A practical approach to disinfectant tests

With increased interest from the regulatory bodies on the validation of cleaning procedures and more specifically the efficacy of disinfectant and sanitising agents, an agreed framework of methods and a harmonised approach has been adopted. However, exactly how harmonised the approach is and whether there scope for methods that would better meet the specific requirements of the pharmaceutical industry are the issues discussed here.

Directive 98/8/EC of the European Parliament and of the Council defines biocidal products as "Active substances and preparations containing one or more active substances, put up in the form in which they are supplied to the user, intended to destroy, deter, render harmless, prevent the action of, or otherwise exert a controlling effect on any harmful organism by chemical or biological means" 1.

To the pharmaceutical industry such products are known as disinfectants and are key to the sanitisation of production areas in which there is a clear and defined regulatory requirement, to control the level of potential microbial contamination to a minimum. The Good Manufacturing Practices (cGMP) for medicinal products details the importance of hygiene in the manufacture of pharmaceutical products. Where critical processes are involved, validation of cleaning procedures is required. An adequate cleaning and disinfection programme must be in place to minimise or remove the favourable conditions required for microbial growth. Cleaning and disinfection is key to pharmaceutical product quality and more importantly, patient safety.

Regulatory authorities are increasingly asking for validation data to support sanitisation and disinfection procedures used in cleanroom areas.2 The challenge for the testing is to provide a means of assessing the ability of the disinfectant to render a situation or surface microbiologically safe for intended purpose. The selection process will require the 'effectiveness' of the chosen disinfectants to be determined.

The BS EN methods established by the European Committee for Standardisation (CEN) Technical Committee (TC 216) are used in a three-phased approach to determine the effectiveness of the disinfectant product. However, the scope of these BS EN methods has been developed to cover a wide range of industries. Not all aspects of the methodology within the CEN methods are suitable or applicable to the pharmaceutical industry. Therefore, when the pharmaceutical industry applies these methods to its disinfectant validation programme, the method is "modified". This then leads to the issue of non-compliance with the CEN methods by applying a modified method, even though it is more meaningful to the pharmaceutical industry.

This article looks specifically at the BS EN methods and focuses on the areas where the pharmaceutical industry modifies the methodology and proposes an approach that could be adopted by the industry for disinfectant efficacy testing.

TC 216 & AOAC

There has been greater awareness and concern by regulatory authorities in the area of cleaning and disinfection. In particular the validation, or more appropriately the efficacy, of disinfectants used in the pharmaceutical industry has been, and continues to be, a hot topic with the regulatory authorities.

In 1990 the technical committee, TC 216, was created with the objective of standardising the various methods that existed at that time for testing the activity of disinfectants and antiseptics, more latterly with the adoption of the Biocidal Products Directive this has changed to "take into account a perspective of the benefits offered, the possible environmental and human safety risks and primarily for disinfectants, their efficacy".3 The resultant activity of this committee has been a tiered approach to disinfectant testing which starts with assessing the biocidal effectiveness of the disinfectant through to the real life application of the disinfectant following a standard operating procedure.

The aim of the TC 216 approach is clear; it establishes whether a product has an antimicrobial activity and meets with the evaluation requirements of Directive 98/8. An application of this testing strategy can also be used as a means of quantifying the efficacy of the products under the Medical Devices Directive.

In the US, the AOAC (association of analytical communities) methods for disinfectant efficacy testing are available and, like the European CEN methods, are applicable to a wide range of industries. Deficiencies have been identified with the AOAC methods used to assess the microbial activities of chemical disinfectants on medical devices and environmental surfaces.4 It is suggested that the AOAC surface test attempts to answer too many questions through a single design. Springthorpe and Sattar proposed a quantitative test through a tiered approach, similar to the CEN approach.4 Furthermore, the general information chapter in the USP-NF 29, chapter 'Disinfectants and Antiseptics' (1072), states that the current process for the registration of a disinfectant within the US, does not address how disinfectants are used in the pharmaceutical, biotechnology and medical device industries. The USP general chapter offers guidance and background on a disinfectant efficacy testing process with information relevant to the pharmaceutical industry (see table 1).

BS EN methods

The CEN standards consist of a number of BS EN methods each designed to test disinfectants in a phased approach (see table 2).

