EN 17111:2018

SLOVENSKI STANDARD
01-januar-2019
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Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of
virucidal activity for instruments used in the medical area - Test method and
requirements (phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Keimträgerversuch zur
Prüfung der viruziden Wirkung für Instrumente im humanmedizinischen Bereich -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Essai quantitatif de porte germe pour
l'évaluation de l'activité virucide pour instruments utilisés en médecine - Méthode d'essai
et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 17111:2018
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 17111
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2018
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative
carrier test for the evaluation of virucidal activity for
instruments used in the medical area - Test method and
requirements (phase 2, step 2)
Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de porte-germe pour l'évaluation de Quantitativer Keimträgerversuch zur Prüfung der
l'activité virucide pour instruments utilisés en viruziden Wirkung für Instrumente im
médecine - Méthode d'essai et exigences (phase 2, humanmedizinischen Bereich - Prüfverfahren und
étape 2) Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 18 June 2018.

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, Serbia, 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: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17111:2018 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Requirements . 6
5 Test method . 8
5.1 Principle . 8
5.2 Materials and reagents . 8
5.2.1 Test organisms . 8
5.2.2 Culture media, reagents and cell cultures . 9
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment . 12
5.4 Preparation of test organism suspensions and product test solutions . 14
5.4.1 Test organism suspensions (test suspension) . 14
5.4.2 Product test solution . 14
5.5 Procedure for assessing the virucidal activity of the product . 15
5.5.1 General . 15
5.5.2 Method . 16
5.5.3 Cytotoxicity caused by product solutions . 18
5.5.4 Control of efficiency for suppression of disinfectant activity . 19
5.5.5 Reference test for virus inactivation . 19
5.5.6 Titration of the virus control . 19
5.5.7 Titration of test samples . 19
5.6 Experimental data and calculation . 20
5.6.1 Protocol of the results . 20
5.6.2 Calculation of infectivity titre (TCID PFU) . 20
50 –
5.7 Verification of the methodology . 20
5.8 Explanation of terms and abbreviations . 21
5.9 Expression of results . 21
5.9.1 General . 21
5.9.2 Calculation of the virucidal activity of products . 21
5.10 Calculation . 21
5.10.1 Virucidal activity . 21
5.10.2 Claims . 22
5.11 Test report . 22
Annex A (informative) Example of a typical test report . 24
Annex B (informative) Examples of viruses sorted according to their presence in the
human body in case of virus infection . 26
Annex C (normative) Detoxification of test mixtures by molecular sieving . 28
C.1 Molecular sieving with Sephadex™ LH 20 . 28
C.1.1 Principle . 28
C.1.2 Sephadex suspension . 28
C.1.3 Procedure . 28
C.2 Molecular sieving using MicroSpin™ S 400 HR. 30
C.3 Determination of the residual virus titre by the large-volume-plating (LVP) method . 30
C.3.1 General . 30
C.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method . 31
Annex D (informative) Calculation of the viral infectivity titre . 33
D.1 Quantal tests - Example of TCID determination by the Spaerman-Kärber method . 33
D.2 Plaque test . 34
D.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 34
D.3.1 General . 34
D.3.2 Calculating the virus titre with 95 % confidence interval . 35
D.3.3 Calculating the reduction and its 95 % confidence interval . 35
D.3.4 Calculating the average reduction (R ) and its 95 % confidence interval . 36
(mi)
D.3.5 Practical example . 37
Bibliography . 40

European foreword
This document (EN 17111:2018) has been prepared by Technical Committee CEN/TC 216 “Chemical
desinfectants 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 2019, and conflicting national standards shall be
withdrawn at the latest by April 2019.
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.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
This European Standard specifies a carrier test for establishing whether a chemical disinfectant for use
on instruments (surgical instruments, anaesthesia material, endoscopes etc.) has a virucidal activity in
the fields described in the scope.
The laboratory test closely simulates practical conditions of application including pre-drying viruses on
a carrier, contact time, temperature, test organisms and interfering substances, i.e. conditions which
may influence the action of chemical disinfectants in practical situations. Each utilization concentration
of the chemical disinfectant found by this test corresponds to defined experimental conditions.
