By Crystal M. Booth, PSC Biotech
Disinfectants play an important role in controlling microbial contamination on surfaces, and disinfectant efficacy testing is a means to measure the effectiveness of disinfectants that are employed to clean, sanitize, or disinfect surfaces. Establishing a compliant disinfectant efficacy testing package aids in providing clear documentation that the disinfectants employed in a facility have been analyzed as required by applicable regulations.
There are several important concepts to consider when establishing a compliant disinfectant efficacy testing package. Part 1 of this two-part article discussed the first two recommended components of a testing package: the master disinfectant efficacy testing protocol and use-dilution tests. This article explains the final three components that can be used to establish a compliant disinfectant efficacy testing package in the U.S. — surface challenge tests, the statistical comparison test, and master disinfectant efficacy testing summary report — along with tips for assembling the package.
3. Surface Challenge Tests (in vitro)
The third key component in establishing a compliant disinfectant efficacy testing package is the surface challenge test, which determines how efficient a selected disinfectant is in reducing the microbial load on a coupon or a carrier.1 The execution of the surface challenge test should be governed by a protocol, and the employees must be properly trained. The test should be performed on three separate lots of disinfectant, and there should be a minimum of three antimicrobial agents qualified, including a sporicide. This in vitro test should be performed in-house or by a qualified contract testing laboratory.2 The disinfectants used for the testing should be close to or beyond their stated in-use expiration date.2
There are many variations of the surface challenge test. PDA Technical Report (TR) 70 describes two methods that can be adapted as needed. These methods include the total kill method and the enumeration method. The expected result from the total kill method is that all of the microorganisms will be destroyed, whereas the enumeration method describes a method where a log reduction is calculated.2 Regardless of which method is utilized, it should be appropriately validated to ensure that the neutralizing agent selected does not prevent the growth of the microorganisms.2
Neutralizers that inactivate the disinfectants should be included in either the diluent, the microbiological media used for microbial enumeration, or both.3 The efficacy of the neutralizers and their ability to recover inoculated microorganisms from the material should also be demonstrated3. USP <1227> Validation of Microbial Recovery from Pharmacopeial Articles contains information regarding neutralizers.4
In regards to coupons or carriers, PDA TR 70 recommends using carriers that are not more than 1.5” x 1.5”, so the coupons can fit into test tubes. This size can also aid in preventing possible contamination from holding the coupons.2 Conversely, USP <1227> recommends carriers that are 2” x 2” in size. Both of these documents are guidance documents.
The swab method, surface rinse method, or contact plate method may be employed when enumerating the test microorganisms. In general, a panel of six to 10 microorganisms including bacteria, yeast, mold, and environmental isolates — or their American Tissue Culture Collection (ATCC) strain, if environmental isolates are not available — should be utilized in the study.2
Following the execution of the test, the data should be reviewed and summarized in a report linked to the execution of the surface challenge test protocol. The report, protocol, data, and employee training documentation should be gathered, organized, and stored with the master DET protocol.
4. The Statistical Comparison Test (in situ)
Laboratory surface challenge tests used to qualify the disinfectant contact time and the microbial reduction on surface samples (i.e., coupons) are sometimes supplemented by an in situ study known as the statistical comparison test.5 Thus, the forth key component in establishing a compliant disinfectant efficacy testing package is the statistical comparison test. Statistical comparison tests are employed to analyze the application method of the disinfectant, the chemical properties of the disinfectant, and the calculated contact time’s ability to reduce or destroy microorganisms in the actual environment. The test compares the frequency of the microbial isolation and the counts of microorganisms isolated prior to and after the implementation of a new disinfectant.3 This assay is considered necessary because critical process steps, like the disinfection of aseptic processing areas, need to be validated as required by good manufacturing practice (GMP) regulations.3
The execution of the statistical comparison test should be governed by a protocol, and the employees must be properly trained.
According to the FDA guidance Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, “To prevent introduction of contamination, disinfectants should be sterile, appropriately handled in suitable (e.g., sterile) containers and used for no longer than the predefined period specified by written procedures.”6 Microorganisms are not inoculated in the facility during the execution of the test. Instead, environmental monitoring (EM) surface samples are collected and processed following worst-case conditions (e.g., shutdown after construction activity).7 These EM samples are intended to provide a baseline of the microbial flora present in the environment.
After the collection of the baseline EM samples, the area is disinfected following standard operating procedures. Care should be taken to adhere to the calculated wet contact time and the proper application method of the disinfectant. After the area has dried, more EM samples of the area (in the same locations) are collected.
Following the execution of the test, the data should be analyzed, summarized, and compared to determine the in situ effectiveness of the disinfectant employed. The data, findings, and conclusions should be reviewed and summarized in a report linking to the execution of the statistical comparison test. The report, protocol, data, and employee training documentation should be gathered and stored with the master DET protocol.
5. Master Disinfectant Efficacy Testing Summary Report
The fifth key component in establishing a compliant disinfectant efficacy testing package is the master DET summary report, which provides an overall summary of analyzes performed, data, deviations, findings, and conclusions. The report may also outline any follow-up requirements, such as the need to update or write new standard operating procedures. In addition, this report may highlight the defined method to apply the disinfectant, the concentration of the disinfectant, and the calculated contact time.3 In general, the master DET summary report will provide a high-level overview and link between the other reports and findings for the entire study. The purpose of this report is to close out the DET package and provide a holistic view of the studies performed.
