SIST EN 14476:2026

SLOVENSKI STANDARD
01-januar-2026
Nadomešča:
SIST EN 14476:2013+A2:2019
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje virucidnega delovanja v humani medicini - Preskusna metoda in
zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of virucidal activity in the medical area - Test method and requirements (Phase 2/Step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l’évaluation de l’activité virucide dans le domaine médical - Méthode d’essai et exigences
(Phase 2/Étape 1)
Ta slovenski standard je istoveten z: EN 14476:2025
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 14476
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2025
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 14476:2013+A2:2019
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of virucidal activity in
the medical area - Test method and requirements (Phase
2/Step 1)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
virucide dans le domaine médical - Méthode d'essai et viruziden Wirkung im humanmedizinischen Bereich -
exigences (Phase 2/Étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 20 July 2025.

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 CEN-CENELEC 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 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14476:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 8
4 Requirements . 9
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 11
5.2.1 Test viruses . 11
5.2.2 Culture media, reagents and cell cultures . 12
5.3 Apparatus and glassware . 16
5.4 Preparation of test organism suspensions and product test solutions . 17
5.4.1 Test organisms suspensions (test virus suspension) . 17
5.4.2 Product test solutions . 18
5.5 Procedure for assessing the virucidal activity of the product . 18
5.5.1 General . 18
5.5.2 Test procedure . 19
5.5.3 Modified method for ready-to-use products . 21
5.5.4 Cytotoxicity caused by product test solutions . 21
5.5.5 Interference control – control of cell susceptibility . 22
5.5.6 Control of efficiency of suppression of product’s activity . 23
5.5.7 Reference test for virus inactivation . 23
5.5.8 Titration of the virus control . 25
5.5.9 Titration of test samples . 25
5.6 Experimental data and calculation . 25
5.6.1 Protocol of results. 25
5.6.2 Calculation of infectivity titre (TCID or PFU) and reduction . 25
5.7 Verification of the methodology and test validity . 33
5.8 Expression of results . 34
5.8.1 General . 34
5.8.2 Calculation of the virucidal activity of products . 34
5.8.3 Control of active and non-active product test solution . 34
5.9 Test report . 35
Annex A (informative) Examples of viruses sorted according to their presence in the human
body in case of virus infection . 37
Annex B (informative) Detoxification of test mixtures by molecular sieving . 39
TM
B.1 Molecular sieving with Sephadex LH 20 . 39
B.1.1 Principle . 39
B.1.2 Sephadex suspension . 39
B.1.3 Procedure . 39
TM
B.2 Molecular sieving using MicroSpin S 400 HR . 41
Annex C (informative) Example of presentation of data results for one active concentration . 42
Annex D (informative) Quantitative determination of formaldehyde and PAA concentrations . 45
D.1 Quantitative determination of formaldehyde . 45
D.2 Determination of PAA concentration . 46
Annex E (informative) Preparation of the glutaraldehyde test solutions (v/v) . 49
Bibliography. 50

