EN 13704:2002

SLOVENSKI STANDARD
01-september-2002
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Chemical disinfectants - Quantitative suspension test for the evaluation of sporicidal
activity of chemical disinfectants used in food, industrial, domestic and institutional areas
- Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der
sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittel,
Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen
(Phase 2, Stufe 1)
Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité
sporicide des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire,
dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et
prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 13704:2002
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 13704
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2002
ICS 71.100.35
English version
Chemical disinfectants - Quantitative suspension test for the
evaluation of sporicidal activity of chemical disinfectants used in
food, industrial, domestic and institutional areas - Test method
and requirements (phase 2, step 1)
Désinfectants chimiques - Essai quantitatif de suspension Chemische Desinfektionsmittel - Quantitativer
pour l'évaluation de l'activité sporicide des désinfectants Suspensionsversuch zur Bestimmung der sporiziden
chimiques utilisés dans le domaine de l'agro-alimentaire, Wirkung chemischer Desinfektionsmittel in den Bereichen
dans l'industrie, dans les domaines domestiques et en Lebensmittel, Industrie, Haushalt und öffentliche
collectivité - Méthode d'essai et prescriptions (phase 2, Einrichtungen - Prüfverfahren und Anforderungen (Phase 2,
étape 1) Stufe 1)
This European Standard was approved by CEN on 11 November 2001.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13704:2002 E
worldwide for CEN national Members.

Contents
page
Foreword.3
Introduction .4
1 Scope .5
2 Normative references .6
3 Terms and definitions.6
4 Requirements .7
5 Test method.7
5.1 Principle.7
5.2 Materials and reagents .8
5.3 Apparatus and glassware .10
5.4 Preparation of spore test suspension and test solutions .11
5.5 Procedure .12
5.6 Calculation and expression of results.15
5.7 Conclusion.17
5.8 Test report .18
Annex A (normative) Preparation of Bacillus spore stock suspensions .20
A.1 Material and reagents .20
A.2 Preparation of Bacillus spore stock suspensions .20
Annex B (normative) Validation of dilution-neutralization and membrane filtration methods .21
B.1 Principle.21
B.2 Preparation of spore suspension.21
B.3 Preparation of product test solution.21
B.4 Test for validation .21
B.5 Validation.24
Annex C (informative) Preparation of Clostridium sporogenes spore stock suspension.25
C.1 Culture media and reagents.25
C.2 Apparatus and glassware .26
C.3 Preparation of regenerated media and incubation conditions .26
C.4 Preparation of Clostridium spore stock suspension .26
Annex D (informative) Neutralizers.27
Annex E (informative) Rinsing liquids.28
Annex F (informative) Example of a typical test report .29
Annex G (informative) Referenced strains in national collections.32
G.1 Bacillus subtilis.32
G.2 Bacillus cereus.32
G.3 Clostridium sporogenes.32
Annex H (informative) Information on the application and interpretation of European standards on
chemical disinfectants and antiseptics .33
H.1 General guidelines for the application and interpretation of test methods in accordance with
European Standards for chemical disinfectants and antiseptics.33
H.2 Guide to interpretation of tests for chemical disinfectants and antiseptics .34
Foreword
This European Standard has been prepared by Technical Committee CEN/TC 216 "Chemical disinfectants and
antiseptics", the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an identical text or
by endorsement, at the latest by August 2002, and conflicting national standards shall be withdrawn at the latest by
August 2002.
In this standard the annexes A and B are normative. The annexes C, D, E, F, G and H are informative.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,
France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain,
Sweden, Switzerland and the United Kingdom.
Introduction
This European Standard describes a suspension test method for establishing whether a chemical disinfectant has
or does not have a sporicidal activity in the fields described in clause 1.
The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time,
temperature, in suspension, etc.) reflect parameters which are found in practical situations including conditions
which can influence the action of disinfectants. Each utilization concentration found from this test corresponds to
defined experimental conditions.
The conditions are intended to cover general purposes and to allow reference between laboratories and product
types.
However for some applications the recommendations of use of a product can differ and therefore additional test
conditions need to be used.
1 Scope
This European Standard specifies a test method (phase 2/step 1) (see annex H) and the minimum requirements for
sporicidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation in hard
water and that are used in food, industrial, domestic and institutional areas, excluding areas and situations where
disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in
the above considered areas.
This European Standard applies at least to the following :
a) processing, distribution and retailing of :
1) food of animal origin :