1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectant products that form a homogeneous, physically stable preparation when diluted with hard
water – or in the case of ready-to-use products – with water.
This document applies to products that are used in the medical area for disinfecting instruments by
immersion.
This document 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 2 test.
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 14476, 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)
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
The product, when diluted with hard water or – in the case of ready-to-use products – with water, and
tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution)
or simulated dirty conditions (3 g/l bovine albumin solution, plus 3 ml/l washed sheep erythrocytes)
according to its practical applications and under the minimum test conditions shall demonstrate at least
a 4 decimal log (lg) reduction.
Table 1 — Minimum and additional test conditions
Virucidal activity
against enveloped a
Virucidal activity
Virucidal activity
b
viruses
Test
(Instrument
(Instrument
Conditions
disinfection when
(Pre-cleaning products disinfection when
temperature is ≥ 40 °C)
with a combined temperature is < 40 °C)
cleaner/disinfectant)
modified vaccinia virus
Minimum Ankara Adenovirus
spectrum of or and murine parvovirus
test organisms vaccinia virus strain murine norovirus
Elstree
additional Any relevant test organism
according to the manufacturer’s recommendation, but at / between
Test
temperature
20 °C 20 °C and < 40 °C ≥ 40 °C and 70 °C
Contact time according to the manufacturer’s recommendation, but no longer than
60 min 60 min 60 min
Interfering
substance
clean
0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin
conditions
solution solution solution
and/or and/or and/or
3,0 g/l bovine albumin 3,0 g/l bovine albumin 3,0 g/l bovine albumin
dirty solution plus 3,0 ml/l solution plus 3,0 ml/l solution plus 3,0 ml/l
conditions washed sheep washed sheep washed sheep
erythrocytes erythrocytes erythrocytes
Additional
any relevant substance any relevant substance any relevant substance
c
conditions
a
Poliovirus (as used in the corresponding suspension test) cannot be used for surfaces, because of drying
problems. To claim the virucidal activity the product shall pass standards EN 14476 with polio-, adeno- and
murine norovirus.
b
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only
(Annex A).
c
For the additional conditions, the concentration defined as a result can be lower than the one obtained
under the minimum test conditions.
5 Test method
5.1 Principle
5.1.1 A test suspension of test viruses in a solution of interfering substances is spread on a glass
carrier (5.3.2.23). After drying the carrier is immersed into a sample of the product as delivered and/or
diluted with hard water (for ready to use products: water). In parallel, a second carrier is treated with
(hard) water instead of the product applying the same test conditions (water control). The carriers are
maintained at one of the temperatures and contact times specified in Clause 4 and 5.5.1.1. At the end of
this contact time, the carriers are transferred into a maintenance medium containing glass beads. The
viruses are to be severed from the surface by shaking (5.3.2.17). The numbers of surviving viruses in
each sample are determined and the reduction is calculated by comparing the results of the product and
the water control.
5.1.2 The test is performed using modified vaccinia virus Ankara or vaccinia virus strain Elstree (pre-
cleaning products with a combined cleaner/disinfectant), adenovirus type 5 and murine norovirus as
test-organisms (minimum spectrum of test organisms); in case of test temperatures of 40 °C or higher
only murine parvovirus shall be used.
5.1.3 Other contact times and temperatures within the limits specified in Clause 4 may be used.
Additional interfering substances may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The virucidal activity shall be evaluated using the following strains as test organisms selected according
1)
to Clause 4.
a) Enveloped DNA virus
Vaccinia virus, strain modified vaccinia virus Ankara (MVA), ATCC VR-1508 or vaccinia virus strain
Elstree, ATCC VR-1549
2)
b) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
c) Non-enveloped DNA virus
Adenovirus type 5, strain Adenoid 75, ATCC VR-5
Murine parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346

1)
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections
(ATCC). 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. Murine Norovirus may be
obtained from Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit, Hauptsitz Insel Riems
Südufer 10, 17493, Greifswald-Insel Riems; phone: +49 (0) 38351 7-0, fax: +49 (0) 38351 7-1219.

