310 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS lOO go 80 70 60 50 4.0 3o 20 lO o i i i i i i i i i [ i i i i i i i 0 2 4 6 8 10 12 14 16 18 MONTHS ON STABIUTY TEST Figure 1. D-values for E. coli in shampoo after storage at 3 ø, 20 ø, 3 , and 49øC for l, 3, 6, 12, and 18 . 8 ø mo. Explanation of symbols: i,__i,, shampoo stored at 49øC O--O, shampoo stored at 3 C I--I, I--•, shampoo stored at 20øC and }--{, shampoo stored at 3øC. obtained with E. coli however, comparison of Figures 1 and 2 reveals that the test strain of E. coli was slightly more resistant to the preservative system than the test strain of P. aeruginosa used in these studies. lOO 90 80 70 60 50 •o 30 20 lO o , 0 2 • 6 i i i i i i i i i 8 10 12 14 16 18 MONTHS ON STABILITY TEST 8 o Figure 2. D-values for P. aeruginosa in shampoo after storage at 3 ø, 20 ø, 3 , and 49øC for 1, 3, 6, 12, and 18 mo. Explanation of symbols: i,__i,, shampoo stored at 49øC O--•, shampoo stored at 38øC •--•, shampoo stored at 20øC and [•--I, shampoo stored at 3øC.

SHAMPOO PRESERVATIVE TESTING 311 Figure 3 illustrates the stability profile of the shampoo when challenged with Bacillus sp. The D-values obtained with this gram-positive spore former at several points in time were similar to those obtained with P. aeruginosa. Although S. aureus was much less resistant to the preservative system than the other test organisms, storage of the shampoo decreased the potency of the preservative system for this organism. Thus, the D-values changed from around 4 hr at the outset of the stability study to 7.6 hr after storage for 6 mo. at 49øC and to 9.8 hr after storage for 12 mo. at 38øC (Figure 4). The preliminary HPLC analyses for MP, CMIT, and MIT revealed that the concentra- tion of MP was unchanged and that the concentration of isothiazolinones appeared to have decreased in all shampoo test samples that had been used in the stability study. A shampoo spiked with MP, CMIT, and MIT and assayed by HPLC gave the chromato- gram shown in Figure 5. Here, the CMIT and MIT peaks are clearly evident. This may be contrasted with the chromatogram obtained from a shampoo aged for 18 mo. at 38øC (Figure 6), in which no MIT peak is evident. Similar findings were obtained with shampoo samples stored at the higher temperatures. The Ea' values for the change in the shampoo preservative system were calculated by substituting 2.303/D-values for the reaction rate constants (k) in the Arrhenius equa- tion (7). The results presented in Table I show that the Ea' values for the shampoo preservative system increased negatively from - 7, - 6, and - 4 Kcal/mole at 1 mo. to - 16, - 10, and -9 Kcal/mole at 12 mo. when performing preservative efficacy tests with E. coli, P. aeruginosa and Bacillus sp., respectively. Insufficient data were available for calculating a change in Ea' values during the first 6 mo. of the study when testing with S. aureus because this organism was inactivated so quickly in the shampoo. The 1 oo 8o 7o i 60 -.., 50 ! 3O 20 o o 2 4 8 8 lO 12 14 16 18 MONTHS ON STABILITY TEST 8 ø Figure 3. D-values for Bacillus sp. in shampoo after storage at 3 ø, 20 ø, 3 , and 49øC for 1, 3, 6, 12, and 18 mo. Explanation of symbols: &--&, shampoo stored at 49øC O--O, shampoo stored at 38øC I--I, shampoo stored at 20øC and }--{, shampoo stored at 3øC.