Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems R. Campana, C. Scesa, V. Patrone, E. Vittoria and W. Baffone Istituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Università di Urbino Carlo Bo, Urbino, Italy Keywords cosmetics, health risk, in-use contamination, preservative system. Correspondence Wally Baffone, Istituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Università di Urbino Carlo Bo, Via S. Chiara 27, Urbino, Italy /1517: received 22 December 2005, revised and accepted 28 March 2006 doi: /j x x Abstract Aim: To evaluate the microbial contamination of 91 cosmetics (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use (intact, in-use, ending product) and the protection efficacy of the preservative systems most frequently used in the analysed cosmetic formulations. Methods and Results: Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10Æ6% of tensiolytes (13Æ5% bath foam, 6Æ7% shampoo, 10% liquid soaps) were contaminated by Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The efficacy of the preservative systems of two cosmetic products, tested against standard micro-organisms (Staphylococcus aureus ATCC 4338 and Pseudomonas aeruginosa ATCC 9027) and two isolates from cosmetics in this study (S. epidermidis and P. putida), satisfied the Cosmetics, Toiletries, and Fragrance Association and Official Italian Pharmacopeia criteria, while only one tested cosmetic respected the Rapid Challenge Test criterion. Conclusions: Contaminated cosmetic products are relatively uncommon, but some products, unable to suppress the growth of several micro-organisms, represent a potential health hazard. Significance and Impact of the Study: The challenge test may be performed not only during the preparation of the preservative system in the intact cosmetics, but also be used to evaluate the protection efficacy during their use. Introduction Contaminating micro-organisms in cosmetics may cause a spoilage of the product and, when pathogenic, they represent a serious health risk for consumers worldwide (Becks and Lorenzoni 1995; Behravan et al. 2005). Over the last 30 years, implementation of Good Manufacturing Practices has been the foundation for improving industrial quality control analyses. For this purpose, the United States Pharmacopoeia (USP) Microbial Limits Test provides several methods for the determination of total microbial count for bacteria, yeast and mould (Anon 2006). In particular the USP specifies four bacterial indicators (Escherichia coli, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus) while the European Pharmacopoeia establishes additional analyses for ascertaining the levels of enterobacteria (Anon 1998). In spite of these guidelines, microbial contamination is still one of the major causes for product recalls in the world, in particular in developing tropical countries (Okeke and Lamikanra 2001). Therefore, it is important to improve the preservative system (Farrington et al. 1994; Linter and Genet 1998) in order to inhibit the growth of contaminating micro-organisms during manufacturing, storage and use by consumers, also by using non-invasive packaging (Brannan and Dille 1990). The 713/86 Italian Law and the 76/768 European Regulation are being modified to conform to the European Directive 2003/15/CE regarding the Period After Opening of cosmetic products. Therefore, in accordance with the Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)

2 Cosmetic contamination during use R. Campana et al. legislation in force, the aim of this study was to evaluate the possible microbial contamination of several cosmetic products in three different states of their use (intact, in-use, ending product). Moreover, the protection efficacy of preservative systems, those most frequently used in the analysed cosmetic formulations, was also evaluated using three microbiological challenge tests. Materials and methods Cosmetic products Ninety-one commercially available cosmetic products (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use, the intact product (at the time of purchase), the in-use product (after 14 days of use) and the ending product (post-use, after 30 days), were collected and analysed in order to verify the degree of possible microbiological contamination during their use by consumers. The microbial species investigated are Escherichia coli, P. aeruginosa, Salmonella spp., S. aureus and enterobacteria, as suggested by USP and EP. Consumers, randomly selected among the students of the University of Urbino, gave an aliquot (1 ml of liquid product or 1 g of paste) of the cosmetic product, collected it in a sterile tube, in the three states mentioned above. Moreover, each consumer gave the cosmetic description (label and ingredients) which allowed a comparison of the preservative systems. Microbiological analyses such as total bacterial count and the isolation and identification of bacteria were performed on the collected cosmetic aliquots. Microbiological analysis Total bacterial count The collected samples of cosmetic products (intact, in-use and ending product) were analysed for the determination of total bacterial count. Each sample, serially diluted (from 10 )1 to 10 )4 cell ml )1 ) in physiological buffer and opportunely homogenized, was spread on Tryptone Soya Agar (TSA; Oxoid, Milan, Italy) and incubated at 37 C. The plates were observed after 24 h and after 5 days and the number of colony-forming units (CFU ml )1 ) was determined. Each assay was performed in duplicate. Media and isolation of pathogenic micro-organisms To determine the presence of pathogenic micro-organisms, 1 ml of each 10 )1 diluted samples was spread on mannitol salt agar (Oxoid), McConkey (Oxoid) and cetrimide agar (Oxoid) to allow the growth of Staphylococcus spp., enterobacteria and Pseudomonas spp. respectively. The plates were then incubated at 37 C for 24 h. Isolates were identified by conventional biochemical tests (Barrow and Feltham 1993; Murray et al. 1995). Laboratory efficacy test methods (challenge tests) The following challenge tests, CTFA test (Cosmetics, Toiletries, and Fragrance Association, Inc. 2001), FUI test (Official Italian Pharmacopeia: Anon 1994), and the Rapid Challenge Test (RCT) (Cagliani et al. 1990), were performed on two cosmetic products, a body cream and a bath foam, selected among the assayed cosmetics, which contained the same mix of preservatives (phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben) differing only for the presence of imidazolidinyl urea in the body cream. Uninoculated samples of each cosmetic served as control. CTFA test Two reference strains, S. aureus (ATCC 4338) and P. aeruginosa (ATCC 9027), and two isolated from the analysed products, Pseudomonas putida and Staphylococcus epidermidis, were used to perform the CTFA test, with slight modification. The cultures were maintained in TSA and transferred twice in Tryptone Soya Broth (TSB, Oxoid) (inoculum 10%) at 37 C for 24 h. The last transfer was the inoculum suspension (10 6 cell ml )1 ). At time zero, four samples (50 g) of each test products were inoculated respectively with 0Æ1 ml of each inoculum suspensions. Samples were shaken and maintained at room temperature (RT). After a contact time of 0, 3, 7, 14, 21 and 28 days, 1-ml aliquots were removed and placed onto 9 ml of neutralizing medium Leethen broth (Difco, Milan, Italy) (Nostro et al. 2002). Cell viability was determined by the plate count method on TSA and CFU were counted after 24 h incubation at 37 C. All determinations were performed in duplicate. A reduction in the number of each micro-organism of 99Æ9% by 7 days was required in order for the formulation to pass the test. FUI test The same micro-organisms were utilized in the FUI test. The cultures, maintained in TSA (Oxoid), were transferred in TSB (Oxoid) and incubated at 37 C to reach an OD 610 ¼ 0Æ13 0Æ18, corresponding to 10 8 cell ml )1 (as measured by sprectrophotometer). Four samples (20 g) of the two test products were inoculated respectively with 0Æ1 ml of each bacterial suspensions. Inoculated samples, after shaking, were maintained at RT; after a contact time of 0, 2, 7, 14 and 28 days from challenge, sample aliquots (1 ml) were removed and counted according to the method described above (Nostro et al. 2002). The acceptance criterion for the FUI test was a logarithmic (log 10 ) 302 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)

3 R. Campana et al. Cosmetic contamination during use reduction of 3 of the viable micro-organisms by the 7th day from challenge. Rapid Challenge Test (only for Gram-negative bacteria) A 50-g portion of the two mentioned cosmetic products were challenged with 0.1 ml of broth suspension containing more than 10 6 cell ml )1 of Gram-negative bacteria (P. putida isolated from our cosmetic products and the reference strain P. aeruginosa ATCC 9027). Twenty grams of the two test products were diluted in order to obtain three final concentrations: 100%, 90% and 80% of the product. Aliquots (1 ml) were removed from each concentration of samples after 24 h and 7 days from challenge and counted according to the method described above (Nostro et al. 2002). The acceptance criterion is established as follows: i the product showing more than 10 2 CFU ml )1 after 24 h from challenge both in the undiluted product and in the diluted product, is considered a high contamination risk product; ii if a progressive decrease in the CFU values at the different dilutions (at least the 80% dilution) after 24 h and absence of bacterial growth after 7 days from challenge was observed, the product is considered a medium low contamination risk product; iii in complete absence of bacterial growth after 24 h and 7 days from challenge in all the dilutions, the product is considered well preserved. Statistical analyses Fisher s exact P test was performed to evaluate the statistical significance in bacterial growth reduction by the three challenge tests used. Results The microbiological contamination of 91 cosmetic products, in three different states of use (intact, in-use, ending product) was