Disinfectant manufacturers would have expected, as a minimum, to perform the phase 1 tests, BS EN 1275 and BS EN 1040. Martinez proposed that disinfectant manufacturers would perform the qualification of a disinfectant by means of suspension testing.5 Pharmaceutical manufacturers then would perform surface testing to develop procedures for the sanitation processes.

For the purposes of this article only phase 2, step 1 suspension tests and phase 2, step 2 surface tests will be considered.

The general principle of the suspension methods are along the lines of a suspension of organisms (bacterial, fungal or sporicidal) are added to a prepared sample of the disinfectant, diluted in hard water. After a defined contact time at 20ºC, an aliquot is taken and the bactericidal, fungicidal or sporicidal action is neutralised. The surviving organisms are quantified and the reduction in viable counts calculated.

The principle of the surface test is similar to the suspension test except that the organisms (excluding sporing organisms) are applied to stainless steel and dried. The number of surviving organisms is quantified and compared to a control test using water in place of disinfectant.

It is evident that the current methods offer a reliable means of evaluating the efficacy of disinfectant products under laboratory conditions. However, laboratory-based evaluation of disinfectants cannot guarantee field performance. It has been shown that real life conditions are variable and almost impossible to standardise.6

There are a number of areas within the standards where a review, in line with the current practices in the pharmaceutical industry is possible. These main areas would be; contact time, use of environmental organisms, clean/dirty conditions of the test, use of hard water and the surfaces used in the BS EN 13679 test. Furthermore, other areas for review for BS EN 13679 could include the drying time in relation to biofilm formation and the physical cleaning process. These points as well as re-validation are considered in more detail below.

Contact time

A contact time of 60 min is required in the BS EN 13704 test but in a cleanroom environment a 60 min contact time would be unachievable due to the air changes within the room or the need for constant repeat application to keep surfaces wet. The tests should be modified to mimic, as closely as possible, what is actually performed in the production areas.

Environmental organisms

The reference organisms described in the standards cover the required ranges for vegetative and sporing bacteria, yeasts and moulds. However, these organisms are grown under ideal conditions, i.e., within a nutrient-rich environment. It is known that many of the target organisms for the cleaning and disinfection process within the manufacturing facility of a pharmaceutical are stress-survivors usually growing under nutrient-poor conditions.

Some bacteria or fungi may also be a problem in manufacturing processes where specific substrates are used. Moreover, it has been described that disinfectants are less effective against higher concentrations of organisms used in the laboratory challenge tests than they are against the number found in cleanrooms4,7, in addition environmental contamination organisms against which disinfectants are required to be specific are likely to be site-specific.8

The reference organisms used in the standards do not accurately mimic either in terms of viability or growth conditions the organisms likely to be recovered from the environment. A representative range of environmental organisms should be used in the tests but it is likely that costs would be too prohibitive if reference and environmental organisms are to be used in these tests. Therefore, it is recommended that only actual environmental organisms should be tested against the disinfectants. This data will provide a true picture of the disinfectants sanitisation process. The data from the environmental flora should also be trended as this will provide indications as to whether any strains are proving to be resistant to the cleaning and disinfection programme over the course of time.

Hard water

The methods stipulate the use of hard water for the dilution of the disinfectants. Within the pharmaceutical industry, the quality of the water used in preparation of disinfectants is either purified water or water for injection (WFI). The application of WFI within the pharmaceutical industry is usually in classified clean areas, A and B with Purified water used in non-classified areas. Furthermore, cGMP guidelines Annex 1, paragraph 38 states, "disinfectants and detergents used in Grades A and B areas should be sterile prior to use"; therefore it is assumed that any dilution of the disinfectants should be performed with sterile water for injection or sterile filtered, e.g., into an aseptic filling facility. There are some products available now that are supplied ready-to-use, removing the need to dilute the product.

It is recommended, where appropriate, that purified water or WFI are used in the preparation and dilution of the disinfectants under test.

Test conditions

The phase 2, step 1 and 2 parts of the standards use the disinfectant under "clean" and "dirty" conditions. The purpose of the clean condition is to represent areas where there has been a satisfactory cleaning programme with minimal levels of organic and inorganic materials. The dirty condition is representative of an area known to contain levels of organic and inorganic materials.