milk and milk products ;

meat and meat products ;

fish, seafood, and related products ;

eggs and egg products ;

animal feeds ;

etc. ;
2) food of vegetable origin :

beverages ;

fruits, vegetables and derivatives (including sugar, distillery, etc.) ;

flour, milling and baking ;

animal feeds ;

etc. ;
b) institutional and domestic areas :

catering establishments ;

public areas ;

public transports ;

schools ;

nurseries ;

shops ;

sports rooms ;

waste containers (bins, etc.) ;

hotels ;

dwellings ;

clinically non sensitive areas of hospitals ;

offices ;

etc. ;
c) other industrial areas :

packaging material ;

biotechnology (yeast, proteins, enzymes, etc.) ;

pharmaceutical ;

cosmetics and toiletries ;

textiles ;

space industry, computer industry ;

etc.
Using this European Standard, it is not possible to determine the sporicidal activity of undiluted product as some
dilution is always produced by adding the inoculum and interfering substance. Products can only be tested at a
concentration of 80 % or less.
NOTE The method described is intended to determine the activity of commercial formulations or active substances on
spores in the conditions in which they are used.
2 Normative references
This European Standard incorporates by dated or undated reference, provisions from other publications. These
normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For
dated references, subsequent amendments to or revisions of any of these publications apply to this European
Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the
publication referred to applies (including amendments).
EN 1040, Chemical disinfectants and antiseptics – Basic bactericidal activity – Test method and requirements
(phase 1).
3 Terms and definitions
For the purposes of this European Standard, the following terms and definitions apply.
3.1
product (for chemical disinfection and/or antisepsis)
chemical agent or formulation used as a chemical disinfectant or antiseptic [EN 1040]
3.2
sporicide (in this standard)
product which kills spores of the genus Bacillus
NOTE The adjective derived from « sporicide » is « sporicidal ».
3.3
sporicidal activity (in this standard)
capability of the product to produce at least a reduction of 10 in the number of bacterial spores belonging to
reference strain of Bacillus subtilis under conditions defined by this European Standard
3.4
clean conditions
conditions representative of surfaces which have received a satisfactory cleaning programme and/or are known to
contain minimal levels of organic and/or inorganic materials
4 Requirements
The product, when diluted in hard water and tested in accordance with clause 5 under simulated clean conditions
(0,3 g/l bovine albumin see 3.4) according to its practical applications and under the required test conditions (20 °C,
60 min, 1 selected reference strain), shall demonstrate at least a 10 reduction in viable counts.
The sporicidal activity shall be evaluated using the test organism Bacillus subtilis.
The determined sporicidal concentration of the test product is suggested as being suitable for practical situations of
use.
Where appropriate, additional specific sporicidal activity shall be determined under other conditions of time,
temperature and additional strains (see 5.2.1 and 5.5.1) in accordance with 5.5.1 in order to take into account
intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the
initial test conditions of 20 °C, 60 min, 1 selected reference strain.
5 Test method
5.1 Principle
5.1.1 A test suspension of bacterial spores in a solution of interfering substance, simulating clean conditions, is
added to a prepared sample of the product under test diluted in hard water. The mixture is maintained at
20 °C ± 1 °C for 60 min ± 10 s (required test conditions).
At this contact time, an aliquot is taken ; the sporicidal action in this portion is immediately neutralized or
suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot
be found, membrane filtration is used. The number of surviving bacterial spores in each sample are determined and
the reduction in viable counts is calculated.
5.1.2 The test is performed using spores of Bacillus subtilis. Additional and optional exposure times,
temperatures and strains are specified.
5.2 Materials and reagents
5.2.1 Test organisms
The sporicidal activity shall be evaluated by using spores of the following strain :
1)

