j. Soc. Cosmet. Chem., 44, 329-336 (November/December) Preservative efficacy testing by a rapid screening method for estimation of D-values D. S. ORTH and D. C. ENIGL, Neutrogena Corporation, Los Angeles, CA 90045 (D.S.O.) and Watson Pharmaceuticals, Inc., Corona, CA. 91720 (D.C.E.)o Received July 20, 1993. Synopsis This report describes a rapid screening method for estimating D-values to determine whether products are adequately preserved. Estimated D-values (ED-values) are determined using aerobic plate counts of test organisms immediately after inoculation into test samples and at 24 hr for pathogenic microorganisms or at 7 days for non-pathogenic bacteria, yeasts, or molds. Products are judged to be adequately preserved if they meet the acceptance criteria of the linear regression method. There was excellent agreement between D-values and ED-values for 60 sets of data (correlation coefficient -- 0.98). The mean D-values and ED-values for the 60 samples differed by 0.5 hr (6.6%) even though the D-values ranged from 0.1 hr to 39 hr. Where differences were observed, the ED-values generally were larger (i. e., more conservative) than D-values for the same samples. The rapid screening method offers about 50% savings in the labor and materials required for preservative efficacy testing by the original linear regression method. INTRODUCTION Preservative efficacy testing is used to determine whether experimental formulas, sta- bility test samples, and finished products are adequately preserved. The goal of preser- vative efficacy testing is to determine the type and minimum effective concentration of preservatives required fbr adequate preservation of the formula during manufacturing, distribution, and use by consumers. The methods of preservative efficacy testing currently in use include official methods such as the United States Pharmacopeia (USP) method (1) and the British Pharmacopeia (BP) method (2) trade association methods such as the Cosmetic, Toiletry & Fragrance Association (CTFA) method (3) and rapid methods such as the linear regression method (4). The procedures used in these methods are similar however, the times at which samples are taken for analysis and the interpretation of test results--the acceptance criteria by which products are judged to be effectively preserved-- are different (5). The acceptance criteria of the USP, BP, and CTFA methods were converted to decimal reduction times (D-values) by Orth (5,6). Use of D-values enables a laboratory to determine the effect of the product preservative system on rates of death of test organ- isms, to compare rates of death in different products tested in different labs, to use 329

330 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS statistical controls of preservative efficacy tests, to determine synergistic effects of for- mula components, to set rational acceptance criteria, and to determine required D-val- ues (4-9). As originally outlined, the linear regression method specified that aerobic plate counts (APCs) be performed immediately after inoculation and at various times afterwards- typically at 2, 4, and 24 hr for bacteria and at 4, 8, and 24 hr for yeasts and molds. Additional samples were taken at 3, 5, or 7 d after inoculation unless the previous APC was 10/ml (4). These APCs were then used to determine the D-values for each test organism in product samples. It would be desirable to be able to determine the rate of microbial death--to determine D-values--using fewer APCs than required by the original linear regression method (4) because this would be less labor-intensive and would save on the cost of materials. This is now possible. This report describes a rapid screening procedure that allows calculation of D-values from two APC data points for each test organism. Our laboratory has performed several hundred rapid screening tests by use of this procedure to determine whether cosmetic and OTC-drug formulas meet acceptance criteria (5). EXPERIMENTAL TEST ORGANISMS The American Type Culture Collection (ATCC) organisms used in this study were received directly from the ATCC and consisted of Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), P. cepacia (ATCC 13945), Escherichia coli (ATCC 8739), Aspergillus niger (ATCC 16404), and Candida albicans (ATCC 10231). The Bacillus sp. was isolated from a product. The bacteria were maintained by weekly transfer on tryptic soy agar (TSA) with 0.07% lecithin and 0.5% Tween 80 (TSALT). A. niger and C. albicans were maintained by weekly transfer on potato dextrose agar (PDA). The bacteria and C. albicans were incubated at 32øC, and A. niger was incubated at 25øC. The bacteria were grown for 24 hr on TSALT agar prior to use in preservative efficacy testing. C. albicans was grown for 24 hr on PDA, and A. niger was grown for 7 d on PDA prior to use in preservative efficacy testing. TEST SAMPLES The test samples used in this study included proprietary formulations of o/w emulsions (creams, lotions, facial moisturizers), anionic surfactants (shower gels and cleansers), and OTC-drug products (sunscreens, anti-dandruff shampoos). TEST PROCEDURES Preservative efficacy tests were performed using saline suspensions from surface growth of each test organism as described elsewhere (4), with the following exceptions: 1) bacterial and C. albicans cultures were incubated at 32øC, and 2) APC determinations were made using single surface-spread plates of each o-ganism or each group of organ-