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Many of these methods have been in use for decades and have a long history and extensive data for multiple classes and types of disinfectants.  This data is very valuable for a regulatory agency when evaluating new products and comparing performance between new and older products to ensure safe and effective use. However, many of these methods predate advancements made in microbiology method development and can be limited in their scope outside of base claim generation. For example, most of the EPA methods are qualitative in nature and provide only a pass or a fail as an experimental endpoint.  Although helpful to determine whether a formula is effective or not, the qualitative methods offer little other information and are challenging to use for comparisons or relative evaluations. Further, the methods present a very high challenge to ensure effectiveness in multiple application areas, from industrial to hospital environments. They may be too great of a challenge for what might be encountered in a cleanroom or controlled environment.  A starting titer, well over one million microorganisms is not representative of a cleanroom or controlled environment’s bioburden levels.

In Europe, the registration process falls under the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) guidelines which also require demonstration that the products are safe and efficacious ⁽⁸⁾. Like the US EPA and corresponding AOAC and ASTM methods, the European Norms also have standard methodologies established for disinfectants and require multiple phases and steps based on labeling and product usage. The EU methods include both basic suspension-based studies for initial evaluation of disinfectant activity via a step one approach and then quantitative hard surface methods for specific applications for step two, phase two in the registration process. Because these methods are newer and provide log10 reduction values versus simply a pass/fail result, they are more easily adaptable to end-user applications. Still, do not perfectly mimic the real-world conditions or challenges met in a cleanroom or controlled environment ^(6-8).

For the rest of the world, the test methods and regulations can differ by region. Regulations may differ in the rest of the world and may conform in part, in whole, or not at all with US and EU guidelines. However, the desire for disinfectants that work as intended, with minimal safety concerns, and well-documented efficacy claims, remains the same. The testing requirements and methods are similar, however, and have the same limitations regarding real-world applicability whether they are more qualitative or quantitative in nature.

Additional considerations must be evaluated to provide labeling and use instructions incorporated into the required test methods.  These include the use of organic soil during testing, hard water for disinfectant preparation, and the establishment of contact time and temperature.   Developing disinfectants that can meet strict regulatory requirements is a challenge found worldwide. One of the most challenging aspects is consistently and reproducibly passing the various efficacy standards that are required by the different regulatory authorities. Although the methods can have a long history of use, with demonstrable success to ensure safe and effective formulations, their use does not always translate to other applications. Often, the methods are focused on reference strains and surfaces, require extremely high microbial challenges beyond what is found in the pharmaceutical environment, and can have pass/fail endpoints that do not apply to real-world situations.

End-user qualification testing

The regulatory guidance entails (bio)pharmaceutical manufacturers to qualify the disinfecting agents’ wet contact time on specific surfaces and microorganisms^(9-11). Norms and industry technical documents share guidance for in vitro and in situ testing^(12-16), as seen in Figure 1.

2. End-user disinfecting agent in vitro qualification

The pharmaceutical manufacturer is responsible for evaluating the optimal wet contact time, use dilution, use dilution expiration, based on the supplier’s data, their cleanroom surfaces, and most frequent isolate(s) from their historical Environment Monitoring (EM) data ^{(2, 5-7, 9-15)}. The suspension qualification demonstrates the efficacy of the disinfecting agent to inactivate a planktonic microorganism at a determined contact time. The disinfecting agent efficacy from the suspension test may differ when a microorganism is adsorbed on a surface. Therefore, coupons that are representative of surfaces in the cleanrooms should be tested in vitro.

In vitro qualification of a disinfecting agent is based on the ⁽²⁾: 

• wet contact time, 
• log reduction acceptance criteria, 
• microorganism type, 
• disinfectant concentration, 
• water quality, 
• clean or soiled surfaces,
• surface types,
• Temperature of application.

The in vitro testing will confirm the efficacy of the disinfecting agent against a microorganism type adsorbed on a specific surface at a defined wet contact time. In-situ testing (real-world) is essential to assess the impact of the element listed below on the disinfecting agent efficacy ⁽²⁾:  

• Temperature and humidity in the cleanroom: that may interact with the disinfecting agent kinetics of kill
• Air exchange in the cleanroom: that may impact the time of the disinfecting agent to dry
• Application technique (e.g., vaporization, spraying, mopping, wiping): that impact the removal of residue or microorganism on the surface
• Surface cleanliness: that may interact with the disinfecting agent’s efficacy.

3. Disinfectant Efficacy Testing

Efficacy is demonstrated by testing the disinfectant efficiency in reducing the microbial bioburden in either suspension (planktonic state) or on cleanroom surfaces (sessile) to an acceptable level.

The points to consider when developing a disinfectant efficacy test (DET) are: 

• Log reduction: A disinfectant efficacy study is built on a microorganism logarithmic reduction that a disinfecting agent can achieve. A different set of logarithmic reduction is proposed for in vitro disinfecting agent qualification. ⁽³⁾

There is no compendial or harmonized regulatory requirement on the logarithmic reduction required for pharmaceutical manufacturers⁽¹⁵⁾. Therefore, pharmaceutical manufacturers should define the most appropriate log reduction based on their activities and historical EM data analysis. In many cases, the USP <1072> log reduction criteria are most suitable for the pharmaceutical industry. The EN documents are subject to various industries (food, industrial, domestic, and institutional areas), including pharmaceutical operations. Therefore, it is logical to observe that pharmaceutical manufacturers use a different type or combination of standards (Figure 2).

