BSI Standards Publication: BS EN 16777:2018

BS EN 16777:2018
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BSI Standards Publication
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)
BS EN 16777:2018 BRITISH STANDARD
National foreword
This British Standard is the UK implementation of EN 16777:2018.
The UK participation in its preparation was entrusted to Technical
Committee CH/216, Chemical disinfectants and antiseptics.
A list of organizations represented on this committee can be obtained on
request to its secretary.
This publication does not purport to include all the necessary provisions
of a contract. Users are responsible for its correct application.
© The British Standards Institution 2018
Published by BSI Standards Limited 2018
ISBN 978 0 580 95873 1
ICS 11.080.20
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 31 December 2018.
Amendments/corrigenda issued since publication

Date Text affected
BS EN 16777:2018
EUROPEAN STANDARD EN 16777

NORME EUROPÉENNE
EUROPÄISCHE NORM December 2018
ICS 11.080.20
English Version
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)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface non poreuse sans action Quantitativer Versuch auf nicht porösen Oberflächen
mécanique pour l'évaluation de l'activité virucide des ohne mechanische Einwirkung zur Bestimmung der
désinfectants chimiques utilisés dans le domaine viruziden Wirkung im humanmedizinischen Bereich -
médical - Méthode d'essai et exigences (phase 2/étape Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
2)
This European Standard was approved by CEN on 24 September 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 16777:2018 E
worldwide for CEN national Members.
BS EN 16777:2018
EN 16777:2018 (E)
Contents Page
European foreword....................................................................................................................................................... 4
Introduction .................................................................................................................................................................... 5
1 Scope .................................................................................................................................................................... 6
2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 7
4 Requirements for virucidal activity on surfaces.................................................................................. 7
5 Test methods .................................................................................................................................................... 8
5.1 Principle ............................................................................................................................................................. 8
5.2 Materials and reagents, including cell cultures ................................................................................... 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 ................................................................................... 13
5.3.3 Test surfaces .................................................................................................................................................. 14
5.4 Preparation of test organism suspensions and product test solutions .................................... 14
5.4.1 Test organisms suspensions (test virus suspension) ..................................................................... 14
5.4.2 Product test solution................................................................................................................................... 14
5.5 Procedure for assessing the virucidal activity of the product ..................................................... 15
5.5.1 Experimental conditions ........................................................................................................................... 15
5.5.2 Test procedure .............................................................................................................................................. 16
5.5.3 Cytotoxicity caused by product solutions ........................................................................................... 17
5.5.4 Control of efficiency for suppression of disinfectant virucidal activity ................................... 18
5.5.5 Reference test for virus inactivation..................................................................................................... 19
5.5.6 Titration of the virus control ................................................................................................................... 19
5.6 Experimental data and calculation ........................................................................................................ 19
5.6.1 Protocol of the results ................................................................................................................................ 19
5.6.2 Calculation of infectivity titre (TCID50 – PFU) .................................................................................. 19
5.7 Verification of the methodology ............................................................................................................. 20
5.8 Expression of results................................................................................................................................... 20
5.8.1 General ............................................................................................................................................................. 20
5.8.2 Calculation of the virucidal activity of products ............................................................................... 20
5.9 Test report ...................................................................................................................................................... 21
Annex A (informative) Examples of viruses sorted according to their presence in the
human body in case of virus infection .................................................................................................. 23
Annex B (normative) Detoxification of test mixtures by molecular sieving ........................................ 25
B.1 Molecular sieving with Sephadex™ LH 20 .......................................................................................... 25
B.1.1 Principle .......................................................................................................................................................... 25
B.1.2 Sephadex suspension.................................................................................................................................. 25
B.1.3 Procedure........................................................................................................................................................ 25
B.2 Molecular sieving using MicroSpin™ S 400 HR .................................................................................. 27
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B.3 Determination of the residual virus titre by the large-volume-plating (LVP) method ....... 27
B.3.1 General ............................................................................................................................................................. 27
B.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method .............................................................................................................................. 28
Annex C (informative) Calculation of the viral infectivity titre................................................................. 30
C.1 Quantal tests - Example of TCID50 determination by the Spaerman-Kärber method ........ 30
C.2 Plaque test ....................................................................................................................................................... 30
C.3 Biometrical evaluation of experimental approaches and assessment of the

disinfecting effect on the virus (reduction [R]): ................................................................................ 31
C.3.1 General ............................................................................................................................................................. 31
C.3.2 Calculating the virus titre with 95 % confidence interval - Example ........................................ 32
C.3.3 Calculating the reduction and its 95 % confidence interval ......................................................... 32
C.3.4 Calculating the average reduction (R(mi)) and its 95 % confidence interval ........................ 33
C.3.5 Practical example ......................................................................................................................................... 34
Bibliography ................................................................................................................................................................. 37
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EN 16777:2018 (E)
European foreword
This document (EN 16777:2018) 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 June 2019, and conflicting national standards shall be
withdrawn at the latest by June 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.
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Introduction
This document describes a surface test method for establishing whether a product proposed as a
disinfectant in the fields described in Clause 1 has or does not have virucidal activity on non-porous
surfaces.
The laboratory test closely simulates practical conditions of application. Chosen conditions (contact
time, temperature, organisms on surfaces etc.) reflect parameters which are found in practical
situations including conditions which may influence the action of disinfectants. Each use concentration
found from this test corresponds to defined experimental conditions.

The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined experimental conditions.
However for special applications the recommendations of use of a product can differ and therefore
additional test conditions might be needed, which cannot be covered by this document.
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1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectants 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 non-porous
surfaces including surfaces of medical devices without mechanical action.
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 on viruses in 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 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 10088-1, Stainless steels — Part 1: List of stainless steels
EN 10088-2, Stainless steels — Part 2: Technical delivery conditions for sheet/plate and strip of corrosion
resisting steels for general purposes
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3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14885 and EN 14476 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 for virucidal activity on surfaces

The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre of the adenovirus
and murine norovirus test strains when tested in accordance with Table 1 and Clause 5. As described in
EN 14885, to claim virucidal activity against enveloped virus the product shall pass both EN 14476 and
this standard with vaccinia virus and to claim limited spectrum virucidal activity the product shall pass
both EN 14476 and this standard with adenovirus and murine norovirus. However, to claim the
virucidal activity the product shall pass standards EN 14476 with poliovirus, adenovirus and murine
norovirus and this standard with adenovirus and murine norovirus, because poliovirus is not resistant
to drying.
Table 1 — Minimum and additional test conditions
Minimum spectrum of test Virucidal activitya

organisms
adenovirus
murine norovirus
Limited spectrum virucidal activityb
adenovirus
murine norovirus
Virucidal activity against enveloped virusesc
vaccinia virus
Test temperature between 18 °C and 25 °C
Additional temperature between 4 °C and 30 °C

