Validating the microbiological integrity of cosmetic products through consumer-use testing

j. Soc. Cosmet. Chem., 37, 481-488 (November/December 1986) Validating the microbiological integrity of cosmetic products through consumer-use testing SUSAN M. LINDSTROM and JOYCE D. HAWTHORNE, Avon Products, Inc., Suffern, NY 10901. Received March 20, 1986. Presented at the Society of Cosmetic Chemists Annual Meeting, December I985, New York. Synopsis To ensure the development of microbiologically safe cosmetics, an in vitro microbial challenge test was developed which accurately predicts the preservativefficacy of a product after consumer use. Over the past five years, 143 products which had met the in vitro challenge test criteria were subjected to consumer testing. These products included mascaras, creams, lotions, liquid makeup, eyeshadows, eyeliners, and bath and hair preparations. Criteria for formula acceptability after use were: recovery of organisms at less than 100 cfu/g upon initial test or no recovery upon retest of the product and no recovery of Pseudomonas species, Escherichia coli, or Staphylococcus aureus. Over 99% of the products tested met these requirements, while only nine out of more than 4300 samples tested did not meet these test criteria. As a result of these failures, the products were reformulated with increased preservative concentrations before release for sale. Further steps were taken to increase the predictability of the in vitro challenge test by including a higher concentration of challenge organisms, a greater number of preservative-resistant product isolates, and more stringent inoculum reduction requirements. These procedures have resulted in an in vitro test which accurately predicts preservativefficacy of a cosmetic product after consumer use. INTRODUCTION Cosmetic products should be proven safe and effective before market sale. In order to accomplish this goal, each company performs numerous evaluations of product integrity including a microbiological assessment. Several in vitro preservative adequacy tests are available (1-6). These challenge tests should be predictive of preservativeffectiveness under ordinary consumer use. However, these procedures are proven valid only when the microbiological integrity of the product after consumer use is confirmed. This paper will review the results of 143 products which represent over 4300 samples tested using in vitro challenge testing followed by consumer-use testing during the period of 1980 to early 1985. The strong correlation of in vitro and in vivo test results will become evident. Over the course of testing, the in vitro test has evolved into a more predictive test through several procedural modifications which will also be discussed. These modifications include higher inoculum concentrations, more stringent microbial reduction requirements, development of the accelerated preservation test (7), and additional test parameters in the consumer-use test itself such as preservative analysis. As the cumulative history was acquired, new directions in preservative philosophies developed. 481

482 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS METHODS PRE-TEST EVALUATION Initially, the adequacy of the preservative system is evaluated through in vitro challenge testing. An accelerated preservation test (APT) is conducted to rapidly assess preservative efficacy (Table I). The product is inoculated with 106 cfu/gram of product for bacteria and 105 cfu of mold and yeast/gram of product. At the time of inoculation, extra nutrients in the form of glucose and a minimal salts buffer are added to the product sample. Assays are performed at 2 and 7 days after inoculation. If mold is recovered at Day 7, an additional Day 12 assay is made. A reduction of bacteria to < 10 cfu/g in 7 days and greater than 99.9% reduction of mold and yeast in 12 days are the criteria for passing the APT. A long-term in vitro challenge test is initiated on those formulations which meet the criteria of the APT. Selected microorganisms are inoculated into the product at Week 0 and again at Week 2; microbiological assays are performed weekly. At the completion of 8 weeks of testing, the product is deemed adequately preserved if bacteria are reduced to < 10 cfu/g one week after each challenge inoculation (Weeks 1 and 3) and maintained at that level throughout the test period. The reduction of fungal recoverieshould initially be greater than 99.9% of the original inoculum concentration and continue to < 10 cfu/g of mold and yeast by test completion. When the formulation has demonstrated in vitro preservation efficacy, it is then tested for in vivo preservation efficacy. CONSUMER-USE TEST REGIMEN A consumer-use panel representative of the target audience for the product's intended use is selected. A dermatologist will supervise studies as required. Panel size is selected for statistical significance, averaging 15-30 panelists per product. Mascara panels are larger, with 40-60 people per product. The test panels designed for mascarand other eye products have more stringent requirements because concern exists in the industry over ocular infections associated with improperly preserved mascara (8,9). The association of Pseudomonas-induced infection leading to corneal ulceration is also documented (10). A microbiological prescreening of the microflora type and level in the area around the panelist's eyelids and lashes is performed. The microflora is then monitored throughout the test and after panel completion as a measure of the potential microbial load which may be introduced into the product. Table I In Vitro Challenge Testing Accelerated Preservation Test Standard Challenge Test Test Length 7 days 8 weeks Challenges 1 2 Organism Concentration 106 cfu/g bacteria Same 105 spores/g of mold and yeast Nutrification Glucose and minimal salt buffers None Assay Intervals 0 time, 2 days, 7 days 0 time, Weeks, 1, 2, 3, (12 days if mold is recovered 4, and 8 at Day 7)