Bacillus subtilis ATCC 6633 .
If required for specific applications, additional strains may be chosen from, for example :
1)

Bacillus cereus ATCC 12826 ;
1)

Clostridium sporogenes CIP 7939 .
NOTE 1 See annex G for corresponding strain numbers in some other culture collections.
NOTE 2 See annex C for particular culture and handling conditions for Clostridium sporogenes.
If additional strains are used, they shall be incubated under optimum growth conditions (temperature, time,
atmosphere) and noted in the test report.
If the additional strains selected do not correspond to the specified strains, their suitability for supplying inocula of
sufficient concentration shall be verified. If the additional strains tested are not classified at a reference centre their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national
culture under a reference for 5 years.
5.2.2 Culture media and reagents
5.2.2.1 General
The reagents shall be of analytical grade and/or appropriate for microbiological purposes.
NOTE To improve reproducibility, it is recommended that commercially available dehydrated material is used for the
preparation of culture media. The manufacturer’s instructions relating to the preparation of these products should be rigorously
followed.
5.2.2.2 Water
The water shall be free from substances that are toxic or inhibiting to the bacterial spores or to the bacteria. It shall
be freshly glass distilled water and not demineralized water.
Sterilize in the autoclave (see 5.3.1).
NOTE 1 If the water is sterilized during the sterilization of the reagents, this is not necessary.
NOTE 2 If distilled water of adequate quality is not available, water for injectable preparation (European Pharmacopoeia) can
be used.
1)
ATCC 6633 and ATCC 12826 are the collection numbers of strains supplied by the American Type Culture Collections. CIP
7939 is the collection number of spores supplied by the Collection de l'Institut Pasteur. This information is given for the
convenience of users of this standard and does not constitute an endorsement by CEN of the product named. Corresponding
strains supplied by other culture collections may be used if they can be shown to lead to the same results.
5.2.2.3 Glucose Yeast Extract Agar (GYA)
For counting of viable Bacillus spores :
Amino-acids, without vitamins, obtained by acid hydrolysis of casein. 1,0 g
Soluble starch. 1,0 g
Glucose . 2,5 g
Yeast extract . 5,0 g
FeSO . 0,1 g
MnSO H O 0,000 1 g
4, 2
Agar .15,0 g
Water (see 5.2.2.2) 1 000,0 ml
Sterilize in the autoclave (see 5.3.1). After sterilization the pH of the medium shall be equivalent to 6,8 ± 0,2 when
measured at 20 °C.
5.2.2.4 Neutralizer
The neutralizer shall be validated for the product under test in accordance with annex D. The neutralizer shall be
sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in annex D.
5.2.2.5 Rinsing liquid (for membrane filtration)
The liquid shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane
under the test conditions described in annex B.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in
annex E.
5.2.2.6 Hard water for dilution of products
Hard water for dilution of products shall be prepared as follows :

Solution A : Dissolve 19,84 g anhydrous magnesium chloride (MgCl ) or an equivalent of hydrated
magnesium chloride and 46,24 g anhydrous calcium chloride (CaCl ) or an equivalent of hydrated calcium
chloride in water (see 5.2.2.2) and dilute to 1000 ml.
Sterilize in the autoclave (see 5.3.1). Store the solution at 2 °C - 8 °C for no longer than one month.

Solution B : Dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (see 5.2.2.2) and dilute to 1 000 ml.

