Preservative Efficacy Testing Vs. Microbiology Testing
What are preservatives?
Preservatives are substances mixed into creams, gels, and other liquids. Preservatives are used to prevent the growth of bacteria, fungi, and other microorganisms within a medical, cosmetic, household, or food product. Preservatives also help keep the freshness of the appearance of a product and keep its consistency intact over time. Preservative tests are available for products that contain preservatives such as antimicrobial agents. Preservative efficacy test methods are used to evaluate the performance of a preservative when exposed to microbes. Professional microbiology test labs are capable of performing preservative tests and determining preservative efficacy test microbiology after microbial challenge(s).
What is preservative efficacy testing (PET)?
Preservative efficacy testing, also known as preservative challenge testing, determines the effectiveness of a preservative during its shelf life and evaluates how well a product withstands microbial contamination during use.
What is microbiology?
Microbiology contains the Greek and Latin roots of “micro,” “bio,” and “ology.” The “micro” stands for small, the “bio” stands for biology, and the “ology” is the study of something. Microbiology can also be defined by “microbial,” which refers to microorganisms. Thus, microbiology is the study of microorganisms, where microorganisms are small living components of the broad field of biology.
What is microbiology testing?
Microbiology testing identifies the presence and the type of microbes in a manufacturing environment, medical product, or medical device. Specific regulatory testing for microbiology may include assays such as microbial enumeration testing, particulate analysis, yeast analysis, antifungal activity assessment, growth promotion testing, microbial limits testing, zone of inhibition testing, water analysis, bacteriostasis/fungistasis testing, bacteria identification, yeast identification, fungi identification, gram-negative staining, product inoculations, biological indicator tests, and BI incubation time reduction studies. Microbiology tests are governed by USP 61 and USP 62.
What products require preservative efficacy testing?
Preservative efficacy test methods can be used to determine the best preservative to use in your topical formulation and the minimum effective concentration needed to preserve the pharmaceutical, food, or biotechnology product being assessed. Topical formulations (gels, creams, ointments, and lotions) comprise a water or oil base that delivers various medications, natural substances (such as herbs), or moisture through the skin. Topical cosmetics contain preservatives to support the stability of the formulation and prevent microbial growth during repeated product use. Preservative efficacy test methods are also used for parenteral products containing multiple doses. In these products, antimicrobial preservatives inhibit the growth of any microorganisms introduced during repeated insertion and withdrawal to load individual amounts.
Preservative efficacy testing can evaluate four categories of products with preservative challenge testing. These product categories are detailed in Table 1 of USP 51, reproduced below.
What products require Microbial bioburden testing?
Bioburden testing is an important quality control step that detects the level of contamination of a product at any stage, from initial product manufacture to final distribution. As microorganisms exist on every surface (including our body), bioburden can be accidentally introduced during the manufacturing or packaging process in many ways. Some of the most common examples are contamination through the raw materials used, technicians, tubing/piping used to transfer product between development stages in a process, or the manufacturing environment itself. With such abundant sources of contamination, regular bioburden testing of all sterile products reduces microbial contamination and recall risks.
How is preservative Efficacy testing performed?
Three preservative efficacy test methods are used. These approaches come from the Schülke KoKo Test, United States Pharmacopeia (USP), and the European Pharmacopeia.
Schülke KoKo Test Vs. U.S. Pharmacopeia Vs. European Pharmacopeia
Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger, and Candida albicans must be tested for in all cosmetic products sold in the European Union. In addition to the microbes above, testing with microbes known to lead to spoilage of cosmetic products is recommended but not required. In the United States, the USP guidelines require testing of Escherichia coli in addition to Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger, and Candida albicans. Spoilage microbes are not needed for PET. In contrast to the pharmacopeia tests, which only evaluate pathogenic microbes, the Schülke KoKo test evaluates product spoiling microorganisms. Schülke KoKo test’s spoiling microorganisms are based on decades of cosmetic testing experience from Schülke & Mayr in Germany.
USP preservative efficacy testing evaluates cosmetic products by exposing them to a single microbial strain at a time. Additional details on USP preservative challenge testing, which follows similar procedures to the European Pharmacopeia, can be found through reading our articles on Preservative Challenge Testing And USP 51 and Preservative Efficacy Testing For Medical Devices. In contrast, for the Schülke KoKo test, the single cultivated microbes are brought together into a mixed suspension to challenge the cosmetic product. A new mixed suspension is prepared for each of the six inoculation cycles for Schülke KoKo testing. Since the Schülke KoKo test uses a mixed microbe inoculation, it simulates microbial exposure of a product during production, filling, and use. Indeed, a product would likely be exposed to multiple microbes at once instead of one at a time. Overall, though different from the USP and European Pharmacopeia methods, the Schülke KoKo Test is a reliable test method for assessing the efficacy of antimicrobial preservation of cosmetic products.