MICROBIOLOGICAL INTEGRITY OF COSMETICS 483 A microbiological assay is performed prior to product distribution. In the case of eye product panels, microbiological assays of the product are conducted at several time intervals throughout the test rather than only at test initiation and completion. Products are coded and distributed randomly to the panelists, using a double crossover study design in which panelists first use one test product and are then "crossed over" to use the second test product. Panelists are given instructions for product use and a calendar to record the frequency of product use. Panels are conducted for 3-8 weeks. The actual length is dependent on the product and its intended use. More intensive evaluation of sensitive eye area products require longer test panels; mascara in use testing averages eight weeks or greater in length. At the conclusion of the use test, the microbiological evaluation is performed first, followed by any additional physical/chemical testing. MICROBIOLOGICAL EVALUATION/CRITERIA Following the consumer-use test, an assay is performed on the used product 24 hours after the last application of the product. Preservativefficacy is more accurately measured by allowing 24 hours before assaying since this more closely simulates ordinary use. Assays are performed on nutrient agar (BBL 12174) with 2% polysorbate 80, which is added for neutralization of the preservative, Vogel-Johnson agar (BBL 11812; for detection of Staphy/ococcus species), Pseudomonas Isolation agar (Difco 0927-01), and Mycophil agar (BBL 11450; for detection of yeasts and molds). Plates are incubated for 48 hours at 35øC and the test results are recorded. Enrichmentests are performed for mascar and eye products. A broth dilution of the product [1/10 in AOAC Letheen Broth (BBL 10914)] is incubated at 35øC for 24 hours. These broth dilutions are then streaked onto enrichment plates of Vogel-Johnson agar, MacConkey agar (BBL 11387; for the detection of gram negatives), and Pseudomonas Isolation agar, which in turn are incubated for 48 hours at 35øC. Criteria for formula acceptability after use is recovery of < 100 cfu/g total aerobic plate count including yeasts and molds and < 10 cfu/g gram negatives on initial assay or recovery of < 10 cfu/g total plate count on retest of the product. In addition, the product should not contain Pseudomonas species, Escherichia coli, or Staphylococcus aureus, All microorganisms recovered after retest are identified. RESULTS One-hundred forty three products were consumer-tested from 1980 through early 1985. A full complement of cosmetic and toiletry products containing a wide range of cosmetic preservatives were evaluated after consumer use. Preservative efficacy under various use conditions was measured. Table II itemizes the creams and lotions which have been tested. Twenty-eight creams were panel-tested; these include night creams, cleansing creams, moisturizers, sunscreens, and eye creams. Seventy-three lotions were evaluated, including moisturizers, cleansers, skin lotions, milky lotions, fresheners and toners, foundations, sunscreens, and body lotions. Other product types evaluated during this five-year period are listed in Table III. The complete history of consumer-use testing is summarized in Table IV. Only four of the 143 products failed to meet the test criteria, i.e., three lotions and one shower gel. With 2182 lotion samples assayed, five samples (0.22%) were unacceptable. With 254

484 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table Consumer Tests--Creams & Lotions II Product Category Product Type Creams Lotions Night Cream 11 -- Cleanser 7 7 Moisturizer 5 32 Freshener/Toner -- 15 -- Foundation -- 5 Skin Lotion/Milky Lotion -- 9 Other 5 5 Total 28 73 shampoo/shower gel samples assayed, four samples (1.6%) were unacceptable. These product samples did not meet the criteria for acceptability of < 10 cfu/g upon retest and absence of S. aureus, E. co/i, and Pseudomonas species. Lotion A (Table V) was consumer-tested for four weeks early in 1980. Bacteria were recovered from three out of 189 samples (1.6%). Microbiological testing in early 1980 was not performed by a standard pour plate method. The product was directly streaked onto plates of nutrient agar and MacConkey agar. At this time in the test program, recovered organisms were not identified according to species. For Lotion A, growth occurred from streaks on nutrient agar only and not on MacConkey, indicating the absence of gram-negative contaminants. Microbial recoveries from the used product persisted for four weeks after test completion. After daily (2 x ) use of Lotion B for three weeks, Enterobacter cloacae was recovered ( 103 cfu/g) from one out of twelve samples (8.3%). Recoveries of the organism remained after retest. One sample out of twenty-six (3.8%) of Lotion C had recovery of 1.2 X 103 cfu/g gram-positive Bacillu species upon retest. Bacillu species are not considered pathogenic, but these high recoveries of microbes were not acceptable. Table III Consumer Tests--Other Products Product Category No. Cleansing Foams 3 Toothpastes 3 Lip Treatment 2 Masks 2 Roll-On Deodorant/AP 1 Concealing Sticks 1 Cheek Blush/Pdr. Foundation 5 Lipstick/Lip Gloss 4 Eyeshadows/Eyeliner 7 Shampoos/Shower Gels 9 Hair Conditioners 4 Water-Based Mascara 1 -- Total 42