Sterilize by membrane filtration (see 5.3.2.7). Store the solution at 2 °C - 8 °C for no longer than one week.
Hard Water :
For the preparation of 1 litre, place at least 600 ml water (see 5.2.2.2) in a 1000 ml volumetric flask (see 5.3.2.12)
and add 6,0 ml of solution A, then 8,0 ml of solution B.
Mix and dilute to 1 000 ml with water (see 5.2.2.2).
The pH of the hard water shall be 7,0 ± 0,2.
If necessary adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH)
or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCI).
The hard water shall be freshly prepared under aseptic conditions and used within 12 hours.
NOTE When preparing the product test solutions (see 5.4.2) the addition of the product to this hard water produces a
different final water hardness in each test tube.
In any case the final hardness is lower than 300 mg/kg of calcium carbonate (CaCO ) in the test tube.
5.2.2.7 Interfering substance
5.2.2.7.1 General
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
5.2.2.7.2 Bovine albumin solution
Bovine albumin solution for the test conditions shall be prepared as follows :

dissolve 0,30 g of bovine albumin (Cohn fraction V for Dubos medium) in 100 ml of water (see 5.2.2.2) ;

sterilize by membrane filtration.
The final concentration of the bovine albumin in the test procedure (see 5.5.2) is 0,3 g/l.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or
the sample, except those which are supplied sterile, by one of the following methods :
3
a) in the autoclave (see 5.3.2.1) by maintaining it at (121 ) °C for a minimum holding time of 15 min ;
b) in the dry heat sterilizer (see 5.3.2.1) by maintaining it at 180 °C for a minimum holding time of 30 min, at
170 °C for a minimum holding time of 1 h, or at 160 °C for a minimum holding time of 2 h.
2)
5.3.2 Usual microbiological laboratory equipment and, in particular, the following :
5.3.2.1 Apparatus for sterilization
3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum holding time
of 15 min ;
b) for dry heat sterilization, a hot air oven capable of being maintained at 180 °C for a minimum holding time of
30 min, at 170 °C for a minimum holding time of 1 h, or at 160 °C a minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, 45 °C ± 1 °C and at additional test
temperatures ± 1 °C (see 5.5.1).
5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C.
, having an accuracy of calibration of ± 0,1 pH units at 25 °C.
5.3.2.4 pH-meter
5.3.2.5 Stopwatch
3)
5.3.2.6 Vortex mixer (electromechanical agitator, i.e. Vortex® mixer )

2)
Disposable equipment is an acceptable alternative to reusable glassware.
5.3.2.7 Membrane filtration apparatus (if this method is used), constructed of a material compatible with the
product under test, with a filter holder which shall have a usable volume 50 ml minimum, and suitable for use with
filters of diameter 47 mm to 50 mm, of 0,45 μm pore size.
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the
microorganisms over the membrane and in order to prevent overlong filtration, the device shall be set so as to
obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Containers : Test tubes or flasks of suitable capacity.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic pipettes
may be used.
5.3.2.10 Petri dishes of size 90 mm to 100 mm
5.3.2.11 Glass beads (Diameter : 3 mm to 4 mm).
5.3.2.12 Volumetric flasks
5.3.2.13 Glass Roux bottles with straight neck
5.3.2.14 Microscope, preferably, a phase-contrast type, with magnification of at least x 400.
5.4 Preparation of spore test suspension and test solutions
5.4.1 Spore suspensions
5.4.1.1 Stock spore suspension of test organism
Bacillus subtilis spore stock suspension for the specific purposes of this standard can be purchased from a national
culture collection or prepared by the testing laboratory.
If the Bacillus subtilis spore stock suspension is prepared in the testing laboratory, the suspension shall be
prepared according to annex A.
As part of good laboratory practice (GLP), laboratories may want to include an internal reference product (e.g :
sodium hypochlorite) to check the resistance of the spores.
For the preparation of the stock spore suspensions of additionnal strains (see 5.2.1) refer to :

annex A for Bacillus cereus ATCC 12826 ;

annex C for Clostridium sporogenes CIP 7939.
5.4.1.2 Spore test suspension
To prepare the spore test suspension, dilute the spore stock suspension (see 5.4.1.1) with water (see 5.2.2.2). The
6 6
number of spores in the test suspension must be adjusted to 1,5 x 10 to 5 x 10 cfu/ml, estimating the number of
units by any suitable mean.
Maintain the suspension test in the water bath at 20 °C ± 1 °C and use within 2 h.
Microscopic examination under 400 x magnification shall be carried out immediately after the preparation and just
before the test, to show the absence of vegetative cells and germinative spores.