European foreword
This document (EN 14476:2025) 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 April 2026, and conflicting national standards shall be
withdrawn at the latest by April 2026.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 14476:2013+A2:2019.
This document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to
harmonize the structure and wording with other existing tests of CEN/TC 216 or in preparation and to
improve its readability and thereby make it more understandable.
EN 14476:2013+A2:2019:
— de-harmonization of the standard (Annex ZA and references to MDR/MDD deleted);
— for textile disinfection and instrument disinfection a test for virucidal activity against enveloped
viruses with vaccinia virus has been added;
— Glutaraldehyde has been added as an alternative reference substance to formaldehyde;
— for chemothermal disinfection e.g. textile disinfection peracetic acid has been added as reference
substance;
— for the hygienic handrub and handwash claims, a test for virucidal activity against enveloped viruses
with vaccinia virus was added with specific lg reduction requirement for handwash products;
— the calculation was shifted to the main text (harmonized with EN 13727);
— the LVP method was shifted to the main text;
— it was clarified that given cell line numbers are only examples;
— for hygienic handrubs, adenovirus shall be tested at (25 ± 1) °C;
— spelling errors and incorrect references were corrected.
The changes of this revision have no impact on the test results obtained with reference to the version
EN 14476:2013+A2:2019. Those results are still valid with the exception of test reports using gel
filtration detoxification, which do not provide a parallel titration of the non-filtrated test mixture.
Additional comparison between unfiltered and filtrated test mixture shall be added in the test report
using gel filtration detoxification.
Test results for hygienic handrub products tested at 20 °C with adenovirus in accordance with the
previous version are still valid.
Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications
in the medical area are in preparation.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
Introduction
This document specifies a suspension test for establishing whether a chemical disinfectant or an
antiseptic has a virucidal activity in the area and fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including contact
time, temperature, test organisms and interfering substances, i.e. conditions which can influence its
action in practical situations. Each utilization concentration of the chemical disinfectant or antiseptic
found by this test corresponds to the chosen experimental conditions.

1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectant and antiseptic products that form a homogeneous physically stable preparation when diluted
with hard water – or in the case of ready-to-use products, i.e. products that are not diluted when applied,
– with water. Ready-to-use-products can only be tested at a concentration up to 80 % (97 %, with a
modified method for special cases) as some dilution is always produced by adding the test organisms and
interfering substance.
This document is applicable to products that are used in the medical area in the fields of hygienic handrub,
hygienic handwash, instrument disinfection by immersion, surface disinfection by wiping, spraying,
flooding or other means and textile disinfection.
This document is applicable to areas and situations where disinfection is medically indicated. Such
indications occur in patient care, for example:
— in hospitals, in community medical facilities, and in dental institutions;
— in clinics of schools, of kindergartens, and of nursing homes;
and can occur in the workplace and in the home. It can also include services such as laundries and
kitchens supplying products directly for the patient.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active
substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2, step 1 test.
NOTE 3 EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
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
EN 12353, and EN 14885 are updated on an ongoing basis as the standards to which they refer are developed and evolve. As the
revision of both standards is much more frequent than the updating of this standard, the standards are not dated. Users should refer to
the latest version of the referenced standards.
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
cytotoxicity
morphological alteration of cells and/or their destruction caused by the product
3.2
plaque forming units
PFU
number of infectious virus particles per unit volume
Note 1 to entry: The unit volume is given in ml.
3.3
reference test for virus inactivation
test with a defined reference product in parallel with a product under test for the internal control of the
test and to determine the stability of the test organism
Note 1 to entry: As defined reference product e.g. glutaraldehyde or formaldehyde could be used.
3.4
TCID
50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (3.5)
in 50 % of cell culture units
3.5
viral cytopathic effect
CPE
morphological alteration of cells and/or their destruction as a consequence of virus multiplication
3.6
viral plaque
area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication
of a single infectious virus particle
3.7
virus titre
amount of infectious virus per unit volume present in a cell culture lysate, cell culture supernatant or in
a solution
Note 1 to entry: The unit volume is given in ml.
4 Requirements
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre (for hygienic
handwash at least a 2 lg reduction) when tested in accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Test Hygienic Hygienic Instrument Surface Textile
conditions handrub handwash disinfection disinfection disinfection
Minimum Virucidal  Virucidal activity Virucidal Virucidal
spectrum of activity activity activity

test organisms
poliovirus
poliovirus
adenovirus poliovirus murine
adenovirus
parvovirus
murine norovirus adenovirus
murine norovirus
murine norovirus
when
temperature is
40 °C or higher:
only murine
parvovirus
Limited   Limited
spectrum spectrum
virucidal virucidal
a a
activity activity
adenovirus adenovirus
murine norovirus murine norovirus

