- Analytical Quality by Design: the required integration for Quality by Design
- Conception d’un isolateur de production. Du besoin utilisateur à la réalisation.
- Cahier Pratique – Essais de stérilité sous-isolateur – Qualification de performance et optimisation des plans de charge
- Advanced vaporized H2O2 decontamination technology for pharmaceutical isolators. Reduction of H2O2 decontamination cycle time using direct injection nozzles.
- Sécurisez le confinement de vos gants.
- Isolator Technology and Automation Enhanced Contamination Control in the Manufacture of Cell and Tissue Culture Derived Regenerative Medicine Products.
- The European approach to disinfectant qualification.
Contamination control is of great importance to healthcare facilities and to pharmaceutical cleanrooms. One way of ensuring the hygiene is maintained through a cleaning and disinfection regime. After a disinfectant has been chosen based on its chemical properties and expected performance/effectiveness, each disinfectant should be validated to ensure its efficacy.
EFFICACY IS DEMONSTRATED THROUGH PERFORMANCE TESTING TO SHOW THAT THE DISINFECTANT IS CAPABLE OF REDUCING THE MICROBIAL BIOBURDEN IN EITHER SUSPENSION (PLANKTONIC STATE) OR FROM CLEANROOM SURFACES TO AN ACCEPTABLE LEVEL(1).
The European approach for the evaluation of disinfectants differs slightly from the approach outlined in the USP <1072> or through the AOAC. This article outlines the European approach to disinfectant qualification.
The European standards were outlined by the European Committee for Standardization Technical Committee 216 (CEN TC 216) in 1991, which began with guidance on disinfectant selection (EN 7152 24) and the first European disinfectant standard was issued in 1997: BS EN 1276 for the quantitative suspension test and several other standards then followed. These new standards replaced former methods for disinfectant validation, such as the once dominant Kelsey-Sykes test. For a full list of European disinfectant standards, refer to Appendix 1 of this chapter. The standard European approach for disinfectant validation consists of a basic suspension test, a quantitative suspension test (with low and high levels of organic material added to act as ‘interfering substances’) and a two-part simulated-use surface test. The standard European approach for disinfectant validation is divided up into three phases:
1. Phase 1 → Basic Suspension Tests
2. Phase 2 → Part 1: Suspension and surface tests to simulate practical usage: Bactericidal and fungicidal (sporicidal and virucidal)
3. Phase 2 → Part 2: Surface test
4. Phase 3 → Field Trial
5. A separate phase exists for the validation of hand sanitizers
The basic suspension test is a simple test to determine if the test disinfectant possesses any antimicrobial properties against microorganisms held in suspension (that is the microorganisms are added to the disinfectant solution). The quantitative suspension and surface tests are tests to determine the most effective concentration and conditions for the disinfectant as a simulation of practical conditions. The field trials show the effectiveness of a chosen disinfectant in-loco conditions (the pharmaceutical cleanrooms). With each stage an important consideration is the selection of an appropriate neutralizer. A neutralizer counter acts any residual disinfectant and allows microorganisms to be recovered which might otherwise have been inhibited.
Basic suspension test
Phase 1 – Basic Suspension Test (Standards EN 1275 and EN 1040)
A suspension test is a test designed to measure the efficacy of a disinfectant against selected microorganisms in the planktonic state after a predetermined contact time. Two standards are published within Europe in order to examine this: EN 1040 to measure bactericidal activity and EN 1275 to measure fungicidal activity. The basic suspension test is a simple, limited test of the product and is performed in order to determine minimum standards. In many ways the basic suspension test only serves to confirm the manufacturer’s data within the testing laboratory. Indeed, many facilities elect to audit the manufacturer and to review the manufacturer’s data in lieu of conducting the basic suspension test at their own premises.
Before undertaking the test, the selection of a suitable sterile neutralizer is required. Selection involves spiking neutralizers of different activity with a range of microorganisms and measuring the recovery. The neutralizer with the optimal recovery should be selected. Some neutralizers have general properties, such as, lecithin. Other neutralizers are compatible with specific disinfectants, such as, polysorbate-80 for biguanides and sodium thiosulphate for hypochlorites.
