414 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS been recovered from the product during any of the previous seven weeks of incubation and sampling. The minimum inhibitory concentration (MIC) of imidazolidinyl urea was determined for the organism taken directly from the product and compared to the MIC for the same organism after it was routinely maintained on a laboratory medium. The laboratory grown strain was killed by a 2% solution of imidazolidinyl urea, while the same organism, taken directly from the product, required 4% imidazolidinyl urea to be killed. In addition, both strains demonstrated a lag time of 72 hours before growing at the subcidal concentrations. Several product isolates from our culture collection were subsequently examined to as- sess their resistance to imidazolidinyl urea. Kill rate studies with six typical Pseudomonas strains produced a variety of resistance patterns. The study was extended to examine growth patterns in simple cosmetic emulsions preserved with imidazolidinyl urea. Ana- lytical methods for the detection of imidazolidinyl urea in growth media were used to support the hypotheseis that the resistant Pseudomonads were able to metabolize or chemically bind the imidazolidinyl urea compounds, thereby allowing the organisms to reproduce in the presence of the preservative. METHODS Pseudomonas aeruginosa (ATCC 9027) and five product isolates Pseudomonas putida (P6), Pseudomonas aeruginosa (P5), Pseudomonas cepacia (445), Pseudomonas putida (495), and Pseudomonas cepacia (RC) were selected for these studies. Stock cultures were maintained on nutrient agar slants at 25øC or 4øC. The inoculum for each study was prepared by growing the cultures for 18-24 hours in Brain Heart Infusion broth at 35øC. The cell suspensions were centrifuged, washed, and resuspended in 0.85% saline and adjusted to the proper optical density using a Bausch & Lomb Spectronic 20 Colorimeter. The inoculum levels were reconfirmed with a total plate count using nutrient agar. In all studies, the assay diluent was dilute (1/10 strength) nutrient broth and the plating medium was nutrient agar containing 2% Tween 80. MINIMUM INHIBITORY CONCENTRATION TEST (MIC) All test organisms were first screened for resistance to imidazolidinyl urea using a modi- fication of the phenol coefficient test, similar to that described by Berke & Rosen (11). Solutions containing from 0.25% to 8% imidazolidinyl urea in dilute nutrient broth were challenged with 105 cfu/ml. These inoculated solutions were incubated at 35øC and examined for turbidity at 2, 7, and 14 days after inoculation. Additionally, at each test interval, a loopful of the test solutions was transferred into AOAC letbeen broth which was incubated for two weeks at 35øC and examined for turbidity at 24-hour intervals for 14 days. Additional MIC tests were performed with other cosmetic preservatives including: dia- zolidinyl urea (0.05, 0.1, 0.2, 0.5%), formalin (0.05, 0.1, 0.2, 0.4%), quaternium 15 (0.05, 0.1, 0.2, 0.5%), and chlorhexidine gluconate (0.05, 0.1, 0.2, 0.4, 0.8%). GROWTH PATTERNS IN IMIDAZOLIDINYL UREA BROTH Imidazolidinyl urea solutions, at concentrations of 0.5, 1.0, 2.0, 4.0, and 6.0% were

IMIDAZOLIDINYL UREA RESISTANT PSEUDOMONAS 415 prepared in dilute nutrient broth. The solutions were inoculated with the test or- ganisms at an initial concentration of 105 cfu/ml, and incubated at 35øC. Growth assays were performed at 0, 2, 4, and 6 hrs and 1, 7, 14, and 28 days. Similar kill rate studies were performed with strains 495 and 445 and quaternium 15 (0.05-0.5%) and diazo- lidinyl urea (0.025-0.25%). Growth assays were performed at 0 and 4 hours and 1, 2, 7, and 14 days. GROWTH PATTERNS IN A SIMPLE EMULSION A peanut oil emulsion developed previously by O'Neill and Mead (12) was prepared to simulate simple cosmetic products preserved with 0.5, 1.0, or 2.0% imidazolidinyl urea (Table I). Each product was inoculated with an initial concentration of 10 4- 10 5 cfu/gram of each of the six Psezidomonas strains. The products were incubated at 25øC and sampled at 0 and 4 hours and 1, 2, 7, and 14 days. Similar challenge studies were also performed in dilute nutrient broth containing 10% or 30% of the peanut oil emul- sion. The diluted emulsions were preserved with 0.5, 1.0, and 2.0% imidazolidinyl urea. An attempt was made to further adapt the strains to imidazolidinyl urea and the emul- sion by pre-incubating the cells for 72 hours under 3 different conditions: (1) in dilute nutrient broth, (2) in dilute nutrient broth containing 1% imidazolidinyl urea, and (3) in 10% emulsion in dilute broth containing 1% imidazolidinyl urea. After pre-incuba- tion, the solutions were centrifuged, washed, and the precipitate was resuspended in 0.85% saline and inoculated into 100% emulsion containing 0.5, 1.0, and 2.0% imid- azolidinyl urea. The inoculated product was incubated at 25øC and sampled at 0, 1, 2, 3, 7, and 14 days. Strains 445 and 495 were selected to measure resistance to a dual preservative system of methyl paraben and imidazolidinyl urea. Peanut oil emulsions were prepared with the following preservative systems: 0.5% imidazolidinyl urea, 0.5% imidazolidinyl urea plus 0.3% methyl paraben, 0.5% imidazolidinyl urea plus 1.0% methyl paraben, and 0.5% imidazolidinyl urea plus 2.0% methyl paraben. An inoculum concentration of 105 cfu/gram was used to challenge the emulsions which were incubated and tested at 0 and 4 hours and 1, 2, 7, and 14 days. SPECTROPHOTOMETRIC ASSAY FOR IMIDAZOLIDINYL UREA The test organisms were inoculated into dilute nutrient broth containing 0.5%, 1.0%, or 2.0% imidazolidinyl urea and incubated at 25øC for 21 days. After incubation, the broth solutions were filtered using a 0.22 p, membrane filter (Millipore) to remove Table I Peanut Oil Emulsion Ingredient g/kg Peanut Oil (Planters', 100%) 200 Stearyl Alcohol, 2 Mole Ethoxylate 15 Stearic Acid, 20 Mole Ethoxylate 20 Preservative q.s. Water q.s. to 1000