Virucidal Virucidal Virucidal activity Virucidal Virucidal
activity against activity against against enveloped activity against activity against
enveloped enveloped b enveloped enveloped
viruses
b b b b
viruses viruses viruses viruses
(Pre-cleaning
vaccinia virus vaccinia virus products with a vaccinia virus vaccinia virus
combined cleaner/
disinfectant)
vaccinia virus
additional Any relevant test organism
Test according to the manufacturer’s recommendation, but at / between
temperature
(20 ± 1) °C (20 ± 1) °C d 4 °C and 30 °C d
20 °C and 70 °C 30 °C and 70 °C
[alcohol dis. with
adenovirus at
c
(25 ± 1) °C]
Contact time according to the manufacturer’s recommendation
but between but between but no longer than but no longer but between
than
30 s and 120 s 30 s and 120 s 60 min 10 min and
60 min 20 min
Test Hygienic Hygienic Instrument Surface Textile
conditions handrub handwash disinfection disinfection disinfection
Interfering substance
clean conditions 0,3 g/l bovine – 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
albumin solution albumin solution albumin solution albumin solution

(hygienic and/or and/or (for pre-washed
handrub) process)
dirty conditions – 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
albumin solution albumin solution albumin solution albumin solution
plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l
erythrocytes erythrocytes erythrocytes erythrocytes
(hygienic (for non-pre-
handwash) washed process)
Additional dirty any relevant any relevant any relevant any relevant
conditions substance substance substance substance
any relevant
substance
a
The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus, rotavirus
and adenovirus.
b
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only (Annex A).
c
For hand rubs, adenovirus shall be tested only at (25 ± 1) °C. It has long been known that adenoviruses, unlike other
non-enveloped viruses (polio + entero viruses), exhibit a temperature effect in alcoholic formulations. [16], [17] This is
most likely due to the special structure with antenna-like fibre proteins, which could cause precipitation of the virus,
which then dissolves again at (37 ± 1) °C. The viruses are then released into the solution. Thus, the hand disinfectant
appears to be false-negative ineffective, although only a few degrees difference makes the viruses capable of
inactivation again in this artificial suspension test. 25 °C is still in the range of the hand skin temperature.
d
For further information regarding validity at elevated temperatures see EN 14885.

5 Test methods
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test virus suspension in a solution of an interfering substance. The mixture is
maintained at one of the temperatures and the contact times specified in Clause 4 and 5.5.1.1. At the end
of this contact time, an aliquot is taken; the virucidal action in this portion is immediately suppressed by
a validated method (dilution of the sample in ice-cold cell maintenance medium). The dilutions are
transferred into cell culture units i.e. wells of microtitre plates either using monolayer or cell suspension.
Infectivity tests are done either by plaque test or quantal tests. After incubation, the titres of infectivity
are calculated according to Spearman and Kärber (quantal tests, 5.6.2.1), by plaque counting (plaque test,
5.6.2.5) or large-volume-plating (LVP) method (5.6.2.6) and evaluated. Reduction of virus infectivity is
calculated from differences of lg virus titres without (virus control) and after treatment with the product.
Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded
as a ready-to-use product (5.4.2).
5.1.2 The test is performed using the test organisms as specified in Clause 4, Table 1.
5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Table 1 may be used.
Additional interfering substances and test organisms may be used.
5.2 Materials and reagents, including cell cultures
5.2.1 Test viruses
The virucidal activity shall be evaluated using the following strains as test organisms selected according
to Clause 4, Table 1. Virus strains shall be obtained from a national or international culture collection :
a) non-enveloped RNA virus :
1) poliovirus type 1, LSc 2ab (Picornavirus);
Regarding poliovirus only virus material that passed the requirements for the production of
oral polio vaccine of the competent authority shall be used (Other stocks derived from LSc-2ab
cannot be used any longer).
NOTE 1 See the list of vaccines of the World Health Organisation (WHO).
NOTE 2 See also WHO Global Action Plan for Poliovirus Containment.
2) murine norovirus, strain S99 Berlin;
b) non-enveloped DNA virus:
1) adenovirus type 5, strain Adenoid 75, ATCC VR-5;
2) murine parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346;
c) enveloped DNA virus:
1) modified vaccinia virus Ankara (MVA), ATCC VR-1508, or vaccinia virus strain Elstree, ATCC VR-
1549.
The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.1.3). The
same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its
control and validation.
If additional test organisms are used, they shall be kept and used under optimum growth conditions
(temperature, time, atmosphere, media, susceptible cell line) noted in the test report. If these additional
test organisms (viruses and cell line) 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
collection under a reference for five years.

