762 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table VI Challenge of 0.3 % Imidazolidinyl Urea Plus 0.2 % Methylparaben Plus 0.1% Propylparaben 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 - one organism that survived 7 d at 25øC, P. fluorescens, was killed within 3 d at 25øC in 0.5% Imidazolidinyl Urea solution. Screening the six most highly resistant "house" pseudomonads at incubation temperatures of 25, 35 and 45øC showed the same general behavior, although in this case survival times were approximately the same at incubation temperatures of 25 and 35øC (Table IX). Survival times at 45øC were shorter in all cases. The one organism that survived 7 d at 25 or 35øC not only was killed in screening tests by 0.5% Imidazolidinyl Urea solution, but also was surprisingly easy to kill with 0.3% Imidazolidinyl Urea solution in an actual cosmetic product. This latter observation reminds us once again that screening experiments may provide interesting general patterns of behavior, but they cannot substitute for the testing of actual cosmetic formulations. APPLICATIONS TO COSMETICS Using this same rigorous screening procedure, where anything other than complete kill is survival and therefore "growth" in the subculture, three cosmetic products now on the market from a major cosmetic company were challenged with five different pseudomon- ads. All three of these products as sold today are preserved with parabens alone. The results recorded in Table X represent subcultures after 2 d incubation. The cleansing cream with parabens alone showed survival of both types of ATCC P. aeruginosa and two of three "house" pseudomonads. When 0.3% Imidazolidinyl Urea was added, all five organisms were killed completely within 2 d. With the moisturizing lotion and the nutrient emulsion, three of the five pseudomonads survived after 2 d. Addition of 0.3% Imidazolidinyl Urea gave products which showed no growth on subculture, that is, complete kill of all five pseudomonads, within 2 days. Table VII Challenge of 0.3% Imidazolidinyl Urea Solutions with ATCC Pseudomonads at Different pHs (+ = Growth, - = No Growth) Species ATCC Number 1 Subcultures of Different pH Incubations (Time in Days) 5.0 6.0 7.0 8.0 9.0 2 3 7 1 2 3 ! 2 3 1 2 3 ! 2 P. aueruginosa 15442 P. cepacia 25416 P. putida 12633 P. fluorescens ! 3525 P. aureofaciens 13985 + + +

IMIDAZOLIDINYL UREA ACTIVITY 763 Table VIII Challenge of 0.3% Imidazolidinyl Urea Solutions with ATCC Pseudomonads at Different Incubation Temperatures (+ = Growth, - = No Growth) Species ATCC Number 1 Subcultures of Different Incubation Temperatures (Time in Days) 25øC 35øC 45øC 2 3 7 1 2 3 7 1 2 3 P. aueruginosa 15442 + + ..... P. cepacia 25416 + ...... P. putida 12633 + + - - + + + P. fluorescens 13525 + + + + - - - P. aureofaciens 13985 + + - - + + + This same test procedure was used on a model shampoo formulation (Table XI) recently proposed (24) by an ASTM Cosmetic Preservative Task Force for use in preservative testing. Challenges were carried out on the shampoo as described and also with added parabens or Imidazolidinyl Urea, or both. Each formulation was challenged not only with P. aeruginosa, but also with Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger. The results are shown (Table XII) for subcultures after incubation for 2 d. The shampoo alone and the shampoo containing parabens were both able to kill E. coli and S. aureus, but not the other three organisms. When 0.3% Imidazolidinyl Urea was added to either one of those formulations, the product killed all five organisms. In this series, with these specific microorganisms, the Imidazolidinyl Urea alone was capable of killing the organisms that the unpreserved shampoo did not kill. However since variations exist even among different types of the same species and since the Imidazolidinyl Urea-paraben preservative system has wide-range effectiveness, it would be wise to include the parabens in the preservative system as added insurance. Another example of the parabens' inability to withstand Pseudomonas challenge was reported recently (25) with a cosmetic lotion. The cosmetic lotion was developed by Amerchol (26) and used by the CTFA Preservation Subcommittee (11) for evaluation of methods of determining preservative efficacy. The Subcommittee tested (11) the formulation without any added preservative, and with 0.2% methylparaben plus 0.1% propylparaben. After inoculation of both lotions with approximately 106 organisms/ml of four different microorganisms, they did microbial counts over a 28-d period. The lotion without any added preservative killed S. aureus within 7 d, but failed to kill the other three Table IX Challenge of 0.3 % Imidazolidinyl Urea Solutions with "House" Pseudomonads (+ = Growth, - = No Growth) Pseudomonas Code Number Subcultures of Different Incubation Temperatures (Time in Days) 25øC 35øC 45øC 1 2 3 7 1 2 3 7 1 2 34A + + ........ 37-3 + + + + + + + + + + + 38-1 + + - - + + + .... 41A + + - - + + + .... 82A + + + - + ..... 83A + + + - + + + ....