Best Practices for Disinfectant Validation in Cleanrooms

Disinfectants used in cleanrooms in the pharmaceutical, biotechnology, and medical device industry must be validated for intended use. This is a regulatory expectation to ensure the appropriate disinfectant is employed and microbial control is maintained in the environment. Disinfectant efficacy testing is one component of the validation, where the disinfectant is challenged for antimicrobial effectiveness in the laboratory. The following Technical Tip provides best practice recommendations for the laboratory disinfectant efficacy testing.

General Expectations

The use dilution, contact time, and exposure temperature of the disinfectant being evaluated in the study should match the actual, intended conditions of use of the disinfectant in the classified area.

It is critical, from a regulatory perspective, to evaluate site isolates from Environmental Monitoring (EM). Select isolates to use in the disinfectant efficacy testing after baseline EM conditions have been established. 

These should be based on:

• Frequency of occurrence
• Anticipated and/or demonstrated difficulty to disinfect (e.g. Bacillus cereus)
• Occurred in high numbers, when recovered

Testing Coupons

Work with manufacturers of cleanroom surfaces to procure representative classified area surface material coupons. Surfaces with well-defined specifications (e.g. 304 SS, 316L SS) may be procured from general sourcing options.

Coupon condition can have a significant impact on disinfectant efficacy testing. Coupons should:

• Not have rust or pitting that is not representative of classified area surfaces
• Be flat
• Not be on a backing that is absorbent, shedding, or linting

Evaluate surface material coupons for heat tolerance, if processing in a moist heat sterilizer. Chemical decontamination with a sporicide, such as Spor-Klenz™ RTU Sterilant, may also be used. Improper coupon processing can negatively impact testing.

Microorganisms

Increase probability of viable vegetative bacterial cultures, by:

• Incubating cultures for 18-24 hours, for most vegetative bacteria
• Use suspensions within 2 hours, if stored at room temperature or within 24 hours, if stored in refrigeration
• Use a spectrophotometer curve to estimate suspension Colony Forming Unit (CFU) levels, rather than to await population results

Ensure that concentration of the inoculum suspension is sufficient to demonstrate the required log10 reduction. Consider the detection limit of the recovery method and potential loss of viable cells, due to desiccation mortality. Preliminary drying studies of problematic species, such as Pseudomonas aeruginosa and Candida albicans, may prevent the need for low recovery related repeat testing.

If working with a fungal spore (conidial) suspension, filtration of the spore suspension to remove mycelial fragments, is necessary. Filtration can be achieved by using a variety of filtration materials, such sterile glass wool or a fritted filter. The presence of mycelial fragments or hyphae may shield the fungal spores from contact with the disinfectant or sporicide.

Ensure that appropriate log10 reduction acceptance criteria are used to evaluate the efficacy of disinfectants. USP <1072> Disinfectants and Antiseptics calls for:

• 3 log₁₀ reduction of vegetative bacteria
• 2 log₁₀ reduction of bacterial spores

These are the appropriate acceptance criteria for disinfectant efficacy testing for an aseptic manufacturing environment.

Laboratory Procedure

If storing a use dilution of a concentrated disinfectant (e.g. in a holding tank or spray bottle), it is a regulatory expectation to have data to support the internally set expiry date of the use dilution.

Take extreme caution to avoid depositing organisms outside of the intended test product exposure area. As little as 0.01 µL of inoculum not being exposed to test product can lead to a test failure.

A neutralization validation must be conducted to qualify a discrete wet contact time. USP <1227> Validation of Microbial Recovery from Pharmacopeial Items, USP <61> Microbial Examination of Nonsterile Products: Microbial Enumeration Tests, and EN 13697 Chemical Disinfectants and Antiseptics, provide potential study designs for neutralization validation. Below is a list of common chemical neutralizers for specific active ingredients.

TABLE 1. Chemical Neutralizers of Common Disinfectant Active

Evaluate appropriate culture media and incubation conditions for each organism prior to study initiation.

Recovery by immersion of the coupons is typically the most efficient recovery method. ASTM E2197 Standard Quantitative Disk Carrier Test Method and EN 13697 Chemical Disinfectants and Antiseptics provide potential immersion recovery methods. Surface coupon size can have a significant impact upon recovery method. The appropriateness of recovery methods should be evaluated with specific study surface coupons.

Additional Resources:

• USP <1072> Disinfectants and Antiseptics
• PDA TR 70 Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities
• EN 13697 Chemical Disinfectants and Antiseptics
• ASTM E2197 Standard Quantitative Disk Carrier Test Method
• ASTM E2614 Standard Guide for Evaluation of Cleanroom Disinfectants