First, cultures of Candida albicans (ATCC No. 10231), Aspergillus brasiliensis (ATCC No. 16404), Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538) are prepared. Stock cultures of these organisms are prepared by centrifuging an ATCC culture removing residual media from the prior culture and resuspending the microorganisms in a sterile suspension fluid. The suspension fluids for each microorganism referenced above are prepared at a microbial count of about 1 × 108 colony-forming units per milliliter (CFU/mL).
Preservative efficacy testing is performed in five sterile, capped containers. Suppose the product’s original container is sterile, can be entered aseptically, and holds an appropriate product volume. In that case, the original filled product containers may be used. Each of the five product samples is injected with a test suspension of either Candida albicans, Aspergillus brasiliensis, Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus. Each product sample is exposed to only a single microbe of the five listed, and all microbes are exposed to the product during testing. The microbial test suspension injected is between 0.5%- 1% of the volume of the product under assessment and is at a concentration of 1 × 105 and 1 × 106 CFU/ml of the product for category 1-3 products. The final concentration per ml of category 4 products is between 1 × 103 and 1 × 104 CFU/mL.
Each of the five product samples with their respective microbial injections is incubated at a simulated room temperature of 22.5 ± 2.5°C or 32.5 ± 2.5°C depending upon the microbe the product sample is exposed. Microbial counts are taken at 7, 14, and 28 days for most microorganisms tested. The plate-count method determines the number of CFU present in each inoculated product sample. This plate-count method is completed in duplicate. Once all counts are taken, the change in log10 of the concentration of CFU/ml is calculated for each microorganism. The requirements for antimicrobial effectiveness are met if no increase in microbial growth for the various organisms tested occurs within the timeframes specific to each organism. are met. No increase in microbial growth is defined as not more than 0.5 log10 units more than the previous microbial growth value.
How are microbiology tests performed?
Microbiology testing often requires sample collection from a product, a surface, a water source, or the air. Once samples are obtained, various microbiology testing can be performed. Below are links to articles related to multiple microbiology tests and testing methods. Firstly, you can find further details on USP 788 particulate matter testing here. Microbial characterization methods can be found here, whereas the top techniques for microbiology examination are here. Lastly, information on how to record and evaluate microbiology data is here.
What are the differences between preservative efficacy testing and microbiology testing?
Microbiology testing is used for environmental monitoring and sterility assurance. In contrast, Preservative challenge testing determines the best preservative for use and the minimum effective concentration needed to preserve multidose parenteral products, topicals products, food, or other healthcare items that contain preservatives. Both preservative and microbiological tests are quantitative examinations. Further, microbial counting methods for both tests are similar. However, preservative challenge testing evaluates a product’s antimicrobial properties, while microbiology testing supports the environmental monitoring and sterility assurance of products.
Summary
Overall, preservative efficacy testing (PET) and microbiology testing are imperative for the regulatory approval of pharmaceuticals, cosmetics, and medical devices. These tests ensure that parenteral products, topicals, and medical devices have sufficient sterility and antimicrobial activity to keep patients safe during product use. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate microbiology and preservative efficacy testing for your unique cosmetic, pharmaceutical, or medical device product needs.
MycoScience is a contract manufacturing organization specializing in Preservative Efficacy Testing and Microbiology Testing. MycoScience also offers Sterilization Validations, Microbial Aerosol Challenge Testing,Cleaning Validations, Accelerated Aging, Bioburden Testing, Cytotoxicity Testing, Bacterial Endotoxin Testing, EO Residual Testing, Package Integrity Testing & Environmental Monitoring services medical devices and allied industries. MycoScience is an ISO 13485 certified facility.
References
Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.
United States Pharmacopeial Convention. <51> Antimicrobial Effectiveness Testing. Rockville, MD, USA. 2021. (USPC <51>).
United States Pharmacopeial Convention. <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. Rockville, MD, USA. 2021. (USPC <61>).
United States Pharmacopeial Convention. <62> Microbiological Examination Of Nonsterile Products: Tests For Specified Microorganisms. Rockville, MD, USA. 2021. (USP <62>).