ATCC microorganism: A disinfecting agent should be qualified against ATCC microorganisms⁽⁴⁾. Such qualification may be transposable from site to site or from supplier testing if the coupons tested to represent the cleanroom’s surfaces ⁽¹⁵⁾.

(1) for specific application addition strains may be chosen if required: Salmonella typhimurium ATCC 13 311; Lactobacillus brevis DSM 6 235; Enterobacter cloacae DSM 6 234; Saccharomyces cerevisiae (for breweries) or ATCC 9 763 or DSM 1 333; Saccharomyces cerevisiae var. diastaticus (for breweries) DSM 70 487. (2) note that ASTM E2197 list virus as organisms that could be tested depending on the applications.

• Isolate microorganisms (in-house microorganisms): To adequately evaluate the performance of a disinfectant in a real-world environment, it is essential to consider environmental isolates from the controlled environment in which the product is used. These isolates should be collected from environmental monitoring and test cultures selected based on the frequency of isolation, location, and general microbiological characteristics ^(4-15). For a broad-spectrum evaluation of in-house microorganisms, representative microorganisms from different classes, i.e., Gram-positive, Gram-negative, fungal or bacterial spores, may need to be considered.
• Disinfectant concentration: The definition of the optimal concentration of use should be designated by the supplier. It is advised that the end-user comply with the recommended supplier concentration.  The end-user may elect to evaluate a range of concentrations and their efficacy against a set of microorganisms. 
• Water quality: during the qualification of the disinfectants pharmaceutical manufacturers may elect to choose the lowest water quality used for the disinfectant dilution.  It is considered good practice to use purified water or water for injection for disinfectant dilution. 
• Clean or soiled situation: the draft guidance on the BPR supports that cleanroom end-users not including a soil load “As an exception to the rule, products to be used in cleanrooms do not require additional soiling in the test. A cleanroom has a controlled level of contamination that is specified by the number of particles per cubic meter at a specified particle size. The soiling level in cleanrooms is so low that even testing under clean conditions for the EN tests is still overdosing on soiling compared to cleanrooms. For these uses the high load of test organisms can be seen as soiling. Tests without soiling will only be accepted when the label states the specific use in clean rooms which are classified according to ISO 14644-1 in class 1 to 9 or according to GMP EU classification in Grade A to D. Generally, soiling will reduce the efficacy of the disinfectant, and where soiling is present, longer contact times, higher concentrations, pre-cleaning or a combination of these elements may be necessary.”⁽¹⁸⁾. A visual inspection of the cleanroom surfaces should justify either a ‘clean’ or ‘dirty’ condition to test⁽¹⁶⁾. 
• Surface types and selection: The disinfectant qualification involves in vitro coupon (small part of surfaces) testing.  The material of construction and porosity of the surface will affect the efficacy of the disinfectant by influencing the wetness of the microorganism to be killed. The coupons are spiked with a known amount of microorganism aliquot, and the disinfectant is then left for a predefined wet contact time. Then, the disinfectant is neutralized, and the log reduction is calculated. 

It can be challenging to choose the representative and correct surfaces to test. There is no harmonized approach between pharmaceutical manufacturers; some may select the surfaces based on: 

1.The most common surfaces in the cleanrooms.

2.A grouping matrix that includes surfaces of similar porosity and material of construction. 

3.An assessment (Figure 3) that takes into consideration several elements, high probability to the parameters indicates that the surface should be tested: 

a) the surfaces prevalent in the cleanrooms. The surfaces tested should be in good condition; therefore, surfaces with poor conditions must not be integrated into the testing⁽¹⁵⁾.

b) increase in surface porosity and roughness can be a challenge in disinfection efficacy. 

c) cleanroom classification where the surface sits. It is critical to ensure that disinfection of surfaces in critical classification (Class 100 and 1,000) are optimal to prevent contamination. 

d) surfaces that are often touched by the manufacturing personnel when activities or interventions are frequent. 

• Temperature of application: various standards mandate at least to test the disinfecting agent at a temperature within 15° to 25°C^(7-27). The efficacy of a disinfecting agent at a defined concentration may increase with elevated temperature and vice versa⁽²). Pharmaceutical manufacturers should explore testing their disinfecting agent at different temperatures when most cleanrooms are maintained at temperatures below ambient.

4. Requirements and minimal information in protocol

The goal of in vitro efficacy testing is to demonstrate that the chosen chemistries are effective against environmental isolates on representative cleanroom surfaces, using test conditions that mimic standard operating procedures (water quality, presence of organic soil load, and use-dilution hold times– Figure 4).

5. Conclusion

Disinfectants are a critical element in cleanroom contamination control and must be well-suited for the applications for which they are intended.

Testing a disinfecting agent during product development can differ greatly from the testing required to validate a finished formula by the end user. The testing performed by the disinfecting agent manufacturer is to register the disinfecting agent and claims based upon a specific wet contact time, microbial kill, and the disinfecting agent concentration. In contrast, end users achieve disinfectant testing in vitro and in situ to demonstrate bioburden control in pharmaceutical cleanrooms. Therefore, the testing conditions may differ from the one used by the supplier or the end user.