The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given for the convenience
of users of this document and does not constitute an endorsement by CEN.
Poliovirus type 1, LSc-2ab can be obtained either from NIBSC (https://nibsc.org/ ) or Friedrich-Loeffler-Federal Research Institute for
Animal Health, Hauptsitz Insel Riems Südufer 10, 17493 Greifswald-Insel Riems; phone: +49 38351 7-0; fax: +49 38351 7-121.
http://www.fli.de/. Murine norovirus can be obtained from Friedrich-Loeffler-Federal Research Institute for Animal Health . This
information is given for the convenience of users of this document and does not constitute an endorsement by CEN.
5.2.2 Culture media, reagents and cell cultures
5.2.2.1 General
All weights of chemical substances given in this document refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
To improve reproducibility, it is recommended that commercially available material is used for the
preparation of culture media. The manufacturer's instructions relating to the preparation of these
products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at (20 ± 1) °C.
5.2.2.2 Water
The water shall be freshly glass-distilled water or demineralized water, which meets the requirements of
ASTM D 1193 (Type I or II, see Table 2) or use water for injections (see bibliographic reference [1]).
Sterilize in the autoclave [5.3.1.1 a)]. Sterilization is not necessary if the water is used e.g. for preparation
of culture media and subsequently sterilized. Alternatively, cell culture grade water is suitable for
preparation of cell culture media and other solutions used in cell culture.
Table 2 — Consolidated classification of water qualities
Characteristic Unit Type I Type II
(ultrapure (purified
water) water)
Resistance (M/cm)Ω > 18 > 1
Conductivity µS/cm < 0,056 < 1
Silicates(SiO ) ppb or µg/L 3 3
TOC ppb or µg/l 50 50
Bacteria CFU/ml < 1 < 100
Endotoxin EU/ml < 0,001 0,03
Type I Required for critical laboratory applications (e.g. for
HPLC, GC, MS etc.). Also, for the production of buffers for
cell culture, as well as for applications in molecular
biology.
Type II Used for buffers and microbiological culture media.
Used as system water for laboratory automats and for
dissolving controls and reagents

