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 + + + - + + + ....

764 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS' Table X Challenge of Cosmetic Emulsions Containing Parabens (+ = Growth, - = No Growth After 2 Days) "House .... House .... House" P. Aeruginosa P. Aeruginosa Pseudomonad Pseudomonad Pseudomonad ATCC 15442 ATCC 13388 34A 37-3 82C Cleansing Cream Cleansing Cream + 0.3 % Imidazolidinyl Urea Moisturizing Lotion Moisturizing Lotion q- 0.3% Imidazolidinyl Urea Nutrient Emulsion Nutrient Emulsion q- 0.3% Imidazolidinyl Urea + + organisms. The lotion with 0.2% methylparaben plus 0.1% propylparaben killed S. aureus, E. coli and C, albicans, but failed to kill P. aeruginosa, which remained at high levels throughout the 28-d study period. Addition of 0.3% Imidazolidinyl Urea to the lotion already containing the parabens gave a lotion which was capable of killing P. aeruginosa within 2 d and which showed no growth over the full 28-d test period (25). A second and third re-challenge of the lotion with P. aeruginosa also showed no survival within 2 d of the re-challenge, and the pseudomonad count stayed 10/ml throughout each 28-d test period. SUMMARY It has been shown in both screening experiments and in actual cosmetic products that Imidazolidinyl Urea is effective against a wide variety of pseudomonads. The few resistant strains that Imidazolidinyl Urea alone did not kill quickly enough at the indicated concentrations were killed quickly using an Imidazolidinyl Urea-paraben combination system. It is recommended that the combination preservative system of Imidazolidinyl Urea-methylparaben-propylparaben be used in proportions of 3:2:1, and it is strongly suggested that all microbial testing be carried out on the finished cosmetic product. Different pseudomonads vary in their susceptibility to antimicrobials, so good housekeep- Table XI Model Shampoo Formulation (Water Base Detergent System), ASTM E-35. ! 5 Cosmetic Preservative Task Force Ingredient % By Weight Grams/Kilo Triethanolamine Lauryl 25.00 250.0 Sulfate (40%) Lauryl Diethanolamide • 5.00 50.0 Amphoteric-2 5.00 50.0 Polyoxyethylene Lanolin (50%) 3.00 30.0 Phosphoric Acid 0.20 2.0 Demineralized Water qs to ! 00% Detergent system containing an amphoteric (Miranol). Add all ingredients to mixing vessel and warm to !50øF. Sweep stir to 90øF.