The test evaluates the activity of a disinfectant against a range of microorganisms under conditions which simulate use. After challenging a disinfectant solution with a microbial population the mixture is plated out, after the required contact time, and the surviving microorganisms enumerated. No organic material is introduced to this test, unlike the quantitative suspension test described below. In addition to the microorganisms prescribed in the standards, the microbiologist may elect to include representative organisms isolated from the cleanroom environment.
Quantitative suspension test
Phase 2, step 1 – Bactericidal suspension test (Standard: EN 1276: 1997) and Fungicidial suspension test (Standard EN 1650: 1998)
The purpose of the quantitative suspension test is to evaluate the activity of a disinfectant against a range of microorganisms under conditions which more closely simulate practical use. The practical conditions make the test more sophisticated than the basic suspension test. The test consists of adding a test suspension of bacteria or fungi to a prepared sample of the disinfectant under test in simulated ‘clean’ and ‘dirty’ conditions. After a specified contact time an aliquot is taken and the bactericidal / fungicidal action is immediately neutralized by the addition of a proven neutralizer (as identified in the basic suspension test). Following this, the number of surviving microorganisms in each sample is determined and the reduction in viable counts is calculated (expressed in logarithms to base 10).
To achieve neutralization the standard recommends dilution but if this is ineffective then membrane filtration maybe used where the filter may trap microorganisms but filter through the disinfectant by the application of rinse solutions. Thus dilution; addition of a chemical neutralizer, and membrane filtration are the three standard methods for inactivation of antimicrobials(2).
The suspension test permits challenges of different concentrations of the disinfectant against a range of set test microorganisms. The concentrations need to be constructed to cover the manufacturer’s recommendations for the active and non-active ranges. This is to demonstrate whether the manufacturer’s recommended concentration is effective and to understand the margin of failure (where the disinfectant solution is too dilute to effective). The set organisms are: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus faecium / hirae, for the bactericidal test, and Aspergillus niger and Candida albicans for the fungicidal test. The bactericidal standard also makes provision for additional microorganisms to be used in specific industries. These are: Salmonella typhimurium (which would be used for the food industry), Lactobacillus brevis (which would be used for breweries) and Enterobacter cloacae. To achieve a ‘pass’, the concentration of disinfectant, at a temperature of 20oC and a contact time of 5 minutes, must produce a minimum five log reduction of the challenge bacteria and a minimum of a four log reduction for the challenge fungi. The time and temperature may be varied depending upon the application, although once established the disinfectant should not be used outside of the verified ranges.
In addition to the standard, it would seem that many regulatory inspectors would expect the inclusion of environmental isolates found from the manufacturing environment. The addition of spore bearing microorganisms can also be introduced to challenge disinfectants with sproricidal properties. Research from Payne et al(3) indicates that of all of the test microorganisms it is Pseudomonas aeruginosa that is generally the most resistant.
In addition to testing the differing concentrations, the standard also requires that the disinfectant is made up in the ‘worst case’ condition by using ‘water of standard hardness’ (which contains ions like magnesium and calcium, as well as other salts). A further condition is the simulation of ‘soiling’, by the addition of bovine serum albumin (at 0.03%, representing ‘clean’ conditions and at 0.3% representing ‘dirty’ conditions). Some manufacturers will also introduce an additional organic load, which is representative of residues likely to be found within their cleanrooms, as well as other in-use temperatures and variations to contact times from one to sixty minutes.
10.6 Surface tests
Phase 2, step 2 – surface test (Standards EN 13713: 1999 and EN 13697: 1999) and AOAC standard AOAC 991.47:1991 Hard surface carrier test method.
Surface tests are sometimes referred to as carrier tests. It is at this stage that the European and US disinfection tests have a level of similarity. With surface tests, representative manufacturing surface samples are inoculated with a selection of microbial challenge organisms. A disinfectant is applied to the inoculated surfaces and exposed for a predetermined contact time after which the surviving organisms are recovered using a qualified disinfectant-neutralizing broth and test method (surface rinse, contact plate, or swab). The number of challenge organisms recovered from the test samples (exposed to a disinfectant) is compared to the number of challenge organisms recovered from the corresponding control sample (not exposed to a disinfectant) to determine the ability of the disinfectant to reduce the microbial bioburden. Successful completion of the validation qualifies the disinfectant evaluated for use.
