Disinfectants that are used in an environment where good manufacturing practice (GMP) is employed are required to be validated to demonstrate that they can reduce typical and anticipated bioburden to an acceptable level.

The FDA Aseptic Processing Guidance for Industry Sterile Drug Products Produced by AsepticProcessing – Current Good Manufacturing Practice September 2004 states: “routinely used disinfectants should be effective against the normal microbial vegetative flora recovered from the manufacturing facility”¹.

In addition, EudraLex Vol 4. “The rules governing medicinal products in the European Union” Annex 1, Aug 2022 states: “4.34 The disinfection process should be validated. Validation studies should demonstrate the suitability and effectiveness of disinfectants in the specific manner in which they are used and on the type of surface material, or representative material if justified, and should support the inuse expiry periods of prepared solutions.”²

In 2018, Ecolab Life Sciences launched its Validex™ Disinfectant Efficacy Program. The Validex™ data package provides a clear, and reproducible test method for the evaluation of disinfectants for use in pharmaceutical cleanrooms. The program is designed to ensure an appropriate method and acceptance criteria for the industry that can be used globally. The method was then used to develop a comprehensive data set using relevant cleanroom microflora and surfaces.

The Validex™ approach reduces complexity and helps pharmaceutical manufacturers gain greater efficiency by giving specific guidance around cleanroom disinfectant validation processes.

Validex™ methodology is based upon the principles of the standard 'European Norm' (EN) method EN 13697. This is a quantitative non-porous surface test3 developed by The European Committee for Standardization (CEN - Comité Européen de Normalization). CEN Technical Committee 216 produces harmonized standards and test methods for assessment of the efficacy of disinfectants and antiseptics.

These ‘European Norm’ (EN) methods are widely used and accepted across Europe and PIC/S member countries in the validation of cleanroom disinfectants but are not specifically designed for the pharmaceutical industry.

To illustrate this, EN13697 the test method that is commonly used to validate cleanroom disinfectants and is arguably one of the most relevant methodologies is actually intended to demonstrate ‘activity of chemical disinfectants used in food, industrial, domestic and institutional areas’. These types of environments would typically have very different levels of soiling, bioburden levels and microflora compared to those typically encountered in a cleanroom environment.

Due to the lack of a specific test method designed for the pharmaceutical industry, the Validex™ method was developed with the consideration of this need, applying parameters suitable for the validation of cleanroom disinfectants and the criteria of the United States Pharmacopeia Chapter 1072 ‘Disinfectants and Antiseptics’ (USP<1072>)⁴. The Validex™ development has also considered variation in differing regional standards, such as the EN methods and AOAC/ASTM which includes variation in organism strains, biocide and inoculum volumes and test surface size. Further information on the Validex™ Program can be found within the November 2019 issue of Cleanroom Technology Article ‘Call for a global disinfectant standard’⁵.

Although there is a regulatory requirement for end users to validate disinfectants used in cleanrooms, there is no guidance on which specific methodology to use. Many end users opt to use modified versions of the EN standard methods as they are well designed and robust. They also choose to use acceptance criteria that reflect the requirements of pharmaceutical industry such as those given in USP <1072>.

The use of modified standards for disinfectant efficacy methods is a recognized approach, widely accepted by regulators. This same approach was taken in the development of the Ecolab Validex™ Program.

Following the introduction of Validex™, and after 6 years of successful implementation across a wide variety of life sciences sectors, Ecolab have further developed the data package to include the assessment of disinfectant wipes.

The incorporation of mechanical action (wiping) into the testing method is significant, as disinfectants used in the cleanroom will usually be applied using some form of mopping or wiping. In 2015, a new method EN 16615: Quantitative Non-Porous Surface Test with Mechanical Action⁶ for wipes used in the medical area was introduced to demonstrate the efficacy of disinfectants and the effect of mechanical action on this eUicacy.

In August 2022, the update to EudraLex Vol.4 Annex 1 was issued. This directed end users to validate disinfectants ‘in the specific manner in which they are used’. As a result, this method has become a popular choice for validation of cleanroom disinfectants.

In 2020 EN 16615 was included in the European Chemicals Agency (ECHA) Technical Agreement for Biocides v2.2⁷ as the recommended method for efficacy testing of disinfectants applied in practice via mopping and wiping. Despite this endorsement, the EN 16615 standard remained primarily designed to assess application of mopping and makes use of a heavy, standardized weight of application (2.4kg) to mimic the pressure applied when using this technique. As weight application (pressure) is not a common factor assessed in efficacy evaluation of biocide wipes, the further development of Validex™ to assess a wiping motion also investigated this factor. The method development work found that a heavier application pressure increased the level of microbial kill observed. For this reason, a lighter, more appropriate weight to reflect hand wiping was established for the Validex™ method, which can be utilized for both mopping and wiping application.

As already touched on, and in line with other EN standards, EN 16615 is not designed specifically for the pharmaceutical industry. As a result, its standard methodology incorporates a high initial bioburden (starting inoculum) and dictates wiping in a bi-directional motion. Whilst EN 16615 may be considered more representative of 'real world' disinfectant application, the best practice application techniques typically used within the cleanroom environment are not incorporated into its methodology.

The Ecolab new wipe disinfectant validation method is specifically designed for the cleanroom environment. The Validex™ wiping method incorporates best practice application technique for the cleanroom (unidirectional overlapping wiping). Other factors assessed within the method development, designed to better represent cleanroom techniques include optimal folding, layers, pressure and saturation of wipes. The Ecolab method has also been developed to assess the removal and movement of organisms as well as kill (log reduction).

In combination with the microbial efficacy assessment, it is important to consider the test methods validity. One of the common pitfalls that can result in ineffective efficacy evaluation is failure to demonstrate how certain stages of a test method can impact microbial viability and detection. Without supporting method validation, it is not always possible to ensure that the microbial log reduction detected is due to biocidal efficacy alone, or due to steps within the test method itself that could influence this result.

As an example, most test methods, including the EN standards and Validex™, will include a stage of organism inoculation onto a surface which is then dried prior to biocide application. Understanding the impact of this drying step is crucial in ensuring enough viable organism remains on the test surface to demonstrate desired log reduction, but also to ensure that the organisms are not being killed by this process alone rather than the eUect of the disinfectant that is subsequently applied.

The level of starting inoculum and drying conditions have been assessed and developed for Validex™ to ensure a valid and repeatable test can be achieved. Further test validity will include assessment of a neutralizer capable of stopping biocidal action beyond the validated contact time whilst not causing any toxic effect to the test organism - all considerations made within the development of Validex™.

With a combination of both non-mechanical action and mechanical action (wiping) studies, the Validex™ Program provides a comprehensive data set, using a wide variety of common cleanroom surfaces and microbiological flora as summarized in Figure 1.0.