oSIST prEN 13704:2026

SLOVENSKI STANDARD
01-maj-2026
Kemična razkužila - Kvantitativni suspenzijski preskus za vrednotenje
sporocidnega delovanja kemičnih razkužil v živilski in drugih industrijah,
gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja
1)
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 Suspensionversuch 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: prEN 13704
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.

DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2026
ICS Will supersede EN 13704:2018
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 Chemische Desinfektionsmittel - Quantitativer
suspension pour l'évaluation de l'activité sporicide des Suspensionversuch zur Bestimmung der sporiziden
désinfectants chimiques utilisés dans le domaine de Wirkung chemischer Desinfektionsmittel in den
l'agro-alimentaire, dans l'industrie, dans les domaines Bereichen Lebensmittel, Industrie, Haushalt und
domestiques et en collectivité - Méthode d'essai et öffentliche Einrichtungen - Prüfverfahren und
prescriptions (phase 2, étape 1) Anforderungen (Phase 2, Stufe 1)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.
If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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 CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13704:2026 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 7
5 Test method . 8
5.1 Principle . 8
5.2 Materials and reagents . 8
5.2.1 Test organisms . 8
5.2.2 Culture media and reagents . 9
5.3 milk.Apparatus and glassware .11
5.3.1 General .11
5.3.2 Usual microbiological laboratory equipment and, in particular, the following .11
5.4 Preparation of spore test suspension and test solutions .12
5.4.1 Spore suspensions .12
5.4.2 Product test solution .14
5.5 Procedure.15
5.5.1 Choice of experimental conditions .15
5.5.2 Test procedure for assessing the sporicidal effect of the product .15
5.5.3 Validation of dilution neutralization and membrane filtration method .17
5.6 Calculation and expression of results .18
5.6.1 Overview of the different suspensions and test mixtures .18
5.6.2 Calculation .18
5.7 Verification of methodology .23
5.7.1 General .23
5.7.2 Control of weighted mean counts .23
5.7.3 Basic limits .23
5.7.4 Expression of results .24
5.8 Conclusion.24
5.9 Test report .24
Annex A (normative)  Preparation of Bacillus subtilis and Bacillus cereus spore stock
suspensions .26
A.1 Material and reagents .26
A.2 Preparation of Bacillus spore stock suspensions .26
Annex B (normative)  Validation of dilution-neutralization and membrane filtration methods 28
B.1 Principle .28
B.2 Preparation of spore suspension .28
B.3 Preparation of product test solution .28
B.4 Test for validation .28
B.4.1 Dilution-neutralization method .28
B.4.1.1 General . 28
B.4.1.2 Validation . 29
B.4.2 Membrane filtration method . 30
B.4.2.1 General . 30
B.4.2.2 Validation . 30
B.5 Validation . 32
Annex C (informative) Preparation of Clostridium sporogenes spore stock suspension . 33
C.1 Culture media and reagents . 33
C.2 Apparatus and glassware. 34
C.3 Preparation of regenerated media and incubation conditions . 34
C.4 Preparation of Clostridium spore stock suspension . 35
Annex D (informative)  Neutralizers and rinsing liquids . 36
Annex E (informative)  Example of a typical test report . 38
Annex F (informative)  Referenced strains in national collections . 40
F.1 Bacillus subtilis . 40
F.2 Bacillus cereus . 40
F.3 Clostridium sporogenes . 40
Annex G (informative)  Guide for preparation of reference test solution . 41
Annex H (informative)  Determination of sporistatical activity . 42
H.1 General . 42
H.2 Reagents . 42
H.3 Procedure . 43
Annex I (informative)  Example for a titration Peracetic acids and hydrogen peroxide . 44
I.1 General . 44
I.2 Equipment . 44
I.3 Procedure . 44
Bibliography . 46

European foreword
This document (prEN 13704:2026) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 13704:2018.
