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Janvier 2017 I La Vague N° 52 I 45
4
The European approach to
disinfectant qualification.
By JTim SANDLE - www.pharmamicroresources.com
timsandle@btinternet.com
Contamination control
is of great importance
to healthcare facili-
ties and to pharmaceutical
cleanrooms. One way of
ensuring the hygiene is main-
tained is through a cleaning
and disinfection regime. Af-
ter a disinfectant has been
chosen based on its chemi-
cal properties and expected
performance/effectiveness,
each disinfectant should be
validated to ensure its effica-
cy. 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 diers slightly from the
approach outlined in the USP <1072> or
through the AOAC. This article outlines
the European approach to disinfectant
qualication.
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
rst 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 Ê Basic Suspension Tests
2. Phase 2 ÊPart 1:
Suspension and surface tests to simulate
practical usage: Bactericidal and fungicidal
(sporicidal and virucidal)
3. Phase 2 ÊPart 2: Surface test
4. Phase 3 Ê 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
Techno/Process
46 I La Vague N° 52 I Janvier 2017
4
Techno/Process
suspension and surface tests are tests to determine the most eective
concentration and conditions for the disinfectant as a simulation of
practical conditions. The eld trials show the eectiveness 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 ecacy 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 conrm 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
dierent 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 specic 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 specied contact time an aliquot is taken and
the bactericidal / fungicidal action is immediately neutralized by the
addition of a proven neutralizer (as identied 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 ineective then membrane ltration maybe used where the
lter may trap microorganisms but lter through the disinfectant by
the application of rinse solutions. Thus dilution; addition of a chemical
neutralizer, and membrane ltration are the three standard methods
for inactivation of antimicrobials (2).
The suspension test permits challenges of dierent 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 eective and to understand the margin of failure (where the
disinfectant solution is too dilute to eective). 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 specic 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 ve 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 veried 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 diering 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'
Janvier 2017 I La Vague N° 52 I 47
4
Techno/Process
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 qualied 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 qualies
the disinfectant evaluated for use.
Prior to initiating disinfectant ecacy validation, a comprehensive
survey of the materials comprising the room surfaces (oors, 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 dierent surfaces is important because the
rates of inactivation on microorganisms on dierent 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 dier depending
upon the degree of nishing 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 ecacy 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 diers 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 ecacy 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 dierent 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 specied contact time (ve 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 ecacious
48 I La Vague N° 52 I Janvier 2017
disinfectants do not require any mechanical action when the
disinfectant and the surface come into contact. For the surface test,
mechanical action is very dicult to reproduce. It is preferable to
evaluate a disinfectant without mechanical action and this aspect can
be examined during the Phase 3 eld trials. Furthermore, mechanical
action is a very variable procedure and is dicult 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 nal pass or fail
result. The suspension test has been shown to be dicult 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 aect of a range
of environmental factors like temperature, pH, detergent residues,
mechanical stress and attachment. For these reasons a disinfectant
which appears eective for the surface test can show marked
variability when applied to practical conditions. The reasons for this
are due to problems in drying and dierences 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 signicantly 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 eective. Some of these limitations can be
addressed through eld trials.
Hand sanitisation
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 dicult to test against the standard in
terms of eectively reducing microbial populations and several types
have compared unfavourably to straightforward hand washing with
simple soaps. Some alcohols are more eective 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 dierent 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 microora
under simulated practical conditions. The hand sanitiser under test is
compared against a reference standard (60% propan-1-ol) using fteen
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 eective the test hand sanitizer
must produce a ve 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 diculties
exist in comparing dierent laboratories. For practical use there is a
signicant eect on the survival of microora based on the frequency
of application, the degree of hand rubbing and the quantity applied.
References
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 reect in vitro activity of antiseptic against clinically chosen signicant
organisms’, Letters in Applied Microbiology, 1999, 28, pp7-12
4. Bloomeld, 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
