J. Cosmet. Sci., 56, 167-174 (May/June 2005) Use of triphenyltetrazolium chloride in preservative efficacy testing D.S. ORTH and K. S. ECK, Neutrogena Corporation, 5760 West 96th Street, Los Angeles, CA 90045. Accepted for publication March 1, 2005. Synopsis Preservative efficacy testing without counting colonies was done by determining growth in dilutions of inoculated product following enrichment in Letheen broth with 0.001 % triphenyltetrazolium chloride (TTC) in 96-well microtiter plates. Bacterial growth was indicated by the development of a red/pink color in the enrichment broth. The method was used to determine log reductions of bacteria at specified times after inoculation, and D-values were calculated using the reciprocal of the highest dilution showing growth (pink color) as the log CFU/ml bacteria at each time point. The method using TTC was validated by demonstrating that D-values for Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, and Esche- richia coli in 44 aqueous cosmetic and OTC-drug products were virtually identical to those obtained when using Alamar Blue® in the miniaturized system (1). Plotting D-values obtained using TTC as a function of D-values obtained using Alamar Blue gave a line with a slope of 0.98, which shows excellent agreement of results obtained by the two methods. This miniaturized assay system has been used for more than three years for preservative efficacy testing of several hundred cosmetic and OTC-drug product samples in our laboratory. It is recommended for laboratories that conduct large numbers of preservative efficacy tests. INTRODUCTION Preservative efficacy testing or "challenge testing" is performed on aqueous cosmetic and drug products to determine the minimum effective concentration of antimicrobial pre- servatives required for adequate preservation. Products are satisfactorily preserved if they meet appropriate acceptance criteria (2,3). Methods of preservative efficacy testing in- clude compendia! methods, such as the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP) methods trade association methods, such as the Cos- metic, Toiletry & Fragrance Association (CTFA) method and rapid procedures such as the linear regression method (4-7). All of these methods have a number of similarities, including test organisms used, recovery systems, and the method of performing aerobic plate counts (APCs). Although these test methods are similar, they have differences in growth temperatures, procedures for preparing inocula, times at which APCs are determined, use of rechallenge testing, and acceptance criteria. These differences may produce variations in test results and affect 167

168 JOURNAL OF COSMETIC SCIENCE whether a product passes or fails the challenge test and whether it has microbiological problems in production and when being used by consumers. The decimal reduction time (D-value) is the time required for killing 90% of the population of test organisms (a 1-log reduction) and is determined by calculating the negative reciprocal of the slope of the survival curve. D-values are used in all areas of microbiology (i.e., antibiotic testing, food microbiology, sterilization by heat/ irradiation, etc.) and have been used to "standardize" or relate the different initial rates of killing allowed by the above-mentioned challenge test methods (8). The slowest initial rates of killing currently allowed for bacteria used in preservative efficacy testing (i.e., the largest D-values) are ::Sl68, ::S56, 24, ::S4, and ::S28 h for the USP, CTFA, and EP methods (based on 2-log reduction at two days [topical products}), and for pathogens (linear regression method) and non-pathogens (linear regression method), respectively. It is apparent that the USP and CTFA methods allow a slower rate of killing than the EP and linear regression methods. Orth et al. (3) reported that the maximum allowable D-values for gram-negative bacteria were around 30 h because these bacteria persisted or grew if they were not killed with initial D-values of 30 h. Orth and Eck (1) reported preservative efficacy testing could be done without counting colonies by use of a miniaturized enrichment system using 96-well microtiter plates followed by streaking onto agar or measuring fluorescence of Alamar Blue® added following enrichment. These workers found that the Alamar Blue color change that revealed microbial metabolism/ growth tended to drift and become difficult to interpret when the dye system was added to the enrichment broth for the 48-h enrichment. This problem was circumvented by addition of the Alamar Blue following enrichment and incubation for 2 h prior to making fluorescence measurements. It was considered de- sirable to have a stable, inexpensive redox dye for use in the miniaturized system. The goal of this work was to determine whether 2,4,5-triphenyltetrazolium chloride (TTC) could be used as a reliable indicator of growth in the enrichment broth in wells in the 96-well microtiter plates as indicated by similar log reductions with Alamar Blue at specified times after inoculation or calculated D-values. MATERIALS AND METHODS TEST PRODUCTS The 44 aqueous cosmetic and OTC drugs tested included shampoos, conditioners, face washes, eye creams, antidandruff shampoos with salicylic acid or coal tar, acne treatment products containing benzoyl peroxide or salicylic acid, sunless tanning products with dihydroxy acetone, mascaras, foundations, and sunscreens. TEST ORGANISMS The microorganisms used for testing included test bacteria used in routine preservative efficacy testing, including Staphylococcus aureus ATCC 65 38, Pseudornonas aeruginosa ATCC 9027, Burkholderia (Pseudornonas) cepacia ATCC 13945, and Escherichia coli ATCC 8739. The bacteria were transferred no more than five times. The test organisms were grown 24--48 h at 35°C on trypticase soy agar with 0.03% lecithin and 0.5% polysor-