It was revised in order to include the spore susceptibility controls, to better detail spore preparation
procedures and to harmonize counting and elaboration of the results with the other CEN/TC 216 current
standards.
- modification of spore suspension susceptibility controls, as described in TR (Technical Report) ;
- modification of spore preparation procedures, aligning this part with the provisions of standard
EN 17126:2018;
- alignment of interfering conditions for dairies with the provisions of standard EN 1276:2019;
Results obtained with the previous version are valid.
Introduction
This document 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 document specifies a test method (phase 2/step 1) 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 document 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 document, 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
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN 12353, Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of
bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including
bacteriophages) activity
EN 14885, Chemical disinfectants and antiseptics - Application of European Standards for chemical disinfectants
and antiseptics
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
4 Requirements
The product shall demonstrate at least 3 decimal log (lg) reduction, when tested in accordance with Table 1
here below and Clause 5.
Table 1 — Minimum and additional test conditions
Test Conditions for Surface disinfection
Minimum spectrum of test organisms Bacillus subtilis
Additional sporicidal activity vs Clostridium sporogenes
anaerobes for specific uses
Additional sporicidal activity vs Bacillus cereus
aerobes for specific uses
Required reduction ≥ 3 lg
Test temperature according to the manufacturer’s recommendation,
but between 4 °C and 75 °C
Contact time (in minutes) according to the manufacturer’s recommendation, but between 1
min and 60 min (only contact times of 1, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55 and 60 min are allowed in this range)
Interfering substance Clean conditions:
0,3 g/l bovine albumin solution
or
Dirty conditions:
3,0 g/l bovine albumin solution
Additional interfering substance for 10,0 g / l of reconstituted milk
dairies
Other additional strains and additional test conditions may be tested according to product claim.
5 Test method
5.1 Principle
A test suspension of bacterial spores in a solution of interfering substance, simulating clean and/or dirty
conditions, is added to a prepared sample of the product under test diluted in hard water (in water for ready-
to-use products). The mixture is maintained at specific test temperature ± 1 °C for the specific test contact
(time ± 10) s (required test conditions). In case the contact time is 1 min, the tolerance allowed shall be ± 5 s
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.
5.2 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 .

1)
ATCC 6633 and ATCC 19404 are the collection numbers of strains supplied by the American Type Culture Collections. CIP 79.3 is
the collection number of spores supplied by the Collection de l'Institut Pasteur. This information is given for the convenience of users
If required for specific applications or products, additional strains may be chosen from, for example :
— Bacillus cereus CIP 105151;
1)
— Clostridium sporogenes ATCC 19404, CIP 79.3 .
NOTE 1 See Annex F for corresponding strain numbers in some other culture collections.
NOTE 2 See Annex C for particular culture and handling conditions for Clostridium sporogenes.
NOTE 3 It has been noted that different sources of Bacillus cereus strain can lead to different sporulation behaviour,
in particular CIP 105151 strain seems to sporulate better.
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.
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 or deionized water.
Sterilize in the autoclave (5.3.2.1). Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.See 5.2.2.6 for preparation of hard water.
5.2.2.3 Tryptone Soja Agar (TSA)
For counting of viable Bacillus spores :
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of Soybean meal 5,0 g
Sodium Chloride (NaCl) 5,0 g
Agar 15,0 g
Water (see 5.2.2.2) 1 000,0 ml
Sterilize in the autoclave (5.3.2.1). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2
measured at (20 ± 1) °C
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.
5.2.2.4 Neutralizer
The neutralizer shall be validated for the product under test in accordance with Annex B. 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 D.