https://www.fli.de/en/home/.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.12).
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 incubated under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains, their suitability for supplying the required inocula
shall be verified. 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. The source of the strains shall be indicated.
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 (5.3.2.4) are measured at 20 °C ± 1 °C.
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 [1]) may be used.
Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.
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 x 12H O) 2,89 g
2 4 2
Potassium phosphate, monobasic (KH PO ) 0,20 g
2 4
Water (5.2.2.2) to 1000,0 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
0,45 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 % w/V solution) (TCA)
Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), and 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 (5.3.2.11) or in
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
(5.3.2.6) 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 (5.3.2.11). Store the solution in the refrigerator (5.3.2.6)
for no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.9) and add 6,0 ml of
solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The pH of the
hard water shall be 7,0 ± 0,2. (5.3.2.4). If necessary, adjust the pH by using a solution of
approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), 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.
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.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids, detergents) shall be defined.
NOTE 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 (5.3.2.11), keep in a refrigerator (5.3.2.6) 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 solutions – 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 (5.3.2.11).
Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.9). Centrifuge the sheep blood at
800 g for 10 min (5.3.2.18). After discarding the supernatant, resuspend erythrocytes in PBS (5.2.2.3).
N
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 contamination this mixture should be split in portions probably needed per day and
kept in separate containers (5.3.2.8) for a maximum of 7 days in a refrigerator (5.3.2.6).
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.
5.2.2.9 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 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 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 [2]. See EN 12353 for a detailed description.
5.2.2.11 Cell cultures
Cell monolayers shall be > 90 % confluent before inoculation. 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). The density shall 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 5.5.1.1e).
5.2.2.12 Reference glutardialdehyde (Glutaral, 1,5-Pentanedial) CAS Number 111-30-8
Required chemical and physical parameters for use as reference standard for testing disinfectant
preparations are defined in Table 2.
Table 2 — Required chemical and physical parameters
Parameters Specifications
Solution 50 % Solution 25 %
a
clear liquid clear liquid
Appearance
pH-value 3,1 to 4,5 3,1 to 4,5
Concentration of glutardialdehyde (by
50,0 % to 52,0 % 25,0 % to 26,0 %
titration)
Stability (ambient conditions) 12 months 12 months
a
Note: Yellowish colour indicates a beginning poarimerization
The specification above (see Table 2) should be checked on the certificate of analyses. The pH values
shall be tested and confirmed regularly by the laboratory. In case pH values and/or appearance are out
of specification the glutardialdehyde cannot be used anymore.
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
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.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, and at additional test
temperatures ± 1 °C (5.5.1).
5.3.2.3 Inverted microscope for reading cell cultures microscopically
5.3.2.4 pH meter, having an accuracy of calibration of 0,1 pH units at 20 °C.
5.3.2.5 Stopwatch
3)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.6 Refrigerator, capable of being controlled at 2 °C to 8 °C.
4)
5.3.2.7 Electromechanical agitator e.g. Vortex ® mixer
5.3.2.8 Containers: Petri plates, sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.2.9 Volumetric flasks, calibrated at 20°C.
5.3.2.10 Sterile microtitre plates, 96 well plates for cell culture, and flasks for cell culture use.
5.3.2.11 Membrane filtration apparatus for filtration of media, 0,22 μm pore size
5.3.2.12 CO incubator (95 % air, 5 % CO ), capable of being controlled at 36 °C ± 1 °C, for
2 2
incubation of cell cultures. An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not
available.
5.3.2.13 Graduated sterile pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.
NOTE Calibrated automatic pipettes can be used.
5.3.2.14 Magnetic stirrer for keeping cells in suspension before seeding
5.3.2.15 Ice producing machine or commercially available ice to cool the cell maintenance
medium and the reaction mixtures during the test [5.5.2.1b), 5.5.4.1]
5.3.2.16 Basin as ice bath with ice and water
5.3.2.17 Mechanical shaker
5.3.2.18 Centrifuge
5.3.2.19 Biological safety cabinet, class II
5.3.2.20 Freezer, −70 °C
5.3.2.21 Glass beads (Diameter: 3 mm to 4 mm and 0,25 mm to 0,5 mm).
5.3.2.22 Cylindrical plastic screw cap tubes, contents of about 15 ml, diameter about 18 mm (for
the carrier).