MICROBIOLOGICAL INTEGRITY OF COSMETICS 485 Table IV Consumer Use-Testing History Total Number of Samples Product Category Products Acceptable Unacceptable % Failures Creams 28 1121 0 0.00 Lotions 73 2177 5 0.22 Mascaras 1 79 0 0.00 Shampoos/Shower Gels 9 250 42 1.60 Hair Conditioners 4 140 0 0.00 Eyeshadows/Eyeliner 7 123 0 0.00 Lipsticks & Other Makeup 12 200 0 0.00 Miscellaneous 9 232 0 0.00 These unacceptable samples represent three products, or five out of a total of 232 samples. See Table V. These four samples are representative of one product panel of 23 consumers. Shower Gel D was used by a panel of 19 once or twice daily for three weeks. Four of the samples were unacceptable with high recoveries of > 103 cfu/g upon retest. The contaminant organisms included Citrobacter freundii, Serratia marcescens, and Serratia liquefaciens. Each of the four products which were unacceptable were reassessed. All three lotions had previously passed our in vitro challenge criteria. Lotions A and C were reformulated with preservative system changes. Imidazolidinyl urea was added to methyl paraben in Lotion A; Lotion C was changed from a methyl paraben, benzoic acid system to methyl paraben with imidazolidinyl urea. The consumer-use panel was repeated for Lotion B in order to assess the statistical significance of the recovery of E. c/oacae. The panel size was increased to 34 people and the duration of the test increased to six weeks. After continual daily use with increased panel size, all samples met the criteria for acceptability with no recoveries of microbes from the used product upon initial testing. The results of the first panel for Lotion B could not be reproduced; therefore, this formula was considered acceptable. The in vitro challenge testing of Shower Gel D resulted in low level recoveries (<200 cfu/g) of two challenge organisms on week 3, one week after rechallenge. Replates were < 10 cfu/g. Results indicate a product that is marginally preserved. The consumer-use testing results validated the formula as marginal. A change in the preservative system Table V Consumer Use-Testing Product Failures Samples Samples Product Category Tested Failed Preservative System A Lotion 189 3 B Lotion 12 1 C Lotion 27 1 D Shower Gel 19 4 Methyl paraben Methyl paraben, diazolidinyl urea Methyl paraben, benzoic acid Benzoic acid, phenoxyethanol

486 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS from benzoic acid, phenoxyethanol to methychloroisothiazolinone/methylisothiazolinone was the result of both the in vitro and in vivo testing. A consumer panel test was conducted with the new preservative system and the product shown to be acceptable in use. Table I contains data on an in-use mascara test for a water-based formulation. This has been included as an example of the mascara panel-testing program. This study was conducted for a period of four weeks. Panelists were divided into subgroups that used the mascara daily for one, two, three, and four weeks. Thirty-six percent of the 79 samples returned after use had low level recoveries, averaging 106 cfu/g, on nutrient agar only. There were no gram-negative recoveries. There were no recoveries on enrichment of S. aureus on Vogel-Johnson agar or Pseudomonas species on Pseudomonas isolation agar. Upon replate, all positive recoveries were < 10 cfu/g, which met our criteria for acceptability after consumer use. Swabs taken of the eye area both before and during product use indicated an average count of 1.27 x 104 cfu/swab (cm 3) surface skin microflora (gram-positive coagulase negative cocci). There was no change in the level or type of microorganisms recovered throughout the test period. This mascara passed our acceptance criteria for in-use testing. DISCUSSION The majority of cosmetic sample subjected to consumer-use testing passed the criteria for acceptability. The low incidence of unacceptable results (0.22% lotions and 1.6% shampoos/shower gels) demonstrates that good correlation can be achieved between in vitro preservation testing and in-use consumer testing. Shower Gel D also demonstrates that the consumer use test will validate the results of a standard challenge test when a preservative system is marginal. Essentially, those products not meeting the acceptance criteria for consumer-use testing are reviewed and preservative system changes are recommended and incorporated into the formulation. Consumer-use testing is an integral part of the development cycle. Several modifications have been made during the course of testing to improve both the predictability and validation of the in vitro preservation test. As a result of product failures which occurred early in the consumer-use testing program Table Mascara In-Use VI Evaluation Test Parameters Microbiological Test Results Test Number of Average Product Panelists Length Positive Samples 2 Count (cfu/g) 2O 1 Wk 9 176 18 2 Wk 7 154 17 3 Wk 7 27 24 4 Wk 6 70 All replates were < 10 cfu/g and therefore met the criteria of acceptability. All recoveries on nutrient agar only.