Contact time according to the manufacturer’s recommendation, but not longer than
5 min or 60 mind
Interfering substances 0,3 g/l bovine serum albumin
a) clean and/or
b) dirty 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
Additional conditionse Further contact time(s), interfering substance(s) or virus(es)

a Poliovirus (as used in the corresponding suspension test) cannot be used for surfaces, because of drying
problems.
b The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus,
rotavirus and adenovirus.
c The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only (Annex A).
d The contact times for surface disinfectants stated in this table are chosen on the basis of the practical
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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.
e Where appropriate (specific purposes), additional specific virucidal activity shall be determined under other
conditions of time, temperature, and interfering substances (see 5.2.2.8) in accordance with 5.5, in order to take
into account intended specific use conditions. Additional virus(es) can be tested, if relevant. For the additional
conditions, the concentration defined as a result can be lower than the one obtained under the minimum test
conditions.
The determined virucidal concentration of the test product is suggested as being suitable for practical
situations of use.
5 Test methods
5.1 Principle
5.1.1 A test suspension of viruses in a solution of interfering substances is inoculated onto a test
surface and dried. A prepared sample of the product under test is applied in a manner which covers the
dried film.
The test surface is maintained at a specified temperature for a defined period of time. The test surface is
transferred to cell maintenance medium so that the action of the disinfectant is immediately
neutralized. The titre of the virus recovered from the test surface is determined.
The titre of the inoculum on a test surface treated with hard water in place of the disinfectant is also
determined and the reduction in virus titre attributed to the product is calculated by difference.
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
to Clause 4, Table 1 1
a) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
NOTE Virus strains can be obtained from a national or international culture collection. Murine norovirus can
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.
b) Non-enveloped DNA virus
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.
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Adenovirus type 5, strain Adenoid 75, ATCC VR-5
c) Enveloped DNA virus
Vaccinia virus, strain modified vaccinia virus Ankara (MVA), ATCC VR-1508 or vaccinia virus strain
Elstree, ATCC VR-1549
The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.12).
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 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 ± 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 (Na2HPO4 x 12H2O) 2,89 g
Potassium phosphate, monobasic (KH2PO4) 0,20 g
Water (5.2.2.2) to 1000,0 ml
5.2.2.4 Neutral Red (1:1000 solution)
Prepare neutral red (Sigma N7005 2 stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a
0,44 µm pore size filter and store at 4 °C in the dark.
2 Sigma N 7005 is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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5.2.2.5 Foetal calf serum (FCS)
FCS shall be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may
interfere with cell and virus growth resulting in false results.
For RAW 264.7 cells, special FCS shall be used due to the cells’ high sensitivity to endotoxins.
5.2.2.6 Trichloroacetic acid (10 % solution) (TCA)
Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), 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 (MgCl2) and 46,24 g calcium chloride
(CaCl2) 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 (NaHCO3) 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
(5.3.2.4) 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 (CaCO3) 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 and detergents) shall be defined.
“Diluent” is generally used in the other European Standards in the medical area to prepare the
interfering substance. Since there is no experience in virucidal testing with diluent, water (5.2.2.2) is
used instead.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine serum albumin)
Bovine serum albumin shall be used as commercially available product or shall be prepared as follows:
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— dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water
(5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the test is 0,3 g BSA per litre.
5.2.2.8.3 Dirty conditions
a) bovine serum albumin:
Bovine serum albumin shall be used as commercially available product or shall be prepared as follows:
— dissolve 3 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of

water (see 5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the control is 3 g BSA per litre.
b) sheep erthrocytes:
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at
800 gN for 10 min (5.3.2.18). After discarding the supernatant, resuspend erythrocytes in PBS
(5.2.2.3). Repeat this procedure at least 3 times, until the supernatant is colourless.
c) bovine albumin and erythrocyte solution:
Resuspend 3 ml of packed erythrocytes with 97 ml of 3 % w/v of bovine albumin solution.
The final concentration of sheep erythrocytes and albumin in the test procedure is 3 ml/l and 3 g/l
respectively. To avoid contamination this mixture shall be split in portions probably needed per day
and stored in separate containers for a maximum of 7 days at 2 °C to 8 °C.
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.
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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) in such a density as to enable the
formation of a monolayer no longer than 2 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 5.5.1e).
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 of glutardialdehyde
Parameters Specifications
Solution 50 % Solution 25 %
Appearancea clear liquid clear liquid

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 polymerization.
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)].
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5.3.2 Usual microbiological laboratory equipment 3
And, in particular, the following:

5.3.2.1 Apparatus for sterilization (moist and dry heat)
a) For moist heat sterilization, an autoclave capable of being maintained at ( 1210+3 ) °C for a minimum
holding time of 15 min;
b) for dry heat sterilization, a hot air oven capable of being maintained at ( 1800+5 ) °C for a minimum
holding time of 30 min, at ( 1700+5 ) °C for a minimum holding time of 1 h or at ( 1600+5 ) °C for a
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 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
5.3.2.6 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.7 Electromechanical agitator e.g. Vortex ® mixer 4
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 Microtitre plates or tubes, petri dishes 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 CO2 incubator (95 % air, 5 % CO2), capable of being controlled at (36 ± 1) °C, for
incubation of cell cultures. An incubator at (37 ± 1) °C may be used if an incubator at (36 ± 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 may 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 (see 5.5.2 and 5.5.4)
5.3.2.16 Basin as ice bath with ice and water
5.3.2.17 Mechanical shaker
5.3.2.18 Centrifuge
3 Disposable sterile equipment is an acceptable alternative to reusable glassware.
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.
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5.3.2.19 Biological safety cabinet, class II

5.3.2.20 Freezer, −70 °C or less
5.3.2.21 Desiccator with vacuum manometer
5.3.2.22 Vacuum Diaphragm Pump (e.g. throughput max. 3,8 m3/h final vacuum < 75mbar)
5.3.2.23 Cryotubes
5.3.3 Test surfaces

These shall be 1.4301 (EN 10088-1) stainless steel discs (2 cm diameter discs) with Grade 2 B finish on
both sides, in accordance with the requirements of EN 10088-2. The discs shall be as flat as possible and
this is best achieved by using stainless steel of a gauge of 1,2 mm or 1,5 mm. The discs shall be handled
only with forceps and used only once.
Prior to use the discs shall be placed in a container with an appropriate quantity of 5 % per volume
Decon 90®, or of 1 % Blanisol-Pur 5 for 60 min, in a manner that they don’t stick together and the
surface is not damaged. Rinse the discs with running freshly distilled water (5.2.2.2) or demineralized
water for 10 s, avoiding them to dry at any extent.
Rinse the discs with water (5.2.2.2) for a further 10 s to ensure complete removal of the surfactant. To
supply a satisfactory flow of water, a sterilized fluid dispensing pressure vessel with suitable hose and
connectors or other suitable method can be used and regulated to supply approximately 2000 ml per
min. Dip the disc in a bath containing isopropanol (IPA) for 15 min. Remove the discs and rinse them
with water (5.2.2.2) for at least 10 s. Sterilize by autoclaving.
NOTE Suitable stainless steel discs can usually be purchased from local engineering companies.
5.4 Preparation of test organism suspensions and product test solutions

