j. Cosmet. sci., 54, 411-420 (July/August 2003) The preservative efficacy testing method for powdered eye shadows M. R. S. E. L. SOUZA and M. T. OHARA, Rua da Consola•'•o 3064, Apto. 62A, 01416-000 S•7o Paulo, Brazil. Accepted for publication January 13, 2003. Synopsis Preservative efficacy testing is based on a sample inoculation using a microbial suspension with a determined amount of colony-forming units (CFU). After that, the number of survivors is investigated by periodic evaluations, and the results are compared with specifications. When liquid cosmetics are evaluated, it is easy to obtain homogeneity between the inoculum and the sample, but for a powder sample it cannot be guaranteed. In this context, freeze-dried microorganisms could be used to help the homogenization. In this research, the preservative efficacy is evaluated using a powdered eye shadow. The microorganisms used were Staphylococc•s aureus, Pseudomonas aowginosa, Candida albicans, and Aspergillus niger. The challenge tests were performed in samples with (P) or without (NP) added preservative. The methods used to evaluate the results were the ones described in the official compendia and in the cosmetics guides of international associations, also using linear regression in calculating the D-value. The results showed that it is possible to use freeze-dried microorganisms instead of suspension to evaluate the preservative efficacy of cosmetic solids. The microorganism stability was verified by the determination of the microbial load and the minimum inhibitory concentration after freeze-drying and during the following six months. INTRODUCTION Preservative efficacy testing is an essential part of substantiating the safety of cosmetic products. The correct use of preservatives protects the product against contamination while it is in trade channels and in the hands of the consumer (1-4). When consumers use cosmetic products, they repeatedly challenge the cosmetic with microorganisms in saliva, on dirty hands, and in tap water (5). In this context, preservative efficacy must be evaluated to assure the product's safety. The methodology for the evaluation of preservative adequacy is described in the official compendia for pharmaceuticals (6-9). It is also given in cosmetic guides such as those of the Cosmetic, Toiletry and Fragrance Association (CTFA) (10) and the American Society for Testing and Material (ASTM) (5). All methods are applied to liquid and semi-solid products, and they are based on the challenge test, which consists in the contamination of the product by fresh pure cultures of microorganisms, suspended in saline, followed by periodic evaluations. The CTFA guidelines (10) describe methods for testing eye area cosmetics. The inocu- 411

412 JOURNAL OF COSMETIC SCIENCE lation method of non-aqueous eye products such as loose and pressed powders consists of spraying on or adding microbial suspensions to the cosmetic, followed by mixing. Pressed eye shadows can also be tested by swabbing or spreading an inoculum on the product surface (10-12). There has also been developed a method to evaluate the surface of pressed powders in this method the test organisms on membrane filters are placed in direct contact with the products. The inoculation of microbial suspensions in powders described by the CTFA (10) is a procedure similar to the official ones for pharmaceuticals and cosmetics. However, it is difficult to guarantee homogeneity when mixing a microorganism suspension with a powder, since the volume of the inoculum must be minimal (not more than 1% of the total sample amount). The use of solid inoculum could facilitate the homogeneity between the microorganism and the sample. In this study a solid inoculum was obtained by using the process of freeze-drying. The efficacy preservative testing period is too long, since it lasts 28 days. There is an alternative method that has been used (13,14) that involves the microorganism death curve and the determination of the decimal reduction time (D-value), which is the time required for the reduction of 90% of the microorganisms. This curve can be constructed by determining the number of surviving microorganisms after 2, 4, and 24 hours for bacteria, and 4, 8, and 24 hours for fungi. The aim of this study was to evaluate the use of freeze-dried microorganisms to inoculate eye-shadow samples in preservative efficacy testing. In order to reach this goal, the results obtained were compared to the specifications in all the official compendia men- tioned above, to those of the CTFA, and also to the determination of the D-value. EXPERIMENTAL TEST ORGANISMS The test organisms were Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16 404. STABILITY EVALUATION OF FREEZE-DRIED MICROORGANISMS Microbial suspensions were obtained as described in the US Pharmacopoeia 24 (6). Sus- pension drops of the inocula were used in order to have about 10 7 CFU/vial. They were transferred to five vials, and 1.0 ml of Molico © skim milk (Nestle) with 5% inositol (DIFCO) was added. The samples were frozen at -70øC, and the freeze-drying process began at -55øC using a Supermodulyo 12K © (Edwards) freeze-dryer. The vials were closed with rubber stoppers. The total aerobic count was determined by the plate count technique, using the five vials (6). These determinations were made immediately and one week, 15 days, and one, two, four, five, and six months after the freeze-drying process. MICROORGANISM RESISTANCE EVALUATION BY MINIMUM INHIBITORY CONCENTRATION (MIC) The preservatives used were methylparaben, propylparaben, and a combination of DMDM hydantoin and iodopropinyl butyl carbamate (Glydant plus©). The determina- tion was made by using the broth dilution technique in a 96-well microtiter plate (15).