oSIST prEN 17430:2019
(Main)Chemical disinfectants and antiseptics - Hygienic handrub virucidal - Test method and requirements (phase 2/step 2)
Chemical disinfectants and antiseptics - Hygienic handrub virucidal - Test method and requirements (phase 2/step 2)
This European Standard specifies a test method simulating practical conditions for establishing whether a
product for hygienic handrub reduces the release of virus contamination on hands when rubbed onto the
artificially contaminated hands of volunteers.
NOTE 1 Attention is drawn to the fact that tests on human volunteers are the subject of legal provisions in
certain European countries/regions.
This European Standard applies to products for hygienic handrub for use in 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 kindergardens 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 patient.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”.
NOTE 2 This method corresponds to a phase 2, step 2 test.
Chemische Desinfektionsmittel und Antiseptika - Viruzide hygienische Händedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Dieses Dokument legt ein Prüfverfahren fest, das praktische Bedingungen simuliert um festzustellen, ob ein Produkt für die hygienische Händedesinfektion die Abgabe der auf den Händen befindlichen Virenkontami
nation verringert, wenn es auf den künstlich kontaminierten Händen von freiwilligen Probanden verrieben wird.
ANMERKUNG1 Es wird darauf hingewiesen, dass Versuche mit menschlichen Probanden in bestimmten europäischen Ländern/Regionen gesetzlichen Bestimmungen unterliegen.
Dieses Dokument gilt für Produkte zur hygienischen Händedesinfektion, die in Bereichen und unter Bedingun
gen angewendet werden, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z.B. bei der Patientenbetreuung in
—
Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich,
—
medizinischen Einrichtungen in Schulen, Kindergärten und Heimen
vor und können auch am Arbeitsplatz und im häuslichen Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung des Patienten dienen.
EN14885 legt im Einzelnen die Beziehungen der verschiedenen Prüfungen untereinander und zu den „Anwen
dungsempfehlungen“ fest.
ANMERKUNG2 Das Verfahren entspricht einer Prüfung der Phase2, Stufe2.
Désinfectants chimiques et antiseptiques Traitement hygiénique virucide de mains par frictions Méthode d'essai et prescriptions (phase 2, étape 2)
Le présent document spécifie une méthode d’essai simulant les conditions pratiques permettant de
déterminer si un produit pour traitement hygiénique des mains par frictions réduit la libération d’une
contamination virale sur les mains lorsqu’il est frotté sur les mains de volontaires contaminées
artificiellement.
NOTE 1 L’attention est attirée sur le fait que les essais sur des volontaires humains sont soumis à des dispositions
légales dans certains pays/certaines régions d’Europe.
Le présent document s’applique aux produits pour traitement hygiénique des mains par frictions à
utiliser dans les 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 au patient.
L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les
« recommandations d’emploi ».
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 2.
Kemična razkužila in antiseptiki - Higiensko virucidno razkuževanje rok - Preskusna metoda in zahteve (faza 2, stopnja 2)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17430:2019
01-september-2019
Kemična razkužila in antiseptiki - Higiensko virucidno razkuževanje rok -
Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Hygienic handrub virucidal - Test method and
requirements (phase 2/step 2)
Chemische Desinfektionsmittel und Antiseptika - Viruzide hygienische
Händedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Traitement hygiénique virucide de mains par
frictions - Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 17430
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 17430:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17430:2019
DRAFT
EUROPEAN STANDARD
prEN 17430
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2019
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Hygienic handrub
virucidal - Test method and requirements (phase 2/step 2)
Désinfectants chimiques et antiseptiques ¿ Traitement Chemische Desinfektionsmittel und Antiseptika -
hygiénique virucide de mains par frictions ¿ Méthode Viruzide hygienische Händedesinfektion -
d'essai et prescriptions (phase 2, étape 2) Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
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, Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17430:2019 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Requirements . 4
5 Test methods . 5
5.1 Principle . 5
5.2 Materials and reagents . 5
5.2.1 Test organism . 5
5.2.2 Culture media and reagents . 5
5.3 Apparatus and glassware . 7
5.3.1 General . 7
5.4 Preparation of test organism suspensions and product test solutions . 9
5.4.1 Test virus suspension (test organisms suspensions, contamination fluid) . 9
5.4.2 Cell lines . 9
5.5 Cell culture preparation for virucidal testing . 9
5.6 Infectivity assay . 9
5.6.1 Quantal tests (endpoint titration). 9
5.6.2 Plaque assay . 10
5.6.3 Product test solutions . 10