5.2.2.6 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride (CaCl ) in
2 2
water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
(5.3.2.1 a)). Autoclaving – if used - may cause a loss of liquid. In this case make up to 1000 ml with water
(5.2.2.2) under aseptic conditions. Store the solution in the refrigerator at 5 °C ± 3 °C (according 5.3.2.15)
for no longer than four weeks;
— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator at 5 °C ± 3 °C
(according 5.3.2.15) for no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9)
of solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The pH of the hard
water shall be (7,0 ± 0,2), when measured at (20 ± 1) °C (5.3.2.4). 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 (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.2), the addition of the product to the hard water produces a
different final water hardness in each test tube. In any case the final hardness expressed as calcium carbonate (CaCO )
is in the test tube lower than 375 mg/l.
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 Clean conditions
Bovine albumin solution for the test conditions shall be prepared as follows:
— dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(see 5.2.2.2) ;
— sterilize by membrane filtration (5.3.2.7).
The final concentration of the bovine albumin in the test procedure (5.5.2) is 0,3 g/l.
5.2.2.7.3 Dirty conditions
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(5.2.2.2).
Sterilize by membrane filtration (5.3.2.7).
The final concentration of bovine albumin in the test procedure (5.5.2) shall be 3,0 g/l.
5.2.2.7.4 Additional interfering substance for dairies
Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder
per litre of water (5.2.2.2), shall be prepared as follows:
— prepare a solution of 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90
parts of water. Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3) °C].
The final concentration of reconstituted milk in the test procedure (5.5.1c) is 1,0 % (v/v) of reconstituted
5.3 milk.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 :
a) in the autoclave (see 5.3.2.1);
b) in the dry heat sterilizer (see 5.3.2.1).
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 ± 1) °C, (45 ± 1) °C, (75 ± 1) °C and at test
temperatures ± 1 °C (see 5.5.1).
5.3.2.3 Incubator, capable of being controlled at (37 ± 1) °C.
5.3.2.4 pH-meter, having an accuracy of calibration of ± 0,1 pH units at 20 °C or equivalent.
5.3.2.5 Stopwatch
2)
Disposable equipment is an acceptable alternative to reusable glassware.
3)
5.3.2.6 Vortex mixer (electromechanical agitator, i.e. Vortex® mixer )
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.
5.3.2.15 Fridge, capable of being controlled at (5 ± 3) °C.
5.3.2.16 Jars for anaerobiosis with oxygen removal system or any other system suitable for generating
anaerobiosis.
5.3.2.17 Centrifuge capable of 10 000 g acceleration.
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
The Bacillus subtilis ATCC 6633 CIP 52.62 spore stock suspension shall be prepared according to Annex A.
For the preparation of the stock spore suspensions of additional strains (see 5.2.1) refer to :
— Annex A for Bacillus cereus; CIP 105.151
— Annex C for Clostridium sporogenes ATCC 19404, CIP 79.3.
When testing in accordance with EN 17126 and EN 13704 the following information is taken into account.
Viability is checked and the susceptibility of each spore batch after at least 8 weeks storage at 2 °C to 8°C
for Clostridium spores and 4 weeks storage at 2 °C to 8 °C for Bacillus spores. Glutardialdehyde is used
and peracetic acid at set concentrations. Tests are performed according to the dilution-neutralization
method and use hard water instead of an interfering substance. The validation is performed with a

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.
6 6
suspension adjusted to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml.Pre-examination studies agreed that at (20 ± 1) °C:
a) With Bacillus subtilis ATCC 6633 without interfering substance:
— 3,0 % (v/v) – 30 min: Glutardialdehyde solution (buffered with NaHCO3) is achieve a lg reduction of < 3 lg.
The pH is equivalent to 7,6 ± 0,2.
— 10,0 % (v/v) – 30 min: Glutardialdehyde solution (buffered with NaHCO3) is achieve a lg reduction of ≥ 3
lg.
The pH is equivalent to 7,6 ± 0,2.
— 0,001 % (v/v) – 30 min: Peracetic acid solution (unbuffered) is achieve a lg reduction of < 3 lg.
