Preservation is a critical element in protecting consumer, TABLE 1. SIMILARITIES AND VARIATIONS REPORTED IN THE PET SURVEY RESPONSES

Transcription

1 PRESERVATIVE EFFICACY TESTING Representatives from finished product companies and suppliers provide insight on methods ensuring preservation of consumer, household and industrial products. Vidya Ananth The Clorox Company; Tony Rook The Sherwin-Williams Company; Dolores A. Shaw The Dow Chemical Company; Sangeeta Ganguly-Mink Stepan Company; Phyllis Vitolo Nice Pak Products, Inc.; Milady Brutofsky Lonza, Inc.; and Courtney Detwiler State Industrial Products For the Consumer Specialty Products Association Microbiology Preservative Subcommittee Preservation is a critical element in protecting consumer, household and industrial (CH&I) products from microbial spoilage. Most CH&I products are aqueous based and contain high levels (>90%) of available water that provide an environment conducive to microbial growth. In addition, many essential nutrients required for microbial growth are present in key product formulation ingredients such as surfactants, dispersants, emulsions, rheology modifiers, enzymes and fragrances. 1 Microbial spoilage may compromise products to decrease efficacy, yield undesirable aesthetics or even pose a human health safety risk. For this reason, spoiled products may lead to expensive recalls that may draw negative attention from consumers, media and regulatory bodies, thus causing damage to brand and company reputation. 2 It is necessary to include effective preservatives in products to help mitigate these risks. Preservatives are defined by the US Environmental Protection Agency (EPA) as antimicrobial pesticides that are effective in non-public health products and are used to control the growth of microorganisms. 3 Preservatives that are used in CH&I products must be registered with EPA for their intended use and are regulated by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) 7 U.S.C. 136 et seq. (1996) which provides for federal regulation of pesticide distribution, sale and use. 4 Preservatives are used in almost all water-based liquid cleaners and help increase consumer confidence in the quality of products. 5 Many companies are attempting to move away from traditional or synthetic preservatives while incorporating natural or more sustainable ingredients, thereby making preservation of these products even more challenging. Similarities The ideal characteristics of a preservative include: broadspectrum efficacy, shelf-life stability, product compatibility, safety in use, cost effectiveness, commercial availability, registration with appropriate regulatory agencies, and an environmentally favorable profile. Other factors that may impact the robustness of the preservative system include raw material quality, packaging design, exposure conditions (warehouse/transit) and consumer use habits. 6 Many companies use in-house or compendial Preservative Efficacy Test (PET) methods to evaluate the mitigation and control of spoilage organisms in products. 7,8 The PET method is used to demonstrate a product s ability to prevent microbial growth TABLE 1. SIMILARITIES AND VARIATIONS REPORTED IN THE PET SURVEY RESPONSES Pooling of challenge inocula Multiple ( 2) microbial challenges Type of recovery media Test product incubation conditions Use of neutralizing agents Neutralization validation included Neat product evaluated Single product lot evaluated Testing conducted on lab scale batches Stability sample test products (room temperature and accelerated temp) PET variations are due to unique product types Variations Selection of microorganisms Frequency of product challenge Ratio of inoculum volume to finished product Inoculum concentration Methods for determining inoculum concentration Preparation of mold spore inoculum Sample replicates Justification for PET method modifications Test sample quantity and containers Acceptance criteria PET studies conducted internally and externally Selection of samples through development and commercialization stages 2 happi happi.com April 2015

2 through the shelf life of the product. PET studies determine the effective concentration of a preservative system that is necessary to protect a formulated product from microbial spoilage. Currently, a standardized guidance for conducting PET evaluations of CH&I products does not exist. In order to understand the current industry practices, the CSPA Microbiology Preservative Subcommittee (MPS) conducted a survey in August 2013 of all CSPA member companies within the Cleaning Products and Antimicrobial Product Divisions, including product manufacturers, raw material suppliers and contract labs. Seventeen responses were collected and summarized and are presented in this paper. Approach and Results As the initial step to understand industry practices, the CSPA s MPS commissioned a PET task force who conducted a 29-question survey. The goal was to gather current information from the member companies on PET study parameters and conditions including acceptance criteria. Data from the survey will