5.7 Procedure for assessing the virucidal activity of the product on volunteers' hands . 11
5.7.1 General . 11
5.7.2 Test procedure with volunteers . 12
6 Experimental data and calculation . 13
6.1 Protocol of the results . 13
6.2 Calculation of virus titre . 13
6.3 Calculation of PFU . 13
6.4 Calculation of the lg reduction R (lg prevalue minus lg postvalue) . 13
6.5 Verification of the methodology - Test validation . 14
6.6 Statistical evaluation (significance testing), expression of results and precision . 14
6.7 Conclusion. 14
6.8 Test report . 14
Annex A (normative) Standard handrub procedure . 17
Annex B (informative) Calculation of the viral infectivity titre . 18
B.1 Example of TCID determination by the Spearman-Kärber method . 18
50
B.2 Calculation of plaque-forming units (PFU) . 18
Annex C (informative) Quality control of soft soap . 20
Annex D (informative) Examples of reporting of results and significance testing . 21
Annex E (normative) Test for non-inferiority . 28
Bibliography . 30
2
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European foreword
This document (prEN 17430:2019) 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.
3
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1 Scope
This document specifies a test method simulating practical conditions for establishing whether a
product for hygienic handrub reduces the release of virus contamination on hands when rubbed onto
the artificially contaminated hands of volunteers.
NOTE 1 Attention is drawn to the fact that tests on human volunteers are the subject of legal provisions in
certain European countries/regions.
This document applies to products for hygienic handrub for use in 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 kindergardens 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.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
NOTE 2 This method corresponds to a phase 2, step 2 test.
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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Requirements
When tested in accordance with Clause 5, the mean reduction of the release of the test organism Murine
Norovirus strain S99 Berlin achieved by the hygienic handrub with the product under test shall be at
least not inferior to that achieved by a specified reference hygienic handrub (a volume fraction of 70 %
concentration of ethanol).
4
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5 Test methods
5.1 Principle
Hands of volunteers are artificially contaminated with test organisms. The number of test organisms
released from their fingertips into sampling fluids is assessed before and after the hygienic handrub.
The ratio of the two resulting values (virus titres) represents a measure for the antimicrobial activity of
the product tested. The necessary precision is achieved by repeating the test on 18 to 22 volunteers. To
compensate for extraneous influences it is compared with the reduction obtained by a reference
handrub which is performed with the same volunteers, on the same day and under comparable
environmental conditions.
5.2 Materials and reagents
5.2.1 Test organism
1
Murine Norovirus strain S99 Berlin
This test organism has been specifically chosen to meet health and safety guidance and ethical
committee considerations. According to the German Safety Ordinance on Gene Technology [4], the
Murine Norovirus is classified as a risk group 1 organism.
5.2.2 Culture media and reagents
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 dehydrated material is used for the preparation of culture
media. The manufacturer's instructions relating to the preparation of these products should be
rigorously followed. For each culture medium and reagent, a time limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [3]) may be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.
If the water is sterilized during sterilization of the reagents, this is not necessary.
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 2,89 g
1
Murine Norovirus can be obtained from Friedrich-Loeffler-Institut Bundesinstitut für Tiergesundheit, Hauptsitz
Insel Riems Südufer 10, 17493 Greifswald-Insel Riems; phone: +49 38351 7-0; fax: +49 38351 7-121.
http://www.fli.de/. This information is given for the convenience of users of this document and does not constitute
an endorsement by CEN of the product named.
5
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(Na2HPO4 × 12H2O)
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.
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 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. Sterilize by membrane filtration (5.3.2.12) or in
2
the autoclave [5.3.2.1 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
(5.3.2.16) 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. Sterilize by membrane filtration (5.3.2.12). Store the solution in the refrigerator (5.3.2.16)
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 (5.3.2.10) and add 6,0 ml
(5.3.2.9) 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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of
approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.6.3), 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.
