PRESERVATIVE REQUIREMENTS OF O/W EMULSIONS 111 Pseudomonas aeruginosa-O.02 ml of 24-hour broth culture (3.7 x 107 or- ganisms/10 ml product) Candida albicans-O.05 ml of 24-hour broth culture (3.5 x 10 • organisms/ 10 ml product) Aspergillus niger-0.1 ml of a freshly prepared spore suspension (4.5 x 105 organisms/10 ml product) P. aeruginosa, C. albicans, and A. niger were recovered in large numbers in all unpreserved test lotions for at least 14 days after challenge. S. faecalis survived in high numbers in all lotions except the acid anionic, where no organisms were detected after a 3-day contact period. Subcultures of Challenged Lotions At the following time intervals after challenge, each sample was thorough- ly mixed and a sterile inoculating loop of ca. 0.05-ml capacity was used to streak the lotion onto a Letheen agar (Difco) plate--0 hour, 3 days, and 7 days. Bacterial and yeast plates were incubated at 37øC for a maximum of 3 days fungal plates were incubated at 30øC for 3-7 days before discarding. Storage of Challenged Lotions Lotions inoculated with challenge organisms were stored at 24øC during the subculture period. After the final subculture, they were discarded. Stages in the Evaluation of Preservative Adequacy Freshly Prepared Unaged Lotions-Three decimal dilutions of each pre- servative were incorporated into each lotion to determine the approximate level of preservative required. The highest concentration tested was usually that recommended by the supplier or noted in the literature however, in some cases, the maximum level tested was twice the recommended level. Kill or marked reduction of 3 or more challenge organisms within 3 days of in- oculation was considered mandatory for additional study. Recovery of high levels of organisms at any preservative level precluded further examination of that level. Aged Lotions-Samples stored at 52øC for one month were subjected to challenge by the 4 test organisms if they had been proven satisfactory in preservative activity by the previous test. Kill of all challenge organisms with- in 3 days indicated potentially adequate preservation after storage at ele- vated temperature. The satisfactory levels determined by the preceding two studies became the starting point for MIC studies. These levels were desig- nated "effective levels." MIC Studies-Preservatives were incorporated into freshly prepared test lotions in 25% decrements from the "effective level" determined in aged lo-

11'2 JOURNAL OF THE SOCIETY OF COSMETIC CItEMISTS Table V. Effects of Combining Preservatives on Effective Concentrations and Microbial Spectrum (Individual Preservatives Precede Preservative Mixtures) Anionic a Nonionic a Acid Alkaline Acid Alkaline Preservative -Result % Result % Result % Result % Bronopol S 0.05 U e 0.1 S 0.1 U e 0.1 Sodium Oreadinc S 0.1 U s.a 0.1 S 0.1 S 0.1 Bronopol q- S 0.1 S 0.1 U e,a 0.1 Ue, a 0.1 Sodium Oreadinc 0.05 0.05 0.05 0.05 Methylparaben q- S 0.15 U s 0.2 U a 0.2 Us,p, a 0.2 Propylparaben 0.04 0.05 0.05 0.05 Bronopol q- S 0.018 S 0.013 S 0.05 U a 0.1 Methylparaben q- 0.038 0.025 0.1 0.2 Propylparaben 0.009 0.006 0.0•5 0.05 Formaldehyde S 0.05 S 0.05 S 0.075 $ 0.075 Formaldehyde q- S 0.013 S 0.018 S 0.038 S 0.03S Methylparaben q- 0.05 0.075 0.15 I 0.15 Propylparaben 0.013 0.018 0.038 0.038 Gemsall U e,a 0.5 U c,a 0.5 U e,a 0.5 U e,a 0.5 Germall q- S 0.38 S 0.5 S 0.5 S 0.5 Methylparaben q- 0.15 0.2 0.2 0.05 Propylparaben 0.038 0.05 0.05 Us,l•,c, a 0.1 Methylparaben q- Us,e,a 0.1 Us,P,e, a 0.1 Us,v,e,a 0.1 0.02 Butylparaben 0.02 (I.02 0.02 U e 0.5 Germall q- S 0.5 S 0.5 Ue 0.5 0.1 Methylparaben q- 0.1 0.1 0.1 0.02 Butylparaben 0.02 0.02 0.02 0.2 MDMH NT NT Uc 0.5 U c 0.5 MDMH q- NT NT U c 0.,5 U e 0.5 Methylparaben q- 0.25 0.25 Propylparaben 0.05 0.05 Vancide 89RE U* 0.2 Up,e, a 0.2 Up,a 0.9. UP, a 0.2 Phenoxetol S 1.0 S 1.0 S 1.0 U* 1.0 Vancide 89RE q- U* 0.2 S 0.075 U* 0.2 U* 0.2 Phenoxetol 1.0 0.375 1.0 1,0 Preservative 68 U s 0.5 U s 0.5 Us,P 0.5 Us,v 0.5 Preservative 68 q- S 0.13 S 0.19 S 0.25 S 0.19 Formaldehyde 0.013 0.019 0.025 0.019 Symbols used: * = Organoleptic problems S = Satisfactory U = Unsatisfactory NT: Not Tested. Subscript figure denotes sin'rival of: s :- S. [aecalis p: P. aeruginosa c: C. albicans a = A. niger.