Step by step disinfectant evaluation

The evaluation and validation of disinfectant products used in the pharmaceutical industry has been closely attended by the regulatory authorities in recent years. Organisations are expected to provide adequate data to support their cleaning and disinfection programmes, including disinfectant efficacy data relevant to the process in question.

Until relatively recently there has been limited published guidance on suitable test procedures for use in such a validation study; although alternative disinfectant test methods exist, all have limitations and tend to be narrow in scope. In 1989, the European Committee for Standardisation (CEN) set up technical committee 216 to produce harmonised test methods for disinfectants and antiseptics. Under the main committee were three working groups covering different applications; only the methods developed by working group three will be discussed here as they are applicable to the pharma-ceutical industry. The methods developed by the other two working groups are applicable to the medical and veterinary industries. The main purpose of the family of CEN standards is to demonstrate that disinfectants have biocidal activity under defined conditions and establish the effectiveness of the products under simulated in-use conditions. The EN test methodology has a modular approach, as follows: Phase 1 – Basic suspension tests Phase 2, Step 1 – Quantitative suspension tests Phase 2, Step 2 – Surface test and hand wash/rub methods Phase 3 – Field tests under practical conditions The standards provide a series of test methods that can be applied to a variety of cleaning and disinfection practices, with each individual test method contributing data towards the overall picture of how a disinfectant product will potentially perform in practice. The phase 3 tests are yet to be published.

Basic suspension test EN 1040 and EN 1275 describe basic suspension tests for establishing whether or not chemical disinfectants have bactericidal or fungicidal activity under defined laboratory conditions. The test procedures use Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538 for the bactericidal test, and Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 for the fungicidal test. The pass criteria for this test are at least a 5 log reduction within 60 minutes or less at 200°C for bacteria and at least a 4 log reduction within 60 minutes or less at 200°C for fungi. If a product meets the criteria, then it is capable of reducing the number of viable organisms under the conditions defined by the standard. This test is a preliminary screening to reduce the number of products taken on to the next stage of testing¹, and provides data more relevant to the disinfectant manufacturer than the end-user. It is worth noting however, that the standard 60 minute contact time is rather generous, although the methods do provide the option of using alternative contact times. Also, a limited range of organisms are used, including one gram positive organism and one gram negative for the bactericidal test, a yeast and the conidiospores of a filamentous fungus for the fungicidal test.

Quantitative suspension test BS EN 1276 and BS EN 1650 are based on the same principle. They are intended to take into account some of the environmental parameters, such as water hardness and organic soiling, as well as contact time, all of which may influence the performance of the disinfectant under in-use conditions.² Testing is performed under clean or dirty conditions using sterile filtered 0.3g/l and 3.0g/l solutions of Bovine Serum Albumin (BSA) respectively as interfering substances. BS EN 1276 offers two methods for evaluating bactericidal activity. Where possible the dilution neutralisation method should be used, but for products that do not respond to neutralisation a membrane filtration method is adopted. In both cases validation is performed in parallel to the test. These additional controls are one of the key features of the quantitative suspension tests and are performed at three levels: • Disinfectant neutralisation validation determines the effectiveness of the neutraliser and is vital as artificial results will be obtained, overstating the efficacy of the product, if the neutraliser is not effectively neutralising the activity of the disinfectant • Neutraliser toxicity validation assesses the toxicity of the neutraliser to the organisms • Experimental conditions validation ensures that the experimental conditions are not having a detrimental effect on the organisms. In the dilution neutralisation method a bacterial suspension is added to an equal volume of BSA solution. Disinfectant solution is then added. When the specified contact time has elapsed the disinfectant activity is neutralised. The solution is enumerated using standard microbiological techniques and the result compared with that of the original bacterial suspension. The membrane filtration method is performed in an identical manner until the contact time has elapsed. At this point an aliquot of test mixture is transferred to sterile diluent contained in the filtration apparatus. Following filtration and rinsing the membrane is transferred to a Tryptone Soy agar plate for incubation and enumeration. For both methods the log reduction achieved is calculated taking dilution factors into account. A log reduction of 5 or greater is required to pass the test, using the specified organisms – Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, E. coli ATCC 10536 and Enterococcus hirae ATCC 10541. BS EN 1650 is identical in principle to BS EN 1276. The key points of difference include the use of a lower target inoculum strength - Malt Extract agar in place of TSA - incubation conditions suited for fungi and a lower pass criteria of a 4 log reduction or greater. Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 are the organisms specified in EN 1650. EN 13704 is the quantitative suspension test for the evaluation of sporicidal activity, which is evaluated using a spore suspension of Bacillus subtilis ATCC 6633. It is performed in the same manner as the other suspension tests, but generally using only "clean conditions" and a contact time of 60 minutes. In the standard, sporicidal activity is defined as the capability of the product to produce at least a 3 log reduction in the number of bacterial spores belonging to the test strain of B.subtilis. The acceptance criteria for the sporicidal method are less stringent than the other suspension tests, and certainly in a pharmaceutical cleanroom it would be virtually impossible to achieve a contact time of 60 minutes due to the number of air changes. Suspension testing can offer a reliable means of evaluating the efficacy of disinfectant products under laboratory conditions; but as with many microbiological methods, repeatability and reproducibility can be a problem. It has been shown that the use of standardised BSA improves reproducibility.³ This is one of the many reasons why the majority of organisations choose a contract testing laboratory, such as MGS, to perform these methods, while a company's experience and routine performance of these methods will obviously help improve reproducibility.