3
5.2.2.8 Growth and maintenance media
Dulbecco's Modified Eagle’s Medium (DMEM) or equivalent, supplemented with appropriate
concentration of heat inactivated (e.g. 56 °C for 30 min) and mycoplasma-free fetal calf serum FCS
(5.2.2.5), antibiotics, and other growth factors as needed shall be used.
a) A growth medium for cell multiplication is supplemented with 10 % FCS (5.2.2.5). Add 10 parts of
FCS to 90 parts of MEM.
6
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b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell
proliferation is 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 also bibliographic reference [5]. See EN 12353 for a detailed description.
NOTE Materials and reagents for cell culture can be purchased from biological supply companies.
5.2.2.9 Diluted soft soap
Linseed oil 50 parts by weight
Potassium hydroxide [3] 9,5 parts by weight
Ethanol (min. 95 %) [3] 7 parts by weight
Hot distilled water (75 °C ± 5 °C) as needed
Prepare a solution of 9,5 parts potassium hydroxyde in 15 parts water (5.2.2.2) and add 50 parts
linseed oil. Heat up to approximately 70 °C while constantly stirring. Add the ethanol and continue
heating while stirring until the saponification process is completed and a sample dissolves clearly in
water and almost clearly in alcohol. The weight of the soft soap is then brought up to 100 parts by
addition of water (5.2.2.2), heated up to 75 °C ± 5 °C to dilute the soft soap. Take 200 g of the soft soap,
fill up to 1 000 g with water (5.2.2.2) and sterilize in the autoclave [5.3.2.1 a)]. The pH of the final
diluted soft soap shall range between 10,0 and 11,0.
For quality control of the soft soap, see Annex C.
5.2.2.10 Ethanol as reference handrub [volume fraction of 70 % at 20°C]
Fill 547,2 g ethanol with a purity of min volume fraction of 99,5 % (determined by gas chromatography;
density 0,790) in a 1 000 ml flask equipped with a glass stopper on the weighing platform of a scale
(precision 0,1 g). Add 322,7 g water (5.2.2.2). Close the flask with the matching glass stopper and shake
the contents of the flask thoroughly. During mixing the solution warms up about to 30 °C. After cooling
down to 20 °C, it will give a volume of approximately 980 ml.
NOTE This solution can be kept indefinitely at approximately room temperature if protected from light.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
2
5.3.2 Usual microbiological laboratory equipment and, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat).
2
Disposable sterile equipment is an acceptable alternative to reusable glassware.
7
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+3
a) For moist heat sterilization, an autoclave capable of being maintained at ( 121 ) °C for a minimum
0
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
0
+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.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C.
5.3.2.3 Inverted microscope for reading cell cultures microscopically.
5.3.2.4 pH meter, having an inaccuracy of calibration of 0,1 pH units at 20 °C ± 1 °C.
5.3.2.5 Stopwatch
5.3.2.6 Deep freezer (−20°C and −70°C or less).
® 3
5.3.2.7 Electromechanical agitator e.g. Vortex mixer .
5.3.2.8 Containers: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml, 1 ml and 0,1 ml. Calibrated automatic
pipettes, with disposable tips, may be used.
5.3.2.10 Volumetric flasks, calibrated at 20°C.
5.3.2.11 Sterile microtitre plates, six well plates for cell cultures, and flasks for cell cultures.
5.3.2.12 Membrane filtration apparatus for filtration of media, 0,2 μm pore size.
incubator (95 % air, 5 % CO ), capable of being controlled at either 36 °C ± 1 °C, or at
5.3.2.13 CO
2 2
37°C ± 1 °C for incubation of cell cultures.
5.3.2.14 Biological safety cabinet, class II.
5.3.2.15 Centrifuge (400 g to 1000 g ).
N N
5.3.2.16 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.17 Cylindrical polypropylene screw cap tubes, volume 25 ml, length 54 mm, diameter
27 mm (for the stainless steel discs), e.g. Sarstedt AG & Co. D-51582 Nümbrecht, Art.No.: 60.9922.115
4
PS .