3)
Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users
of this standard and does not constitute an endorsement by CEN of this product.
If there is any evidence of spore germination, the suspension shall be discarded.
-4 -5
For counting of the spore test suspension prepare 10 and 10 dilutions of the test suspension (see 5.4.1.3) using
water (see 5.2.2.2). Mix (see 5.3.2.6). Take a sample of 1,0 ml of each dilution in duplicate and transfer each 1,0 ml
sample into separate Petri dishes (see 5.3.2.10) and add 12 ml to 15 ml melted GYA (see 5.2.2.3), cooled to
45 °C ± 1 °C.
5.4.1.3 Counting of spore test suspension
Incubate the Petri dishes at 30 °C ± 1 °C (see 5.3.2.3) for 3 days. Determine the higher number of colonies V for
c
4)
each plate. Calculate the number of cfu/ml (see 5.4.1.3).
in the test suspension (N) using the method given in 5.6.1.1.
5.4.2 Product test solution
Details of samples of the product as received shall be recorded.
Product test solutions shall be prepared in hard water (see 5.2.2.6) at three different concentrations to include one
concentration in the active range and one concentration in the non-active range. The concentration of the product
test solution shall be 1,25 times the required test concentration.
For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a volumetric
flask and filling up with hard water (see 5.2.2.6). Subsequent dilutions shall be prepared in volumetric flasks
(see 5.3.2.12) on a volume/volume basis in hard water (see 5.2.2.6).
For liquid products, dilutions of the product shall be prepared in hard water (see 5.2.2.6) on a volume/volume basis
using volumetric flasks (5.3.2.12).
For products supplied in a ready to use state, water (see 5.2.2.2) shall be used to prepare dilutions.
When the product is diluted in hard water it shall give a physically homogeneous stable preparation.
The product test solutions and dilutions of it shall be prepared freshly and used within 60 min.
NOTE If the product is of low stability this period should be shortened.
The concentration of the product stated in the test report shall be the test concentration. Record the test
concentration in terms of mass per volume or volume per volume.
5.5 Procedure
5.5.1 Choice of experimental conditions
The selection of contact temperature and contact time shall be carried out according to the practical use considered
for the product (see clause 4) as follows :
a) temperature :
in °C :

the temperature to be tested is 20 °C ± 1 °C ;

the additional temperatures may be chosen from 4 °C ± 1 °C or 10 °C ± 1 °C or 40 °C ± 1 °C or
75 °C ± 1 °C ;
b) contact time : t in min :
4)
cfu/ml = Colony forming unit per ml.

the contact time to be tested is 60 min ± 10 s ;

the additional contact times may be chosen from 5 min ± 10 s or 15 min ± 10 s or 30 min ± 10 s ;
c) strains :

strains shall be as stated in 5.2.1 ;
d) interfering substance :

