SYNERGY OF PRESERVATIVES 357 Table VI Use of STs and MPSTs to Determine Antimicrobial Synergy for Several Species of Pseudomonas in 0.2% MP, 0.01% Na2EDTA, and 0.2% MP q- 0.01% Na2EDTA (data from Table V) 0.2% MP solution: Test organism ST MPST D-value MPD-value Slope MPSlope P. aeruginosa 9027 -- 48 -- 9.5 -- - 0. 105 P. aeruginosa 27853 -- 48 -- 9.3 -- - 0. 108 P. aeruginosa 9721 -- 48 -- 9.1 -- - 0. 109 P. aeruginosa 10145 -- 48 -- 9.1 -- - 0.110 P. cepacia 13945 48 -- 9.2 -- - 0. 109 -- P. cepacia 25416 -- 48 -- 9.6 -- - 0. 104 P. fluorescens 13525 -- 48 -- 9.2 -- - 0. 108 P. putida 12633 48 -- 9.2 - 0.108 -- P. stutzeri 17588 -- 48 -- 9.2 -0. 109 Pseudomonas sp. 9230 -- 48 -- 10 - 0.100 S. aureus 6538 -- 48 -- 9.3 -0. 107 E. coli 8739 -- 48 -- 10 - 0. 100 0.01% Na=EDTA solution: Test organism ST MPST D-value MPD-value Slope MPSlope P. aeruginosa 9027 -- 48 -- 9.5 -- - 0. 105 P. aeruginosa 27853 -- 48 -- 9.3 -- - 0. 108 P. aeruginosa 9721 -- 48 -- 9.1 -- - 0.109 P. aeruginosa 10145 -- 48 -- 9.1 -- - 0.110 P. cepacia 13945 48 -- 9.2 -- -0. 109 -- P. cepacia 25416 -- 48 -- 9.6 -- - 0. 104 P. fluorescens 13525 -- 48 -- 9.2 -- - 0. 108 P. putida 12633 48 -- 9.2 -- - 0. 108 -- P. stutzeri 17588 24 -- 4.6 -- - 0.218 -- Pseudomonas sp. 9230 -- 48 -- 10 -- - 0.100 S. aureus 6538 -- 48 -- 9.3 -- -0.107 E. coli 8739 -- 48 -- 10 --• - 0.100 0.2% MP q- 0.01% Na=EDTA solution: Test organism ST MPST D-value MPD-value Slope MPSlope P. aeruginosa 9027 4 -- 0.8 -- - 1.260' -- P. aeruginosa 27853 4 -- 0.8 -- - 1.294' -- P. aeruginosa 9721 24 -- 4.6 -- - 0.219' -- P. aeruginosa 10145 4 -- 0.8 -- - 1.325' -- P. cepacia 13945 48 -- 9.2 -- - 0. 109 -- P. cepacia 25416 2 -- 0.4 -- - 2.498* -- P. fluorescens 13525 2 -- 0.5 -- - 2. 102' -- P. putida 12633 2 -- 0.4 -- -2.602* -- P. stutzeri 17588 0.1 -- 0.02 -- - 52. 304* -- Pseudomonas sp. 9230 24 -- 5.0 -- - 0.200 -- S. aureus 6538 -- 48 -- 9.3 -- - 0. 107 E. coli 8739 -- 48 -- 10 -- - 0. 100 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-• MPSlope, maximum possible slope of the virtual survivor curve, in hr-•. * 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 MP and Na=EDTA taken separately.

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