3
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.
4
These "Art. No." from Sarstedt AG & Co. D-51582 Nümbrecht are examples of suitable products available
commercially. This information is given for the convenience of users of this document and does not constitute an
endorsement by CEN of these products.
8
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5.3.2.18 Cylindrical polypropylene screw cap tubes, volume 10 ml, length 79 mm, diameter
4
16 mm (for the glass carrier), e.g. Sarstedt AG & Co., D-51582 Nümbrecht, Art.No.: 60.551.001) .
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test virus suspension (test organisms suspensions, contamination fluid)
The test organisms and their stock cultures 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
N
called “test virus suspension”.
It is suggested that the minimum titre of the virus suspension - determined by a quantal test (5.6.1) or
8 4
by plaque test (5.6.2) - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at least 10
50
TCID /ml may be recovered from the hands.
50
In exceptional cases the test virus suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
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.
The test virus suspension is used undiluted for the test procedure (5.7).
The titre of the virus suspension is determined as described (see 6.2 and 6.3).
5.4.2 Cell lines
Murine Norovirus is multiplied in RAW 264.7 cells (ATCC TIB-71) or other cell lines of appropriate
sensitivity.
5.5 Cell culture preparation for virucidal testing
Cells used as suspension in quantal tests shall be added to the dilutions of the test mixture in such a
density to enable the formation of a monolayer in at least two days in the cell control (see 5.6.1.1).
Cell monolayers shall be > 90 % confluent before inoculation. Cell lines and culture media will be
selected in accordance with the test virus (see 5.2.1).
5.6 Infectivity assay
5.6.1 Quantal tests (end point titration)
5.6.1.1 Virus titration on cells in suspension
Dilute the test virus suspension (5.4.1) by tenfold dilution steps with a volume of 0,5 ml or 0,1 ml of test
virus suspension plus an appropriate volume of maintenance medium (see 5.2.2.8). Pipettes shall be
changed after each dilution step.
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate, beginning with the highest
dilution. Add 0,1 ml of cell culture in such a density to enable the formation of a monolayer in at least 2
days in the cell control. The last row of six or eight wells shall not receive any test virus suspension and
serve as the cell control.
After the appropriate incubation time, according to the virus type, the viral cytopathic effect is read
using an inverted microscope (see 5.3.2.3).
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5.6.1.2 Virus titration on cell monolayers
Transfer 0,1 ml of each dilution (see 5.6.1.1) into six or eight wells of a microtitre plate containing a
confluent (>90 %) cell monolayer. The last row of six or eight wells will receive 0,2 ml of maintenance
medium (see 5.2.2.8) and will serve as cell control. If the cells were inoculated without any medium
after 1 h of incubation at 37 °C, 0,1 ml of maintenance medium is added to each well.
The plates are incubated at 37 °C for the appropriate incubation time. The viral cytopathic effect is read
using an inverted microscope (see 5.3.2.3).
5.6.2 Plaque assay
Plastic tray wells (surface diameter 30 mm to 35 mm) with confluent cell monolayers are washed once
with phosphate buffered saline (PBS) (5.2.2.3) and inoculated with 0,2 ml of serial dilutions of virus in
DMEM + 2 % FCS (see 5.2.2.8). Three wells are generally used per dilution. After absorption period of 1
h at 37 °C, during which the cell monolayers are kept moist by tilting the dishes every 8 min to 10 min,
the inoculum is removed and the cell monolayers are washed once with PBS. Subsequently, the wells
are overlaid with 3 ml of a mixture consisting of 2 % melted agarose or another appropriate semisolid
medium and 2times concentrated DMEM with 4 % FCS. The cultures are incubated for 5 days to 6 days
at 37 °C in a CO incubator (see 5.3.2.13). Plaques can be counted after addition of 2 ml of a second
2
overlay with the same composition of the first and also containing 5 % of a 1:1 000 solution of neutral
red and further incubation (in the dark) at 37 °C for 24 h to 48 h in a CO incubator (see 5.3.2.13).