Prior to initiating disinfectant efficacy validation, a comprehensive survey of the materials comprising the room surfaces (floors, walls, windows) and equipment (stainless steel, acrylic, vinyl) present in the facility which could potentially be exposed to the disinfectant should be conducted. The use of different surfaces is important because the rates of inactivation on microorganisms on different surfaces can vary considerably. One study demonstrated that bactericidal activity reduced on PVC compared with stainless steel. This was a factor both of the material type and the surface conditions, such as, the number of pores or ridges. Surfaces of the material can also differ depending upon the degree of finishing with smoother surfaces, like stainless steel or Formica, giving greater repeatability and reproducibility(4).
Most facilities will not use every type of surface but instead will select the most common types of surfaces. Should this bracketing strategy be employed, it is crucial that the rationale for surface selection be detailed in the efficacy validation protocol as regulators will seek evidence that representative surfaces have been challenged. Once appropriate surfaces have been selected, 2” x 2” coupons of the surface material should be obtained. These coupons, referred to as “surface carriers,” serve as the representative surfaces for the testing(5).
The European standards that describe the test are EN 13713, for the basic surface test, and EN 13697, for a quantitative surface test, which includes the presence of interfering substances. The standards are largely similar to previous German DGHM methods. The surface test is based on the suspension test with the variable parameters of interfering substances, temperature and contact time. However, the required log reduction differs from the suspension test in that, to pass, a 4 log decrease for bacteria and a 3 log decrease for fungi. must be obtained The required test organisms are identical to the suspension test: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Aspergillus niger and Candida albicans. For this test, fungi are incorporated within the one standard. The microbiologist will also consider the inclusion of environmental isolates and spore bearing microorganisms (arguments as to when an environmental isolate becomes a ‘laboratory culture’ and problems in creating adequate spore suspensions notwithstanding(8)).
With the AOAC use-dilution test (a carrier-based test), the organisms used are: Salmonella cholerasuis, Staphylococcus aureus and Pseudomonas aeruginosa. The principles are generally similar to the European methods but there are some variations. The European and AOAC methods vary.
The surface test is by far the most important, challenging and representative of the tests of disinfectant efficacy and this chapter examines this test in greater detail. The surface test is more relevant than the suspension test because it is truer to practical conditions and theoretically, microorganisms attached to a surface will be more resistant than those in a suspension, therefore this presents the greatest challenge. The quantitative surface test evaluates test suspensions of bacteria and fungi in a solution of interfering substances, designed to simulate clean and dirty conditions, which are inoculated onto a test surface and dried. The test aims to acquire quantitative information about the ability of a disinfectant to kill microorganisms attached to hard surfaces.
The test works by examining preparations of microorganisms dried onto surfaces. To such a dried suspension a prepared sample of the disinfectant is added. The surface is then transferred to a previously validated neutralization medium and tests performed to measure the reduction in viable counts. The test involves drying 0.05 ml suspensions of the microorganisms (with interfering substances such as bovine serum albumin) onto different surfaces. The microorganisms should have a population range of 1.5 – 5.0 x 108 for bacteria and 1.5 – 5.0 x 107 for fungi and are equilibrated to 25oC before use. Once applied to the surface the drying of the microorganisms maybe accelerated using an incubator operating at 36-38oC. Disinfectant solutions (where disinfectants are made with Water of Standard Hardness) are added to the surfaces. After the specified contact time (five minutes is the target) the surfaces are transferred to the validated neutralization medium and then pour plates are prepared for incubation and counting.
A variation of the surface test involves the use of mechanical action. Mechanical action is more akin to practical conditions (such as the application of a cloth or a mop). However, the more efficacious disinfectants do not require any mechanical action when the disinfectant and the surface come into contact. For the surface test, mechanical action is very difficult to reproduce. It is preferable to evaluate a disinfectant without mechanical action and this aspect can be examined during the Phase 3 field trials. Furthermore, mechanical action is a very variable procedure and is difficult to evaluate.
It may arise that the disinfectant concentration shown to be optimal for the suspension test needs to be increased to meet the requirements of the surface test. The suspension test has further weaknesses in that it enhances the potential for small dilution errors made in the preparation of disinfectant solutions in relation to the final pass or fail result. The suspension test has been shown to be difficult to reproduce both between and within laboratories and often lacks precision. The suspension test can also pose problems when disinfectants with a high viscosity are challenged due to their distribution in the test suspension.