the interfering substance to be tested is the bovine albumin solution (see 5.2.2.7) under clean conditions
(0,3 g/l bovine albumin).
The product shall not cause the formation of any precipitate in the experimental conditions used.
Each selected experimental condition (
, t, strains) shall be validated in accordance with annex B.
5.5.2 Test procedure for assessing the sporicidal effect of the product
5.5.2.1 General
The method of choice is the dilution-neutralization method. To determine a suitable neutralizer the following
procedure shall be adopted. Carry out the validation of the dilution neutralization method (B.4.1) using a suitable
neutralizer, chosen according to laboratory experience and published data.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer containing a combination of
polysorbate 80 30 g/l, saponin 30 g/l, L-histidine 1 g/l, lecithin 3 g/l, sodium thiosulphate 5 g/l in either water (see
5.2.2.2) or in phosphate buffer 0,0025 mol/l. If both neutralizers are found to be invalid, the membrane filtration may
be used in place of the dilution-neutralization method.
The inactivation of the sporicidal activity of the product shall be validated for each of the tested strains and for each
of the chosen experimental conditions (see 5.5.1).
5.5.2.2 Dilution-neutralization method
5.5.2.2.1General
a) if
is lower or equal to 40 °C ± 1 °C :
Prior to testing, equilibrate all reagents (product test solutions, spore test suspension, interfering substance) to
the test temperature of
°C ± 1 °C using the water bath (see 5.3.2.2) controlled at
°C ± 1 °C. Check that the
temperature of the reagents is stabilized at
°C ± 1 °C. The neutralizer and water (see 5.2.2.2) shall be
equilibrated at a temperature of 20 °C ± 1 °C.
b) if
is higher than 40 °C ± 1 °C :
Prior to testing, equilibrate the product test solutions to the test temperature of
°C ± 1 °C using the water bath
(see 5.3.2.2) controlled at
°C ± 1 °C. Check that the temperature of the reagents is stabilized at
°C ± 1 °C.
The neutralizer, the spore test suspension, the interfering substance and the water (see 5.2.2.2) shall be
equilibrated at a temperature of 20 °C ± 1 °C.
5.5.2.2.2Test procedure for sporicidal activity of products
Pipette 1,0 ml of interfering substance (see 5.2.2.7) into a test tube. Add 1,0 ml of the spore test suspension
6 6 5)
containing 1,5 x 10 to 5 x 10 cfu/ml (see 5.4.1.3).
Start the stopwatch immediately, mix (see 5.3.2.6) and place the test tube in the water bath at
°C ± 1 °C for
2 min ± 10 s. At the end of the contact time, add 8,0 ml of one of the product test solutions. Restart the stopwatch
immediately, mix (see 5.3.2.6) and place the test tube in a water bath controlled at
°C ± 1°C for the appropriate
contact time t ± 10 s.
NOTE When adding spore suspension, care should be taken to avoid touching the upper part of the test tube sides.
Just before the end of the contact time, mix (see 5.3.2.6). At the end of the contact time pipette 1,0 ml of the test
mixture into a tube containing 8,0 ml neutralizer (see 5.2.2.4) and 1,0 ml water (see 5.2.2.2). Mix (see 5.3.2.6) and
place in a water bath controlled at 20 °C ± 1 °C.
After a neutralization time of 5 min ± 10 s, immediately take a sample of 1,0 ml of neutralized mixture (neutralizer,
product test solution, interfering substance, spore test suspension) in duplicate and transfer each 1,0 ml sample
into separate Petri dishes (see 5.3.2.10) and quickly add 12 ml to 15 ml melted GYA (see 5.2.2.3), cooled to
45 °C ± 1 °C.
Perform this procedure using the other product test solutions.
5.5.2.2.3Counting of the test mixture
Incubate the Petri dishes at 30 °C ± 1 °C (see 5.3.2.3) for 3 days.
Determine the higher number of colonies V for each plate.
c
5)
Calculate the number of cfu/ml in the test mixture (N ) using the method given in 5.6.1.2.
a
-1
For calculating the viable count of the test mixture, the dilution factor is 10 .
5.5.2.3 Membrane filtration method
5.5.2.3.1General
a) if  is lower or equal to 40 °C ± 1 °C :
Prior to testing, equilibrate all reagents (product test solutions, spore test suspension, interfering substance) to
the test temperature of
°C ± 1 °C using the water bath (see 5.3.2.2) controlled at
°C ± 1 °C. Check that the
temperature of the reagents is stabilized at °C ± 1 °C. The rinsing liquid (see 5.2.2.2) shall be equilibrated at a
temperature of 20 °C ± 1 °C.
b) if
is higher than 40 °C ± 1 °C :
Prior to testing, equilibrate the product test solutions to the test temperature of
°C ± 1 °C using the water bath
(see 5.3.2.2) controlled at
°C ± 1 °C. Check that the temperature of the reagents is stabilized at
°C ± 1 °C.
The rinsing liquid, the spore test suspension, the interfering substance and the water (see 5.2.2.2) shall be
equilibrated at a temperature of 20 °C ± 1 °C.
5.5.2.3.2Test procedure for sporicidal activity of products
Pipette 1,0 ml of interfering substance (see 5.2.2.7) into a test tube. Add 1,0 ml of the spore test suspension
(see 5.4.1.3).
5)
cfu/ml = Colony forming unit per ml.
Start the stopwatch immediately, mix (see 5.3.2.6) and place the test tube in the water bath at
°C ± 1 °C for
2 min ± 10 s. At the end of the contact time, add 8,0 ml of one of the product test solutions. Restart the stopwatch
immediately, mix (see 5.3.2.6) and place the test tube in a water bath controlled at
°C ± 1 °C for the appropriate
contact time t ± 10 s.
Just before the end of the chosen contact time, mix (see 5.3.2.6). At the chosen contact time pipette two samples
of 0,1 ml of the test mixture and transfer each sample into a separate membrane filtration apparatus equipped with
a membrane and containing 50 ml of the rinsing liquid (see 5.2.2.5). Filter immediately. The time required for
transfer and filtration should not exceed 1 min. If greater than 1 min, this time shall be recorded in the test report.
Rinse with at least 150 ml but not more than 500 ml of rinsing liquid (see 5.2.2.5). If the rinsing liquid is not water,
complete the procedure by filtering 50 ml of water (see 5.2.2.2). Then transfer the membranes to the surface of two
separate GYA plates.
NOTE When transferring, care should be taken to ensure that the spores are on the upper side of the membrane when
placed on the GYA and to avoid trapping air between the membrane and agar surface.
Perform this procedure using the other product test solutions.
5.5.2.3.3Counting of test mixture
Incubate the Petri dishes at 30 °C ± 1 °C (see 5.3.2.3) for 3 days.
Determine the higher number of colonies V for each plate.