2
Counting can be performed also after addition of crystal violet. The cell monolayers are fixed by adding
2 ml of 10 % trichloroacetic acid (TCA) (5.2.2.6) over the agar overlay for 10 min to 15 min at room
temperature. The agar overlay is then removed and 2 ml of 0,1 % crystal violet in 20 % ethanol are
added. After 10 min to 15 min at room temperature, the wells are extensively washed with water and
the plaques (white spots) are counted.
Product test solutions
The product, as received, shall be used as product test solution if recommended by the manufacturer.
Product test solutions of products recommended by the manufacturer to be diluted shall be prepared in
hard water (5.2.2.7).
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 (5.3.2.10) 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
(5.3.2.10) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 3 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculant, it shall be recorded in
the test report.
NOTE Counting microorganisms embedded in a precipitate or flocculant is difficult and unreliable.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
10
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...
SLOVENSKI STANDARD
oSIST prEN 17430:2022
01-september-2022
Kemična razkužila in antiseptiki - Higiensko razkuževanje rok z drgnjenjem z
virucidnim sredstvom - Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Hygienic handrub virucidal - Test method and
requirements (phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Viruzide hygienische Händedesinfektion
- Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Traitement hygiénique de mains par frictions
virucide - Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 17430
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 17430:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17430:2022
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oSIST prEN 17430:2022
DRAFT
EUROPEAN STANDARD
prEN 17430
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2022
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Hygienic handrub
virucidal - Test method and requirements (phase 2, step 2)
Désinfectants chimiques et antiseptiques ¿ Traitement Chemische Desinfektionsmittel und Antiseptika -
hygiénique de mains par frictions virucide ¿ Méthode Viruzide hygienische Händedesinfektion -
d'essai et prescriptions (phase 2, étape 2) Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
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, Turkey 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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17430:2022 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Requirements . 5
5 Test methods . 5
5.1 Principle . 5
5.2 Materials and reagents . 5
5.2.1 Test organism . 5
5.2.2 Culture media and reagents . 6
5.3 Apparatus and glassware . 8
5.3.1 General . 8
5.3.2 Usual microbiological laboratory equipment and, in particular, the following: . 8
5.4 Preparation of test organism suspensions and product test solutions . 9
5.4.1 Test virus suspension (test organisms suspensions, contamination fluid) . 9
5.4.2 Cell lines . 10
5.5 Cell culture preparation for virucidal testing . 10
5.6 Infectivity assay . 10
5.6.1 Quantal tests (end point titration). 10
5.6.2 Plaque assay . 10
5.6.3 Product test solutions . 11
5.7 Procedure for assessing the virucidal activity of the product on volunteers' hands . 11
5.7.1 General . 11
5.7.2 Test procedure with volunteers . 12
6 Experimental data and calculation . 14
6.1 Protocol of the results . 14
6.2 Calculation of virus titre . 14
6.3 Calculation of PFU . 14
6.4 Calculation of the lg reduction R (lg prevalue minus lg postvalue) . 14
6.5 Verification of the methodology — Test validation . 14
6.6 Statistical evaluation (significance testing), expression of results and precision . 14
6.7 Conclusion. 15
6.8 Test report . 15
Annex A (normative) Standard handrub procedure . 17
Annex B (informative) Calculation of the viral infectivity titre . 18
B.1 Example of TCID determination by the Spearman-Kärber method . 18
50
B.2 Calculation of plaque-forming units (PFU) . 18
Annex C (informative) Quality control of soft soap . 20
Annex D (informative) Examples of reporting of results and significance testing . 21
Annex E (normative) Test for non-inferiority . 28
Bibliography . 30
2
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European foreword
This document (prEN 17430:2022) 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 CEN-Enquiry.
3
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1 Scope
This document specifies a test method simulating practical conditions for establishing whether a product
for hygienic handrub reduces the release of virus contamination on hands when rubbed onto the
artificially contaminated hands of volunteers.
NOTE 1 Attention is drawn to the fact that tests on human volunteers are the subject of legal provisions in certain
European countries/regions.
This document applies to products for hygienic handrub for use in 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 kindergardens 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.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
NOTE 2 This method corresponds to a phase 2, step 2 test.
