EN 14675:2015

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthodes d'essai et prescriptions - (Phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14675:2015SIST EN 14675:2015en,fr,de01-julij-2015SIST EN 14675:2015SLOVENSKI
STANDARDSIST EN 14675:20061DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14675
April 2015 ICS 71.100.35 Supersedes EN 14675:2006English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (Phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 14 February 2015.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
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Referenced strains of national collections . 23 Annex B (normative)
Cytotoxicity, reference inactivation test, test virus titration and detoxification of test mixtures . 24 B.1 Cytotoxicity caused by product solutions . 24 B.2 Reference inactivation test . 24 SIST EN 14675:2015

Calculation of the viral infectivity titre . 28 C.1 Quantal tests - Example of TCID50 determination by the Spearman-Kärber method . 28 C.2 Plaque test . 28 Annex D (informative)
Example of a typical test report . 30 Annex E (informative)
Presentation of test results of one active concentration . 32 Bibliography . 34
PFU number of infectious virus particles per unit volume (ml) 3.3 reference test for virus inactivation test with a defined reagent (e.g. formalin) instead of a product for the internal control of the test Note 1 to entry: Results of reference virus inactivation test should be within limits for validating the method. 3.4 reference virus suspension virus suspension of a defined virus strain which is not passaged more than 10 times, is maintained in national culture collection centres and kept in small volumes (less than 1 ml) at a temperature of -70°C or preferably at about -196 °C under liquid nitrogen Note 1 to entry: Stock virus suspensions are prepared from reference virus suspensions. SIST EN 14675:2015

TCID50 viral dose that induces a cytopathic effect (CPE) (3.8) in 50 % of inoculated cell culture 3.8 viral cytopathic effect
CPE morphological alteration of cells and/or their destruction as a consequence of virus multiplication 3.9 viral plaque area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication of a single infectious virus particle 3.10 virus titre amount of infectious virus per unit volume present in a cell culture lysate or in a solution 4 Requirements The product when diluted with hard water (5.2.2.3) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with Table 1 and Clause 5 shall demonstrate at least a lg reduction in virus titre of 4. It is possible to test also the product as delivered (highest test concentration is 80 %). Table 1 — Obligatory and additional test conditions
Obligatory conditions Additional conditions Test organism Bovine enterovirus Type 1 (ECBO)
Test temperature a 10 °C 4 °C, 20 °C or 40 °C Contact time b 30 min 1 min, 5 min and 60 min Interfering substance–low level soiling c 3,0 g/l bovine albumin
Interfering substance-high level soiling c 10 g/l bovine albumin plus 10 g/l yeast extract
a
Allowed deviation ± 1 °C. b
The allowed deviation for each chosen contact time is ± 10 sf except for ≤1 min for which it is ± 5 s. c
To be chosen according to practical applications. SIST EN 14675:2015

1) ATCC VR-248, is a strain supplied by the American Type 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. A corresponding strain supplied by other culture collections may be used if they can be shown to lead to the same results. SIST EN 14675:2015

2) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. SIST EN 14675:2015

in water (5.2.2.2) 1 000 ml Preparation Make up solution to 1 l with water (5.2.2.2) [omitting the sodium bicarbonate and antibiotics (5.2.2.5 and 5.2.2.6)]. Filter through a membrane filter (0,22 m pore size) using positive pressure. Add 76,2 ml 7,5 % sodium bicarbonate solution in water (5.2.2.2) and 5 ml antibiotics (5.2.2.5) to the sterile filtrate. Dispense aseptically into 50 ml aliquots and store at - 20 °C. For use add 50 ml of thawed ATV 10 × concentrate to 450 ml of water (5.2.2.2). The working strength solution contains trypsin 0,05 %, versene 0,02 %, pH = 7,8. 5.2.2.7 Dulbecco’s Phosphate Buffered Saline pH 7,2 – 7,4 (PBS) Sodium chloride (NaCl) 8,00 g Potassium dihydrogen phosphate (KH2PO4) 0,12 g Disodium hydrogen phosphate anhydrous (Na2HPO4) 0,91 g Potassium chloride (KCl) 0,2 g Water (5.2.2.2) to 1 000 ml SIST EN 14675:2015

per litre of 10x medium C 10 ml D 10 ml E 10 ml d) In a separate flask containing 160 ml of water (5.2.2.2), 2 g anhydrous calcium chloride are dissolved and then added to the pool with vigorous shaking. e) 200 ml of Solution A are added to the pool and the volume is brought to approximately 950 ml with water (5.2.2.2); the mixture can be held in the refrigerator (5.3.2.16) overnight. f) The total volume is brought to exactly 1 000 ml with water (5.2.2.2) and the solution is sterilized by membrane filtration [5.3.2.1 c)]. This 10x medium is stored at 4 °C. 5.2.2.12 Preparation of 1X Eagle MEM For use, the 10x medium is diluted to 1x with sterile water (5.2.2.2), and 1 % of the 3 % glutamine solution (5.2.2.10, f) and 2,5 % of an 8,8 % sodium bicarbonate solution in water (sterilized by membrane filtration) are added. a)
A growth medium for cell multiplication is supplemented with 10 % FCS. Add 10 parts of FCS (5.2.2.13) 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 (5.2.2.13) to 98 parts of MEM. 5.2.2.13 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. SIST EN 14675:2015

m pore size. 5.3.2.2 Water baths, capable of being controlled at 4 °C ± 1 °C and 10 °C ± 1 °C and at additional temperatures ± 1 °C (5.5.1). 5.3.2.3 CO2 incubator, (95 % air, 5 % CO2), capable of being controlled at either 36 °C ± 1 °C or 37 °C ± 1 °C. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C. 5.3.2.5 Inverted microscope, for reading cell cultures microscopically. 5.3.2.6 Stopwatch. 5.3.2.7 Shakers a) Electromechanical agitator, e.g. Vortex® mixer4) b) Mechanical shaker
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 European Standard and does not constitute an endorsement by CEN of this product. SIST EN 14675:2015

5) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. SIST EN 14675:2015
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