PRESERVATIVE EFFICACY TESTING 335 ED-values may be determined using saline suspensions of pure cultures or mixed inocula (i.e., where more than one test organism was used in the saline suspension added to a test sample). We have found that use of pooled inocula is convenient when using organisms with similar ED-values (i. e., P. aeruginosa and S. aureus E. coli and P. cepacia) and/or recovery media (i.e., C. albicans and A. niger). Microbiologists generally use duplicate plates for each dilution when determining APCs. This is done to improve the reliability of the plating method and to help overcome human factors, which decrease the precision of the method (i.e., inaccuracies in pipeting). Preliminary data in our laboratory indicate that use of single plates and duplicate plates give essentially the same D-values. The difference in D-values was 0.5 hr for those tests in which the maximum D-values were no larger than 12 hr. Although the differences in D-values may be greater with larger D-values, it is believed that the percentage difference would be 10% as long as the assays are "in control" (7-9). Laboratories should implement statistical control procedures to ensure that assays are in control. Our preliminary data suggest that in some instances a laboratory may be able to use single plates for rapid screening studies. This work describes a rapid screening method for estimating D-values. This method is similar to the linear regression method however, intermediate samplings and APCs are omitted. This allows determination of an ED-value using APCs at 0 hr and 24 hr (for pathogens) and at 0 hr and 7 d (for non-pathogenic bacteria, yeast, and molds). When using an inoculum of 10 6 organisms/g product, recovery of 10 organisms/g product at 24 hr or 7 d indicates that the MPST is 424 hr or 4 168 hr, respectively, and that the ED-value is 44 hr or 428 hr, respectively. The reliability of the rapid screening method was good over the range of D-values one finds in both satisfactorily and unsatisfactorily preserved products (i.e., D-values 4 hr to 39 hr). There was excellent agreement between ED-values and D-values (correlation coefficient = 0.98), and the difference between mean D-values and mean ED-values was well below 10%. This is considered to be suitable for a rapid screening method. Where differences in estimated and D-values were observed, the ED-values generally were larger (i.e., more conservative) than D-values for the same samples. The physicochemical make-up of each formula, the type of packaging, and conditions of use by consumers determine the risk of microbial contamination and spoilage (10). Several formulas frequently must be tested in the process of selecting the final formula. The rapid screening method provides a convenient means of selecting the preservative system of a product and offers about 50% savings in terms of labor and materials required for testing. It is recommended that ED-values be confirmed by determining D-values for finished formulations. ACKNOWLEDGMENT We thank Ms. Fariba Jashnian for her assistance with the microbiological analyses. REFERENCES (1) Microbiological Tests, Antimicrobial Preservatives--Effectiveness, United States Pharmacopeia XXII, United States Pharmacopeial Convention, Rockville, MD, pp. 1478-1479 (1990).

336 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (2) Efficacy of antimicrobial preservatives in pharmaceutical products, British Pharmacopoeia 1988, Vol. II., Her Majesty's Stationery Office, London, England, pp. A200-A208 (1988). (3) Preservation Subcommittee of the CTFA Microbiological Committee, A guideline for the determi- nation of adequacy of preservation of cosmetics and toiletry formulations, TGA Cosmet. J., 2, 20-23 (1970). (4) D. S. Orth, Linear regression method for rapid determination of cosmetic preservative efficacy., J. Soc. Cosmet. Chem., 30, 321-332 (1979). (5) D. S. Orth, Standardizing preservative efficacy test data, Cosmet. Toiletr., 106(3), 45-48,51 (1991). (6) D. S. Orth, C. M. Lutes Anderson, D. K. Smith, and S. R. Milstein, Synergism of preservative system components: Use of the survival curve slope method to demonstrate anti-Pseudomonas synergy of methyl paraben and acrylic acid homopolymer/copolymers in vitro, J. Soc. Cosmet. Chem., 40, 347-365 (1989). (7) D. S. Orth and L. R. Brueggen, Preservative efficacy of cosmetic products. Rechallenge testing and reliability of the linear regression method, Cosmet. Toiletr., 97(5), 61-65 (1982). (8) D. S. Orth, "Principles of Preservation," in S. P. Denyet and R. M. Baird, Eds., Guide to Microbi- ological Control in Pharmaceuticals (Ellis Horwood, London, 1990), pp. 241-250. (9) D. S. Orth, Preservative efficacy testing: Rationale, acceptance criteria, and determination of syner- gism. Oral presentation at The First Pan European Conference on the Significance and Validation of Test Methods for the Efficacy of Preservatives in Cosmetics, Toiletries and Pharmaceuticals, held November 20-21, 1991, at the Commonwealth Institute, London, England. (10) D. S. Orth, The required D-value: Evaluating product preservation in relation to packaging and consumer use/abuse. Cosmet. Toiletr., 107, 39-43 (1992).