5.4.1 Test organisms suspensions (test virus suspension)
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 gN for 15 min). This preparation
is called “test virus suspension”.
It is suggested that the minimum titre of the virus suspension - determined by a quantal test [5.5.2 a)]
or by plaque test [5.5.2 b)] - is at least 108 TCID50/ml. In any case, it shall be sufficiently high to at least
enable a titre reduction of 4 lg to verify the method.
If necessary 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 between −135°C and −196°C in
liquid nitrogen vapor phase or liquid nitrogen (with suitable tight cryotubes, 5.3.2.23).
Due to safety reasons, and – in some cases – to avoid 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
Detail sample information of the product as received from the producer or from any other source shall
be recorded.
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.
5 Decon 90® and Blanisol-Pur are examples of suitable products available commercially. This information is given
for the convenience of users of this standard and does not constitute an endorsement by CEN of those products.
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Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared
in water (5.2.2.2).
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 (5.3.2.9) 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 on a volume/volume
basis using volumetric flasks (5.3.2.9).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous 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 substance, it shall be recorded in the test report.
The concentration of the product stated in the test report shall be the desired test concentration.
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 Experimental conditions
The selection of contact temperature, contact time and interfering substances shall be carried out
according to the practical use considered for the product (Clause 4) as follows:
a) Test temperature, θ in °C:
The test temperature shall be room temperature [between (18 ± 1) °C and (25 ± 1) °C]; additional
temperatures may be chosen between (4 ± 1) °C and (30 ± 1) °C and shall be reported in the test
report (5.9).
b) Contact time, t (min):
The contact times to be tested are specified in Clause 4, Table 1.
The allowed deviation for each chosen contact time is ± 10 s; additional times may be chosen and
shall be reported in the test report (5.9).
c) Virus strains:
Strains shall be as specified in Clause 4, Table 1.
Additional strains may be chosen and shall be reported in the test report (5.9).
d) Interfering substances:
The interfering substance to be tested is the bovine albumin solution (5.2.2.8.2) under clean
conditions or albumin and sheep erythrocytes under dirty conditions (5.2.2.8.3), according to
practical applications.
Additional interfering substance can be selected and shall be recorded in the test report (5.9)
e) Cell line(s):
Adenovirus 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.
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Modified vaccinia virus Ankara is multiplied in BHK-21 cells (ATCC CCL-10) or other cell lines of
appropriate sensitivity. Vaccinia virus strain Elstree is multiplied in Vero cells (ATCC CCL-81), CV-1
cells (ATCC CCL-70) or other cell lines of appropriate susceptibility.
5.5.2 Test procedure
The test shall be performed at a temperature of between (18 ± 1) °C and (25 ± 1) °C. Place each test
surface (5.3.3) aseptically in a Petri plate (5.3.2.8) and ensure that the plate is in a horizontal position.
Nine volumes of test virus suspension (5.4.1) are mixed with one volume of interfering substance
solution (5.2.2.8).
For each test organism, for each concentration of the product and contact time prepare 2 test surfaces
plus 2 test surfaces for the water control (5.5.6). The 2 test surfaces shall be used in the same working
session, in parallel. Place the test surfaces in a sterile Petri dish under the laminar air flow and ensure
that the dish is in a horizontal position. Prepare the test surfaces by inoculating 50 μl of the virus
suspension plus interfering substance on to each test surface, paying attention to deliver the suspension
it in the centre of the test surface (5.3.3) (avoiding to touch the test surface edges). Dry the surfaces
until they are visibly dry.
It is understood that drying of the test surfaces will occur at different rates due to the ambient
conditions of the laboratory and the design of the laminar air flow cabinet. For this reason no time
duration is given and the minimum required time for the surfaces to become visibly dry should be
established for each laboratory. The drying time should not exceed 60 min and if it does, alternative
drying conditions shall be used. Allow the test surfaces to equilibrate with the chosen test temperature
(θ ± 1)°C.
Use the test surfaces within 60 min, to avoid virus inactivation with time.
Immediately after drying, carefully pick up each test surface and place it, inoculated side up, in a sterile
Petri plate and ensure that the test surface is in a horizontal position. Cover the dried inoculum on the
test surfaces with 100 µl of the test solution. Care shall be taken, not to touch pipette tip to carrier. For
the water control, place 100 µl of hard water (5.2.2.7), or water (5.2.2.2), if the product has been diluted
in water, onto the other test surfaces ensuring that the dried inoculum is totally covered.
After the chosen contact time transfer immediately each of the test surfaces to a separate container (flat
bottom between 4 cm and 5 cm at the base, with cap) and add 0,9 ml of ice-cold cell culture medium
without FCS e.g. Eagle's minimal essential medium (MEM) or Dulbecco’s Modified Eagle Medium
(DMEM) according to the cell line used. Mix (5.3.2.7) each container for 60 s (or as long as visibly no
dried inoculum can be seen) to re-suspend the virus.
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.
Reduction of virus titre is calculated from titre differences between those treated with disinfectant and
those treated with hard water.
After treatment with the product or water, the infectivity is tested with one of the following procedures:
a) Quantal tests (end point titration) – [the procedures 1) to 2) are alternatives]
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
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formation of a monolayer (>90 %) no longer than 2 days 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 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 b)) 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 a)) is added to each well. Change pipettes
for tubes or wells when adding medium.
After the appropriate incubation time, according the virus type, the viral cytopathic effect is read using
an inverted microscope.
b) 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
MEM + 2 % FCS. Six to eight 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 2 times concentrated MEM with 4 % FCS. The cultures are incubated for 5 to 6 days at 37°C
in a CO2 incubator (5.3.2.12). Plaques can be counted after addition of 2 ml of a second overlay with the
same composition of the first and also containing 5 % of a 1:1000 solution of neutral red and further
incubation (in the dark) at 37°C for 24 h to 48 h in a CO2 incubator (5.3.2.12). 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.
NOTE Determination of CPE can be used as an alternative to plaque assay.
5.5.3 Cytotoxicity caused by product solutions
5.5.3.1 Cytotoxic effect
To check for possible morphological alteration of cells by the disinfectant, mix 45 µl MEM +
2 % FCS + 5 µl of interfering substance, inoculate on a carrier and dry. Add 100 µl of product (at the
dilutions used in the test) and leave for 5 min at test temperature. Elute with 0,9 ml MEM + 2 % FCS mix
30 s (5.3.2.7). Serial dilutions (dilution step: 1:10) are prepared in the culture medium and are
inoculated into cell cultures (monolayers or suspended cells). This shall be done in parallel with the
test. Any microscopic changes in the cells are recorded when reading the tests for CPE (2 test surfaces).
5.5.3.2 Interference control – control of cell susceptibility
The aim of the interference control is to verify that the susceptibility of the cells for the virus infection is
not influenced negatively by the treatment with the disinfectant.
Comparative virus titrations are performed on cells that have or have not been treated with
disinfectants to check the reduction of the sensitivity to viruses as follows:
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Treatment of cells:
a) monolayers: 0,1 ml of the lowest apparently non-cytotoxic dilution (no microscopic alteration) of
the product test solution (5.4.2) or PBS (5.2.2.3) and 0,1 ml of culture medium are distributed onto
each of 6 established cell cultures in microtitre plates with 96 wells (5.3.2.10). After 1 h at 37 °C the
supernatant is discarded;
b) suspension cells: a volume of the lowest apparently non-cytotoxic dilution of the product or PBS
(5.2.2.3) is added to a volume of double concentrated cell suspension. After 1 h at 37 °C the cells are
centrifuged and resuspended in culture medium [5.2.2.10 a)].
Comparative titration of viruses:

The virus is diluted from 10−1 to 10−10 and titrated on the treated or untreated cells in parallel as in 5.5.2
a) or b).
Only those dilutions of the product solution can be used for the determination of the residual infectivity
which:
a) show a low degree of cell destruction (<25 % of monolayer);
b) produce a titre reduction of the virus of < 1 lg.
5.5.3.3 Elimination of cytotoxicity
If the cytotoxicity is so great that the reduction (5.8.2) cannot demonstrate the range of 4 lg TCID50,
special techniques shall be used, such as:
a) molecular sieving with a molecular sieve filter, i.e. SephadexTM LH 20 6 (Annex B.1);
b) ultrafiltration with Minicon® (Millipore) or with ready to use i.e. MicroSpinTM S 400 HR
columns6) (GE Healthcare) (Annex B.2);
c) Determination of the residual virus titre by the large-volume-plating (LVP) method (Annex B.3).
Appropriate controls are required to demonstrate that the procedure does not reduce the infectivity of
the virus. The virucidal activity is measured by comparing the titres of the treated and untreated viral
suspensions. Use according to the instructions of the manufacturer.
5.5.4 Control of efficiency for suppression of disinfectant virucidal activity
5.5.4.1 Dilution in ice-cold medium
100 μl of the test solution (test concentration) and 850 μl of ice-cold medium with 2 % FCS are mixed.
Afterwards 50 μl of the virus inoculum are added and the mixture is incubated in an ice-bath for the
period between end of contact time and the preparation of dilutions for titration (see 5.5.2). A serial
dilution in log10- steps is prepared and cell cultures are inoculated with the dilutions. The titre is
determined.
The difference of titre with the test suspension shall be ≤ 0,5 lg.
6 SephadexTM LH 20, Minicon® and MicroSpinTM S 400 HR are examples of suitable products available
commercially. This information is given for the convience of user of this standard and does not constitute an
endorsement by CEN of these products.
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5.5.4.2 Filtration technique
If the difference is > 0,5 lg a 1/100 ice-cold dilution can be tried or another technique shall be used:
molecular sieving with SephadexTM LH 20 or ultrafiltration with Minicon® (Millipore) or with ready to
use MicroSpinTM S 400 HR columns (GE Healthcare) (Annex B).
5.5.5 Reference test for virus inactivation
A control of the test system shall be included using the test organisms, 5.5.2 applies with
glutardialdehyde (5.2.2.12) as a reference (see Table 3).
5.5.6 Titration of the virus control
The infectivity of the test organism suspension shall be determined under test conditions at the
beginning of the contact time and at the maximum contact time used in the test. The product test
solution (5.4.2) is substituted by hard water (see 5.2.2.7) or water (5.2.2.2) for ready-to-use products.
5.5.2 applies with the following modification:
100 µl of hard water (5.2.2.7) - or water (5.2.2.2) for ready-to-use products - is added instead of the
product test solution.
5.6 Experimental data and calculation
5.6.1 Protocol of the results
The cell culture results are recorded as 0 (cells without CPE), and 1 (25 % of cells with CPE) to 4 (all
cells with CPE) (quantal tests) or as number of plaques.
Calculation of the estimated virus titres shall be carried out by appropriate methods (Annex C).
Reduction of virus titre is calculated from differences between the titre after treatment with hard water
(5.2.2.7) - or water (5.2.2.2) for ready-to-use products - and after treatment with the product.
The titres shall be calculated with their 95 % confidence intervals from which the reduction factor with
the 95 % confidence interval shall be calculated (C.3.1).
5.6.2 Calculation of infectivity titre (TCID50 – PFU)
5.6.2.1 General
Calculation of the estimated virus concentration shall be carried out by appropriate methods (Annex C).
The titres shall be calculated with their 95 % confidence intervals from which the reduction with a 95 %
confidence interval shall be calculated (C.3.1).
5.6.2.2 Calculation of TCID50
An appropriate way of calculation is the Spearman-Kärber method (Annex C.1).

5.6.2.3 Calculation of PFU
A practical way to calculate the number of plaque-forming units per millilitre (PFU/ml) is described in
Annex C.2.
Only plates with non-confluent plaques shall be used for calculation. PFU should be calculated using
three plates. If plates from 2 dilutions fall within this range, calculate the number of PFU/ml as the
weighted mean count. If plates from only one dilution fall within this range, calculate the arithmetic
mean.
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5.7 Verification of the methodology
A test is only valid, if the following criteria are fulfilled:

a) test virus suspension (5.4.1) has TCID50 108 /ml, or possesses at least a concentration which allows
the determination of a 4 lg reduction of the virus titre;
b) detectable titre reduction is at least 4 lg;

c) difference between the logarithmic titre of the virus control minus the logarithmic titre of the test
organism in the reference inactivation test (5.5.5) is between the values given in Table 3 (when all
tests shall be done for 5 min under clean conditions).
Table 3 — Limits for reference substances
Test organism
Concentration / reduction
Reference substance
Adenovirus type 5 strain Vaccinia virus Murine norovirus
adenoid 75
Glutardialdehyde 125 ppm / 2,0 to 3,5 lg 50 ppm / < 3 lg 1000 ppm / 1,5 to 3 lg
d) cytotoxicity of the product solution does not affect cell morphology and growth or susceptibility for
the test organism in the dilutions of the test mixtures which are necessary to demonstrate a 4 lg
reduction of the virus;
e) comparative virus titration on cells treated with test mixture dilutions (or without, i.e. only addition
of PBS) result in a difference of < 1 lg of virus titre;
f) control of efficiency for suppression of product’s activity (5.5.4: the difference to the test suspension
shall be ≤ 0,5 lg).
5.8 Expression of results

