By Crystal M. Booth, PSC Biotech
Microbial contamination control is essential in ensuring consumer safety, especially in controlled environments. Disinfectants play an important role in controlling microbial contamination on surfaces. The term disinfectant is often broadly used to describe chemical agents used to reduce the microbial load of controlled environments.
Disinfectant efficacy testing is utilized to measure the effectiveness of chemicals that are used clean, sanitize, or disinfect surfaces. There are many regulations and guidance documents that discuss disinfectant efficacy testing, and 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 these regulations.
There are several important concepts to consider when establishing a disinfectant efficacy testing package. This two-part article discusses and recommends five key components that can be used to establish a compliant disinfectant efficacy testing package in the United States. (Note: The disinfectant efficacy testing package is only one component of a contamination control policy.)
Disinfectant Efficacy Testing Regulations and Concepts
Cleaning and sanitization procedures are critical segments of an overall contamination control strategy within a facility. Proper control over microbial contamination is necessary to ensure products are produced safely and properly, and are effective as intended. Chemical agents used to maintain the microbial control must be effective in destroying microorganisms.
There are many regulations and guidance documents that discuss disinfectant efficacy testing (DET), including the following:
- PDA Technical Report 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities1
- PDA Technical Report 13 – Fundamentals of an Environmental Monitoring (EM) Program2
- ISO 14464-1 – Cleanrooms and Associated Environments3
- USP <1072> Disinfectants and Antiseptics4
- U.S. Code of Federal Regulations (CFR), Title 21 Part 211.42 (c)5
- U.S. Code of Federal Regulations (CFR), Title 21 Part 211.67 Sec. 211.675
- FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practices6
- EudraLex Volume 4 – EU Guidelines to Good Manufacturing Practice (GMP) for Human and Veterinary Medicinal Products, Annex 1 – Manufacture of Sterile Medicinal Products7
- EudraLex Volume 4 – EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Part 1, Chapter 3: Premises and Equipment8
- EudraLex Volume 4 – EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Part 1, Chapter 5: Production9
Failure to properly adhere to these and other regulatory guidance documents may lead to observations. The FDA posts warning letters to its website, and these warning letters can be reviewed in order to gain insight into the current regulatory expectations. This knowledge can then be utilized to prevent similar findings in the future.
Many different types of chemicals are available for cleaning, sanitization, or disinfection. United States Pharmacopeia (USP) <1072> Disinfectants and Antiseptics defines and describes some of the available chemicals on the market. According to USP <1072>, the following properties can have an impact on the effectiveness of the disinfectants being utilized in a facility:
- The pH of a disinfectant
- The concentration of the disinfectant
- The contact time
- An important item to note regarding the testing is that “contact times” refers to wet contact times. In other words, the material being disinfected must remain wet with the disinfectant for the period of the validated contact time.2 In general, contact times should not exceed 120 seconds for alcohols or 10 minutes for disinfectants and sporicides.1
- The method of the disinfectant application
- For example, spraying, wiping, mopping, etc.10
- The nature of the surface disinfected
- The hardness of the water used to dilute the disinfectant
- The temperature of the environment
- The amount of organic materials present on the surface being cleaned
- Soil or debris present on the surface
- The type and the number of microorganisms present
The properties described in USP <1072>, along with the regulation and guidance document information, should be considered when developing a disinfectant efficacy test and facility cleaning procedures. Some additional concepts to consider when planning the disinfectant efficacy testing strategy include the following:
- The types of disinfectants to be examined
- USP <1072> provides a list of the different types of disinfectants commonly used in the industry.
- The microbial isolates to be utilized to be used in the study
- There is a regulatory expectation that environmental isolates be included in the study whenever possible.4
- Also include rationale for why the microorganisms were chosen for use in the study.10
- The types of material coupons (stainless steel, glass, plastic, etc.) to test10
- USP <1072> provides a list of the different types of common cleanroom materials.
- The environmental conditions (e.g. airflow, temperature, humidity)10
- Proper training of employees
- Realistic acceptance criteria
- At least a 2 log reduction for bacterial spores and a 3 log reduction for vegetative bacteria during a predetermined contact time (i.e., 10 minutes over and above the recovery observed with a control disinfectant application) should be acceptable.4
The 5 Components of a Compliant Disinfectant Efficacy Testing Package
1. Master Disinfectant Efficacy Testing Protocol
The first key component to establishing a compliant disinfectant efficacy testing package is researching and planning the contents of the final package. To this end, it is recommended to develop an overarching protocol (or master DET protocol) for the study. This protocol is a way to capture and document the research performed during the planning phase and to outline the final appearance of the DET package. This protocol is similar to a master validation protocol and will outline subsequent protocols and information to be included in the DET package.