— 0,05 % (v/v) – 30 min: Peracetic acid solution (unbuffered) is achieve a lg reduction of ≥ 3 lg.
b) With Bacillus cereus CIP 105151 without interfering substance:
— 0,5 % (v/v) – 15 min: Glutardialdehyde solution (buffered with NaHCO3) is achieve a lg reduction of < 3 lg.
The pH is equivalent to 7,6 ± 0,2.
— 3,0 % (v/v) – 15 min: Glutardialdehyde solution (buffered with NaHCO3) is achieve a lg reduction of ≥ 3 lg.
The pH is equivalent to 7,6 ± 0,2 .
— 0,05 % (v/v) –30 min: Peracetic acid solution (unbuffered) is achieve a lg reduction of < 3 lg.
— 0,50 % (v/v) – 30 min: Peracetic acid solution (unbuffered) is achieve a lg reduction of ≥ 3
lg.Glutaraldehyde 50 % with pH between 3,1 and 4,5 shall be used.
For the validation a specific Standard-Biocide should be used. Appropriate substances are glutardialdehyde
and peracetic acid.:
TM 4
Commercial products ofGlutardildehyde are available, e.g. BIOBAN GA 50 Antimicrobial , Glutardialdehyde
50 % by weight in H O from Sigma-Aldrich.
Commercial products for peracetic acid are available, e.g.Lerasept ® Spezial (peracetic acid 15 % pure from
AppliChem), Stockmeier Chemie Eilenburg GmbH and Co. KG, Gustav-Adolf-Ring 5, D-04838 Eilenburg. Stored
at 2 °C to 8 °C.
The standard test solutions are prepared strictly according to guidance given in Annex G.
For each test of a product solution test in parallel a reference product in low concentration (GA/PAA)
which cover the active ingredients of the test product. In the case of chlorine test product, it is
recommended to carry along the reference product PAA. The actual active ingredients of peracetic acid
are determined by titration the day before (see Annex I) and are considered for the preparation of
standard test solutions (see Annex G).
5.4.1.2 For Clostridium sporogenes no susceptibility check shall be done as there are no data
available in order to define a susceptibility range. The C. sporogenes spore suspensions may be
stored for a maximum of 12 months. Spore test suspension (N)
To prepare the spore test suspension, dilute the spore stock suspension (see A.2) with water (see 5.2.2.2). The
6 6
number of spores in the test suspension shall be adjusted to 1,5 × 10 to 5 × 10 cfu/ml, estimating the
number of units by any suitable mean.
Maintain the suspension test in the water bath at (20 ± 1) °C and use within 2 h.

to be obtained through: DOW Chemical Company Ltd, Diamond House
Microscopic examination under 400 × magnification shall be carried out immediately after the preparation of
the spore test suspension and just before the test, to show the absence of vegetative cells and germinative
spores.
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 TSA (see 5.2.2.3),
cooled to (45 ± 1) °C.
5.4.1.3 Counting of spore test suspension
Incubate the Petri dishes at (37 ± 1) °C for 20-24 h and plate count. Re-incubate for 20-24 h more and re.count.
5)
(see 5.3.2.3). Determine the highest number of colonies V for each plate. Calculate the number of cfu/ml
c
(see 5.6) in the test suspension (N) using the method given in 5.6.1.2.
5.4.1.4 Validation suspension (“N ”, “N ”)
v v0
a) To prepare the validation suspension (“Nv”), dilute the test suspension (5.4.1.2) with the water (5.2.2.2) to
2 3 −3
obtain 3,0 x 10 cfu/ml to 1,6 x 10 cfu/ml [about one fourth (1+3) of the 10 dilution].
b) Maintain and use this validation suspension (Nv) the same way as the test suspension (5.4.1.2).
−1
For counting Nv prepare a 10 dilution with water (5.2.2.2), mix and take a sample of 1,0 ml in duplicate and
inoculate using the pour plate technique or spread plate technique (only Bacillus).
For incubation and counting see 5.5.2.2.3.
