j. Soc. Cosmet. Chem., 30, 321-332 (November 1979) Linear regression method for rapid determination of cosmetic preservative efficacy D. S. ORTH The Andrew Jergens Co., 2535 Spring Grove Ave., Cincinnati, OH 45214. ReeivedApd120, 1979. Synopsis A method is described for determining preservative efficacy of cosmetic products in 48 hr for bacteria and in 7 days for molds. The rate of inactivation of selected test organisms in a product is given by the decimal reduction time (D-value), which is calculated from the plot of the log number of surviving organisms/g as a function of the time after inoculation into the product. The D-value provides a quantitative expression of the rate of death of a specific test organism in a product and enables comparison of the rates of inactivation of different organisms in one or more products. The time for complete destruction of any size population of organisms may be calculated when the D-value is known. Examples are given to show how the method was used to determine preservative efficacy of creams, lotions and shampoos. Performance criteria using D-values for PRESERVATIVE EFFICACY ASSESSMENT are proposed for different classes of organisms in different types of COSMETIC PRODUCTS. INTRODUCTION Preservative efficacy testing is an essential part of substantiating the safety of cosmetic products. Testing is necessary to demonstrate that finished products are free from excessive numbers of microorganisms and that they are free from pathogenic organisms. In addition, microbiological analyses are performed to establish that the preservative system is potent enough to prevent the growth of yeasts, molds and bacteria in the product during conditions of use by the consumer. Methods for determining preservative efficacy of cosmetic products were reviewed by Cowen and Steiger (1). Test methods usually follow the guidelines published by the United States Pharmacopeia (2) or the Cosmetic, Toiletry and Fragrance Association (CTFA) (3). In addition, capacity tests and streak methods have been used for preservative testing (4,5). These methods involve challenging the test material with several microorganisms and checking for the number of surviving organisms at subsequent time intervals to determine if the preservative system inactivates the challenge organisms. It is not uncommon for preservative efficacy testing to require several weeks, and, if rechallenge studies are performed, the testing may take two months. It would be desirable if the time required for preservative efficacy testing could be 321

322 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS shortened to a few days. The objectives of this study were: (i) to develop a rapid method that provides a quantitative expression of the rate of death of a specific test organism in a particular product (ii) to provide examples of how the method has been used to assess preservative efficacy in creams, lotions, shampoos and hair conditioners and (iii) to propose minimum performance criteria for preservatives in different classes of cosmetic products. EXPERIMENTAL TEST ORGANISMS The test organisms used in this study were taken from the Jergens culture collection and consisted of Staphylococcus aureus (FDA 209 strain), Pseudomonas aeruginosa (PRD 10 strain), Bacillus sp. (isolated from a contaminated cosmetic product), Aspergillis niger and A. flavus. The bacteria were grown on Standard Methods Agar (Baltimore Biological Laboratories, Baltimore, Md.) slants for 24 hr at 37øC, and the molds were grown on Potato Dextrose Agar (PDA, Difco Laboratories, Detroit, Mich.) slants for 7 days (d) at 25øC. The inocula were prepared by transferring growth from the slants to sterile saline and mixing using a Vortex Genie Mixer (Scientific Products, Columbus, Ohio). The percentage of sporulation of the Bacillus sp. was typically 30-50% at 24 hr. TEST SAMPLES The test samples used in this study were proprietary formulations of lab and production samples of face cream, lotions, shampoos and hair conditioners. The bacteriological properties of the face creams were compared using face cream with borate (control), face cream with borate and 0.12% methyl- and 0.08% propyl-paraben, and face cream with borate and 0.2% methyl- and 0.1% propyl-paraben. All percentages are % (wt/vol) unless otherwise specified. The lotions studied contained methyl- and propylparaben and Quaternium-15 (Dowicil © 200 Preservative The Dow Chemical Co., Midland, Mich.). The effect of formulation change on the preservative efficacy of lotion was studied using lotion containing glyceryl dilaurate (standard formulation) and lotion containing glyceryl monolaurate (Lauricidin ©, Med-Chem Laboratories, Okemos, Mich.) substituted for the dilaurate. The shampoos and conditioners were preserved with formaldehyde. TEST PROCEDURE A 0.1-ml amount of test organism suspension was added to 50-ml portions of one to four samples in 100-ml screw-capped bottles. Immediately thereafter, the bottles were shaken vigorously 100 times, and 11 ml were pipetted into 99 ml of Letheen Broth (Difco Laboratories) with 0.01% (vol/vol) Triton X-100 (Sigma Chemical Co.) in milk dilution bottles. This diluent was designated LBT. The bottles were shaken vigorously 50 times, and 1:100 dilutions were made by transferring 1 ml to 99 ml of LBT and shaking 25 times. Then, 0.1 and 1.0 ml amounts of appropriate dilutions were pipetted into duplicate Petri dishes, and pour plates were prepared by adding Tryptic Soy Agar (Difco Laboratories) with 0.5% (vol/vol) tween 80 and 0.07% (vol/vol) lecithin. This plating medium was designated TSALT agar.