c
6)
Calculate the number of cfu/ml in the test mixture (N ) using the method given in 5.6.1.2.
a
5.5.3 Validation of dilution neutralization and membrane filtration method
The dilution-neutralization and membrane filtration methods shall be validated for each of the test organisms
according to annex B.
The validation test (see annex B) shall also be carried out at the same time as the test procedure (see 5.5) using
only the highest concentration and the same conditions (spore test suspension, product test solution and
neutralizer or rinsing liquid) as used in the test (see 5.5.2.2 or 5.5.2.3).
5.6 Calculation and expression of results
7)
5.6.1 Calculation of viable counts (cfu/ml)
5.6.1.1 Spore test suspension
Viable counts of the spore test suspension (see 5.4.1.3) should be calculated according to the principles described
as follows :
a) only colony counts which are less than 300 cfu/set of plates should be used for calculation of viable counts.
For a result to be valid at least one set of plates has to contain 15 or more colonies ; viable counts should be
calculated using at least one pair of the set of plates, where one or both are containing more than 15 colonies
and both contain less than 300 colonies. If the set of plates from 2 dilutions fall within this range calculate the
number of cfu/ml as the weighted mean count. If the set of plates from only one dilution fall within this range
calculate the arithmetic mean.
For calculation of the weighted mean count, use the following formula (1) :

6)
cfu/ml = Colony forming unit per ml.
7)
cfu/ml = Colony forming unit per ml.
c
cfu/ml = (1)
(n + 0,1 n ) d
1 2
where
c is the sum of the colonies counted on all the plates taken into account ;
n is the number of the sets of plates taken into account at the first dilution ;
n is the number of the sets of plates taken into account at the second dilution ;
d is the dilution factor corresponding to the first dilution taken into account.
Round off the results calculated to two significant figures. For this, if the last figure is below 5, the preceding figure
is not modified ; if the last figure is more than 5 the preceding figure is increased by one unit ; if the last figure is
equal to 5, round off the preceding figure to the next nearest even figure. Proceed stepwise until two significant
figures are obtained.
b) as a result the number of cfu/ml is expressed by a number between 1,0 and 9,9 multiplied by the appropriate
power of 10.
168 21514 25 422
6 6
EXAMPLE   1,9182 x 10  1,9 x 10
in cfu/ml
4 4