Nv is the number of cells per ml in the validation suspension. It is tenfold higher than the counts in terms of Vc
−1
values due to the dilution step of 10 [5.4.1.4 b)].
Nv is the number of cells per ml in the mixtures A, B and C at the beginning of the contact time.
5.4.2 Product test solution
Details of samples of the product as received shall be recorded.
Solutions of the test product shall be prepared in hard water (see 5.2.2.6) or distilled water (5.2.2.2) in the
case of ready-to-use products 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 1,0 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 the second and third
dilutions.
When the product is diluted in hard water (or in water, see 5.2.2.2) it shall give a physically homogeneous
stable preparation. If precipitate or flocculation appears during the assay, it shall be mentioned in the test
report.
The product test solutions and dilutions of it shall be prepared freshly and used within 2h.

5)
cfu/ml = Colony forming unit per ml.
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 experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature θ (in °C):
The temperatures to be tested are specified in Clause 4, Table 1.
The allowed deviation for each chosen temperature is ± 1 °C.
b) contact time t (in min):
The contact times to be tested are specified in Clause 4, Table 1.
The allowed deviation for each chosen contact time is ± 10 s (±5 s when the contact time is 1 min).
c) interfering substance:
The interfering substance to be tested is 0,30 g/l bovine albumin (5.2.2.7.2) under clean conditions or
3,0 g/l bovine albumin (5.2.2.7.3) under dirty conditions – according to Clause 4, Table 1 and practical
applications. Additional interfering substance may be tested according to the specific intended uses of the
product. The product shall not cause the formation of any precipitate in the experimental conditions used.
Each selected experimental condition (θ, t, strains) shall be validated in accordance with Annex B.
The longest contact time and the highest concentration shall be validated.
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 unsuitable, repeat the validation test with another neutralizer taking into account the
information given in Annex D.
If neither of the two neutralizers is considered valid, the membrane filtration method (5.5.2.3) may be used.
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
5.5.2.2.1 General
a) if the test temperature 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 θ °C ± 1 °C using the water bath (see 5.3.2.2) controlled at
θ °C ± 1 °C. Check that the temperature of the reagents is stabilized at θ °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 the test temperature is higher than 40 °C ± 1 °C:
Prior to testing, equilibrate the product test solutions to the test temperature of θ °C ± 1 °C using the water
bath (see 5.3.2.2) controlled at θ °C ± 1 °C. Check that the temperature of the reagents is stabilized at
θ °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.
5.5.2.2.2 Test 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 6)
containing 1,5 x 10 to 5 x 10 cfu/ml (see 5.4.1.2).
Start the stopwatch immediately, mix (see 5.3.2.6) and place the test tube in the water bath at θ °C ± 1 °C for
2 min ± 10 s. At the end of the contact time, add 8,0 ml of each 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 θ °C ± 1°C for
the appropriate contact time (t ± 10) s (±5 s when the contact time is 1 min).
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 ± 1) °C.
After a neutralization time of 5 min ± 10 s (in case of contact times of 10 min or shorter only 10s ± 1s),
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 TSA (see 5.2.2.3), cooled to (45 ± 1) °C.
An additional control is included to differentiate between sporistatical and sporicidal activity, following phase
1 EN 14347 norm. This control is relevant to claim sporicidal activity of a product. The determination of the
sporistatical activity is determined strictly according to Annex H.
5.5.2.2.3 Incubation and counting of the test mixture
Incubate the Petri dishes at (37 ± 1) °C (see 5.3.2.3) for 20-24 h and plate count. Re-incubate for 20-24 h more
and re.count.
Determine the highest number of colonies V for each plate.
c
Calculate the number of cfu/ml in the test mixture (N ) using the method given in 5.6.2.
a
For calculating the viable count of the test mixture, the dilution factor is 1:10. The text mixture N and two
a
1:10 dilutions are performed and counted.

6)
cfu/ml = Colony formin
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