evaluated. The results of this first phase of the investigation are reported in Table 1. As shown, no contamination was observed in o/w emulsions and in aqueous pastes, while among the 47 tensiolytes, 13Æ5% bath foam (3/22), 6Æ7% shampoo (1/15) and 10% liquid soaps (1/10) resulted contaminated, presenting total counts ranging between and CFU ml )1. In all cases the contamination was found in the ending products, while in one case it was observed in the in-use product as well. The species isolated were identified as Staphylococcus warneri (two cases), S. epidermidis (two cases) and P. putida (one case). Table 1 Contaminated samples, total bacterial counts and bacterial species recovered from several cosmetic products in three states of use (A, intact product; B, in-use product; C, ending product), subdivided on the basis of their contamination risk Positive samples N (%) Total bacterial count (CFU ml )1 ) Cosmetic product Type of closure/containers No. samples A B C A B C Isolated species High contamination risk o/w emulsions Hair mask Vases Body cream Vases Broad orifice diameter Dispenser Face cream Vases Hand cream Vases Shaving cream Vases Cleansing lotion Narrow orifice diameter Solar lotion Narrow orifice diameter Tensiolytes Bath foam Broad orifice diameter (4Æ5) S. warneri Narrow orifice diameter (4Æ5) P. putida Dispenser (4Æ5) S. epidermidis Shampoo Broad orifice diameter (6Æ7) 1 (6Æ7) S. epidermidis Narrow orifice diameter Liquid soap Dispenser (10) S. warneri Medium contamination risk: aqueous pastes Toothpaste Tubes Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)

4 Cosmetic contamination during use R. Campana et al. The efficacy of preservative systems, assayed by challenge tests recommended by the CTFA, FUI and RCT, is reported in Tables 2 4. A 99Æ99% reduction in bacterial cell number (P < 0Æ01) was observed after 7 days in the two tested cosmetic products according to the CTFA procedure (Table 2); similarly, a logarithmic reduction of 3 (P <0Æ01) by the 7th day was noted in the two tested cosmetics, as requested by the FUI procedure (Table 3). The rapid decline in bacterial counts, observed for P. putida and P. aeruginosa ATCC 9027 (Table 4) after 1 and 7 days (P <0Æ01) from challenge in the three utilized dilutions of bath foam, identify this product as medium low contamination risk product, according to the criterion of the RCT. On the other hand, in the body cream P. aeruginosa ATCC 9027, opposite to P. putida, showed CFU values more than 10 2 CFU ml )1 in the undiluted, in the 90% and 80% diluted product after 1 and 7 days from challenge, thus this product can be considered as a high contamination risk product. Discussion The level of microbiological contamination in a nonsterile product, such as cosmetic formulations, is made clear in the microbial limit standards (Official Italian Pharmacopeia: Anon 1994; Cosmetics, Toiletries, and Fragrance Association, Inc. 2001). These values should be maintained in the products during their use, in spite of the inevitable contamination by the users, through the addition of a suitable preservative in the products which guarantees the control of microbial growth even before they are marketed (Linter and Genet 1998). However, there is poor information in the literature regarding the efficacy of preservative systems contained in cosmetic products to control the microbial contamination of these products during their use by consumers (Farrington et al. 1994; Okeke and Lamikanra 2001). In accordance with the European Directive 2003/15/CE which obliges cosmetic industries to sign the post-opening expiration date of cosmetic products, in a first phase Table 2 The percentage reduction of bacterial cell number for body cream and bath foam samples evaluated by CTFA test CTFA test (reduction %) Strains Inoculum (CFU ml )1 ) 0 h 3 days 7 days* 14 days 21 days 28 days Tested cosmetic: body cream S. aureus ATCC Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 S. epidermidis Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 P. aeruginosa ATCC Æ00 90Æ00 99Æ99 99Æ99 99Æ99 99Æ99 P. putida Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 Tested cosmetic: bath foam S. aureus ATCC Æ00 99Æ90 99Æ99 99Æ99 99Æ99 99Æ99 S. epidermidis Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 P. aeruginosa ATCC Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 P. putida Æ00 99Æ99 99Æ99 99Æ99 99Æ99 99Æ99 *Reduction >99Æ9% as requested by CTFA procedure compared with inoculum (P < 0Æ01). Table 3 The average counts in CFU ml )1 for body cream and bath foam samples evaluated by FUI test FUI test Strains Inoculum 0 h 2 days 7 days* 14 days 28 days Tested cosmetic: body cream S. aureus ATCC S. epidermidis P. aeruginosa ATCC P. putida Tested cosmetic: bath foam S. aureus ATCC S. epidermidis P. aeruginosa ATCC P. putida *Logarithmic reduction factor of 3 of viable micro-organisms as requested by FUI procedure compared with inoculum (P < 0Æ01). 304 Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)