2 0,1x 2 10 2,2 x 10
5.6.1.2 Test procedure and test for validation
For the test procedure (see 5.5.2.2.2 and 5.5.2.3.2) and test for validation (see B.2, B.4.1 and B.4.2) the viable
count should be calculated using the following method. Only colony counts which are less than 300 cfu per plate
should be used for calculation of viable counts. Viable counts should be calculated using colony counts from both
plates. When at least one plate contains 15 or more colonies, use the following formula (2) for calculation of the
viable counts in cfu/ml :
c
(2)
n dV
where
c is the sum of the colonies counted on both plates ;
n is the number of plates taken into account ;
d is the dilution factor corresponding to the dilution taken into account. For the dilution-neutralization test
-1
procedure (see 5.5.2.2.) and the spore suspension (see B.2) the dilution factor is 10 ;
V is the sample volume. For the dilution neutralization and validation procedures (see 5.5.2.2.3 and B.4.1)
and the spore suspension (see B.2) the sample volume is 1,0 ml. For the membrane filtration test and
validation procedure (see 5.5.2.3.3 and B.4.2) the sample volume is 0,1 ml.
For the test procedure (see 5.5) where the number of cfu on all plates counted is lower than 15, record the viable
2 2
count of the test mixture as lower than 1,5 x 10 cfu/ml (< 1,5 x 10 cfu/ml). Where the number of cfu on all plates
counted is higher than 300 record the viable count of the test mixture as higher than 3 x 10 cfu/ml
(> 3 x 10 cfu/ml).
5.6.2 Verification of methodology
For each test organism check that :
6 6
a) N is between 1,5 x 10 cfu/ml and 5 x 10 cfu/ml ;
2 3
b) N is between 6 x 10 and 3 x 10 cfu/ml ;
v
c) B is equal to or greater than 0,05 times N ;
v
d) C is equal to or greater than 0,5 times B ;
e) A is equal to or greater than 0,05 times N .
v
where
N is the number of cfu/ml of the spore test suspension (see 5.4.1.3) ;
N is the number of cfu/ml of the spore suspension (see B.2) ;
v
B is the number of cfu/ml of the neutralizer toxicity validation (see B.4.1.2 b)) or of the filtration control
(see B.4.2.2 b)) ;
C is the number of cfu/ml of the dilution-neutralization validation (see B.4.1.2 c)) or of the membrane filtration
test control (see B.4.2.2 c)) ;
A is the number of cfu/ml of the experimental conditions validation (see B.4.1.2 a) or B.4.2.2 a)).
5.6.3 Expression of results
For the test organism record the number of cfu/ml in the spore test suspension (N) (see 5.4.1.3) and after the test
procedure for sporicidal activity of the product (N ) (see 5.5.2.2.3 or 5.5.2.3.3).
a
For the validation of neutralization (see annex B) record the number of cfu/ml (N ) in the spore suspension
v
(see B.2).
For validation of the dilution neutralization method (see B.4.1) record the number of cfu/ml in the neutralizer toxicity
control (B), the dilution neutralization control (C) and the experimental conditions control (A).
For validation of the membrane filtration method (see B.4.2), record the number of cfu/ml in the filtration control (B),
the filtration test control (C) and the experimental conditions control (A).
For each test organism and product test concentration calculate and record the reduction in viability as follows :
1
N 10
R  reduction in viability  (3)
N
a
5.7 Conclusion
Sporicidal activity for general purposes is characterized by the concentration of the tested product for which criteria
5.6.1 and 5.6.2 are met and for which a 10 or more reduction in viability is demonstrated under the required test
conditions : 60 min ± 10 s, 20 °C ± 1 °C and clean conditions (see clause 4), and when the test organisms are
spores of Bacillus subtilis.
Sporicidal activity for specific purpose is characterized by the concentration of the tested product for which criteria
5.6.1 and 5.6.2 are met and for which a 10 or more reduction in viability is demonstrated, when the test organisms
Bacillus subtilis and if required (see clause 4) additional test organisms, under additional conditions : t
are spores of
in min and
in °C.
It is accepted that for certain applications, this suspension test may
...