j. Soc. Cosmet. Chem., 38, 307-319 (September/October 1987) Determination of shampoo preservative stability and apparent activation energies by the linear regression method of preservative efficacy testing D. S. ORTH, C. M. LUTES, S. R. MILSTEIN, and J. J. ALLINGER, The Andrew Jergens Company, 2535 Spring Grove Avenue, Cincinnati, OH 45214. Received October 3 O, 1986. Synopsis Preservative efficacy tests were performed on stability samples of a shampoo during storage for 18 mo. at 3 ø, 20 ø, 38 ø, and 49øC. The shampoo preservative system deteriorated with increasing storage time and temperature, as measured by the "linear regression method." Thus, the decimal reduction time (D-value), which was used as the measure of the rate of death for Escherichia coli, Pseudomonas aeruginosa, and Bacillus sp., increased from about 4 hr to over 30 hr after accelerated aging at 49øC for 3 mo. The D-values for $taphylococcus aureus increased more slowly. Apparent activation energies (Ea') for the change in shampoo preservative potency were calculated from the D-values at different temperatures. The Ea' values decreased from -7, -6, and -4 Kcal/mole at 1 mo. to -16, -10, and -9 Kcal/mole at 12 mo. for shampoo challenged with E. coli, P. aeruginosa, and Bacillus sp., respectively. This loss of preservative system potency appeared to follow first-order or pseudo first-order reaction kinetics, while the magnitude of the Ea' varied with the length of storage and the test organisms used. This work illustrates the need for using different microorganisms when conducting preservative efficacy tests of cosmetic products and shows how the quantitative values obtained by the linear regression method are well-suited to monitoring stability tests. The use of this method for determining the apparent molar concentration of a preservative from D-values and for predicting the stability of cosmetic preservative systems from Ea' values is discussed. INTRODUCTION Stability testing is the final step in the development of cosmetic products. The objective of this testing is to demonstrate that a product does not change significantly during its expected shelf life. Product stability is necessary because several months or years may elapse between the time a cosmetic is manufactured and used up by the consumer. Recognizing this, current Good Manufacturing Practices (cGMP) for human OTC drug products stipulate that products must be stable for at least 3 yr. and that this must be supported by appropriate stability data (1). In many instances, cGMP are used as a basis for supporting documentation for cosmetic products. Thus, stability tests may be per- 3O7

308 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS formed by storage of products at various temperatures for up to 5 yr. to demonstrate satisfactory shelf life. Microbial spoilage may occur in aqueous products and in anhydrous products that are exposed to water consequently, cosmetic preservatives are included in formulations to inhibit the growth of bacteria, yeasts, and molds while products are in trade channels and in the hands of the consumer. The principles of preservative efficacy testing have been reviewed (2,3), including the need for demonstrating that products have adequate stability (2). Although several methods of preservative testing are available (4-6), our laboratory uses the "linear regression method" because it provides a rapid, quantitative expression of the rate of death of specific test organisms in a product when using defined test conditions (4). The rate of death determined by the linear regression method is ex- pressed as the decimal reduction time (D-value), which is the time required for inacti- vation of 90% of the population of test organisms. The rationale for using D-values is that every organism has a characteristic rate of death when subjected to a specific lethal treatment (4). This enables a laboratory to provide quantitative results on the rate of inactivation of specific test organisms in a product. Thus, the linear regression method can be used to determine the effect of formulation changes and component interaction on the stability of the preservative system. Since the linear regression method was adopted, we have observed changes in preserva- tive efficacy of some formulations during the course of stability studies. This report illustrates the value of performing preservative efficacy tests on stability samples by showing how a shampoo preservative system deteriorates with age. EXPERIMENTAL TEST ORGANISMS The test organisms used in these studies 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), and Escherichia coli (ATCC 8739). These organisms were cultured and used for challenging the test samples, as described in a previous report (4). TEST SAMPLES The test samples consisted of a proprietary formulation of a shampoo in high-density polyethylene containers. The shampoo contained ammonium lauryl sulfate, cocamido- propyl betaine, propylene glycol, polysorbate 20, hydrolyzed animal collagen, tetra so- dium EDTA (and other ingredients), and was preserved with methylparaben IMP], chloromethylisothiazolinone [CMIT], and methylisothiazolinone [MIT]. STABILITY TEST One bottle of freshly prepared shampoo was used for the initial determinations (i.e., 0-mo.). Several bottles of the test samples were stored at refrigerator temperature, room