358 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table VII Determination of Sterilization Times for P. aeruginosa 9027 in Saline Containing 0 to 1.0% Phenoxyethanol and 0 to 0.10% Nipastat Growth response at Preservative 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST Saline control + / + + / + + / + 0.1% P +/+ +/+ +/+ 0.5% P +/+ +/+ +/+ 1.0% P +/+ +/+ +/- 0.005% N +/+ +/+ +/+ 0.01% N +/+ q-/q- +/+ 0.05%N +/+ +/+ +/+ 0.10% N +/+ -/- / 0.1% P + 0.005% N +/+ +/+ 0.1% P + 0.01% N +/+ +/+ 0.1% P + 0.05% N +/+ - - 0.1% P + 0.10% N -/- + + + + -- -- + -- + - + -- 0.5% P + 0.005% N +/+ 0.5% P + 0.01% N +/+ 0.5% P q- 0.05% N +/+ 0.5% P + 0.10% N -/- 1.0% P + 0.005% N +/+ 1.0% P + 0.01% N q-/q- 1.0% P + 0.05% N q-/q- 1.0% P + 0.10% N +/- +/+ +/+ / / +/+ +/+ / / / / / +/+ +/+ +/+ +/- +/+ +/+ +/+ / +/+ +/+ + + _ _ nd** / + / + -- 48 + / + -- 48 +/- -- 48 -/- 48 -- +/+ -- 48 +/+ -- 48 + / + -- 48 -/- 2 -- + / + -- 48 +/+ -- 48 -/- 2 -- -/- 0.1' -- +/- -- 48 -/- 24 -- -/- 2 -/- 0.1- -/- 4 -/- 4 -/- 4 -/- 2 -- Table symbols represent growth of P. aerugtnosa on one ( q- / - ), on both ( + / + ), or neither ( - / - ) TSALT plates inoculated from duplicate tubes containing the indicated concentrations of phenoxyethanol (P) or Nipastat (N). Tubes with P and/or N were incubated at room temperature for the times indicated. ST, sterilization time in hr MPST, minimum possible sterilization time in hr. * Although P. aeruginosa was not recovered in these samples, the ST was set at 0.1 hr because this is the approximate time required for setting up the series of samples after inoculation. ** nd, not done. The P. aeruginosa inoculum APC = 1.7 X 107/mi. DISCUSSION This work was initiated when it was realized that the preservative system in adequately preserved nonionic emulsion systems was inactivating P. aeruginosa much more rapidly than the other test organisms customarily used in preservative efficacy testing (8). All of these emulsions contained MP. The parabens are known to be active against a wide range of gram-positive bacteria and fungi, but they are less active against gram-negative bacteria, especially the pseudomonads (12). Although 0.2% MP did not kill P. aeru- ginosa in 24 hr, combinations of MP and 1342 caused rapid killing of this test organism (Figure 1). The nonionic lotion with 0.2% MP (Table I) was selected for investigating the cause of this rapid inactivation of P. aeruginosa observed in our laboratory. Pseudomonas has been particularly troublesome for the cosmetic and pharmaceutical in- dustries. P. aeruginosa, P. cepacia, P. fluorescens, P. putida, and P. stutzeri can survive and grow in aleionized water, and they have been isolated from contaminated cosmetics (13-15). P. aeruginosa has been recovered from contaminated mascaras and has caused

SYNERGY OF PRESERVATIVES 359 Table VIII Use of STs and MPSTs to Determine Antimicrobial Synergy for P. aeruginosa 9027 in 0 to 1.0% Phenoxyethanol and 0 to 0.10% Nipastat (data from Table VII) Preservative ST MPST D-value MPD-value Slope MPSlope Saline control -- 48 6.6 -- - 0.151 0.1% P -- 48 6.6 -- -0.151 0.5% P -- 48 6.6 -- -0.151 1.0% P 48 -- 6.6 -- -0.151 -- 0.005% N -- 48 6.6 -- -0.151 0.01% N -- 48 6.6 -- -0.151 0.05% N -- 48 -- 6.6 -- - 0.151 0.10% N 2 -- 0.3 -- -3.615 -- 0.1% P + 0.005% N -- 48 -- 6.6 -- -0.151 0.1% P + 0.01% N -- 48 -- 6.6 -- -0.151 0.1% P + 0.05% N 2 -- 0.3 -- -3.615' -- 0.1% P + 0.10% N 0.1 -- -0.01 -- -72.304* -- 0.5% P + 0.005% N -- 48 -- 6.6 -- -0.151 0.5% P + 0.01% N 24 -- 3.3 -- -0.301 -- 0.5% P + 0.05% N 2 -- 0.3 -- -3.615' 0.5% P + 0.10% N 0.1 -- 0.01 -- -72.304* 1.0% P + 0.005% N 4 -- 0.6 -- - 1.808' 1.0% P + 0.01% N 4 -- 0.6 -- -1.808' 1.0% P + 0.05% N 4 -- 0.6 -- - 1.808' 1.0% P + 0.10% N 2 -- 0.3 -- -- -3.615 Explanation of symbols: ST, sterilization time in hr MPST, minimum possible sterilization time in hr D-value, D-value in hr MPD-value, minimum possible D-value in hr Slope, slope of the survivor curve, in hr- 2 MPSlope, maximum possible slope of the virtual survivor curve, in hr- x * Synergy observed, because the slope of the survivor curve was a larger negative number than the sum of the slopes (or MPSlopes) for the same concentrations of P and N taken separately. corneal ulcers (16,17). This organism produces several virulence factors that are be- lieved to contribute to its multifactorial pathogenicity and complicate the clinical course of infections (18-23). P. cepacia has considerable physiological versatility and has broad resistance to antibi- otics (24,25). P. cepacia 13945 was selected for detailed investigation in this work because, generally, it was more resistant than the other pseudomonads in our culture collection to preservative systems containing MP and acrylic acid homopolymer/co- polymers. P. fluorescens and P. putida were selected for detailed studies here because they are nutritionally versatile and are able to grow on a wide variety of substrates (24,26). When the lotion was prepared using 0.2% 1342 and 0.2% MP, preservative efficacy testing revealed significant antibacterial activity against most test cultures of Pseudo- monas (Table II). All fluorescent pseudomonads [P. aeruginosa, P. fluorescens, P. putida and P. stutzeri (24)] were inactivated rapidly, with D-values • 1.1 hr. Both P. cepacia strains were inactivated more slowly in the preservative system than were the fluores- cent pseudomonads. The reasons for the resistance of strains 13945 and 25416 are not known however, P. cepacia is nutritionally versatile and accumulates poly-beta-hy- droxybutyrate (PHB) as a carbon reserve (24,27,28). These physiological characteristics may enable P. cepacia to be more difficult to inactivate in test systems containing che- lating agents and MP than are the fluorescent pseudomonads.