Friday, 26 March 2021

Qualification of a Cleanroom manual Sanitisation programme


Approaches to disinfectant qualification vary. The two main standards are those of the European Union and of the U.S. The approaches are similar, although there are subtle methodological differences.


The U.S. approach is as defined by the US Environmental Protection Agency (EPA) and Association of Official Analytical Chemists (AOAC)[i] methods[ii]. This approach is referred to in USP General Chapter <1072> Disinfectants.


The European standards are produced by the European Committee for Standardisation Technical Committee 216 (CEN TC 216)[iii].


European approach


The standard European approach for disinfectant validation is divided up into three phases:


·         Phase 1            Basic Suspension Tests[iv]

·         Phase 2           

o   Part 1: Suspension and surface tests to simulate practical usage: Bactericidal and fungicidal (sporicidal and virucidal)[v]

o   Part 2:              Surface test[vi]

·         Phase 3            Field Trial (no standard has yet to be produced)

·         A separate phase exists for the validation of hand sanitisers[vii]


The first phases (the basic suspension tests) are tested by manufacturers. All subsequent phases are evaluated by the disinfectant user. This is because the user will need to evaluate the disinfectants on the surfaces which relate to the facility (such as wall covering, floor vinyl, stainless steel and so on) and undertake ‘field trials’ to establish appropriate cleaning frequencies and environmental testing regimes in-loco conditions (the pharmaceutical clean rooms).


Phase 1 - Basic Suspension Test (Standards EN 1275 and EN 1040)


There are two standards published within Europe for the basic suspension test: EN 1040 to measure bactericidal activity, and 1275 to measure fungicidal activity. The basic suspension test is a simple, if somewhat limited test of the disinfectant, in order to determine minimum standards. The test evaluates the activity of a disinfectant against a range of micro-organisms 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 micro-organisms enumerated. Before undertaking the test, the selection of a suitable sterile neutraliser is required. Selection involves challenging (or ‘spiking’) neutralisers of different activity with a range of micro-organisms and measuring the recovery. The neutraliser with the optimal recovery should be selected.


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. These practical conditions make the test more advanced 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 microcidal action is immediately neutralised by the addition of a proven neutralizer (the one identified in the basic suspension test). Following this, the number of surviving micro-organisms in each sample is determined and the reduction in viable counts is calculated.


To show recovery the standard recommends dilution but if this is ineffective then membrane filtration maybe used. With membrane filtration a filter acts to trap micro-organisms whilst allowing the disinfectant to be filtered through[viii].


The suspension test permits challenges of different concentrations of the disinfectant against a range of set test microorganisms. The concentrations tested need to cover the manufacturer's recommendations for the ‘active’ and ‘non-active ranges’ (where the ‘active’ concentration is the concentration at which the manufacturer of the disinfectant considers that microorganisms will be killed). The required microorganisms 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. 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. Where the test is undertaken by the user many regulatory inspectors now expect the inclusion of environmental isolates found from the pharmaceutical cleanroom environment. The addition of spore bearing micro-organisms can also be introduced to challenge disinfectants with sproricidal properties[ix]. Research from Payne et al[x] indicates that of all of the test micro-organisms 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 clean rooms, as well as other in-use temperatures and variations to contact times from one to sixty minutes.


Phase 2, step 2 - surface test (Standards prEN 13713: 1999 and prEN 13697: 1999)


The second part of phase 2 is the surface test. The European standards that describe the test are prEN 13713, for the basic surface test, and prEN 13697, for a quantitative surface test, which includes the presence of interfering substances[xi].


The surface test is based on the outcome of the suspension test, using the established contact time. The required log reduction differs from the suspension test in that it is a 4 log decrease for bacteria and a 3 log decrease for fungi. The required test organisms are identical to the suspension test. The microbiologist will also consider the inclusion of environmental isolates.


The quantitative 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 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 neutralisation medium and tests performed to measure the reduction in viable counts. The test involves drying 0.05 ml suspensions of the micro-organisms (with interfering substances such as bovine serum albumin) onto different surfaces. The micro-organisms 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 micro-organisms 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 neutralisation medium and then pour plates are prepared for incubation and counting.


The surfaces selected should mimic the range of surfaces found in the clean room environment, such as, stainless steel and polyvinyl chloride (PVC).