5 R. Campana et al. Cosmetic contamination during use Table 4 The average counts in CFU ml )1 for body cream and bath foam samples evaluated by Rapid Challenge Test (RCT) RCT for body cream P. aeruginosa ATCC 9027 P. putida RCT for bath foam P. aeruginosa ATCC 9027 P. putida Inoculum 100% sample 0h day * days % diluted sample 0h day * days % diluted sample 0h day * days * *CFU values are greater than those established for the rapid challenge test criterion. of our study we investigated the microbiological contamination of 91 cosmetic products (o/w emulsions, tensiolytes and aqueous pastes), commercially available, in three different states of use (intact, in-use, ending product). The examined intact products showed no bacterial contamination, while species diffused in air, soil and water, such as Staphylococcus spp. and Pseudomonas spp., were recovered in the in-use and ending tensiolytes, which in one case evidenced CFU values more than the standard microbial limit. The likely source of these micro-organisms is from the hands of users, as moisturizers are often used after or independent of washing. The recovery of these potential pathogenic species in the examined tensiolytes suggested that the preservative system was not effective. However, the preservative systems of the tensiolytes do not differ substantially from the system used in the emulsions; so contamination could be due to their different formulations which might have partially inactivated the preservative system. In addition, emulsions contain several substances (surface active agents, bio-mimetic phospholipids) which may contribute through a synergic mechanism to improve product preservation (Nostro et al. 2002). As regards the second part of the investigation, the preservative systems of the two tested cosmetics, a combination of methylparaben and propylparaben, showed longlasting antimicrobial activity, satisfying the CTFA and FUI criteria. However, when the RCT was used, the body cream did not respect the criterion suggested by this procedure, because of the intrinsic resistance of P. aeruginosa to a wide variety of antimicrobial agents. On the basis of our data, supported by the use of a neutralizing solution which avoided the eventual presence of false positives, most of the tested cosmetic products did not represent a risk for the consumer during use. Nevertheless, the recovery of pathogenic species in some tensiolytes in the in-use and ending products, although in a limited number of cases, led us to presume that the challenge test should be performed not only during the preparation of the preservative system in the intact cosmetic products, but should also be used to evaluate the protection efficacy of these systems during their use, as recommended by the European Directive 2003/15/CE. References Anon (1994) Farmacopea Ufficiale della Repubblica Italiana, 9th edn, Suppl. III. pp Rome: Istituto Poligrafico e Zecca dello Stato. Anon (1998) European Pharmacopeia. Strasbourg, France: European Pharmacopeia Secretariat. Anon (2006) US Pharmacopeial Convention. Rockville, MD: US Pharmacopeia. Barrow, G. and Feltham, R. (1993) Cowan and Steel s Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. Becks, V. and Lorenzoni, N. (1995) Pseudomonas aeruginosa outbreak in a neonatal intensive care unit: a possible link to contaminated hand lotion. Am J Infect Control 23, Behravan, J., Bazzaz, F. and Malaekeh, P. (2005) Survey of bacteriological contamination of cosmetic creams in Iran (2000). Int J Dermatol 44, Brannan, D. and Dille, J. (1990) Type of closure prevents microbial contamination of cosmetic during consumer use. Appl Environ Microbiol 56, Cagliani, I., Cerini, R., Citernesi, U., Scampini, M., Trovato, M., Meloni, M. and Pastoni, F. (1990) La microbiologia nell industria cosmetica. RTC-Ed. UNIPRO. Rome: RTC Press. Cosmetics, Toiletries, and Fragrance Association, Inc. (2001) Determination of Preservation Adequacy of Water-Miscible Cosmetic and Toiletry Formulations. CTFA Technical Guidelines. Washington, DC: Cosmetics, Toiletries, and Fragrance Association, Inc. Farrington, J.K., Martz, E.L., Wells, S.J., Ennis, C.C., Holder, J., Levchuk, J.W., Avis, K.E., Hoffman, P.S. et al. (1994) Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic. Appl Environ Microbiol 60, Linter, K. and Genet, V. (1998) A physical method for the preservation of cosmetic products. Int J Cosmet Sci 20, Murray, P., Baron, E., Pfaller, M., Tenover, F. and Yolken, R. (1995) Manual of Clinical Microbiology. Washington, DC: ASM Press. Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)

6 Cosmetic contamination during use R. Campana et al. Nostro, A., Cannatelli, M.A., Morelli, I., Cioni, P.L., Bader, A., Marino, A. and Alonzo, A. (2002) Preservative properties of Calamintha officinalis essential oil with and without EDTA. Lett Appl Microbiol 35, Okeke, I.N. and Lamikanra, A. (2001) Bacteriological quality of skin-moisturizing creams and lotions distributed in a tropical developing country. J Appl Bacteriol 91, Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (2006)