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

In addition, EudraLex The Rules Governing Medicinal Products in the European Union, Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use Annex 1 Manufacture of Sterile Medicinal Products (2022) Section 4.34 states “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 in-use expiry periods of prepared solutions”.

End users should seek out reputable disinfectant manufacturers that sell disinfectants intended for use in  cleanroom areas as described above. 

In order to sell products which make a label claim of bactericidal, fungicidal or sporicidal efficacy there is a legal requirement for manufacturers to demonstrate that their disinfectants are effective. This is achieved by performing disinfectant efficacy testing using a recognised methodology such as the CEN, AOAC or ASTM test methods.

The European Committee for Standardization (CEN – Comité Européen de Normalisation) set up Technical Committee 216 to produce harmonized standards and test methods for assessment of the efficacy of disinfectants and antiseptics. These “European norm” (EN) standards include a mixture of suspension tests and surface test methods to cover bactericidal, fungicidal, viricidal, sporicidal and mycobactericidal activity. The ‘European norm’ (EN) methods are widely used and accepted across Europe and PIC/S member countries. 

Manufacturers perform efficacy testing of their disinfectant products to provide data for in-country registration, registration with the Biocidal Products Regulation (BPR – [EU] 528/2012) or label claims. This testing will use a recognised method such as the EN methods that have not been modified. This testing gives an outline of the product performance and is useful for end users to be able to compare the performance of different biocides in the market or ensure the disinfectants meet a minimum level of efficacy. 

Manufacturers data is usually based on in-vitro (suspension) tests and/or surface tests that are performed in full adherence to the standard tests available, meaning use of defined parameters, such as surfaces, organisms and contact times. It should be noted that the EN standards were developed to test disinfectants used in a wide variety of industries including hospitals, food processing plants and industrial areas where the requirements for performance (efficacy), material compatibility or disinfectant residue levels may be very different when compared to the requirements of a cleanroom. 

As discussed earlier, 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 standard methods (such as the EN 13697 surface test) as they are well designed, robust, repeatable and have suitable controls built into the method. Use of modified standards for disinfectant efficacy methods are also widely recognised and accepted by regulators. 

When numerous disinfectants at various concentrations and/or contact times are being assessed, use-dilution or suspension methods may be useful as screening tests to pre-select the most appropriate disinfectant/concentration/contact time combination to progress for subsequent surface testing. Basic screen testing usually takes the form of quantitative suspension tests (e.g. tests based on EN1276:2019; EN1650:2019; EN13704:2018). In these suspension tests a known number of organisms is mixed in solution with interfering substance, and the test disinfectant added for a set contact time. Following neutralization of the disinfectant, the number of organisms inactivated is calculated. 

End users can compare supplier suspension data if performed by an accredited laboratory for disinfectant selection, however, these tests do not necessarily predict how the disinfectant will perform on contaminated surfaces.

“In order to demonstrate compliance with pre-determined acceptance criteria specified in terms of a log reduction, it is necessary that a quantitative test method is used.”specific conditions.”

Disinfectant surface testing is the most common type of testing performed for the purpose of validation of cleanroom disinfectants and involves application of a known number of organisms, mixed with an interfering substance onto a hard, non-porous surface. After the inoculum has dried the disinfectant is then applied and then left for a set contact time. Following neutralization of the disinfectant and recovery from the surface, the number of organisms inactivated is calculated. 

There are a variety of internationally recognized surface test methods available: both qualitative and quantitative; with or without mechanical action. The reduction in the number of test organisms caused by a disinfectant is generally expressed as decimal logarithm (log), commonly referred to as the log reduction. In order to demonstrate compliance with pre-determined acceptance criteria specified in terms of a log reduction, it is necessary that a quantitative test method is used. 

EN 13697: 2024 is a surface test performed on stainless steel coupons as described above. For a disinfectant manufacturer the minimum requirement of biocidal activity for this test is a 4-log reduction for bacteria and a 3-log reduction for fungi. This is less stringent than the requirements of the Quantitative Suspension Tests (i.e. a 5-log reduction for bacteria and 4 log reduction for fungi). However, this is to reflect the normally expected reduction in the susceptibility of organisms to disinfectants as a consequence of being dried onto the surface. 

It has been recognized that the conditions used to dry the organisms onto the surface of the stainless-steel coupons (37°C for one hour) can result in a loss of viability with Gram negative and yeast cultures. This can be a significant factor and can result in a sufficient loss of viability that it may render it difficult to satisfy the surface test acceptance criteria (4 log reduction). 

One disadvantage of the EN 13697 test method is that it does not take into consideration the manner in which disinfectant will be applied by the end user. The incorporation of mechanical action 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 EN method to demonstrate the efficacy of disinfectants was introduced that included the effect of mechanical action. This new method is EN 16615:2015, for the assessment of disinfectants used in the medical area, and is both quantitative as well as incorporating mechanical action by means of using a wipe to apply the disinfectant to a series of test fields. The method may also be adapted or modified by end users of disinfectants to test biocidal efficacy on a variety of hard, non-porous surfaces. This method has become a popular choice for validation of cleanroom disinfectants and in 2020 was included in the ECHA Technical Agreement for Biocides (December 2023) mopping and wiping.

As previously mentioned, regulatory authorities do not demand that end users of disinfectants comply with unmodified standard test methods. However, validation studies based on adapted published methods appear likely to be accepted as adequate proof of disinfection efficacy, provided they are relevant to the processes in question. The main advantage of using an internationally recognized standard test is that the method is usually “self-validating” due to the inclusion of appropriate system suitability checks and controls that are part of the method, therefore eliminating the need for in-house method validation. 

When end users are writing their protocols there are some test parameters that should be considered for modification to best reflect when disinfectants are used in a cleanroom, these may include: 

• different types of test surface,
• predominant microorganisms isolated from the facility environment, 
• contact times,
• starting inoculum levels,
• acceptance criteria (the majority of end users commonly use the rationale given in USP Chapter <1072>),
• test temperature (i.e. use of product in temperature sensitive environments),
• test reagents (such as a suitable neutralizer for the product being tested).

It is important to note that whilst there is no obligation to test using the unmodified standard method, deviation from the standard should be documented with an appropriate rationale. Modifying the test methods may more accurately reflect actual usage conditions but may also adversely affect test reproducibility. In some instances, it may be more appropriate to modify the test procedures to make them more relevant to particular applications rather than rigidly applying official methods that may not be entirely relevant to the cleanroom environment. 

EN 14885:2022, which describes application of the EN standard methods, provides further guidance with regards to best practice for the control of potential imprecision due to test modification.

Conclusion

the particular methods chosen for a disinfectant efficacy validation depend on the needs of the end user and the intended use of the disinfectant. A validation study must ensure that the disinfectants selected are the most effective for supporting a facility’s contamination control strategy (CCS). Surface testing based on EN 13697 and/or EN 16615 may be considered the most relevant tests to conduct as they are most reflective of end user application, but will often include modification of test parameters, surfaces and organisms. In addition to validation efficacy tests subsequent field trials (also known as ‘Phase III trials) should be performed  to further support implementation via environmental microbial data.