PSEUDOMONADS IN COSMETICS 801 Preservation Regardless of sanitary manufacturing methods, a poorly preserved preparation provides many opportunities for microbial infection. As described earlier an O/W emulsion at a pH of 7-8 containing nonionics provides a particularly suitable menstruum for pseudomonas growth. Preservatives used to restrain growth are the well known phenolics, parabens, bisphenols, and organo-metals. Organic acids can be effective at pH 7 while quaternaries tend to be incompatible with a nonionic slightly alkaline system. It is necessary to bear in mind that few cosmetically acceptable bacteriocides are active against pseudomonads specifically, even in the absence of surfactants. Anionics in general ap- pear to inhibit selectively the metabolism of gram-positive organisms, and those which are bacteriocides tend to have a narrower spectrum than cationics. The bacterial action of anionics is influenced to a greater ex- tent by changes in pH. Some nonionics actually inactivate the bacteriostatic action of bisphenols and Tween © 80,* for example, is a more effective antidote than blood serum for nullifying the antibacterial properties of these com- pounds (17). However, Tween 80 in low concentrations, such as 0.02%, render pseudomonads actually more susceptible to antimicrobial agents. This enhancing effect at low levels is noteworthy since at higher levels the surfactant was found by Kohn et al. (18) to be an antagonist to the very same bacteriostatic agents. Studies have shown that Pseudornonas aeruginosa is capable of growth in solutions and dispersions of nonionic surfactants of the Tween type and can split the ester linkages of these agents (19). Practically all the nonionics of ethylene oxide or propylene oxide condensates of fatty acids and alcohols inactivate many pre- servatives in current use. It was found, in a study of 36 nonionics and 26 preservatives, that the nonionic surfactants reduced the efficiency of all preservatives when the ratio of surfactant to preservative ex- ceeded certain critical values (20). Of course, one is now cognizant that with time, inactivation of many biostats occurs and what appears as a well-preserved preparation today can be spoiled by an inoculum 2-3 months after manufacture (21). Emulsions of the water-in-oil type are relatively resistant to natural pseudomonas attack since the continuous oil phase acts as a barrier to penetration of the organism into the water phase, and impedes the spread of growth through the system. However, the features which pre- * Tween 80 is a registered trademark of Atlas Powder Company, Wilmington, Del.

8O2 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS vent invasion also retard the antimicrobial activity of the biostats, so that a W/O emulsion which is more difficult to infect also appears to be more difficult to preserve. Other factors affecting the activity of pre- servatives in W/O systems are those controlling the availability of the preservatives in the aqueous phase, in particular, the O/W partition co- efficient of the preservative, the phase-volume ratio, and the tempera- ture. Antimicrobial compounds with high oil-water partition coeffi- cients concentrate in the oil often with insufficient quantities being dis- tributed in the aqueous phase to inhibit microbial growth. The parti- tion coefficient of most biostatic agents will be higher for vegetable oils than for mineral oils, making vegetable oil creams more difficult to pre- serve than those prepared from mineral oil. Knowing the concentration of a biostat, the dissociation constant, the concentration of nonionics and the partition coefficient, the amount of the agent dissolved in the aqueous and micellar phases at a given pH can be calculated. Evans (22) found, for example, that a 0.5% concentration of p-hydroxy benzoate in an aqueous 6.0% Tween solution gave a concentration equivalent to 0.1% of the benzoate in the water phase. Noble and Savin (23) reported a hospital pseudomonas outbreak which illustrates these concepts. A well preserved steroid cream was diluted with lipids. The diluted cream containing 0.1% chlorocresol was found contaminated with P. aeruginosa. Although 0.02% chloro- cresol in aqueous solution was sufficient to prevent growth of organism, the chlorocresol migrated into the oil constituents of the cream leaving the aqueous portion with insufficient biostat. Examination of the clini- cal records suggested that the cream had caused minor infections over a period of several months. Similarly diluted creams from two of eight other hospitals were also found contaminated with pseudomonads. In preserving a product one frequently overlooks the nutritive qual- ity of the preparation. Cosmetic and pharmaceutical formulations often range from water solutions to high protein soups. The growth po- tential of the product itself is a prime agent influencing the ease or diffi- culty of preservation, for a nutrient-free preparation is easier to preserve than one containing nutriles. The "magic ingredients" now current in many formulations can on occasion be nutrients, stimulating the growth of organisms and obstructing the action of preservatives. Existing screening tests in agar or broth which determine a com- pound's inherent antimicrobial activity are helpful only in eliminating completely inactive materials. They will not predict, with any reliabil- ity, performance in the actual cosmetic or pharmaceutical emulsion.