362 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS systems. This suggests that the observed synergy is not due to the nonionic emulsions studied here. The effect of 934, 941, or 1342 and MP on the inactivation of Pseudomonas was not tested in anionic or cationic emulsion systems or in various surfactant systems such as anionic shampoos and liquid soaps. The reason for this is that 1342 emulsion systems are sensitive to electrolytes, which cause loss of emulsion stability (35). Adair et al. (36) reported that P. aeruginosa 9027 underwent lysis following metabolism of di- or tricarboxylic acids and sodium lauryl sulfate, and that lysis was not due to chelation. It is evident that the mechanism reported by these workers is not the same as the mechanism observed in the current work. The antibacterial synergy of MP and acrylic acid homopolymer/copolymers against most Pseudomonas test cultures was not observed with E. coli 8739. This gram-negative or- ganism was not inactivated rapidly in nonionic emulsions containing MP and 1342 or in solutions containing MP + Na2EDTA (Tables II, V, and VI). These findings sug- gest a mechanism of action that is relatively specific for pseudomonads and not other gram-negative bacteria however, testing with other strains of E. coli and other gram- negative organisms is necessary to confirm this. The survivor curve slope method of determining synergy of preservative systems has application to both current experimental findings and to data presented in the litera- ture. Application of this method to the ST data of Richards and Hardie (37) revealed synergism for fentichlor/phenylethanol combinations against E. coli and Proteus vulgaris at 0.0015% or 0.0050% fentichlor q- 0.4% phenylethanol, and with 0.0050% fen- tichlor + 0.4% phenylethanol against P. aeruginosa. The survivor curve slope method revealed increased antibacterial activity against S. aureus,' however, this effect was not synergistic. Our use of the survivor curve slope method corroborated the findings of Richards and Hardie. The survivor curve slope method of determining synergy was applied to the D-values for S. aureus, in various combinations of preservatives in saline, reported by Akers et al. (38). Slopes of the survival curves were determined by taking the negative reciprocal of the D-values reported by these workers, and these slopes were used to determine whether mixtures of two preservatives exhibited synergy. Although these workers did not attempt to determine synergy, application of the survival curve slope method to their data for linear analysis of preservatives in saline solutions revealed that systems containing 0.2% phenol q- 0.3% m-cresol, 0.2% phenol q- 0.2% m-cresol, and 0.2% MP + 1.0% benzyl alcohol exhibited synergy. Akers et al. ranked the efficacy of these preservative systems in the top half of the systems tested against S. aureus. The survival curve slope method was used to study synergy in a system reported by Boehm to be synergistic (4). He reported that 0.25% P and 0.09% N were synergistic against P. pyocyanea. Since we did not have the same strain as in Boehm's experiments, it was decided to "bracket" the concentrations of P and N used in Boehm's studies. The results in Table VII show the growth response observed with P. aeruginosa 9027 in 0 to 1% P and/or 0 to 0.1% N. The STs and MPSTs in Table VII were used to calculate the slopes, MPSlopes, D-values, and MPD-values in Table VIII. Our findings show syn- ergy for concentrations that bracket the synergistic combination reported by Boehm. The use of kinetic parameters--the slopes of survivor curves obtained by use of the linear regression method--to demonstrate anti-Pseudomonas synergy of MP and acrylic acid homopolymer/copolymers in vitro has not been reported previously. The survivor
SYNERGY OF PRESERVATIVES 363 curve slope method may be used to determine synergy when STs are known for the test organisms in systems containing combined preservative system components and in which the inoculum level is known. Isobolograms (5,7,39) are not needed when using this method. We propose that the synergy with acrylic acid homopolymer/copolymers and MP is due, at least in part, to the chelation of divalent metal ions by the homopolymer/co- polymers and that it is similar to the potentiation of preservative action by EDTA. No synergy was demonstrated in systems challenged with E. coli, S. aureus, and B. cereus, which suggests that the synergy was specific for the pseudomonads. In general, P. cepacia was inactivated more slowly than the fluorescent pseudomonads in test systems containing acrylic acid homopolymer/copolymers and MP. It is possible that the pri- mary benefit from the polyacrylic acid/acrylic acid copolymer synergy with MP may be obtained in systems in which EDTA cannot be used or in systems with low ionic strength. Experiments were not performed to determine whether acrylic acid homo- polymer/copolymers exert a synergistic effect on MP/EDTA systems, or whether EDTA exerts a potentiating effect on MP/acrylic acid homopolymer/copolymers. Additional experiments are required to define the range of synergy of MP and other paraben esters with these homopolymer/copolymers and to characterize the mechanism of this synergy with certainty. ACKNOWLEDGMENTS We thank Mr. Mark Entrup, of Hill Top Biolabs, Inc., for supplying the culture of E. coli. We thank Mr. Gary Kramzar, of Nipa Laboratories, Inc., for supplying phenoxy- ethanol and Nipastat. REFERENCES (1) D. S. Orth, Principles of preservative efficacy testing. Cosmet. Toilet,, 96(3), 43-52 (1981). (2) J. J. Kabara, "Food-Grade Chemicals in a Systems Approach to Cosmetic Preservation," in Cosmetic and Drug Preservation. Principles and Practice, J. J. Kabara, Ed. (Marcel Dekker, Inc., New York, 1984), pp. 339-356. (3) D. S. Orth, C. M. Lutes, S. R. Milsrein, and J. J. Allinger, Determination of shampoo preservative stability and apparent activation energies by the linear regression method of preservative efficacy testing,J. Soc. Cosmet. Chem., 38, 307-319 (1987). (4) E. E. Boehm, Synergism in vitro of certain antimicrobial agents, J. Soc. Cosmet. Chem., 19, 531- 549 (1968). (5) R. M. E. Richards and R. J. McBride, Phenylethanol enhancement of preservatives used in oph- thalmic preparations, J. Pharm. Pharmac., 23 (Suppl.), 141S- 146S (1976). (6) P. G. Hugbo, Additivity and synergism in vitro as displayed by mixtures of some commonly em- ployed antibacterial preservatives, Cosmet. Toilet., 92(3), 52,55 - 56 (1977). (7) S. P. Denyet, W. B. Hugo, and V. D. Harding, Synergy in preservative combinations, Internat. J. Pharm., 25, 245-253 (1985). (8) D. S. Orth, Linear regression method for rapid determination of cosmetic preservative efficacy, J. Soc. Cosmet. Chem., 30, 321-332 (1979). (9) Betz Laboratories, Analytical Procedure #130, "Hardness Titration Method" (1978). (10) C. C. Garber and R. N. Carey, "Laboratory Statistics," in Clinical Chemistry--Theory, Analysis, and Correlation, L. A. Kaplan and A. J. Pesce, Eds. (C. V. Mosby Company, St. Louis, 1984), pp. 287-300.
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