The surface test, however, cannot demonstrate the affect of a range of environmental factors like temperature, pH, detergent residues, mechanical stress and attachment. For these reasons a disinfectant which appears effective for the surface test can show marked variability when applied to practical conditions. The reasons for this are due to problems in drying and differences between surfaces. In terms of drying microbial suspensions, there is a marked loss in the viability of a population when dried onto a surface and attempts to speed the drying process up do not significantly reduce the variability of the actual number of microorganisms challenged. Surfaces introduce another variation because surfaces, even of the same grade of material, are not truly identical and there have been marked problems in achieving reproducibility and repeatability for the surface test between laboratories particular in estimating the concentration of disinfectant required to be effective. Some of these limitations can be addressed through field trials.
Hand sanitisation (Standard: EN 1500)
An associated part of disinfectant evaluation is the assessment of hand sanitisers. There are many commercially available hand sanitizers, with the most commonly used types being alcohol-based gels. Within Europe there is a standard describing the approach for the validation of hand sanitisers based on two norms: EN1499 (hygienic hand wash), and EN 1500 (hygienic hand disinfection). It is more typical for the EN 1500 standard to be followed. Many commercially available hand sanitisers are surprisingly difficult to test against the standard in terms of effectively reducing microbial populations and several types have compared unfavourably to straightforward hand washing with simple soaps. Some alcohols are more effective than others, based on their molecular weight. The alcohol 1-propanol (C3H8O) (An isomer of isopropanol (2-propanol), that is a compound with the same molecular formula but with a different structural formula) is used as the test standard against which hand sanitizers are compared.
The test for hand sanitisers can be applied to skin and to gloved hands. One problem with the application to gloved hands is that the gloves themselves may either carry a microbial load or be prone to leaks. Some material, such as latex, can trap microorganisms onto the surface. These factors can reduce the reliability of the test results. The test determines if a hand sanitiser can reduce the number of transient microflora under simulated practical conditions. The hand sanitiser under test is compared against a reference standard (60% propan-1-ol) using fifteen test subjects. For tests of gloved hands, several microorganisms can be selected. However, only one microorganism can be used for the study on human skin for health and safety considerations: Eschericia coli K12 (ATCC 10538) which is a non-pathogenic Class I microorganism under Directive 90/679 EEC (Strain K-12 was isolated at Stanford University in 1922 from human faces). To be effective the test hand sanitizer must produce a five log reduction of the test microorganism. The agar plates used to measure recovery contain the additive 0.5g/l of sodium desoxycholate in order to inhibit the growth of any skin Staphylococci.
The act of agitation and rubbing the hand sanitiser into the skin or into the glove presents the greatest variable into the test. This is partly, but not completely, overcome by the large subject size but difficulties exist in comparing different laboratories. For practical use there is a significant effect on the survival of microflora based on the frequency of application, the degree of hand rubbing and the quantity applied.
Tim SANDLE – wwww.pharmamicroresources.com
1. Sandle, T. (2016) The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms, 2nd Edition, Grosvenor House Publishing: Surrey, UK
2. Russell, A.D., Ahonkhai, I. And Rogers, D. T.: ‘Microbiological Applications of the Inactivation of Antibiotics and Other Antimicrobial Agents’, Journal of Applied Bacteriology, 1979, 46, pp207-245
3. Payne, D.N., Babb, J.R. and Bradley, C. R.: ‘An evaluation of the suitability of the European Suspension Test to reflect in vitro activity of antiseptic against clinically chosen significant organisms’, Letters in Applied Microbiology, 1999, 28, pp7-12
4. Bloomfield, S.F., Arthur, M., Van Klingeren, B., Pullen, W., Holah, J.T. and Elton, R.: ‘An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants’, Journal of Applied Bacteriology, 1994, 76, pp86-94
5. Vina, P., Rubio, S. and Sandle, T. (2011): ‘Selection and Validation of Disinfectants’, in Saghee, M.R., Sandle, T. and Tidswell, E.C. (Eds.) (2011): Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices, New Delhi: Business Horizons, pp219-236