See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Phosphate buffered saline (PBS)
Sodium chloride (NaCl) 8,00 g
Potassium chloride (KCl) 0,20 g
Disodium hydrogen phosphate, 12-hydrate (Na HPO × 12H O) 2,89 g
2 4 2
Potassium phosphate, monobasic (KH PO ) 0,20 g
2 4
Water (5.2.2.2) to 1 000,0 ml
5.2.2.4 Neutral Red (1:1 000 solution)
Prepare neutral red (e.g. Sigma N7005 ) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a
0,45 µm pore size filter and store at (4 ± 1) °C in the dark.
5.2.2.5 Foetal calf serum (FCS)
FCS shall be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may interfere
with cell and virus growth resulting in false results.
For RAW 264.7 cells, low endotoxin FCS shall be used due to the cells’ high sensitivity to endotoxins.
5.2.2.6 Trichloroacetic acid (10 % solution) (TCA)
Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), then adjust the volume to 100 ml with water. Stir
to complete solution.
5.2.2.7 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 water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.1.7) or in the
autoclave [5.3.1.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.1.8)
for no longer than one month;
— 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 (5.3.1.7). Store the solution in the refrigerator (5.3.1.8) for
no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.1.12) and add 6,0 ml
(5.3.1.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.1.4) of the hard water shall be 7,0 ± 0,2 (5.3.1.4). 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 (5.4.2), the addition of the product to the hard water
produces different final water hardness in each test tube. In any case, the final hardness in the test tube expressed
as calcium carbonate (CaCO ) is lower than 375 mg/l.
Sigma N 7005 is an example of a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by CEN of this product.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50
times in the case of the modified method, 5.2.2.8.4).
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and detergents) shall be specified.
NOTE 1 “Diluent” is generally used in the other European Standards in the medical area to prepare the
interfering substance. Since there is no experience in virucidal testing with diluent, water (5.2.2.2) is used instead.
NOTE 2 The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(5.2.2.2).
Sterilize by membrane filtration (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one month.
The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.
5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solution – high concentration with sheep
erythrocytes)
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water
(5.2.2.2).
Sterilize by membrane filtration (5.3.1.7).
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g for
N
10 min (5.3.1.13). After discarding the supernatant, resuspend erythrocytes in PBS (5.2.2.3). Repeat this
procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see
above). To avoid later contamination this mixture should be split in portions probably needed per day.
Keep the mixture in separate containers for a maximum of 7 d in a refrigerator (5.3.1.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3 g/l and 3 ml/l respectively.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.3)
Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering
substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems
with the filtration.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of water
(5.2.2.2);
b) dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml (instead of
48,5 ml) of water (5.2.2.2). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml
(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution
to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood
The defibrinated sheep blood shall be sterile (aseptic blood-letting and preparation). The defibrinated
sheep blood can be pooled from more than one sheep and can be acquired from a commercial supplier.
5.2.2.10 Medium for cell cultures
Eagle’s minimal essential medium (MEM) or equivalent, supplemented with FCS (5.2.2.5), antibiotics, and
other growth factors as needed shall be used.
a) A growth medium for cell multiplication is generally supplemented with 10 % FCS. Add 10 parts of
FCS (5.2.2.5) to 90 parts of MEM.
b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell
proliferation is generally supplemented with 2 % FCS. Add 2 parts of FCS (5.2.2.5) to 98 parts of MEM.
Other media may be used if appropriate for certain cell lines.
See EN 12353 for a detailed description.
5.2.2.11 Cell cultures
Before virus inoculation, cell monolayers shall be at appropriate level of confluence based on the specific
virus (see also EN 12353). Cell lines are selected in accordance with their sensitivity to the test organisms
(5.2.1). Cells for virus titration, if used as suspensions in quantal tests, shall be added to the dilutions of
the test mixture (5.5.2) in such a density as to enable the formation of a monolayer in at least two days in
the cell control. Cell cultures can be used as cell monolayers or in suspensions for quantal tests. For details
of cell lines see 5.5.1.1 e).
5.2.2.12 Reference substances
5.2.2.12.1 Glutaraldehyde (Glutaral, 1,5-Pentanedial) CAS Number 111-30-8
Required chemical and physical parameters for use as reference standard for testing disinfectant
preparations are specified in Table 3.
Table 3 — Required chemical and physical parameters
Parameters Specifications
Solution 1 % Solution 25 %
a
clear liquid clear liquid
Appearance
pH-value 3,1 to 4,5 * 3,1 to 4,5
Concentration of glutaraldehyde (by titration) 50,0 % to 52,0 % 25,0 % to 26,0 %
Stability (ambient conditions) 12 months 12 months
* The pH value of the 1 % solution was in a ring trial 3,78 ± 0,5.
a
Yellowish colour indicates a beginning polymerization.