be used in developing the guidance and methodology for conducting PET evaluations. An overall summary of the similarities and variations of the PET methodologies are provided in Table 1. Variations in PET Method Most of the surveyed companies have defined an internal PET method and allow for a number of modifications of these methods. The survey identified previous microbiological issues as the critical reason for modifying their PET method. Other product factors necessitating variations of the method included product ph, viscosity, product component, solvent content, water content or water activity (Aw) and packaging processes. Additional aspects cited by those responding were salt/electrolyte content, product application and end use. Modifications or variations to a company s internal PET method are depicted in Figure 1. The two most frequent modifications to the PET were the inclusion of unique microbes and the number of challenges applied. Sample Considerations The survey addressed multiple characteristics related to samples in PET evaluations. In particular, sample selection based on the stage of product development was considered. Other characteristics included the volume of product tested, the number of lots evaluated, the number of replicates, inclusion of controls and sample container specifics. Responses from the companies surveyed indicated that testing was conducted through various stages of product development. The majority of the companies conducted PET evaluations on the lab scale batch during product development. Some of those surveyed performed PET evaluations on pilot scale batches, manufacturing scale test batches and product launch material. In some cases, the product development team was responsible for determining the stage at which the PET evaluation will occur. Over half of the respondents indicated that testing was completed on neat (undiluted) products. However, a few responded that product was diluted to customer specifications or label claims prior to testing. Dilutions included were 1:1 (50% solution); 1:9 (10% solution); 9:1 (90% solution). One company FIGURE 1: VARIATIONS/MODIFICATIONS IN PET METHODOLOGY VARIATIONS IN PET Unique microbes Number of challenges Recovery media Inoculum volume Incubation conditions Acceptance criteria Incubation Sample volume % RESPONSE FOR EACH VARIATION April 2015 happi.com happi 3

3 indicated that dilutions may be necessary depending on the product viscosity. More than 80% of those surveyed evaluated only one lot of product and about half performed from one to three replicates. Participating companies indicated that PET evaluation sample volumes ranged from 1-250mL or grams per sample. Sterile plastic or glass sample containers were used which ranged from mL. Since controls are a critical aspect of any microbiological evaluation, a question pertaining to the types of controls used during PET evaluation was included. The most common laboratory controls reported were sterility controls (media, unpreserved sample), negative controls (unpreserved samples), positive controls (well preserved samples) and inoculum controls (individual/pooled counts). Other controls employed were neutralizer confirmation, neutralizer sterility and current marketed products as comparative benchmarks. Most surveyed companies reported that the PET product samples were typically stored between C for up to four weeks. A few companies indicated shorter sample incubation TABLE 2. ATCC MICROBIOLOGICAL STRAINS USED FOR CHALLENGE TESTING Microorganisms Gram Negative Bacteria ATCC Number Pseudomonas aeruginosa 9027, 15442, Pseudomonas putida 12633, Pseudomonas fluorescens 13525* Burkholderia cepacia Escherichia coli 8739, 11229, Enterobacter gergoviae Enterobacter aerogenes Citrobacter freundii 8090 Klebsiella pneumoniae 13883, 4352* Klebsiella oxytoca 13182* Serratia liquefaciens Acinetobacter baumannii 19606* Gram Positive Bacteria Yeast Mold Staphylococcus epidermidis 12228, Staphylococcus aureus 6538 Candida albicans Saccharomyces cerevisiae 834* Aspergillus brasiliensis (formerly Aspergillus niger) * Reported only by a few respondents periods of one to three weeks. The number and frequency of microbial challenges were company specific and depended on many conditions including product type, raw materials, manufacturing environment, packaging format, shelf life and intended use. These conditions also impacted the sampling intervals following each challenge inoculation. Interestingly, the survey indicated that the number of challenges performed by companies was evenly split between conducting a single challenge and multiple challenges. Of those responding that multiple challenges were used, two to eight challenges were performed, with two to three challenges as most common. The interval between challenges was most typically reported as one week. The studies generally lasted from one to eight weeks. A respondent inoculated and immediately sampled afterwards to provide a Day 0 evaluation. This ensured that the challenge organisms were recovered in the test substance thus demonstrating that neutralization occurred. Inoculum Choice and Consideration When selecting microbes for PET evaluation, most companies TABLE 3. ENVIRONMENTAL/ PRODUCT ISOLATES USED FOR CHALLENGE TESTING Microorganisms Gram Negative Bacteria Pseudomonas oleovorans Pseudomonas pseudoalcaligenes Pseudomonas alcaliphila Pseudomonas aeruginosa Pseudomonas japonica Pseudomonas sp. Enterobacter gergoviae Aeromonas hydrophila Alcaligenes faecalis Provencia rettgeri Serratia marcescans Halomonas venusta Gram Positive Bacteria Enterococcus avium Enterococcus casseliflavus Microbacterium paraoxydans Bacillus subtilis/spp Yeast Candida xestobii Rhodotorula mucilaginosa Cryptococcus saitoi Cryptococcus magnus Rhodosporidium kratochvilovae 4 happi happi.com April 2015