5.3.2.23 Frosted glass carriers, 15 mm x 60 mm x 1 mm, one surface sandblasted. For preparation,
the glass carrier is boiled 10 min in a suitable detergent, cleaned minimum 3 times with water (5.2.2.2)
and at the end once with ethanol (70 % v/v). Mark a square (lateral length: 10 mm) at one end of the
dried carrier on its sandblasted surface, about 2 mm off the three edges. Sterilize in the heat oven
[5.3.2.1 b)]. After the sterilization process the markings of the “inoculation square” shall be clearly
visible.
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.
Key
1 inoculation square
Figure 1 — Frosted glass carrier with markings
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test suspension)
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
N
is called “test virus suspension”.
It is suggested that the minimum titre of the virus suspension - determined by a quantal test (5.5.2.2) or
by plaque test (5.5.2.3) - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at least
enable a titre reduction of 4 lg to verify the method.
In exceptional cases the test suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
The test 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 suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solution
Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different
concentrations to include one concentration in the active range and one concentration in the non-active
range. The product as received may be used as one of the product test solutions.
Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared
in water (5.2.2.2) instead of hard water.
For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a
volumetric flask (5.3.2.9) and filling up with hard water (5.2.2.7). Subsequent dilutions (i.e. lower
concentrations) shall be prepared in volumetric flasks (5.3.2.9) 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 using volumetric flasks
(5.3.2.9) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 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 (for example through
the addition of the interfering substances), it shall be recorded in the test report.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
5.5 Procedure for assessing the virucidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions (minimum and additional)
The experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature θ (in °C):
The temperatures to be tested are specified in Clause 4.
The allowed deviation for each chosen temperature is ± 1 °C.
b) contact time t (in min):
The contact times to be tested are specified in Clause 4.
The allowed deviation for each chosen contact time is ± 10 s, except for 1 min or less where it
is ± 5 s.
c) interfering substance:
The interfering substance to be tested is 0,30 g/l bovine albumin (5.2.2.8.2) representing clean
conditions or a mixture of 3 ml/l sheep erythrocytes and 3 g/l bovine albumin (5.2.2.8.3)
representing dirty conditions – according to Clause 4 and practical applications.
Additional interfering substances may be tested according to specific fields of application.
d) test organisms:
Strains shall be as specified in Clause 4, Table 1.
Additional strains may be chosen and shall be reported in the test report (5.11).
e) Cell line(s):
Modified vaccinia virus Ankara or vaccinia virus strain Elstree is multiplied in BHK-21cells
(ATCC CCL-10) or other cell lines of appropriate sensitivity. Adenovirus Type 5 is multiplied in
HeLa cells or other cell lines of appropriate sensitivity. Murine norovirus is multiplied in
RAW 264.7 cells (ATCC TIB-71) or other cell lines of appropriate sensitivity. Murine parvovirus is
multiplied in A9 cells (ATCC CCL-1.4) or other cell lines of appropriate sensitivity.
Additional test viruses may be tested.
5.5.1.2 General instructions for validation and control procedures
In the case of ready-to-use-products use water (5.2.2.2) instead of hard water.
5.5.1.3 Equilibration of temperature
Prior to testing, equilibrate all reagents [product test solutions (5.4.2), PBS (5.2.2.3), cell culture
(5.2.2.11), water (5.2.2.2), hard water (5.2.2.7) and interfering substance (5.2.2.8)] to the test
temperature of θ (5.5.1.1 a)) using the water bath (5.3.2.2) controlled at θ. Check that the temperature
of the reagents is stabilized at θ. The test suspension (5.4.1) is kept in the ice bath (5.3.2.16) to avoid
titre loss.
Water (5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C.
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ.