The specification above (see Table 3) should be checked on the certificate of analyses. The pH values shall
be tested and confirmed regularly by the laboratory. If the pH values and / or appearance are out of
specification the glutaraldehyde shall not be used.
5.2.2.12.2 Formaldehyd and peracetic acid
Formaldehyd and peracetic acid see 5.5.7 and Annex D.
5.3 Apparatus and glassware
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) by moist heat, in the autoclave [5.3.1.1 a)];
b) by dry heat, in the hot air oven [5.3.1.1 b)].
5.3.1 Usual microbiological laboratory equipment and, in particular, the following:
5.3.1.1 Apparatus for sterilization (moist and dry heat):
+3
a) For moist heat sterilization, an autoclave capable of being maintained at ( 121 ) °C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilization, a hot air oven capable of being maintained at ( 180 ) °C for a minimum
+5 +5
holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.1.2 Water baths, capable of being controlled at (20 ± 1) °C, and at additional test
temperatures ±1 °C (5.5.1), or thermoblock or thermomixer
5.3.1.3 C0 Incubator (95 % air, 5 % C0 ), capable of being controlled either at (36 ± 1) °C or
2 2
(37 ± 1) °C (5.2.1).
5.3.1.4 pH-meter, having a maximum permissible error of no more than ±0,1 pH units at (20 ± 1) °C.
5.3.1.5 Stopwatch.
5.3.1.6 Shakers:
® 6
a) electromechanical agitator, e.g. Vortex mixer ;
b) mechanical shaker.
5.3.1.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,22 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8.2,
5.2.2.8.3 and 5.2.2.8.4).
Disposable sterile equipment is an acceptable alternative to reusable glassware.
6 ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by CEN of this product.
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 to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml in 20 s to 40 s. The filtration of the interfering substance (5.2.2.8) for
the modified method (5.5.3) may take a longer time.
5.3.1.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.1.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 10 µl or calibrated automatic
pipettes.
5.3.1.10 Ice producing machine or commercially available ice to cool the cell maintenance medium
and the reaction mixtures during the test.
5.3.1.11 Basin as ice bath with ice and water.
5.3.1.12 Volumetric flasks.
5.3.1.13 Centrifuge (400 g to 1 000 g ).
N N
5.3.1.14 Microtitre plates or tubes, petri dishes and flasks for cell culture use.
5.3.1.15 Magnetic stirrer for keeping cells in suspension before seeding.
5.3.1.16 Inverted microscope for reading cell cultures microscopically.
5.3.1.17 Container: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.1.18 Biological safety cabinet, class II.
5.3.1.19 Freezer, −70 °C or less.
5.3.1.20 Equipment for cell storage in liquid nitrogen.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organisms suspensions (test virus suspension)
The test organisms or their stock cultures respectively shall be prepared and kept in accordance with
EN 12353.
The stock virus suspension is multiplied in an appropriate cell line that produces high titres of infectious
viruses. The cell debris is separated by centrifugation (400 g for 15 min). This preparation is called “test
N
virus suspension”.
The test virus suspension is kept in small volumes below −70 °C or preferably at −196 °C under nitrogen.
Due to safety reasons, and – in some cases – to limit the possibility of genetic mutations, only 10 passages
from the original seed (e.g. virus from culture collection) are allowed.
It is suggested that the minimum titre of the test virus suspension - determined by a quantal test [5.5.2
a)] or by plaque test [5.5.2 b)] - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at
least enable a titre reduction of 4 lg (2 lg for hygienic handwash) to verify the method.
The test virus suspension may be concentrated by appropriate methods [e.g. ultracentrifugation or ultra-
® 7
centrifugal filters (Amicon ) ].
NOTE Ultracentrifugation can cause aggregates which can have influence of virus inactivation
The test virus suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration (= real test
concentration) because it is diluted to 80 % during the test (5.5.2).
Product test solutions shall be prepared in hard water (5.2.2.7) at a minimum of three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.8.3). The product as received may be used as one of the product test solutions, in this case the
highest tested concentration is 80 %. Ready-to-use products may be tested at 97 % [modified method
(5.5.3)]. In this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash
products are always pre-diluted with hard water (5.2.2.7) to achieve a 62,5 % solution. This solution
simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a “ready-
to-use product”. The modified method (5.5.3) cannot be used, since 62,5 % represents the highest
accepted concentration (50 %), multiplied by 1,25.
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 (5.2.2.7). Subsequent dilutions (= lower concentrations)
shall be prepared in volumetric flasks (5.3.1.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks
(5.3.
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