5.8.1 General
All results are tabulated as raw data and are presented in addition in negative logarithmic values of
TCID50 or PFU.
In case no virus multiplication is observed [5.5.2 a) or b)] in the highest concentration, this value is
preceded by the sign “≤”. In case virus multiplication can be observed in all dilutions, this value is
preceded by the sign “≥”.
5.8.2 Calculation of the virucidal activity of products
The assessment of the efficacy of the dilution of the disinfectant is carried out by calculation the
reduction. The reduction is the difference between the infectivity titre obtained without exposure to the
disinfectant to TCID50/ml or lg plaque forming units (PFU)/ml of the water control (5.5.6) and the
infectivity titre obtained after exposure to the disinfectant (5.5.2) at the specific contact time chosen.
The product shall be deemed to have passed the test if it demonstrates a 4 lg or more reduction in titre
for adenovirus and murine norovirus at the specific contact time chosen at between 18 °C ± 1 °C and
25 °C ± 1 °C, with the chosen interfering substance (Table 1) under the conditions defined by the test
(5.5.2).
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Confidence interval calculation (e.g. 95 %) may be confirmed (Annex C.3), it is necessary to calculate the
confidence interval from two series of tests.
5.9 Test report
The test report shall refer to this European Standard (EN 16777)
The test report shall, at least, state the following information:
a) Identification of the testing laboratory;
b) Identification of the client;
c) Identification of the sample:
1) Name of the product;
2) Batch number and – if available – expiry date;
3) Manufacturer – if not known: supplier;
4) Date of delivery;
5) Storage conditions;
6) Product diluent recommended by the manufacturer;
7) Active substance(s) and concentration(s) (optional);
8) Appearance of the product;
d) Experimental conditions:
1) Date(s) of tests (period of analysis);
2) Diluent used for product test solution (hard water or distilled water);
3) Product test concentrations;
4) Appearance of product dilutions;
5) Contact times;
6) Test temperature(s);
7) Interfering substance(s);
8) Stability and appearance of the mixture during procedure (note the formation of any
precipitate or flocculant);
9) Temperature of incubation;
10) Method of filtration;
11) Identification of the source of the viral strain and number of passage;
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12) Procedure to stop action of the product;
13) Cell line: name, source, number of passage, medium for cell cultures;
e) Test results, control and evaluation:
1) Titre of test suspension;
2) Maximum detectable virus inactivation;

3) Virus inactivation of the reference virus inactivation test after 5 min;
f) Test results, evaluation of virucidal activity:
1) Description;
2) Table of results (raw data):
i) Virus titre with 95 % confidence interval;
ii) Reduction with 95 % confidence interval;
g) Special remarks;
h) Conclusion;
i) Locality, date and identified signature.
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Annex A
(informative)
Examples of viruses sorted according to their presence in the human body

in case of virus infection
These viruses may contaminate hands, instruments, other surfaces and textiles.
NOTE 1 This list is not exhaustive.
NOTE 2 Enveloped viruses are in bold
Blood
Enterovirus Hepatitis C virus (HCV)
Filoviridae Hepatitis Delta virus (HDV)
Flavivirus Human Immunodeficiency Virus (HIV)
Herpesviridae Human T-Cell Leukaemia Virus (HTLV)
Hepatitis A Virus (HAV) Parvovirus B 19
Hepatitis B virus (HBV)
Respiratory tract
Adenovirus (Mast-) Influenza Virus
Coronavirus Paramyxoviridae
Enterovirus Rhinovirus
Herpesviridae Rubella Virus
Neuronal tissue, ear and nose, eye
Adenovirus (Mast-) Human Immunodeficiency Virus (HIV)
Enterovirus Polyomavirus
Herpesviridae Rabies Virus
Measles Virus Rubella Virus
Gastro-intestinal
Adenovirus(Mast-) Enterovirus
Caliciviridae Hepatitis A Virus (HAV)
Coronavirus Hepatitis E Virus (HEV)
Astrovirus Rotavirus
Skin, breast and/or milk
Enterovirus Human T-Cell Leukaemia Virus (HTLV)
Herpesviridae Papillomavirus
Human Immunodeficiency Virus (HIV) Poxviridae
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Spleen and lymph nodes (see also „Blood“)

Human T-Cell Leukaemia Virus (HTLV)
Human Immunodeficiency Virus (HIV)
Dental procedure
Adenovirus(Mast-) Hepatitis C Virus (HCV)
Enterovirus Hepatitis Delta Virus (HDV)
Herpesviridae Human Immunodeficiency Virus (HIV)

Hepatitis B virus (HBV)
Urogenital tract
Hepatitis B Virus (HBV) Human T-Cell Leukaemia Virus (HTLV)
Herpesviridae Papillomavirus
Human Immunodeficiency Virus (HIV) Polyomavirus
Reference:
Van Regenmortel MHV et al.,Eds.: Virus Taxonomy, Classification and Nomenclature of Viruses, seventh
report of the international committee on taxonomy of viruses.
Academic Press, San Diego, 2000
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Annex B
(normative)
Detoxification of test mixtures by molecular sieving
B.1 Molecular sieving with Sephadex™ LH 20 7

B.1.1 Principle
The samples are sieved through Sephadex™ LH 20 which retains the disinfectant and allows the virus to
pass through. The viral titre of the filtrate is compared with that of the initial viral suspension treated
under the same conditions.
B.1.2 Sephadex suspension
Prepare the suspension of Sephadex™ LH 20 mixing 22 g in 100 ml of PBS. It is sterilized for 30 min at
120 °C. On removal from the autoclave, it is left at room temperature before being dispensed at the rate
of 25 ml per previously sterilized stainless steel bottle or 20 ml per sterile syringe placed in a
previously sterilized centrifuge bottle.
Centrifuge at 1 000 gN for 10 min.
After removal of the centrifuging liquid, the system is ready for use (Figure B.1).
B.1.3 Procedure
The samples are deposited, each at the rate of 1 ml, on the top of a Sephadex™ column. The whole of the
device is centrifuged at 1 000 gN for 10 min in a centrifuge which is preferably cooled to 4 °C. Collect
the filtrate in a sterile manner. The volume collected shall be about 1 ml.
Two similar operations are carried out beforehand for 1 ml of the solution of the product under test and
for the viral suspension.
The virus filtrates are titrated.
The titre of the viral suspension obtained by sieving is compared with the titre of the initial unsieved
viral suspension. Both titres shall be identical (difference not exceeding 0,5 lg).
The virucidal activity of the product is measured by comparing the titres of the treated and untreated
viral suspensions in parallel.
7 SephadexTM LH 20 is an example of a suitable product available commercially. This information is given for
the convenience of user of this standard and does not constitute an endorsement by CEN of this product.
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Dimensions in millimetres
Key
1 Plastic (polypropylene) syringe
2 45 ml (Jouan) glass centrifuge tube
3 SEPHADEX™ LH-20 gel
4 Glass fibre or polyester fabric
5 Tube made of neutral glass
Figure B.1 — Molecular sieving device with syringe
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B.2 Molecular sieving using MicroSpin™ S 400 HR
Toxicity of formaldehyde 0,8 × 1,4 % Infectivity of herpes virusb
100 μl: 107,6 TCID50/ml (before