4 used American Type Culture Collection (ATCC) strains of bacteria and fungi. The ATCC strains used are listed in Table 2. Additionally, environmental isolates recovered from raw materials, finished products or production equipment surfaces are also used by some companies (Table 3). The survey results indicated that microbe selection was a key contributor to variation in a company s standard method. There were no general trends identified for organism isolates with either standard or environmental strains among respondents. However, the surveyed companies focused heavily on Gram negative bacteria since these were the organisms that had the greatest spoilage potential in high water based formulations. Most companies reported using standard growth media for the inoculum preparation. Seventy-seven percent (77%) of the respondents used Trypticase Soy Agar/Broth (TSA/TSB) for bacteria while 35% used Sabouraud Dextrose Agar/Broth (SDA/ SDB) and 41% used Potato Dextrose Agar (PDA) for growing yeast and mold. A few of those surveyed also used other media such as Nutrient Agar/Broth, MacConkey, Tryptone Yeast Glucose (TYG), Plate Count Agar (PCA), Tryptone Glucose Extract (TGE), Brain Heart Infusion, Middlebrook and Synthetic Broth. The typical incubation temperatures used were C for bacteria, C for yeast and C for mold. Approximately half of those surveyed used cultures in the stationary growth phase. A few used cultures in the exponential/log or late log phase of growth. Most companies prepared separate pools of bacterial and fungal strains while some challenged with individual strains of microorganisms in the PET. Most companies targeted a final concentration of 105 to 106 CFU/mL (Colony Forming Unit/mL) for bacteria, yeast and mold. Variations in inoculum concentration were as low as 103 CFU/mL and as high as 109 CFU/mL. Various methods for determining inoculum concentration were used including plate counts, turbidity measurements, Most Probable Number (MPN) and hemacytometer cell counts. Table 4 shows that most companies used plate counts to enumerate inoculum levels, followed by turbidimetric measurements. The survey revealed that currently there is no standard approach to preparing the mold inoculum; however, the following techniques were reported: Grew the mold on SDA or PDA plates or slants; Used phosphate buffer, saline containing a wetting agent (Tween 80, Triton X-100) and/or glass beads to dislodge the mold spores from the agar surface; Filtered the mold suspension through sterile gauze to remove mycelial fragments; and Prepared inoculum from pellets or dehydrated cultures. Lastly, respondents were asked about the quantity of inoculum added to the finished product. The most common response was 1:100 volume to volume ratio of organisms into finished product. The variations reported are presented in Table 5. TABLE 4. METHODS FOR ENUMERATING INOCULUM CONCENTRATION Inoculum Enumeration Methods Number of Responses Plate Count 8 Turbidity (Spectrophotometry) 5 MPN 1 Hemacytometer Cell Counts 1 TABLE 5. RATIO OF INOCULUM TO FINISHED PRODUCT Ratio Percentage of Responses 1: : : :250 8 Aspects of Microbial Recovery An important aspect of conducting PET evaluations is the selection of appropriate media to support the growth of relevant bacteria and yeasts/mold. 9 The majority of the companies utilized TSA or TSA with neutralizers for recovery of bacteria. PDA or SDA, with or without neutralizers, were used for recovery of yeasts and molds. Bacterial recovery was conducted by incubating the plates at a temperature range of C for one to two days. Incubation of mold/yeast recovery media was typically conducted at a temperature range of C for a period of two to seven days. The survey specifically requested companies to comment on their neutralization practices. Sixty-five percent of respondents indicated the use of a neutralizer for recovery of organisms during PET, while 24% do not use a neutralizer. Most commonly utilized neutralizing media reported were Dey-Engley broth (D/E broth), D/E supplemented, Letheen broth (LB), LB modified with thioglycollate, TAT and lecithin. The vast majority of companies that incorporated a neutralizer in their recovery media also conducted neutralization validation (91%). However, the justification for conducting neutralization validation and the methodologies employed varied among responding companies, and no trend could be established. Acceptance Criteria The preservative level recommended by the outcome of the PET study should be predictive of effective protection against spoilage during consumer use. Since the acceptance criteria of a PET study can greatly influence the recommended effective preservative levels within a product, understanding how to accept a April 2015 happi.com happi 5