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 14476, Chemical disinfectants and antiseptics —Virucidal quantitative suspension test for chemical
disinfectants and antiseptics used in human medicine — Test method and requirements (phase 2/step 1)
EN 16777, Chemical disinfectants and antiseptics - Quantitative non-porous surface test without mechanical
action for the evaluation of virucidal activity of chemical disinfectants used in the medical area - Test method
and requirements (phase 2/step 2)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4
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4 Requirements
When tested in accordance with Clause 5, the mean reduction of the test organism murine norovirus
strain S99 Berlin achieved by the hygienic handrub with the product under test shall be at least not
inferior to that achieved by a specified reference hygienic handrub (a volume fraction of 70 %
concentration of ethanol).
Table 1 — Minimum and additional test conditions
Hygienic handrub
a
Test virus Virucidal activity
Murine norovirus
b
Limited spectrum virucidal activity
Murine norovirus
Test temperature room temperature of between 21,5 °C ± 3,5 °C
Contact time according to the manufacturer’s recommendation, but between
30 s and 120 s
Additional Further contact time(s)
d
conditions
a
To claim the virucidal activity the product shall pass standards EN 14476 with Poliovirus, Adenovirus
and Murine Norovirus and EN 16777 with Adenovirus and Murine Norovirus.
b
To claim limited spectrum virucidal activity the product shall pass EN 14476 with Adenovirus and
Murine Norovirus.
5 Test methods
5.1 Principle
Hands of volunteers are artificially contaminated with test organisms. The number of test organisms
released from their fingertips into sampling fluids is assessed before and after the hygienic handrub. The
ratio of the two resulting values (virus titres) represents a measure for the antimicrobial activity of the
product tested. The necessary precision is achieved by repeating the test on 18 to 22 volunteers. To
compensate for extraneous influences it is compared with the reduction obtained by a reference handrub
which is performed with the same volunteers, on the same day and under comparable environmental
conditions.
5.2 Materials and reagents
5.2.1 Test organism
1
Murine norovirus strain S99 Berlin
1
Murine norovirus can be obtained from Friedrich-Loeffler-Institut Bundesinstitut für Tiergesundheit, Hauptsitz Insel
Riems Südufer 10, 17493 Greifswald-Insel Riems; phone: +49 38351 7-0; fax: +49 38351 7-121. http://www.fli.de/. This
5
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This test organism has been specifically chosen to meet health and safety guidance and ethical committee
considerations. According to German Ordinance on Biological Substances (BiostoffV)/TRBA 462 Murine
Norovirus is classified in risk group 1.
5.2.2 Culture media and reagents
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 dehydrated material is used for the preparation of culture
media. The manufacturer's instructions relating to the preparation of these products should be rigorously
followed. For each culture medium and reagent, a time limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of adequate
quality is not available, water for injections (see bibliographic reference [3]) may be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e.g. for preparation
of culture media and subsequently sterilized.
If the water is sterilized during sterilization of the reagents, this is not necessary.
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 (1:1000 solution) or crystal violet solution
2
Prepare neutral red (e.g. Sigma N7005 ) stock solution at 0,1 mg/ml in water (5.2.2.2).
Prepare 0,1 % crystal violet in 20 % ethanol.
Filter through a 0,40 µm pore size filter and store 4 °C in the dark.
5.2.2.5 Fetal 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, special FCS shall be used due to the cells’ high sensitivity to endotoxins.
information is given for the convenience of users of this document and does not constitute an endorsement by CEN of the
product named.
2
Sigma N7005 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.
6
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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 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. Sterilize by membrane filtration (5.3.2.13) or in the
2
autoclave [5.3.2.1 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
(5.3.2.17) 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. Sterilize by membrane filtration (5.3.2.13). Store the solution in the refrigerator (5.3.2.17)
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 (5.3.2.10) and add 6,0 ml
(5.3.2.9) 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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of
approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about
1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.6.3), 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.
