstrated. Data obtained with isolated stratum corneum membranes show that deposition of polymers may be asymmetric, i.e., the uptake on the outside of the membrane may be different from that on the inside. Results from experiments where the membranes are preexposed to surfactant imply a role of surface lipids in this deposition inequality. Experiments on the effect of various protective skin emollients are also reported. We conclude that ESCA, with its unusually high sensitivity, provides a potentially very powerful tool for use in cosmetic science. An original predictive method for in vivo per- cutaneous absorption studies A. Rougier, D. Dupuis, C. Lotte, and R. Roguet, Laboratoires L'Oreal, 1 Avenue de Saint Germain, 93601 Aulany Sous Bois, France A relationship between stratum corneum (SC) reser- voir function and percutaneous absorption has been shown in human volunteers and in hairless rats. In the hairless rat (back), 10 radiolabeled molecules (200 n.mol) were applied (1 cm2). The quantity (x) of drug within the SC reservoir was determined at the end of application time (30 min) using the stripping technique, and the total amount pene- trated (y) was measured after 4 days. A linear correlation exists between these two param- eters and is given by y = 1.644 x -0.536 (r = 0.998, p inf. 0.001). In both rat (back) and human (arm), such relation is still valid when the same molecule (benzoic acid) is applied at different doses (125, 250, 500 and 1000 n.mol). This allows to establish in human, the correlation curve valid for all types of molecules, with only one compound. The relation obtained may be written as follow: y = 1.82 x -0.52 (r = 0.998, p inf. 0.001). It is worth noting that this relation has been found inde- pendent from the application time, the dose ap- plied, and the vehicle used. Thus, the quantity of drug penetrating through rat or human skin in vivo can be predicted by only mea- suring its horny layer concentration. Moreover, the relatively large amounts of product present in the SC at the end of application time should allow per- cutaneous absorption studies using nonradioactive techniques. Cell proliferation rate studies as efficacy indi- cators Fred Burmeister, Tri-K Industries, Inc., 466 Old Hook Road, P.O. Box 312, Emerson, NJ 07630 The use of human cell cultures in cell proliferation rate studies has been accepted as an in vitro model in the evaluation of antitumor agents' cytotoxic ac- tivity. The potential to look directly at cell prolifer- ation and viability led researchers to believe that a properly modified method could yield valuable in vitro data in the assessment of the efficacy of cos- metic agents and whole products. Tri-K industries, in conjunction with Alfacel Cor- poration, has developed a colorimetric method by which the viability and proliferative profile of human epidermal fibroblast cultures can be quanti- fied. As the culture medium is changed by the ad- dition of chemicals or cosmetic products, so is the specific proliferative profile of the cells in culture. We sought to eliminate the handling and storage problems associated with tritiated thymidine in the choice of this colorimetric assay. This assay utilizes a reagent which is cleaved only by active mitochon- dria. Dead or dying cells do not metabolize the re- agent and will not yield the blue chromophore which is read colorimetrically. We are looking at the physiological responses of cells to the modified media as they replicate . . . not factors associated with the synthesis of DNA . . . as is the case with the 'Warburg' assay of oxygen uptake.

ABSTRACTS 293 The data presented are treated statistically via a computer program intended solely for use in bio- chemical assays. They are represented either in tab- ular or graphic format. (examples will be given) They are easily intrepreted and can be used to show stimulatory, null, inhibitory, or toxic responses. Correlation of this in vitro evaluation with "folk- loric" claims for herbal products is exceedingly good. Likewise, the correlation between this evalua- tion and in vivo evaluations demonstrating increased cell turnover with finished cosmetic products sup- ports the use of this test as a screening method for preliminary claims substantiation. Tri-K Industries feels that we have bridged the classical definitions of in vitro and in vivo evaluations with this method. Whereas in the past, claims were initially substan- tiated in laboratory environments using synthetic models or formerly living substrates, we are using living substrates in an elegant and thoroughly prac- tical manner to determine true efficacy of modern cosmetic formulae. SESSION G PRODUCT ANALYSIS A three-day mold assay for cosmetics and toilet- ries Catherine A. Mead and John J. O'Neill, Avon Products, Division Street, Suffern, NY 10901 Cosmetic product mold recovery assays generally call for five or more days of incubation of a nutrifled agar plate in which the product has been dispersed. This paper reports the development and validation of an assay procedure which requires only three days of incubation. It is a refinement of the five-day test in two respects. First, and most importantly, the temperature is carefully controlled in an incubator at 27 to 28øC, replacing "room temperature." Second, a more nutritious agar, buffered near neu- trality, replaces the low pH Mycophil agar (BBL) used in our conventional five-day test. The method was validated by simulating contamination of a wide variety of cosmetic products with environ- mental and ATCC fungal strains. Novel empirical method to calculate HLB by TLC Kazutami Sakamoto, Ph.D., Ajinomoto U.S.A., Glenpointe Centre West, 500 Frank W. Burr Blvd., Teaneck, NJ 07666-6894 HLB is a very practical concept to clarify the prop- erties of surfactant as an emulsifier. Many methods to calculate HLB are proposed, but some are re- stricted for general use and others are too compli- cated practically. It seems noteworthy that thin layer chromatography (TLC) gives hydrophylic and hydrophobic character to the molecule by using normal phase or reversed- phase method. We already reported about applica- tion of reversed-phase TLC for the determination of hydrophobicity of the surfactants based on amino acid as a preliminary test for toxicity and irrigation. Hydrophilicity was also measured by normal phase TLC. New empirical HLB calculation based on hy- drophilicity and hydrophobicity by TLC was pro- posed, and a good relationship is found between new methods and traditional methods for a series of nonionic surfactants. Further application for anionic surfactants such as N-acylamino acids will be discussed. Color precision shading by eye and instrument Raymond K. Reilly, Noxell Corporation, P.O. Box 1799, Baltimore, MD 21203 The traditional method for evaluating cosmetic product color has been visual. An alternative to vis- ual evaluation has been provided by the develop- ment of sophisticated color computer systems. Lighting and application of product are important factors in visual color matching, while presentation of samples to the spectrophotometer is an important factor in instrumental color evaluation. Both methods of color determination are affected by pig- ment quality and processing variables. The color computer offers some advantages in the color evalu- ation of cosmetic products, pigments, and pack- aging components. Scanning electron microscopy of dentifrices Timothy R. Kapsner, and Linda J. Haning, Min- netonka, Inc., P.O. Box 1A, Minnetonka, MN 55343 The objective of this study was to determine if the Scanning Electron Microscope (SEM) could be used to compare the abrasives used in dentrifice products. The abrasives were viewed as raw mate- rials and in finished formulations. The method de- veloped in this study for viewing finished dentifrice formulations employs the use of freeze/fracture tech- nology. Ordinarily, this is a sample preparation technique for transmission electron microscopy. The freeze/fracture process produces a replica of the dentifrice sample, retaining the abrasive particles on the underside of the replica. The intact particles may be viewed on the SEM, where different magni- fications allow a comparison of both size and struc- ture. Results are presented as a visual comparison of SEM photomicrographs. The freeze/fracture tech- nique is a useful method for viewing abrasive par- ticles in a finished dentifrice formt•lation. The vari-