Depending on the company, the master DET summary report may be written in extensive detail to serve as a standalone document. In other words, this report could contain enough detail to be presented to regulatory auditors as proof of testing compliance. The supporting data, reports, and protocols should still be readily available for review if requested.
Assembling the Compliant Disinfectant Efficacy Testing Package
Once all of the data has been gathered, summarized, reviewed, approved, and reported, the DET package may be assembled. There are several ways to organize and present disinfectant efficacy testing data during regulatory audits. Depending on the systems of the facility, this may include electronic files, paper based files, or a combination of electronic and paper based files.
A compliant disinfectant efficacy testing package may appear as follows:
- Section 1 – Approved master DET summary report
- Section 2 – Approved master DET protocol
- Section 3 – Approved use-dilution test report
- Approved use-dilution test protocol
- Reviewed and approved use-dilution test data organized by disinfectant
- Section 4 – Approved surface challenge test report
- Approved surface challenge test protocol
- Reviewed and approved surface challenge test data organized by disinfectant
- Section 5 – Approved statistical comparison test report
- Approved statistical comparison test protocol
- Reviewed and approved statistical comparison test data organized by disinfectant
- Section 6 – Reference documents and checklists organized by disinfectant
- Technical data sheets1
- Certificate of analysis (CoA) 1
- Supplier’s change control policies1
- Recommended directions for use1
- All available testing on substrate compatibility1
- All available testing on stability1
- All available testing on microbial efficacy1
- Description of packaging, label, and container type of the disinfectant2
- Description of the ingredients and concentrations of the disinfectant2
- Lot or batch number of the disinfectant2
- Irradiation or other sterilization verification certification2
- Safety data sheet information1, 2
- Disposal information2
- Section 7 – Employee training documentation organized by employee and analysis
Disinfectant efficacy testing is utilized to measure the effectiveness of chemicals that are used clean, sanitize, or disinfect surfaces. The testing is required by the regulations to ensure that the chemicals employed are effective against microorganisms. The implementation of the disinfection, cleaning, and sanitization procedures is a critical component of overall contamination control within a facility.5
There are many regulations and guidance documents that discuss disinfectant efficacy testing. Two excellent resources include the PDA TR 70 and USP <1072>. In addition, there is a regulatory expectation that environmental isolates be included in the study whenever possible.3 To avoid potential findings, it is recommended to be proactive and research warning letters published online and correct any noted procedural gaps as soon as possible.
There are many important concepts to consider when establishing a compliant DET package. In summary, five key components of a compliant DET package include the following:
- A master disinfectant efficacy testing protocol to guide the entire study and various testing regimes.
- Use dilution tests to quickly analyze the concentrations and contact times of the disinfectants that are effective against microorganisms
- Surface challenge tests to determine how efficient a selected disinfectant is in reducing the microbial load on a coupon or a carrier
- Statistical comparison test to analyze the application method of the disinfectant, the chemical properties of the disinfectant, and the calculated contact time of the disinfectant on surfaces in the facility
- A master disinfectant efficacy testing summary report to provide a holistic view of the testing performed, summary of the data, discussion of any deviations, discussion of the findings, and summary of the conclusions
Establishing a compliant disinfectant efficacy testing package provides clear documentation that the disinfectants employed in a facility are effective against microorganisms as intended. Disinfectants play an important role in cleaning, sanitizing, and disinfecting facilities. They also play a role in preventing, removing, and controlling microbial contamination. Preventing microbial contamination in a facility is essential in producing safe and effective products for consumers.
- Bartnett, C. et al., Control Strategies for Fungal Contamination in Cleanrooms, Controlled Environments, September 2007.
- PDA Technical Report No. 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities, Parenteral Drug Association, Bethesda, MD (2015).
- United States Pharmacopeia (USP) <1072> Disinfectants and Antiseptics.
- United States Pharmacopeia (USP) <1227> Validation of Microbial Recovery from Pharmacopeial Articles
- PDA Technical Report No. 13 – Fundamentals of an Environmental Monitoring Program, Parenteral Drug Association, Bethesda, MD (2014).
- FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice, Food and Drug Administration, Rockville, MD (2004).
- Sartain, E., The ABCs of Disinfectant Validation, Cleanrooms, March 2005.
About The Author:
Crystal M. Booth is president of Azzur Labs, LLC. She has over 19 years of experience in pharmaceutical microbiology, working in quality assurance, CDMOs, R&D, and quality control laboratories, including startup companies. During her career, she has developed and validated methods for antibiotics, otic products, topical creams, topical ointments, oral solid dose products, oral liquid dose products, veterinary products, human parenterals, vaccines, biologics, aseptically filled products, and terminally sterilized products. Those methods include microbial limits testing, bacterial endotoxins testing, particulate testing, sterility testing, pharmaceutical water system validations, EM programs, surface recovery validations, disinfectant efficacy studies, minimum inhibitory concentration testing, antimicrobial effectiveness testing, hold time studies, and various equipment validations. Booth earned her bachelor’s degree in biology from Old Dominion University and her master’s in microbiology from North Carolina State University.