330 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The relationship between the D-values for bacteria, such as S. aureus, and molds needs to be established to determine if the rate of death of molds can be predicted from the D-values obtained for S. aureus in a product. If experiments show that this is possible, the preservative testing could be set up using only bacteria and the results could be obtained in 48 hr. The reliability of any test method depends on the precision, sensitivity and accuracy of the method. In microbiological testing, the precision generally depends on the skill and care exercised by the analyst. The correlation coefficient was used as an indicator of precision and was used to determine the goodness of fit of the data to the linear regression. The running average of the correlation coefficient was -0.99, and the 95% confidence limits were _+0.03. In practice, all analyses were considered to be unac- ceptable and were repeated if the correlation coefficient was outside of limits. An attempt was made to minimize the variation between different analyses by handling all samples in the same manner. The sensitivity of the method depended on the ability of the recovery system to allow growth of the viable organisms present in the test samples. The LBT and TSALT agar were used because they are recommended by the CTFA (3) and because it is believed that this recovery system provides the most reliable recovery of organisms present in the test materials examined. The linear regression method has been used to determine the preservative efficacy of over 150 samples of creams, lotions, shampoos and hair conditioners using S. aureus, P. aeruginosa, Bacillus sp., A. niger or A. flayus as test organisms. A perfect correlation was observed between the predicted time for complete destruction of the test organisms and the time observed to recover 10/ml on product resamplings with all products except for protein-containing shampoos and hair conditioners. Even though the linear regression method predicted complete destruction of the populations of test bacteria in less than 24 hr, low levels of bacteria were recovered at the 24-hr sampling. This observation was made using all of the test bacteria on several occasions and is thought to be due to the protein in these products. It is reported that protein interferes with the preservative efficacy of formaldehyde (8), and it is possible that the protein affords some protection to the bacteria so that there is tailing (i.e., departure from linear die-off in the semi-log plots shown in the survivor curves) that results in recovery of the organisms past the predicted times for complete destruction. Further study is needed to understand the lack of correlation observed here. As with all methods of preservative efficacy testing, the usefulness of the test depends on the choice of appropriate test conditions, test organisms and acceptance criteria. It is desirable to use the organisms with the greatest resistance to the preservative system (i.e., organisms with the largest D-values in the product being examined) in order to establish that the product is adequately preserved. Unfortunately, there is always the possibility that more resistant organisms exist in nature, or that the consumer will abuse the product so that microbial growth can occur. It is possible to set meaningful guidelines for accepting or rejecting a product based on the performance of its preservative system because the linear regression method provides a quantitative measure of the rate of die-off of test organisms. It is proposed that the preservative system for each cosmetic meet minimum requirements for inactivation of three classes of organisms: 1) pathogenic microorganisms, such as

EVALUATION OF COSMETIC PRESERVATIVE EFFICACY 331 5. aureus and P. aeruginosa 2) bacteria not known to be pathogens and 3) yeasts and molds. It is believed that pathogens should be inactivated completely in 24 hr in multiple-use products intended for application in or around the eye and for baby products. Thus, a product should have a D-value of _4 hr so that 106 pathogens/g will be inactivated completely in 24 hr. The 24-hr timing is considered appropriate because this provides time for the product to "cleanse" itself before use one day later. The CTFA guidelines for acceptable microbial levels in cosmetic products allow no recognized pathogens and different levels of nonpathogenic organisms in cosmetics for general use and in cosmetics intended for use in and around the eye or on babies (9). It seems appropriate that general use products would not need to have the same preservative requirements as those used on sensitive skin or in sensitive areas. Although specific guidelines need to be established for each product, it is believed that D-values of _28 hr are realistic and attainable for nearly all nonpathogenic bacteria, yeasts and molds. This would enable the preservative system to inactivate 106 organism/g in one week. The linear regression method is offered as an alternate procedure to current methods of preservative efiScacy testing. The rapid method offers the advantages of obtaining results in a few days, quantitation of the rates of death of specific organisms in products, and estimation of the time required for complete destruction of any size microbial population. It is believed that this method will be useful in determining additive vs. synergistic effects of combinations of cosmetic preservatives, and in other areas where quantitative information about the antimicrobial properties of cosmetic formulations is desired. The data presented in this report used linear regression analysis to determine D-values and to estimate the time for complete destruction of microbial populations. Similar information may be obtained by graphical means. ACKNOWLEDGMENT The author thanks Dr. Jon Kabara for supplying the Lauricidin ©. REFERENCES (1) R. A. Cowen and B. Steiger, Antimicrobial activit¾--a critical review of test methods of preservative efficiency, J. Soc. Cosmet. Chem., 27,467-481 (1976). (2) Anon, "Microbiological tests, antimicrobial preservatives--effectiveness," United States Pharmacopeia XIX, The United States Pharmacopeial Convention, Rockford, Md., 1975 pp 587-592. (3) Preservation Subcommittee of the CTFA Microbiological Committee, A guideline for the determina- tion of adequacy of preservation of cosmetics and toiletry formulations, TGA Cosmet. Journal, 2, 20-23 (1970). (4) M. Barnes and G. W. Denton, Capacity tests for the evaluation of preservatives in formulations, Soap, Perfumery & Cosmetics, 42,729-733 (Oct. 1969). (5) H. S. Bean, Preservatives for pharmaceuticals,J. Soc. Cosmet. Chem., 23,703-720 (1972). (6) O. Rahn, Physical methods of sterilization of microorganisms, Bacteriol. Rev., 9, 1-47 (1945). (7) C. R. Stumbo, Thermobacteriology in Food Processing, Academic Press: New York, N.Y., 1965 pp 56-59. (8) B. Croshaw, Preservatives for cosmetics and toiletries,J. Soc. Cosmet. Chem., 28, 3-16 (1977). (9) Microbial Content Subcommittee of the CTFA Microbiology Committee, Microbiological limit guidelines for cosmetics and toiletries, CTFA (1973).