3
5.2.2.8 Growth and maintenance media
Dulbecco's Modified Eagle’s Medium (DMEM) or equivalent, supplemented with appropriate
concentration of heat inactivated (e.g. 56 °C for 30 min) and mycoplasma-free fetal calf serum FCS
(5.2.2.5), antibiotics, and other growth factors as needed shall be used. Additional supplements like
glucose, glutamine, and sodium pyruvate shall be indicated in the test report.
a) A growth medium for cell multiplication is supplemented with 10 % FCS (5.2.2.5). Add 10 parts of
FCS to 90 parts of DMEM.
b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell
proliferation is supplemented with 2 % FCS. Add 2 parts of FCS (5.2.2.5) to 98 parts of DMEM.
Other media may be used if appropriate for certain cell lines.
See also bibliographic reference [4]. See EN 12353 for a detailed description.
NOTE Materials and reagents for cell culture can be purchased from biological supply companies.
5.2.2.9 Diluted soft soap
Linseed oil 50 parts by weight
Potassium hydroxide 9,5 parts by weight
Ethanol (min. 95 %) 7 parts by weight
7
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Hot distilled water (75 °C ± 5 °C) as needed
Prepare a solution of 9,5 parts potassium hydroxyde in 15 parts water (5.2.2.2) and add 50 parts linseed
oil. Heat up to approximately 70 °C while constantly stirring. Add the ethanol and continue heating while
stirring until the saponification process is completed and a sample dissolves clearly in water and almost
clearly in alcohol. The weight of the soft soap is then brought up to 100 parts by addition of water
(5.2.2.2), heated up to 75 °C ± 5 °C to dilute the soft soap. Take 200 g of the soft soap, fill up to 1 000 g
with water (5.2.2.2) and sterilize in the autoclave [5.3.2.1 a)]. The pH of the final diluted soft soap shall
range between 10,0 and 11,0.
For quality control of the soft soap, see Annex C.
5.2.2.10 Ethanol as reference handrub [volume fraction of 70 % at 21,5 °C ± 3,5 °C ]
Fill 547,2 g ethanol with a purity of min volume fraction of 99,5 % (determined by gas chromatography;
density 0,790) in a 1 000 ml flask equipped with a glass stopper on the weighing platform of a scale
(precision 0,1 g). Add 322,7 g water (5.2.2.2). Close the flask with the matching glass stopper and shake
the contents of the flask thoroughly. During mixing the solution warms up about to 30 °C. After cooling
down to 21,5 °C ± 3,5 °C, it will give a volume of approximately 980 ml.
NOTE This solution can be kept indefinitely at approximately room temperature if protected from light,
provided that the flask is closed with a ground-in glass stopper to avoid any evaporation.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
3
5.3.2 Usual microbiological laboratory equipment and, in particular, the following:
5.3.2.1 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
0
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
0
+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.
5.3.2.2 Water baths, capable of being controlled at 21,5 °C ± 3,5 °C.
5.3.2.3 Inverted microscope for reading cell cultures microscopically.
5.3.2.4 pH meter, having an inaccuracy of calibration of 0,1 pH units at 21,5 °C ± 3,5 °C.
5.3.2.5 Stopwatch
3
Disposable sterile equipment is an acceptable alternative to reusable glassware.
8
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5.3.2.6 Deep freezer (−20°C and −70°C or less).
® 4
5.3.2.7 Electromechanical agitator e.g. Vortex mixer .
5.3.2.8 Containers: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml, 1 ml and 0,1 ml. Calibrated automatic
pipettes, with disposable tips, may be used.
5.3.2.10 Volumetric flasks, calibrated at 21,5 °C ± 3,5 °C.
5.3.2.11 Sterile microtitre plates, six or eight well plates for cell cultures.
5.3.2.12 flasks for cell cultures.
5.3.2.13 Membrane filtration apparatus for filtration of media, 0,2 μm pore size.
5.3.2.14 CO incubator (95 % air, 5 % CO ), capable of being controlled at either 36 °C ± 1 °C, or at
2 2
37°C ± 1 °C for incubation of cell cultures.
5.3.2.15 Biological safety cabinet, class II.
5.3.2.16 Centrifuge (400 g to 1000 g ).
N N
5.3.2.17 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test virus suspension (test organisms suspensions, contamination fluid)
The test organisms and their stock cultures 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 (5.3.2.16) (400 g for 15 min). This preparation is
N
called “test virus suspension”.