MICROBIOLOGICAL INTEGRITY OF COSMETICS 487 (example, Lotion A), changes in the in vitro test methodology were initiated. The challenge inoculum was increased mid-1980 to its current concentration of 106 cfu/gram of product for bacteria and 105 cfu of mold and yeast/gram of product. In addition, more stringent inoculum reduction requirements were instituted. Bacterial recoveries are required to be < 10 cfu/g one week after each challenge inoculation and continue at that level throughouthe test. Low initial recoverable counts of mold and yeast (<0.1% of the original inoculum), which must reduce to < 10 cfu/g by test completion, meet the criteria for passing. The selection of challenge organisms was changed to better simulate the population of organisms encountered during consumer use. Evaluations of organism resistance to standard cosmetic preservatives are frequently made. In addition, the accelerated preservation test was added as a more stringent challenge test for preservativ efficacy. It is used to eliminate marginally preserved formulas earlier in the development cycle. Several additional modifications to the consumer-use testing program have taken place. Whenever failures occur, a chemical analysis of the preservative system in the used sample is compared to an unused sample from the same batch. This enables us to monitor preservative stability over time and after use. We currently isolate and identify organisms recovered from used products and, subsequently, use these contaminants to rechallenge the test product and to monitor its preservative resistance. This information allows a more thorough understanding of preservativefficacy during product use. The consumer-use panels for mascara evaluations now include testing of all new mascara formulations and any applicator or packaging changes. CONCLUSION The microbiological acceptability of a cosmetic is a function of its preservativefficacy during actual use conditions. The microbiologist strives to simulate consumer "challenges" to the product in use by measuring the adequacy of the preservation system by means of an in vitro challenge test. The predictability of the in vitro test procedure is proven only through a well-controlled consumer-use testing program. Several modifications to the in vitro test procedure have been made to increase this correlation and predictability. With all these parameters measured and validated accurately, the consumer is guaranteed a microbiologically safe cosmetic product. REFERENCES (1) A guideline for the determination of adequacy of preservation of cosmetics and toiletries, CTFA Technical Guidelines (1970). (2) Microbial tests, antimicrobial preservatives--effectiveness, United States Pharmacopeia XXI, USP Pharmacopial Convention, Inc., 1151 (1984). (3) Efficacy of antimicrobial preservatives in pharmaceutical products, Appendix XVIC, British Pharmacopeia Volume II, Her Majesty's Stationery Office, London, A192-A194 (1980). (4) W. L. Bryon, E. R. Fizer, and J. K. Fartington, A review of methods for determining the preservative efficacy of cosmetic products, Developments in Industrial Microbiology, 21, 273-276 (1980). (5) D. S. Orth, Establishing cosmetic preservativefficacy by use of D-values, J. Soc. Cosmet. Chem., 31, 165-172 (1980).

488 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (6) M. Chan and H. Prince, A rapid screening test for ranking preservative efficacy, Drug and Cosmetic Industry, 34-37 (December 1981). (7) J. J. O'Neill, C. A. Mead, and E. J. Scibienski, An accelerated preservation test. Presentation SCC Annual Meeting NYC, 12/11/81. (8) R. Bhadauriand D. G. Ahearn, Loss of effectiveness of preservative systems of mascaras with age, Applied & Environmental Microbiology, 39, 665-667 (1980). (9) L. A. Wilson, A. J. Julian, and D. G. Ahearn, The survival and growth of microorganisms in mascara during use, American Journal of Opthalmology, 79, 596 (1975). (10) L. A. Wilson and D. G. Ahearn, Pseudomonas induced corneal ulcers associated with contaminated eye mascaras, American Journal of Opthalmology, 84, 112-119 (1977).