5.5.1.4 Precautions for manipulation of test organisms
Do not touch the upper part of the test tube sides when adding the test or the validation suspensions
(5.4.1).
5.5.1.5 Inoculation of the carriers
a) Place the carriers to be used aseptically in Petri dishes (5.3.2.8) in a horizontal position.
Pipette 1,0 ml of interfering substances (5.2.2.8) into a sterile tube. Add 9,0 ml of the test
suspension N (5.4.1). Mix (5.3.2.7) and pipette 0,05 ml of this mixture on the “inoculation square” of
a carrier (5.3.2.23) and distribute equally inside the square, e.g. with the tip of the pipette. Do not
close the Petri dish completely. Let the inoculum dry under an air laminar flow of a bio-safety
cabinet (5.3.2.19) at 18 °C to 25 °C until visible dryness, maximum 60 min. Use the carrier
immediately after the end of the drying time.
b) Note the drying time in the test report.
5.5.2 Method
5.5.2.1 Test “N ” (Determination of virucidal concentrations), water control “N ”
a W
a) Pipette 10 ml of one of the product test solutions (5.4.2) into a cylindrical screw tube (5.3.2.22)
placed in a water bath controlled at the chosen test temperature of θ [5.5.1.1 a)]. Immerse an
inoculated carrier (5.5.1.5) immediately after the drying process has been finished. Ensure that the
inoculation square is completely covered by the product test solution (5.4.2). Start the stopwatch
(5.3.2.5) and leave for the chosen contact time t [5.5.1.1 b)].
b) At the end of t transfer the carrier into a second cylindrical screw tube (5.3.2.22) filled with
5 ml of medium (5.2.2.10) and approximately 1 ml of glass beads (5.3.2.21) and placed in a water
bath controlled at 20 °C. Restart the stopwatch (5.3.2.5) and mix (5.3.2.7) for 60 s. Immediately
after elution, prepare a series of ten-fold dilutions of the virus suspension in ice-cold maintenance
medium (MEM + 2 % FCS). Pipette tips shall be changed after each dilution to avoid carry-over of
viruses.
The dilutions are inoculated on cell culture. After incubation the titre of virus is calculated.
c) Perform the procedure using the other product test solutions at the same time.
d) Water control N : Perform the procedure but instead of the product test solution, pipette 10 ml of
W
hard water (5.2.2.7) or – in the case of ready-to-use products: water (5.2.2.2).
e) Perform the procedure a) to d) applying other additional experimental conditions (5.5.1.1).
The dilutions are inoculated on cell culture. After incubation the titre of virus is calculated.
Reduction of virus titre is calculated from differences of water control N and after treatment with the
W
product N .
a
After treatment with the product, the infectivity is tested with one of the following procedures 5.5.2.2 or
5.5.2.3.
5.5.2.2 Quantal tests (end point titration) – [the procedures 1) to 2) are alternatives]
Dilute the viral suspension by tenfold dilution steps with a volume of 0,1 ml of viral suspension plus an
appropriate volume of culture maintenance medium (MEM + 2 % FCS). Pipettes (5.3.2.13) shall be
changed after each dilution step.
1) Virus titration on cells in suspension on microtitre plates:
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate (5.3.2.10), beginning
with the highest dilution. Add 0,1 ml of cell culture suspension (5.2.2.11) in such a density as to
enable the formation of a monolayer (>90 %) in at least 2 d in the cell control. In parallel six or
eight wells do not receive any viral suspension and will serve as the cell control.
The viral cytopathic effect (CPE) is read by using an inverted microscope (5.3.2.3) after the
appropriate incubation time (according to the virus type).
2) Virus titration on monolayers of cells on microtitre plates:
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate (5.3.2.10) containing a
confluent (>90 %) cell monolayer (5.2.2.11) without any medium. The last row of six or eight wells
will receive 0,1 ml of culture medium (5.2.2.10) and will serve as the cell control. After 1 h of
incubation at 37 °C, 0,1 ml of cell culture medium (5.2.2.10) is added to each well. Change pipettes
(5.3.2.13) for tubes or wells when adding medium.
After the appropri
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