10-4 a (before microspin) microspin)
centrifugation of microspin centrifugation of microspin
(735 x gN, 2 min) virus-
(735 x gN, 2 min) virus suspension

formaldehyde suspension
< 10−1 (after microspin) 100 μl: 107,5 TCID50/ml (after
microspin)
Key
a highest dilution toxic to freshly seeded Vero cells
b herpes virus was selected to demonstrate that:
1) no spontaneous inactivation occurs with this very unstable virus. and
2) larger viruses pass the sieve.
Figure B.2 — MicroSpin™ S 400 HR for purification of virus samples
B.3 Determination of the residual virus titre by the large-volume-plating (LVP)

method
B.3.1 General
Using the LVP, the lowest apparently non-cytotoxic dilution of the test mixture is added to ice-cold
medium after the specified contact time and this test mixture has to be added to a defined number of
wells containing the indicator cells in 100 μl cell culture medium.
NOTE The detection of residual virus can be improved by the testing of a large sample volume.
The cells are cultivated for a specified incubation period. Then, the cells are inspected for virus-induced
changes in cell morphology. The viral titre has to be calculated as follows:
If no virus is observed, the number infectious virus particles are determined by the Poisson distribution
using the following formula:
ln p
c= (B.1)
−V P
where
c concentration of virus particles in the test mixture;
p denoting the 95 % probability to detect virus;
VP is the plated volume and shall be < Vtot in ml;
Vtot is the total test volume in ml.
If low amount of viruses is detected the most probable average number of TCID50 can be calculated by
the use of the following formula which is derived from the Taylor series:
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D  n − np 
c= ×  − ln  (B.2)
Vw  n 
 
where
c concentration of virus particles in the test mixture;
D dilution factor of pre-diluted sample;
VW is the plated volume per well;
n number of inoculated wells;

np is the number of successfully infected wells
B.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method
If the LVP method is applied, there are two possibilities for the calculation of the titres which are used
for the calculation of the reduction.
a) Calculation referred to the Taylor formula
If low amount of viruses is detected the most probable average number of TCID50 can be calculated
by the use of the formula (B. 2) which is derived from the Taylor series with the following values:
V W = 0,1 ml
n = 96
np = 9
D = 1 000
According to the Taylor formula the result is 984 infectious particles per ml.
However, one TCID50 is equivalent to 0,69 infectious virus particles because the natural logarithm
of a 50 % likelihood (P = 0,5) is 0,69 or ln(0,5) = 0,69. This implies that at least 69 trials are
necessary to infect successfully 50 % of 100 wells. Therefore this factor is needed to calculate the
TCID50 /ml as:
984/ 0,69 = 1426 TCID50/ml.
The titre, which is used for the calculation of the reduction, is the logarithm of this value, namely
3,15 lg TCID50 per ml.
There is no result possible if all wells are positive
b) Calculation referred to Poisson formula
If no virus is observed, the number of infectious virus particles is determined by the Poisson
distribution using the formula (B. 1).
28
BS EN 16777:2018
EN 16777:2018 (E)
In this example, the test mixture is due to high cytotoxicity of the test product diluted to 1:1000.
The diluted test mixture is inoculated due to the cytotoxicity in 96 wells, (0,1 ml/well).
This produces in the following calculation:
P = 0,05 - > ln P = −2,99
VP = 96 × 0,1 ml; the total volume tested is 9,6 ml.

Using the formula (B. 1) the result is c ≤ 0,31 virus particle per ml. Taking into account the test
product dilution of 1:1000 (0,31 × 1 000) produces a value of 310 infectious particles, and a
logarithm of ≤ 2,64 lg TCID50/ml. This value is the titre, which is used for the calculation of the
reduction.
For the calculation of the reduction and its 95 % confidence interval, we assume that the titre of the
control titration is (8,88 ± 0,38) lg.
Applying the Taylor Formula the residual virus titre is 3,15 lg. The reduction is then:
(8,88 - 3,15) lg = 5,73 lg.
Due to formal reasons, the residual virus titres does not have 95 % confidence interval, resulting in
a reduction factor of (5,73 ± 0,38) lg.
If no infectivity by the determination of the residual virus is found, the titre is determined
according to the Poisson formula. The calculation of the reduction factor results is:
(8,88 – 2,65) lg = 6,23 lg.
Due to formal reasons, the residual virus titres does not have 95 % confidence interval, resulting in
a reduction factor of ≥ (6,23 ± 0,38) lg. The character ≥ preceding the reduction factor, shows that
no residual infectivity has been demonstrated; i.e. the reduction factor has at least this value, can
possibly also be higher.
29
BS EN 16777:2018
EN 16777:2018 (E)
Annex C
(informative)
Calculation of the viral infectivity titre
C.1 Quantal tests - Example of TCID50 determination by the Spaerman-Kärber

method
The Spearman-Kärber method is most simple and gives results most compatible with other calculations.
Prerequisite of the evaluation is the use of several dilutions which cover infection of all cell culture units
to those in which no virus multiply. The mean and standard deviation can only be calculated; when
several test results are available.
The formula is:
Negative logarithm of the 50 % end point = Negative logarithm of the highest virus concentration used -
[(Sum of % affected at each dilution / 100 - 0,5) x (lg of dilutions)]
Table C.1 — Example of titration results
Dilution (-lg) Resulta % positive
4 444 444 100

5 444 344 100
6 443 003 66,7
7 400 020 33,3
8 000 000 0
Sum of % positive cultures 300
a
1 to 4 virus present, degree of CPE in 6 cell culture units (tubes, microtitre plates)
0 no virus present
Using the data from Table C.1 the 50 % end point is calculated as follows:
-4 - [{(100 + 100 + 66,7 + 33,3 + 0) / 100} – 0,5] x 1 =
-4 - [300 / 100 – 0,5] x 1 =
-4 – 2,5 x 1 = - 6,5
Therefore, the 50 % end point is 10-6,5 or

lg TCID50 = 6,5
C.2 Plaque test

For calculation of the weighted mean count, use the following formula:
30
BS EN 16777:2018
EN 16777:2018 (E)
PFU c1 +
c2 …cn
= (C.1)
t ( n1 + ×v )× d
n2 × v2 +… nn n
where
t is the test volume that was added per dilution step to a plate;
c1 is the PFU number of all plates of the first dilution step (lowest dilution step) with
nonconfluent plaques;
c2 is the PFU number of all plates of the second dilution step with nonconfluent plaques;
cn is the PFU of all plates of the last dilution (highest dilution step);
n1 is the number of all plates of the first dilution step (lowest dilution step) with nonconfluent
plaques to which c1 corresponds;
n2 is the number of all plates of the second dilution step with nonconfluent plaques (c2);
v2 is the dilution factor between n1/ n 2 (e.g. n 1 = 10−3 and n 2 = 10−4, then v2 = 0,1);
nn is the number of all plates of the last dilution for which PFUs were counted (cn);
vn is the dilution factor between n 1/ n n (e.g. n 1 = 10−3 and n n = 10−6, then vn = 0,001);
d is the dilution step of c1.
Round off the calculated PFU/t to one decimal number. If the last number is under 5, the preceding
number remains unchanged; if the last number is equal to or over 5, one is added to the preceding
number. Proceed from the last decimal number.
EXAMPLE
PFU
= ,
( ) ( ) (
52 + 48 + 49 + 25 + 27 + 31 + 5 + 6 + 7 250
= = 75 075 075
)
t ( ) (
3 + 3 × 0, 1 + 3 × 0, 01  × 10
  )−3
3, 33 × 10 −3
075=
lg 75 4 875 ≈ 4, 9
, 075 ,
If the test volume that was added per dilution to a plate was 0,2 ml, the PFU/ml = 5,6 lg
C.3 Biometrical evaluation of experimental approaches and assessment of the