Phase 3 - field trials (Standard has yet to be produced)


Only disinfectants that have satisfactorily passed the previous phases should be studied for field trials. The purpose of field trials is to test a disinfectant's efficacy in true in-use conditions: the working environment. Although no common European Standard exists, the common approach for field trials involves applying the disinfectant, at the selected concentration, to a selection of surfaces and equipment within the manufacturing facility. To these controlled areas, an intensive level of viable microbiological environmental monitoring is undertaken.


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 their validation (EN 1500).


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-2-ol) using fifteen test subjects. Only one micro-organism 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 micro-organism under Directive 90/679 EEC. To be effective the test hand sanitiser must produce a five log reduction of the test micro-organism. 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[xii].


U.S. approach


The U.S. approach is:


a) Suspension tests (the AOAC Use-Dilution Test).


This method is undertaken to show that the disinfectant can produce a high log reduction of a challenge microorganisms. It does not reflect the conditions in the cleanroom. The outcome of suspension tests might be a poor predictor for the efficacy of a disinfectant on clean room surfaces.


b) Hard surface test (AOAC Hard surface carrier test method)


Hard surface tests are used to mimic cleanroom conditions, including the nature of the surfaces and the application method, be it spraying or mopping.


The method involves testing the disinfectant on coupons, which are approximately 2" by 2" samples of representative surfaces found in the clean room. Examples of surfaces are stainless steel, vinyl, and tile. For the test:


·         The coupons are inoculated with 0.1 mL inoculum of the microorganism challenge.

·         The disinfectant is applied onto the coupon.

·         The disinfectant is left on the coupon for a specified amount of time. (For example 5 or 10 minutes.)

·         The coupons are sampled for recovery.

·         The samples are neutralized and plated or filtered and transferred onto media, and incubated.

·         Colonies are enumerated.

·         Log reduction is determined for treated coupons versus untreated coupons. The use of positive controls (untreated coupons) is important. Log reduction calculations depend upon good control counts.

·         The goal for sporicidal activity is at least a 2 log CFU reduction and for bactericidal activity is at least a 3 log CFU reduction.

[i] Association of Official Analytical Chemists International (AOAC), 1995, Disinfectants. Official Methods of Analysis, 16th edition, Arlington, VA: AOAC

[ii] Reybrouck, G.: The testing of disinfectants, International Biodeterioration and Biodegradation, 41, 1998, pp269-272

[iii] Holah, J. T. Progress report on CEN TC 216 WG3: Disinfectant test methods for food hygiene - institutional and domestic applications, International Biodeterioration and Biodegradation, 1997, 36, 355 – 365

[iv] BS EN 1040 (1997): Chemical Disinfectants and Antiseptics – Basic bactericidial activity – Test Method and Requirements (Phase 1) and BS EN 1275 (1997): Chemical Disinfectants and Antiseptics – Basic fungicidial activity – Test Method and Requirements (Phase 1)

[v] BS EN 1276: Chemical disinfectants and antiseptics - quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional use - test methods and requirements (Phase 2, step1), 1997 and EN 1650 (1998): Chemical Disinfectants and Antiseptics – Quantitative Suspension Test for the Evaluation of Fungicidal Activity of Chemical Disinfectants and Antiseptics used in Food, Industrial, Domestic and Institutional Areas – Test Method and Requirements (Phase 2/1)

[vi] prEN 13697 (1999): Chemical Disinfectants and Antiseptics – Quantitative Surface Test for the Evaluation of Bactercidal and  / or Fungicidal Activity of Chemical Disinfectants used in Food, Industrial, Domestic and Institutional Areas – Test Method Without Mechanical Action and Requirements (Phase 2/2)

[vii] prEN 1500(1994):  Chemical Disinfectants – Quantitative Carrier Test to Evaluate the Bactericidial Activity of a Hygenic Handrub Solution (Phase 2/2)

[viii] 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

[ix] Russell, A. D.: Assessment of sporicidal efficacy, International Biodeterioration and Biodegradation, 41, 1998, pp281-287

[x] 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

[xi] 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

[xii] Best, M. and Kennedy, M.E.: 'Effectiveness of handwashing agents in eliminating Staphylococcus aureus from gloved hands', Journal of Applied Bacteriology, 1992, 73, pp63-66

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (

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