760 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Challenge of 0.5 % Imidazolidinyl Urea Solutions with ATCC Pseudomonads (+ = Growth, - = No Growth) Subculture After Incubation Times iDays) Species ATCC Number 1 2 3 7 14 21 28 P. putida 12633 + - - P. aureofaciens 13985 + - - traditional wisdom in the field is that mutated types are often harder to kill than the "tame" ATCC types. As shown in Table IV, a 0.3% solution of Imidazolidinyl Urea was challenged with 17 different "house" pseudomonads in the usual way (106/ml, pH 7.0, etc.). Twelve of the seventeen were completely killed within 1 d, one survived for 1 d, but not for 2 d, and four survived for 3 d. Extending the incubation time gave complete kill of three of the four resistant strains, but one "house" pseudomonad, number 37-3, survived for the full 28 d. The four "house" pseudomonads that survived for three or more days in 0.3% Imidazolidinyl Urea solution were inoculated in the usual way into 0.5% Imidazolidinyl Urea solutions. As shown in Table V, two of the four pseudomonads were killed by 0.5% solution within 1 d, but the other two survived for 3 d. By 7 d, however, both of these two resistant "house" pseudomonad were also killed by 0.5% Imidazolidinyl Urea solution. The combination preservative system 0.3% Imidazolidinyl Urea plus 0.2% methylparaben plus 0.1% propylparaben killed three of the four resistant "house" pseudomonads within ! d and killed the exceptionally resistant pseudomonad number 37-3 within 2 d (Table VI). Reducing the level of Imidazolidinyl Urea from 0.3% to 0.2% gave a system which killed three of the four organisms within 1 d, but permitted the exceptional pseudomonad number 37-3 to survive for 3 d. Raising the level of Imidazolidinyl Urea from 0.3% to 0.5%, still in combination with 0.2% methylparaben plus 0.1% propylparaben, gave a system which killed all four pseudomonads, including 37-3, within 1 d. These screening tests show that pseudomonads differ considerably in their vulnerability to a given antimicrobial, and that a so-called "adequate preservative system" against Pseudomonas depends in large part on the specific pseudomonad used in the test. Even the most resistant pseudomonad can apparently be killed by the right preservative system but the test results warn that, even after full microbial testing, a mutated pseudomonad might later grow in an otherwise satisfactorily preserved product. Even after production is routine, good housekeeping is essential and constant watchfulness is prudent. After everything is worked out and running, the job of surveillance must continue. Table III Challenge of 0.3 % Imidazolidinyl Urea Plus 0.2 % Methylparaben Plus 0. ! % Propylparaben Solutions with ATCC Pseudomonads(+ = Growth, - = No Growth) Subculture After Incubation Times (Days) Species ATCC Number 1 2 3 7 14 21 28 P. putida 12633 - - - P. aureofaciens !3985 - - -

IMIDAZOLIDINYL UREA ACTIVITY 761 Table IV Challenge of 0.3 % Imidazolidinyl Urea Solutions with "House" Pseudomonads (+ = Growth, - = No Growth) Pseudomonas Subculture After Incubation Times (Days) Code Number 1 2 3 7 14 21 28 34A - - - 37-1 - - - 37-2 - - - 37-3 + + + 37-4 - - - 38-1 + + + 38-2 - - - 41A + + + 82A + - - 82B - - - 82C - - - 83A + + + 83B - - - 87A - - - 87B - - - 99 - - - + + + + In order to test,the effect of pH, a series of challenges were run on 0.3% Imidazolidinyl Urea solutions at pHs of 5.0, 6.0, 7.0, 8.0 and 9.0, using five different ATCC pseudomonad species (Table VII). In this series of challenges, P. aeruginosa, P. putida and P. auerofaciens were all completely killed at all pHs within 1 d. At pH 5.0, P. cepacia appeared to survive for 1 d, but not for 2 d. Interestingly, P. fluorescens survived for 3 d at pH 5.0 even though it was completely killed in 1 d at pH 7.0. At pH 5.0, P. fluorescens was killed within 7 d, but its increased survival at pH 5.0 suggests caution in assuming that optimum growth occurs for all pseudomonads at pH 6-8. It is well documented that antimicrobials act faster (21) and better (22) at elevated temperatures, and that an increase in temperature has a potentiating effect on a preservative system (23). Our studies tended to confirm these generalizations. The five ATCC pseudomonads shown in Table VIII all were capable of surviving for long periods in dilute nutrient broth alone at 25, 35 or 45øC. In dilute nutrient broth also containing 0.3% Imidazolidinyl Urea, pseudomonads were killed at all temperatures, but survival times of the pseudomonads tended to be longer at 25øC and shorter at 45øC than at 35øC. The Table V Challenge of 0.5 % Imidazolidinyl Urea Solutions with "House" Pseudomonads (+ = Growth, - = No Growth) Pseudomonas Subculture After Incubation Times (Days) Code Number 1 2 3 7 14 21 28 37-3 + + + 38-1 + + + 41A - - - 83A - - -