disinfecting effect on the virus (reduction [R]):
C.3.1 General
For the evaluation of disinfectant efficacy the virus titre before and after exposure to the disinfectant is
determined and the reduction (R) calculated including its 95 % confidence interval. The virus titrations
are conducted in such a way that the virus titre exhibits a 95 % confidence interval of ± 0,5 lg for the
Spearman-Kärber method.
If two test runs on different days are performed, the calculation of the average R and its 95 %
confidence interval is done according C.3.4.
The number of replicates per dilution (e.g. 8, 12 or 16) and the dilution factor in the dilution series (e.g.
3, 5 or 10) used for the titration shall be determined respectively. A sufficient titre reduction caused by
the product can be assumed if the average R (see below) is at least 4 lg. The results shall not be affected
by cytotoxicity, interferences or after-effects of the product.
31
BS EN 16777:2018
EN 16777:2018 (E)
The virus titre (TCID50/ml or PFU/ml) can be determined using several methods, e.g. the Spearman-
Kärber Formula (5) and [6].
C.3.2 Calculating the virus titre with 95 % confidence interval - Example
Using the Spearman-Kärber method the logarithmic infectivity titre in TCID50/ml is calculated as
follows:
d
m = xk + − d ∑ pi (C.2)
2
where
m negative decimal logarithm of the titre based on the test volume
xk logarithm of lowest dose (dilution level) at which all test objects exhibit a positive reaction
d logarithm of dilution factor
pi observed reaction rate
∑ pi sum of the value pi from x k to the highest dilution tested

The standard error (S) from m is calculated as follows:
Sm =
{ }
d 2 ∑  pi (1 − pi )  / ( n − 1) (C.3)
where
Sm standard error of logarithmic titre
d logarithm of dilution factor
pi observed reaction rate
n number of test objects per dilution
95 % confidence interval of the titre is approximately 2 Sm. When calculating the titre, take into account
the pre-dilution of the sample and calculate the titre to the same volume.
C.3.3 Calculating the reduction and its 95 % confidence interval
The reduction (R) is calculated as the difference between the logarithmic virus titre before (“virus
control titration”, see 5.5.7, equals titre to a) and after exposure to the product test solution (“rest
virus”, see 5.5.2, equals titre to b).
The reduction (R) is thus calculated as follows:
RT1= a − b (C.4)
where
RT1 reduction from first test run
a lg TCID50/ml of control titration of the first test run
b lg TCID50/ml of “rest virus” titration of the first test run
The 95% confidence interval of R of the first approach (KR(T1)) is calculated as follows:
32
BS EN 16777:2018
EN 16777:2018 (E)
2 × Sa2 + S b2
K R(T1) = (C.5)
where
KR(T1) 95 % confidence interval of the R of the first test run
Sa standard error of control titration of the first test run
2Sa 95 % confidence interval of control titration of the first test run
Sb standard error of “rest virus” titration of the first test run

2Sb 95 % confidence interval of “rest virus” titration of the first test run
If no virus can be detected any more in the test run with product (“rest virus”), the 95 % confidence
interval is calculated according to:
× Sa2
K R(Tkv) = 2 (C.6)
where
KR(Tkv) 95 % confidence interval of the R in the case no virus can be detected in the test run with
product (“rest virus”)
Sa standard error of control titration of the first test run
C.3.4 Calculating the average reduction (R(mi)) and its 95 % confidence interval
The average R from both runs and its 95% confidence interval is calculated as follows:
R=
(mi) ( RT1 +
RT2 ) / 2 (C.7)
where
R(mi) average reduction
RT1 reduction from first test run
RT2 reduction from second test run
The 95% confidence interval of the average R (KR(mi)) is calculated as follows:
2 2
 K R(T1)  +  K R(T2) 
K R(mi) = (C.8)
2
where
KR(mi) 95 % confidence interval of the average reduction
KR(T1) 95 % confidence interval of the reduction of the first test run
KR(T2) 95 % confidence interval of the reduction of the second test run
For the virus control and the active concentration of the product, two test runs on different days are
performed.
The reduction and the 95 % confidence interval for each test run is calculated.
33
BS EN 16777:2018
EN 16777:2018 (E)
C.3.5 Practical example
Table C.2 — a) Raw data for the example of Titration results –

Before (virus control titration = a)
Dose Log % of virus 1-pi

Totala Nb pi
(TCID50) (Dose) presentb
3,16E-08 −7,50 8 0 0 0 1
1,00E-07 −7,00 8 4 50 0,5 0,5

3,16E-07 −6,50 8 6 75 0,75 0,25
1,00E-06 −6,00 8 8 100 1 0
3,16E-06 −5,50 8 8 100 1 0
1,00E-05 −5,00 8 8 100 1 0
3,16E-05 −4,50 8 8 100 1 0
a total number of cell culture units (tubes, microtitre plates)
b number of cell culture units (tube, microtitre plates) with virus presence
Table C.2 — b) Raw data for the example of Titration results –

After exposure to product (rest virus = b)
Dose Log Total 1-pi

Nb % of virus presentb pi
(TCID50) (Dose) a
3,16E-08 −7,50 8 0 0 0 1
1,00E-07 −7,00 8 0 0 0 1
3,16E-07 −6,50 8 0 0 0 1
1,00E-06 −6,00 8 1 12,5 0,125 0,875
3,16E-06 −5,50 8 2 25 0,25 0,75
1,00E-05 −5,00 8 4 50 0,5 0,5
3,16E-05 −4,50 8 8 100 1 0
a total number of cell culture units (tubes, microtitre plates)
b number of cell culture units (tube, microtitre plates) with virus presence
34
BS EN 16777:2018
EN 16777:2018 (E)
Calculating the virus titre with 95 % confidence interval

a) Before (a)
Following the Formula (C.2)

m =−6 + 0, 5 / 2 − 0, 5 × 5, 25 = − 8, 37
The standard error (S) from m is calculated following the Formula (C.3)
0 × 1 + 0, 5 × 0, 5 + 0, 75 × 0, 25 + 1 × 0 + 1 × 0 + 1 × 0 + 1 × 0
0, 52 ×
Sm = =
0, 125
8−1
b) After exposure to the product (b)
Following the Formula (C.2)

m=
−4, 5 + 0, 5 / 2 − 0, 5 × 1, 875 =− 5, 19
The standard error (S) from m is calculated following the Formula (C.3)
0 × 1 + 0 × 1 + 0 × 1 + 0, 125 × 0, 875 + 0, 25 × 0, 75 + 0, 5 × 0, 5 + 1 × 0
0, 52 ×
Sm = =
0, 1396
8−1
Calculating the reduction and its 95 % confidence interval

From the section above it follows that
a = 8,37
b = 5,19
Sa = 0,125
Sb = 0,1396
The reduction (R) is simply calculated as shown in the Formula (C.4)
R T1 = 8, 37 − 5, 19 = 3, 18
And the 95 % confidence interval of the R [KR(T1)] is calculated as showed in the Formula (C.5)
2 × 0, 1252 + 0, 1396 2 =
K R(T1) = 0, 375
If no virus can be detected any more in the test run with product (“rest virus”), the 95 % confidence
interval [KR(Tkv)] is calculated according to the Formula (C.6):
2 × 0, 1252 =
K R(Tkv) = 0, 250
35
BS EN 16777:2018
EN 16777:2018 (E)
Calculating the average reduction [R(mi)] and its 95 % confidence interval.