Is it necessary for the pharmaceutical industry to apply clean and dirty conditions to the disinfectant efficacy tests considering how rigorous the industry applies its cleaning regime? Sterile manufacturing facilities have very strict criteria regarding environmental levels, so it is likely that any levels of organic or inorganic contamination present will be in trace amounts. Increased scrutiny from the regulatory authorities has meant that even non-sterile manufacturing organisations are producing a high standard of cleanliness within their facilities.

It is also important to consider that the pharmaceutical industry will remove a spillage, usually with detergents and water, depending upon the nature of the spillage, before disinfecting the area. Thus, any levels remaining would be in trace amounts. Detergents can remove up to 90% of vegetative bacteria from surfaces so it can be seen that there is clearly no requirement for the pharmaceutical industry to perform disinfecting efficacy testing under the dirty conditions.

There is a reasoned argument to continue to use the clean conditions, as there may be low levels of organic and inorganic material present. Instead of using bovine albumin, pharmaceutical manufacturers should consider using low levels of their product in the disinfectant efficacy tests to provide clean conditions. This would provide a more accurate representation of what could be occurring in the facility.

Surfaces

The surface test represents a more accurate "in-use" type of test. However, the surface method limits the test surface to stainless steel only. All surfaces within a pharmaceutical manufacturer's facility will be exposed to a cleaning and disinfection programme. In order to establish a representative overview of the effectiveness of the disinfectant(s) coming into contact with the surfaces, a pragmatic view should be taken. Where the surface is considered critical in terms of cleaning and disinfection, then it should be considered for disinfectant surface testing.

The USP chapter 1072 lists common materials used in cleanrooms that should be considered when setting up disinfectant surface testing. Along with stainless steel, other surfaces within the manufacturing facility should be tested. For example, different grades of vinyl and stainless steel, different types of plastic, glass from windows and vessels could be tested where there is a tangible risk of contamination.

Other considerations

Biofilm formation within the pharmaceutical industry is widely experienced and there are a number of papers and books on this subject9. Biofilm formation would normally be expected to result in microbial populations that are even less susceptible to disinfectant activity6. The drying time within the surface test is not sufficient to allow anything other than a short-term attachment and definitely not sufficient to allow biofilm formation10. Biofilms could form in pipework, e.g., water or clean-in-place (CIP) systems; biofilms can be reduced through increased contact times. This is an area of disinfectant efficacy testing that may need to be considered.

The cleaning and disinfection programme of any manufacturing or production facility usually involves a significant amount of training in the mopping and wiping technique to provide best practice under cGMP guidelines. However, the standards do not take the actual physical nature of the cleaning technique into consideration. It has been shown that wiping is much more efficient at reducing bioburden in combination with spraying11,12. In a recent paper Martinez5 suggested that, in order to reflect exactly how the disinfectant is used in the manufacturing environment, the actual cleaning process should be included as part of the sanitisation validation.

Another aspect of the standards not considered is the re-validation of the disinfectants. The golden rule within the pharmaceutical industry regarding re-validation is usually 12 months. However, an annual re-validation of disinfectants would be prohibitive due to time and costs. Any re-validation undertaken would be expected to be limited.

It is suggested that re-validation would only be required if one or a combination of the following were to occur; the disinfectants were changed or the formulation of the disinfectants amended, a significant change in the environmental flora away from the flora initially tested or a modification to the surfaces within the facility.

Scope for change

The International Committee on Harmonisation (ICH) is the steering group on h rmonisation of regulatory requirements. Its objective is towards the development of a single market for pharmaceuticals. There are now a number of areas within the pharmaceutical industry where it is harmonised. For example, there are a significant number of pharmacopoeial monographs and some methods within the pharmacopoeia that are now harmonised. It is not unrealistic to consider that disinfectant efficacy testing could become a focus for this or a similar group. Indeed, Springthorpe & Sattar concluded that changing the current AOAC methods to their proposed phased approach Quantitative Carrier Tests (QCT) would be the initial steps in a global harmonisation of disinfectant efficacy testing. Moreover, both European and US regulatory authorities have accepted the CEN standards are the basis for testing.