It is suggested that the minimum titre of the virus suspension - determined by a quantal test (5.6.1) or by
8 4
plaque test (5.6.2) - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at least 10
50
TCID /ml may be recovered from the hands.
50
In exceptional cases the test virus suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
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.
The test virus suspension is used undiluted for the test procedure (5.7).
The titre of the virus suspension is determined as described (see 6.2 and 6.3).
4
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.
9
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5.4.2 Cell lines
Murine norovirus is multiplied in RAW 264.7 cells (ATCC TIB-71) or other cell lines of appropriate
sensitivity.
5.5 Cell culture preparation for virucidal testing
Cells used as suspension in quantal tests shall be added to the dilutions of the test mixture in such a
density to enable the formation of a monolayer in at least two days in the cell control (see 5.6.1.1).
Cell monolayers shall be > 90 % confluent before inoculation. Cell lines and culture media will be selected
in accordance with the test virus (see 5.2.1).
5.6 Infectivity assay
5.6.1 Quantal tests (end point titration)
5.6.1.1 Virus titration on cells in suspension
Dilute the test virus suspension (5.4.1) by tenfold dilution steps with a volume of 0,5 ml or 0,1 ml of test
virus suspension plus an appropriate volume of maintenance medium (see 5.2.2.8). Pipettes shall be
changed after each dilution step.
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate (5.3.2.11), beginning with the
highest dilution. Add 0,1 ml of cell culture in such a density to enable the formation of a monolayer in at
least 2 days in the cell control. The last row of six or eight wells (5.3.2.11) shall not receive any test virus
suspension and serve as the cell control.
After the appropriate incubation time, according to the virus type, the viral cytopathic effect is read using
an inverted microscope (see 5.3.2.3).
5.6.1.2 Virus titration on cell monolayers
Transfer 0,1 ml of each dilution (see 5.6.1.1) into six or eight wells (5.3.2.11) of a microtitre plate
containing a confluent (>90 %) cell monolayer. The last row of six or eight wells will receive 0,2 ml of
maintenance medium (see 5.2.2.8) and will serve as cell control. If the cells were inoculated without any
medium after 1 h of incubation at 37 °C, 0,1 ml of maintenance medium is added to each well.
The plates are incubated at 37 °C for the appropriate incubation time. The viral cytopathic effect is read
using an inverted microscope (see 5.3.2.3).
5.6.2 Plaque assay
Plastic tray wells (surface diameter 30 mm to 35 mm) with confluent cell monolayers are washed once
with phosphate buffered saline (PBS) (5.2.2.3) and inoculated with 0,2 ml of serial dilutions of virus in
DMEM + 2 % FCS (see 5.2.2.8). Three wells are generally used per dilution. After adsorption period of 1
h at 37 °C, during which the cell monolayers are kept moist by tilting the dishes every 8 min to 10 min,
the inoculum is removed and the cell monolayers are washed once with PBS. Subsequently, the wells are
overlaid with 3 ml of a mixture consisting of 2 % melted agarose or another appropriate semisolid
medium and 2times concentrated DMEM with 4 % FCS. The cultures are incubated for 5 days to 6 days at
37 °C in a CO incubator (see 5.3.2.14). Plaques can be counted after addition of 2 ml of a second overlay
2
with the same composition of the first and also containing 5 % of a 1:1 000 solution of neutral red and
further incubation (in the dark) at 37 °C for 24 h to 48 h in a CO incubator (see 5.3.2.14). Counting can
2
be performed also after addition of crystal violet. The cell monolayers are fixed by adding 2 ml of 10 %
trichloroacetic acid (TCA) (5.2.2.6) over the agar overlay for 10 min to 15 min at 21,5 °C ± 3,5 °C. The agar
overlay is then removed and 2 ml of 0,1 % crystal violet in 20 % ethanol are added. After 10 min to 15
min at 21,5 °C ± 3,5 °C, the wells are extensively washed with water and the plaques (white spots) are
counted.
10
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