Supposing that for the second test run we obtained the following results (after the calculations
performed following the sections above):
R T2 = 4, 06
K R(T2) = 0, 295
The average R from both runs is calculated following the Formula (C.7)
R(mi) = ( )
3, 18 + 4, 06 / 2 =3, 62
And the 95 % confidence interval of the average R (KR(mi)) is calculated following the Formula (C.8):
0,3752 + 0, 2952
=K R ( mi ) = 0,337
2
36
BS EN 16777:2018
EN 16777:2018 (E)
Bibliography
[1] EUROPEAN PHARMACOPOEIA. EP-edition 2017
[2] LENNETTE E., SCHMIDT N., eds. Diagnostic procedures for viral and rickettsial infections. American
Public Health Association Inc, 1969
[3] VAN REGENMORTEL M.H.V. et al., eds. Virus Taxonomy, Classification and Nomenclature of Viruses,
seventh report of the international committee on taxonomy of viruses. Academic Press, San Diego,
2000
[4] SPEARMAN C. The method of ‘right and wrong cases’ (‘constant stimuli’) without Gauss’s formulae.
Br. J. Psychol. 1908, 2 pp. 227–242
[5] KÄRBER G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. Exp.
Path. Pharmak. 1931, 162 pp. 480–487
[6] MAGULSKI T., PAULMANN D., BISCHOFF B., BECKER B., STEINMANN E., STEINMANN J. et al. BMC Infect. Dis.
2009, 9 pp. 107–113. Available at: Inactivation of murine norovirus by chemical biocides on
stainless steel.
[7] WHYSHAK C., DETRE K. Estimating the number of organisms in quantal assays. Appl. Microbiol.
1997, 73 pp. 784–790
[8] EN 14820, Single-use containers for human venous blood specimen collection
[9] EGGERS M., EICKMANN M., KOWALSKI K., ZORN J., REIMER K. Povidone-iodine hand wash and hand rub
products demonstrated excellent in vitro virucidal efficacy against Ebola virus and modified
vaccinia virus Ankara, the new European test virus for enveloped viruses. BMC Infect. Dis. 2015,
15 p. 375. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574578/
[10] SIDDHARTA A., PFAENDER ST., VIELLE N.J. Edall: Virucidal Activity of World Health Organization–
Recommended Formulations Against Enveloped Viruses, Including Zika, Ebola, and Emerging
Coronaviruses. J. Infect. Dis. 2017 Mar 15, 215 (6) pp. 902–906. Available at:
https://www.ncbi.nlm.nih.gov/pubmed/28453839
[11] RABENAU H.F., RAPP I., STEINMANN J. Can vaccinia virus be replaced by MVA virus for testing
virucidal activity of chemical disinfectants? BMC Infect. Dis. 2010, 10 p. 185. Available at:
http://www.biomedcentral.com/1471-2334/10/185
[12] The European Agency for the Evaluation of Medicinal Products, Committee for proprietary
medicinal products (CPMP): Note for Guidance on virus validation studies: the design, contribution
and interpretation of studies validating the inactivation and removal of viruses.
CPMP/BWP/268/95. 14. February 1996;
[13] TAYLOR J.R. An Introduction to Error Analysis: The Study of Uncertainties in Physical Measurements.
University Science Books, Second Edition, 1997, 327 p.
37
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BS EN 16777:2018

BSI Standards Publication

Chemical disinfectants and antiseptics - Quantitative

Amendments/corrigenda issued since publication

EUROPEAN STANDARD EN 16777

Chemical disinfectants and antiseptics - Quantitative non-

This European Standard was approved by CEN on 24 September 2018.

EUROPEAN COMMITTEE FOR STANDARDIZATION

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

2 Normative references .................................................................................................................................... 6

C.3 Biometrical evaluation of experimental approaches and assessment of the

found from this test corresponds to defined experimental conditions.

indications occur in patient care, for example:

— in clinics of schools, of kindergartens, and of nursing homes;

and may occur in the workplace and in the home.

NOTE 2 This method corresponds to a phase 2, step 2 test.

EN 10088-1, Stainless steels — Part 1: List of stainless steels

3 Terms and definitions

4 Requirements for virucidal activity on surfaces

Minimum spectrum of test Virucidal activitya

Test temperature between 18 °C and 25 °C

Additional temperature between 4 °C and 30 °C

Additional conditionse Further contact time(s), interfering substance(s) or virus(es)

5.2 Materials and reagents, including cell cultures

Murine norovirus, strain S99 Berlin

b) Non-enveloped DNA virus

Adenovirus type 5, strain Adenoid 75, ATCC VR-5

c) Enveloped DNA virus

and its control and validation.

Sodium chloride (NaCl) 8,00 g

5.2.2.5 Foetal calf serum (FCS)

5.2.2.7 Hard water for dilution of products

For the preparation of 1 l of hard water, the procedure is as follows:

5.2.2.8 Interfering substance

5.2.2.8.2 Clean conditions (bovine serum albumin)

— sterilize by membrane filtration;

— keep in a refrigerator and use within one month.

a) bovine serum albumin:

— dissolve 3 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of

— sterilize by membrane filtration;

— keep in a refrigerator and use within one month.

c) bovine albumin and erythrocyte solution:

Resuspend 3 ml of packed erythrocytes with 97 ml of 3 % w/v of bovine albumin solution.

5.2.2.10 Medium for cell cultures

Other media may be used if appropriate for certain cell lines.

Appearancea clear liquid clear liquid

b) by dry heat, in the hot air oven [5.3.2.1 b)].

5.3.2 Usual microbiological laboratory equipment 3

And, in particular, the following:

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.

5.3.2.6 Refrigerator, capable of being controlled at 2 °C to 8 °C.

5.3.2.7 Electromechanical agitator e.g. Vortex ® mixer 4

NOTE Calibrated automatic pipettes may be used.

5.3.2.14 Magnetic stirrer for keeping cells in suspension before seeding

5.3.2.16 Basin as ice bath with ice and water

5.3.2.17 Mechanical shaker

3 Disposable sterile equipment is an acceptable alternative to reusable glassware.

5.3.2.19 Biological safety cabinet, class II

5.3.2.21 Desiccator with vacuum manometer

5.3.3 Test surfaces

5.4 Preparation of test organism suspensions and product test solutions

b) Contact time, t (min):