When considering the items to include in the study, it is important to gather all of the data and information that will be needed. It is recommended that the master DET protocol include a checklist for the following items:
- Technical data sheets11
- Certificate of analysis (CoA)11
- Supplier’s change control policies11
- Recommended directions for use11
- All available testing on substrate compatibility11
- All available testing on stability11
- All available testing on microbial efficacy11
- Description of packaging, label, and container type of the disinfectant1
- Description of the ingredients and concentrations of the disinfectant1
- Lot or batch number of the disinfectant1
- Irradiation or other sterilization verification certification1
- Safety data sheet information1, 11
- Disposal information1
The above information should be organized by the disinfectant and included in a reference documents section for easy retrieval in the future.
A typical master DET protocol will include the following:
- Reference documents and checklists section (including documents) as previously described
- This section may also include a unit organized by employee for any relevant training documentation required for the execution of the protocols.
- References to any relevant internal operating procedures may also be included in this section, if desired.
- Link to the protocol for the use-dilution tests (in vitro)
- Link to the protocol for the surface challenge tests (in vitro)
- Link to the protocol for the statistical comparison test (in situ)
- Defined requirement to summarize all of the data and findings in a master DET summary report at the conclusion of the study
2. Use-Dilution Tests (in vitro)
The second key component in establishing a compliant disinfectant efficacy testing package is the information regarding the use-dilution test. The use-dilution test screens the concentrations and contact times of disinfectants against microorganisms.4 In addition, the test can also be used to quickly determine the time necessary to achieve an acceptable log reduction of microorganisms without the interference of coupon surfaces.11
The execution of the use-dilution 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.1 The disinfectants used for the testing should be close to or beyond their stated in-use expiration date.1
According to Bartnett et al., most use-dilution test procedures “Tend to employ a standard suspension of the microorganism in appropriate dilutions of the test disinfectant. Tests are carried out at room temperature. At selected time intervals, samples are removed and viable counts are performed following the neutralization of any disinfectant remaining in the sample. Residual disinfectant can be neutralized by dilution or by adding specific agents, such as lecithin or polysorbate 80.”11
There are many variations for the use-dilution test. PDA TR 70 describes a use-dilution test method that may be adapted. 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.1
The efficacy of the neutralizers and their ability to recover inoculated microorganisms from the material should be demonstrated during the use-dilution test.4 Neutralizers that inactivate the disinfectants should be included in either the diluent, the microbiological media used for microbial enumeration, or both.4 USP <1227> Validation of Microbial Recovery from Pharmacopeial Articles contains helpful information regarding neutralizers.12
Following the execution of the test, the data should be reviewed and summarized in a report linked to the execution of the use-dilution test protocol. The report, protocol, data, and employee training documentation should be gathered, organized, and stored with the master DET protocol.
Part 2 of this article will discuss components 3 through 5 — surface challenge tests, statistical comparison tests, and master disinfectant efficacy testing summary report.
- PDA Technical Report No. 70 – Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities, Parenteral Drug Association, Bethesda, MD (2015).
- PDA Technical Report No. 13 – Fundamentals of an Environmental Monitoring Program, Parenteral Drug Association, Bethesda, MD (2014).
- ISO 14644-1:1999 (E) Cleanrooms and Associated Controlled Environments – Part 1: Classification of Air Cleanliness, International Organization for Standardization (1999)..
- United States Pharmacopeia (USP) <1072> Disinfectants and Antiseptics.
- Code of Federal Regulations (CFR) Title 21: Food and Drugs, accessed on November 3, 2016 at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm
- FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice, Food and Drug Administration, Rockville, MD (2004).
- EudraLex, The Rules Governing Medicinal Products in the European Union: Volume 4 – EU Guidelines to Good Manufacturing Practice (GMP) for Human and Veterinary Medicinal Products, Annex 1 – Manufacture of Sterile Medicinal Products, European Commission (2008).
- EudraLex, The Rules Governing Medicinal Products in the European Union – EU Guidelines to Good Manufacturing Practice (GMP) for Medicinal Products for Human and Veterinary Use, Part I, Chapter 3: Premises and Equipment, European Commission (2014).
- EudraLex, The Rules Governing Medicinal Products in the European Union – EU Guidelines to Good Manufacturing Practice (GMP) for Medicinal Products for Human and Veterinary Use, Part I, Chapter 5: Production, European Commission (2014).
- Sartain, E., The ABCs of Disinfectant Validation, Cleanrooms, March 2005.
- Bartnett, C. et al., Control Strategies for Fungal Contamination in Cleanrooms, Controlled Environments, September 2007.
- United States Pharmacopeia (USP) <1227> Validation of Microbial Recovery from Pharmacopeial Articles
About The Author:
Crystal M. Booth, M.M., is an independent pharmaceutical microbiology consultant with Pharmaceutical Advisors, LLC. She has over 17 years of experience in pharmaceutical microbiology, working in both R&D and quality control laboratories, including a startup company.
During her career, Crystal 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, environmental monitoring programs, surface recovery validations, disinfectant efficacy studies, minimum inhibitory concentration testing, antimicrobial effectiveness testing, hold time studies, and various equipment validations. She has experience working with global markets and regulatory bodies.
Crystal is a technical author and public speaker in the microbiology industry, and she also teaches aseptic gowning qualification classes. She earned her bachelor’s degree in biology from Old Dominion University and her masters in microbiology from North Carolina State University.