Surface test method EN 13697 surface test is performed under either clean or dirty conditions represented by the use of sterile filtered 0.06g/l or 0.6g/l solutions of BSA respectively, both containing 1g/l tryptone. For pharma-ceutical applications, especially the manufacture of sterile products, where the level of soiling is expected to be low, it is appropriate to test only under clean conditions. An inoculum suspension appropriate for the organism under test is added to an equal volume of BSA solution. A known small volume of the mixture is transferred to a clean, sterile sample of the surface under test, and allowed to dry under suitable conditions. Once dry the residue on the surface is treated with disinfectant solution of the appropriate concentration and allowed to stand for the required contact time. The surface is then transferred to a previously validated neutraliser solution and agitated to dislodge any remaining organisms from the surface. The solution is enumerated using standard microbiological techniques and the result compared with that obtained in a control experiment, in which water is used in place of the disinfectant solution. The official EN 13697 surface test is performed with specified type cultures – the same cultures specified in both EN 1276 and EN1650 – and a contact time of five minutes for bacteria and 15 minutes for fungi. It has pass criteria of greater than a 4 log reduction for bacteria and greater than a 3 log reduction for fungi. Surface testing presents a more stringent challenge to disinfectant products and is a closer representation of practical in-use conditions. Organisms tend to be less susceptible to disinfectant action when they are attached to a surface than when in suspension,^4,5 although the drying time in the method is relatively short and hence probably allows only a loose attachment.⁶ The drying process also needs to be carefully controlled as it can result in a loss of viability of the organisms and make it difficult to achieve the required log reduction, Gram negative organisms are particularly susceptible. The CEN working group is currently reviewing this, along with other considerations such as the inclusion of a mechanical action step.

Specific applications The standard uses stainless steel as the surface carrier, but most organisations also include other surface materials used in their facilities when validating their disinfection procedures. The results can differ considerably from surface to surface, both in terms of the performance of the disinfectant and also the ability to recover the organisms from the surface. It is possible to adapt the CEN standards to specific applications by changing parameters such as contact time, interfering substance and temperature; also, organisations often choose to use purified water or water for injection from their own facility, rather than water of standard hardness to reflect in-use conditions. When the standards are adapted in this way it can often provide more useful information; however, only when they are included in addition to the standard requirements can a claim of compliance to the standard be made. Care must also be taken with such adaptations as they may have a detrimental effect on the reproducibility or repeatability of the test; this particularly applies to using alternative interfering substances. It is a regulatory expectation, when using all methods to validate a disinfection programme, to include environmental isolates from the facility in question as this provides useful information on the expected performance of the disinfectant in practice. However, if the requirements of the standards are not met using the environmental isolates then a number of questions often arise. For example, "should this data be presented to the regulatory authorities?" or "does this mean we should not use this product?" However, failure to comply with the standard using environmental isolates does not mean the product in question does not have any biocidal activity against the organism, as some of these test methods present quite a stringent challenge. Organisations need to consider all the relevant data and make a reasoned justification to support their individual disinfection programme and product choice. Another question that is frequently asked is "how often should we perform these tests?" The answer is that organisations need to consider carefully the requirement for re-validation. A full validation programme can be a costly exercise, and is routine validation really necessary? Environmental isolates do change over time, both in nature and in susceptibility, and the regulatory authorities expect validation to be kept up to date; but again, whether full validation is repeated or whether part re-validation is performed is dependant on individual circumstances. Other factors should also be considered before embarking on a full validation programme, such as product longevity. Since the introduction of the Biocidal Products Directive (BPD), which is designed to ensure that disinfectants placed on the market are both safe to use and safe for the environment, all active biocidal substances that manufacturers wish to continue using must have been identified and notified to the relevant local authorities. From September 2006 any disinfectant using an active substance that has not been notified must be withdrawn from the market. It would also be prudent to have an agreement in place to ensure any changes the manufacturer plans to make to the formulation, process or packaging are notified in advance to enable the requirement for re-validation to be considered. The CEN standards provide a useful basis for disinfectant validation, and although alternative methods could be used for assessing disinfectant efficacy, following the same basic methods allows not only direct comparison between products but also comparison across various different laboratories. The adaptability of the methods - numerous validation studies based on the CEN methods have been accepted by both the European and US regulatory authorities - allows end- users to customise the methods to their specific requirements.