5 TABLE 6: PET ACCEPTANCE CRITERIA FOR BACTERIA, YEAST AND MOLDS Acceptance Criteria (over test period) Percentage of Responses Bacteria (%) Mold/Yeast (%) Complete log reduction log reduction log reduction 20 0 No increase 0 36 preservative package based on testing is a critical parameter. 10 Participants were questioned regarding the criteria they find acceptable against bacterial, fungal and/or yeast challenges. Survey responses provided a large variation in acceptance criteria, ranging from no increase of the initial microbial challenge concentration to a complete reduction over the course of the PET evaluation (Table 6). Over half of the respondents indicated their acceptance criteria was complete reduction for all three types of microbial challenges (bacteria, mold and yeast). A smaller proportion of the responses indicated a two or three log bacterial reduction from the initial microbial challenge by the end of the testing period was acceptable. Just over a third of the responses indicated it was acceptable when there was no increase of mold or yeast concentrations over the period of the test. Controls are a critical aspect of any microbiological evaluation, and, therefore, the survey questioned participants on the acceptance criteria of any control data generated during PET studies. In general, the majority of respondents indicated sterility controls should demonstrate no growth and inoculum viability controls should exhibit growth. Acceptance criteria for product controls mainly consisted of demonstrating sustained spoilage within an unpreserved product sample (negative control). However, some respondents required a product sample with a sufficient level of preservative to demonstrate no growth (positive control). Conclusions The consumer, household and industrial products industry widely recognizes the potential for microbes to survive within a variety of water-based consumer products. Therefore, preservatives are commonly included in aqueous based CH&I formulations to protect against microbiological contamination during normal consumer use. 7,11 This survey focused on understanding the various aspects of conducting an in-house Preservative Efficacy Test. Based on the responses, it is evident that many companies employ PET evaluations to determine the effectiveness of their preservative systems against microbiological contamination. It was discovered that many variations exist between company methodologies but core similarities were uncovered. Within companies, internal method modifications are often employed based on product characteristics or past performance. Due to the number of variations seen within the industry, there is a clear need for a more standardized guidance when conducting PET evaluations. Therefore, the CSPA MPS will continue to work toward developing a standard guidance document that includes recommendations for best practices when employing PET evaluations within CH&I products. The CSPA Microbiology Preservative Subcommittee The CSPA Microbiology-Preservative Subcommittee (MPS) is committed to establishing best practices and acceptable standards to address the increasing concern for microbiological quality within consumer, household, and industrial products. To support these goals, a Microbial Control Stewardship Task Force communicates the necessity for effective preservation strategies within consumer, household and industrial products. Website: References 1. Dolores A. Shaw, Beth A. Browne, Tony Rook, Phil Geis & Vidya Ananth. (2014). Critical Elements of Household Product Preservation: An Overview. Household and Personal Products Industry May 2, 2014 p Phil Geis & Tony Rook. Microbiological Quality of Consumer Product. Household and Personal Products Industry May 3, 2011 p Retrieved Nov , from 4. Retrieved Nov , from summary-federal-insecticide-fungicide-and-rodenticide-act. 5. Retrieved Nov , from ingredients/preservatives/ 6. Daniel K. Brannan. (1995). Cosmetic Preservation. J. Soc. Cosmetic Chem.46, J. F. Krowka & J. E. Bailey. (2007). CTFA Technical Guidelines - CTFA Microbiology Guidelines The Cosmetic, Toiletry & Fragrance Association. 8. The United States Pharmacopeia and The National Formulary (USP-NF); USP <51> Antimicrobial Effectiveness Testing. 9. Philip A. Geis. (2006) Cosmetic Microbiology: A Practical Approach. 2nd Edition. New York: Taylor & Francis. 10. Wolfgang Siegert. (2013). Comparison of microbial challenge testing methods for cosmetics. Household and Personal Care Today, 8(2), The Cosmetic, Toiletry & Fragrance Association. (2003). CTFA preservative challenge and stability testing survey. Cosmetics and Toiletries. May happi happi.com April 2015 Form No: