SIST EN 14476:2013+A1:2015
(Main)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)
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)
This European Standard 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. Products can only be tested at a concentration of 80 % (97 %, with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies 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 European Standard applies 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 may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
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".
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte als homogene, physikalisch stabile Zubereitung vorliegen, bzw. bei gebrauchsfertigen Produkten, z. B. Produkte, die bei der Anwendung nicht mit Wasser verdünnt werden. Die Produkte können nur bei einer Konzentration von 80 % (97 % bei einem modifizierten Verfahren für Sonderfälle) geprüft werden, da durch Zugabe der Prüfkeime und der Belastungssubstanz immer eine gewisse Verdünnung bewirkt wird.
Diese Europäische Norm gilt für Produkte, die im medizinischen Bereich auf den Gebieten der hygienischen Händedesinfektion, hygienischen Händewaschung, Instrumentendesinfektion durch Eintauchen und Oberflächendesinfektion durch Abwischen, Besprühen, Überfluten oder auf sonstige Weise sowie für die Wäschedesinfektion verwendet werden.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. vor bei der Patientenbetreuung:
in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
in medizinischen Bereichen in Schulen, Kindergärten und Heimen
und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
ANMERKUNG 1 Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit kommerziell erhältlicher Zuberei¬tungen oder Wirkstoffe unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
ANMERKUNG 3 EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour lévaluation de lactivité virucide dans le domaine médical - Méthode dessai et prescriptions (Phase 2/Étape 1)
La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité virucide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans l’eau dure ou – dans le cas de produits prêts à l’emploi, c’est-à-dire de produits qui ne sont pas dilués lors de leur utilisation – dans l’eau. Les produits ne peuvent être soumis à essai qu’à une concentration de 80 % (97 % avec une méthode modifiée dans certains cas particuliers), car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente Norme européenne s’applique aux produits utilisés dans le domaine médical pour le traitement hygiénique des mains par frictions, le lavage hygiénique des mains, la désinfection des instruments par immersion, la désinfection des surfaces par essuyage, pulvérisation, inondation ou d’autres moyens, et la désinfection des textiles.
La présente Norme européenne s’applique aux domaines et situations où la désinfection est indiquée en médecine humaine. De telles indications se produisent pour les soins des patients, par exemple :
- dans les hôpitaux, les centres médicaux communautaires et les institutions dentaires ;
- dans les cliniques d’écoles, de jardins d’enfants et de maisons de soins infirmiers ;
et peuvent se produire sur le lieu de travail et à la maison. Elles peuvent également inclure les services tels que les blanchisseries et les cuisines fournissant directement des produits aux patients.
NOTE 1 La méthode décrite a pour objet de déterminer l’activité de formulations commerciales ou de substances actives dans leurs conditions d’emploi.
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 1.
NOTE 3 L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les «recommandations d’emploi».
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje virucidnega delovanja v medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)
Ta evropski standard določa preskusno metodo in minimalne zahteve za virucidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo ali, pri proizvodih, ki so pripravljeni za uporabo (tj. proizvodih, ki med uporabo niso razredčeni), z vodo. Proizvode je mogoče preskušati samo pri 80-odstotni koncentraciji (s prilagojeno metodo v posebnih primerih 97-odstotni), ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
Ta evropski standard velja za proizvode, ki se uporabljajo na področju zdravstva pri higienskem drgnjenju rok, higienskem umivanju rok, dezinfekciji instrumentov s potapljanjem, površinski dezinfekciji z brisanjem, pršenjem, zalivanjem ali na druge načine in dezinfekciji tekstilij.
Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za dezinfekcijo. Te indikacije se pojavljajo pri negi bolnikov, na primer:
– v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
– v ambulantah šol, vrtcev in domov za starejše;
in lahko nastanejo na delovnem mestu ali doma. Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode dostavljajo neposredno bolnikom.
OPOMBA 1: Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo.
OPOMBA 2: Ta metoda ustreza preskusu stopnje 1 faze 2.
OPOMBA 3: Standard EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 14476:2013+A1:2015
01-november-2015
.HPLþQDUD]NXåLODLQDQWLVHSWLNL.YDQWLWDWLYQLVXVSHQ]LMVNLSUHVNXV]D
YUHGQRWHQMHYLUXFLGQHJDGHORYDQMDYPHGLFLQL3UHVNXVQDPHWRGDLQ]DKWHYH ID]D
VWRSQMD
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)
$QWLVHSWLTXHVHWGpVLQIHFWDQWVFKLPLTXHV(VVDLTXDQWLWDWLIGHVXVSHQVLRQSRXU
OpYDOXDWLRQGHODFWLYLWpYLUXFLGHGDQVOHGRPDLQHPpGLFDO0pWKRGHGHVVDLHW
SUHVFULSWLRQV 3KDVHeWDSH
Ta slovenski standard je istoveten z: EN 14476:2013+A1:2015
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
SIST EN 14476:2013+A1:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST EN 14476:2013+A1:2015
---------------------- Page: 2 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2015
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 14476:2013
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 -
prescriptions (Phase 2/Étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 5 July 2013 and includes Amendment 1 approved by CEN on 27 July 2015.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14476:2013+A1:2015 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
Contents Page
European foreword . 4
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 8
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 10
5.2.1 Test organisms . 10
5.2.2 Culture media, reagents and cell cultures . 11
5.3 Apparatus and glassware . 14
5.4 Preparation of test organism suspensions and product test solutions . 15
5.4.1 Test organisms suspensions (test virus suspension) . 15
5.4.2 Product test solutions . 15
5.5 Procedure for assessing the virucidal activity of the product . 16
5.5.1 General . 16
5.5.2 Test procedure . 17
5.5.3 Modified method for ready-to-use products . 18
5.5.4 Cytotoxicity caused by product test solutions . 18
5.5.5 Control of efficiency of suppression of product’s activity . 20
5.5.6 Reference test for virus inactivation . 20
5.5.7 Titration of the virus control . 20
5.5.8 Titration of test samples . 20
5.6 Experimental data and calculation . 21
5.6.1 Protocol of results . 21
5.6.2 Calculation of infectivity titer (TCID50 or PFU) . 21
5.7 Verification of the methodology . 21
5.8 Expression of results . 22
5.8.1 General . 22
5.8.2 Calculation of the virucidal activity of products . 22
5.9 Test report . 22
Annex A (informative) Examples of viruses sorted according to their presence in the human
body in case of virus infection . 24
Annex B (informative) Detoxification of test mixtures by molecular sieving . 26
TM
B.1 Molecular sieving with Sephadex LH 20 . 26
B.1.1 Principle . 26
B.1.2 Sephadex suspension . 26
B.1.3 Procedure. 26
TM
B.2 Molecular sieving using MicroSpin S 400 HR . 28
Annex C (informative) Calculation of the viral infectivity titre . 29
C.1 Quantal tests — Example of TCID50 determination by the Spearman-Kärber method . 29
2
---------------------- Page: 4 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
C.2 Plaque test . 29
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 30
C.3.1 General . 30
C.3.2 Calculating the virus titre with 95 % confidence interval . 31
C.3.3 Calculating the reduction and its 95 % confidence interval . 31
C.3.4 Calculating the average reduction (R ) and its 95 % confidence interval. . 32
(mi)
C.3.5 Practical example . 33
Annex D (informative) Presentation of test results of one active concentration . 35
Annex E (informative) Quantitative determination of formaldehyde concentrations . 38
Annex ZA (informative) !Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC" . 39
Bibliography . 40
3
---------------------- Page: 5 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
European foreword
This document (EN 14476:2013+A1:2015) 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 March 2016 and conflicting national standards shall be
withdrawn at the latest by March 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes !EN 14476:2013".
This document includes Amendment 1 approved by CEN on 2015-07-27.
The start and finish of text introduced or altered by amendment is indicated in the text by tags !".
!This document has been prepared under a mandate given to CEN by the European Commission and
the European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this
document."
The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to
harmonise the structure and wording with other existing tests of CEN/TC 216 or in preparation and to
improve the readability of the standard and thereby make it more understandable. The following list is
a list of significant technical changes since the last edition:
• The scope was expanded for the following fields of application within the medical area, i.e. products
for textile disinfection.
• “Obligatory test conditions” were replaced by “minimum test conditions” (test temperatures and
contact times can be chosen within limits) that have to be performed to pass the test.
• An additional modified method is described to test ready-to-use products in a higher concentration
than 80 %, i.e. 9 7%;
!
• For the hygienic handrub and handwash method a test for virucidal activity against enveloped
viruses with Vacciniavirus was added.
• The relationship between this European Standard and the MDD was added (Foreword and
Annex ZA).
• The value of v in C.1 was corrected (0,001 instead of 0,0001)."
n
Data obtained using the former version of EN 14476 may still be used.
Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different
applications in the medical area are in preparation.
4
---------------------- Page: 6 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
5
---------------------- Page: 7 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
Introduction
This European Standard 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 may
influence its action in practical situations. Each utilisation concentration of the chemical disinfectant or
antiseptic found by this test corresponds to the chosen experimental conditions.
6
---------------------- Page: 8 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
1 Scope
This European Standard 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. Products can only be tested at a concentration of 80 % (97 %, with a
modified method for special cases) as some dilution is always produced by adding the test organisms
and interfering substance.
This European Standard applies 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 European Standard applies 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 may occur in the workplace and in the home. It may also include services such as laundries and
kitchens supplying products directly for the patients.
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, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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 and the following apply.
3.1
cytotoxicity
morphological alteration of cells and/or their destruction or their reduced sensitivity to virus
multiplication caused by the product
3.2
plaque forming units
PFU
number of infectious virus particles per unit volume (ml)
7
---------------------- Page: 9 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
3.3
reference test for virus inactivation
test with a defined product (e.g. formaldehyde) in parallel with a product under test for the internal
control of the test
3.4
TCID
50
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 or in a solution
4 Requirements
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre when tested in
accordance with Table 1 and Clause 5.
8
---------------------- Page: 10 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
Table 1 — Minimum and additional test conditions
Test Conditions Hygienic handrub and Instrument disinfection Surface disinfection Textile
handwash
disinfection
Minimum Poliovirus Poliovirus Poliovirus Parvovirus
spectrum of
Adenovirus Adenovirus Adenovirus
test organisms
Murine Norovirus Murine Norovirus Murine Norovirus
Limited spectrum when temperature is
a
virucidal activity 40 °C or higher: only
Adenovirus Parvovirus
Murine Norovirus
!Virucidal activity
against enveloped
b
viruses
Vacciniavirus"
additional Any relevant test organism
Test temperature
according to the manufacturer’s recommendation, but at / between
20 °C 20 °C and 70 °C 4 °C and 30 °C 30 °C and 70 °C
Contact time
according to the manufacturer’s recommendation
but between but no longer than but no longer than but no longer than
30 s and 120 s 60 min 5 min or 20 min
c
60 min
Interfering substance
clean conditions 0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin
solution (hygienic solution solution
d
handrub)
and/or and/or
dirty conditions 3,0 g/l bovine albumin 3,0 g/l bovine albumin
solution plus 3,0 ml/l solution plus 3,0 ml/l
3,0 g/l bovine albumin 3,0 g/l bovine albumin
erythrocytes (hygienic erythrocytes
solution plus 3,0 ml/l solution plus 3,0 ml/l
e
handwash)
erythrocytes erythrocytes
d, e
Additional clean or dirty ; any relevant substance any relevant substance any relevant substance
f
conditions any relevant substance
a
The test for limited spectrum virucidal activity will cover all enveloped viruses (Annex A) and the specified test organisms .
b
!The test for virucidal activity against enveloped virus will cover all enveloped viruses only (Annex A)."
c
The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of the product.
The recommended contact time for the use of the product is within the responsibility of the manufacturer. Products intended to
disinfect surfaces that are likely to come into contact with the patient and / or the medical staff and surfaces, which are
frequently touched by different people, leading to the transmission of microorganisms to the patient, shall be tested with a
contact time of maximum 5 min. The same applies where the contact time of the product shall be limited for practical reasons.
Products for other surfaces than stated above may be tested with a contact time of maximum 60 min.
d
Hygienic handrub shall be tested as a minimum under clean conditions.
e
Hygienic handwash shall be tested as a minimum under dirty conditions.
f
For the additional conditions, the concentration defined as a result can be lower than the one obtained under the minimum test
conditions.
9
---------------------- Page: 11 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
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 suspension of viruses 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 (petri dishes, tubes or 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, C.1) or by plaque counting (plaque test, C.2) and evaluated. Reduction of virus infectivity is
calculated from differences of lg virus titres before (virus control) and after treatment with the product.
NOTE 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 organisms
The virucidal activity shall be evaluated using the following strains as test organisms selected according
1)
to Clause 4, Table 1
2)
a) Non-enveloped RNA virus
1) Poliovirus type 1, LSc 2ab (Picornavirus)
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) Vacciniavirus, strain Ankara (MVA), ATCC VR-1508."
1)
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 European Standard and does not constitute an endorsement by CEN of the product named.
2) Virus strains may be obtained from a national or international culture collection. Regarding Poliovirus only virus material that passed the
requirements for the production of oral polio vaccine of the World Health Organisation (WHO) shall be used (Other stocks derived from LSc-
2ab cannot be used any longer). LSc-2ab can be obtained from NIBSC (www.nibsc.ac.uk: contact Dr. Javier Martin) or from Eurovir Hygiene
Institut (www.eurovir.de: contact Dr. Jursch).
Murine Norovirus may be obtained from Friedrich-Loeffler-Insitut !Bundesforschungsinstitut" für Tiergesundheit,
Hauptsitz Insel Riems Südufer 10, 17493, Greifswald-Insel Riems; phone: +49 38351 7-0, fax: +49 038351 7-121.
http://www.fli.bund.de.
10
---------------------- Page: 12 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 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) noted in the test report. If these additional test organisms 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.
5.2.2 Culture media, reagents and cell cultures
5.2.2.1 General
All weights of chemical substances given in this European Standard 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 – if appropriate the 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 °C ± 1 °C.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralised water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [1]) may be used.
Sterilise in the autoclave [5.3.1.1 a)]. Sterilisation is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilised.
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 (Na2HPO4 x 12H2O) 2,89 g
Potassium phosphate, monobasic (KH2PO4) 0,20 g
Water (5.2.2.2) to 1 000,0 ml
5.2.2.4 Neutral Red (1:1000 solution)
Prepare neutral red (Sigma N7005) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a
0,40 µm pore size filter and store 4 °C in the dark.
5.2.2.5 Foetal calf serum (FCS)
FCS has to 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, special FCS has to be used due to the cells’ high sensitivity to endotoxins.
11
---------------------- Page: 13 ----------------------
SIST EN 14476:2013+A1:2015
EN 14476:2013+A1:2015 (E)
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
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilise by membrane filtration (5.3.1.7) or in the
2
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
3
1000 ml. Sterilise 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/
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.