BRITISH STANDARD BS EN 1656:2009 Incorporating corrigendum August 2010 Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area — Test method and requirements (phase 2, step 1) ICS 11.080.20; 11.220; 71.100.35 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN 1656:2009 National foreword This British Standard is the UK implementation of EN 1656:2009, incorporating corrigendum August 2010 It supersedes BS EN 1656:2000 which is withdrawn The UK participation in its preparation was entrusted to Technical Committee CH/216, Chemical disinfectants and antiseptics A list of organizations represented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 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 March 2010 © BSI 2010 ISBN 978 580 73008 Amendments / corrigenda issued since publication Date Comments 30 November 2010 Implementation of CEN corrigendum August 2010 Correction to Annex A, b) Tent disinfectants: ATCC reference for Escherichia coli corrected EN 1656 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM November 2009 ICS 71.100.35 Supersedes EN 1656:2000 Incorporating corrigendum August 2010 English Version Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal 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 bactéricide 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 bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 20 September 2009 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 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 Management Centre has the same status as the official versions CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Ref No EN 1656:2009: E BS EN 1656:2009 EN 1656:2009 (E) Contents Page Foreword 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Terms and definitions 5 4 Requirements 5 5 Test method 6 Annex A (informative) Referenced strains in national collections 25 Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids 27 Annex C (informative) Graphical representations of dilution-neutralization method and membrane filtration method 28 Annex D (informative) Example of a typical test report 32 Bibliography 37 BS EN 1656:2009 EN 1656:2009 (E) Foreword This document (EN 1656:2009) 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 May 2010, and conflicting national standards shall be withdrawn at the latest by May 2010 Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights This document supersedes EN 1656:2000 This document was revised to include the results of a collaborative trial (ANDISTAND), to correct obvious errors and ambiguities, to harmonize the structure and wording with other quantitative suspension tests of CEN/TC 216 (existing or in preparation), and to improve the readability of the standard and thereby make it more understandable According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom BS EN 1656:2009 EN 1656:2009 (E) Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have a bactericidal activity in the fields described in the scope This laboratory test takes into account practical conditions of application of the product, including contact time, temperature, test organisms and interfering substance, i.e conditions which may influence its action in practical situations 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 some applications the recommendations of use of a product may differ and therefore additional test conditions need to be used BS EN 1656:2009 EN 1656:2009 (E) Scope This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or – in the case of ready-to-use products – with water Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance This European Standard applies to products that are used in the veterinary area – e.g in the breeding, husbandry, transport and disposal of all animals except when in the food chain following death and entry to the processing industry EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations” NOTE The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used NOTE This method corresponds to a phase step test Normative references The following referenced documents are indispensable for the application of this document For dated references, only the edition cited applies For undated references, the latest edition of the referenced document (including any amendments) applies EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity EN 14885:2006, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885:2006 apply Requirements The product shall demonstrate at least a five-decimal log (lg) reduction when diluted with hard water (5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with Clause under simulated low-level soiling (3,0 g/l bovine albumin solution – 5.2.2.8.2) or simulated high-level soiling (10 g/l bovine albumin solution and 10 g/l yeast extract – 5.2.2.8.3) (or 10 g/l skimmed milk for teat disinfectants – 5.2.2.8.4) according to its practical applications and under the other obligatory test conditions four or three [for teat disinfectants] selected test organisms, 10 °C [30 ˚C for teat disinfectants], 30 [5 for teat disinfectants]) The bactericidal activity shall be evaluated using the following organisms: a) Products for general disinfection: b) Teat disinfectants: Enterococcus hirae; Escherichia coli; Proteus vulgaris; Staphylococcus aureus; Pseudomonas aeruginosa; Streptococcus uberis BS EN 1656:2009 EN 1656:2009 (E) Staphylococcus aureus Where indicated, additional specific bactericidal activity shall be determined applying other contact times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into account intended specific use conditions NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions Test method 5.1 Principle 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products) is added to a test suspension of bacteria in a solution of an interfering substance The mixture is maintained at (10 ± 1) °C (or (30 ± 1) °C for teat disinfectants) for 30 ± 10 s (5 ± 10 s for teat disinfectants) (obligatory test conditions) At the end of this contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method The method of choice is dilution-neutralization If a suitable neutralizer cannot be found, membrane filtration is used The numbers of surviving bacteria in each sample are determined and the reduction is calculated 5.1.2 For general disinfectant products, the test is performed using Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus as test organisms For teat disinfectants the test is performed using Escherichia coli, Staphylococcus aureus and Streptococcus uberis as test organisms 5.1.3 used 5.2 Additional and optional contact times and temperatures are specified Additional test organisms can be Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms1): a) b) General disinfection products: Enterococcus hirae ATCC 10541 Proteus vulgaris ATCC 13315 Pseudomonas aeruginosa Staphylococcus aureus ATCC 15442 ATCC 6538 Teat disinfectants: Escherichia coli ATCC 10536 Staphylococcus aureus ATCC 6538 Streptococcus uberis ATCC 19436 1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC) This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named BS EN 1656:2009 EN 1656:2009 (E) NOTE See Annex A for strain references in some other culture collections The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3) The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report If the additional test organisms selected not correspond to the specified strains, their suitability for supplying the required inocula shall be verified If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this 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 NOTE To improve reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media The manufacturer's instructions relating to the preparation of these products should be rigorously followed NOTE For each culture medium and reagent, a shelf life should be fixed (see ISO/IEC 17025) 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralized water Sterilize in the autoclave (5.3.2.1 a) NOTE sterilized NOTE NOTE Sterilization is not necessary if the water is used e.g for preparation of culture media and subsequently If distilled water of adequate quality is not available, water for injections (see [1] in the bibliography) can be used See 5.2.2.7 for the procedure to prepare hard water 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein 15,0 g Soya peptone, papaic digest of soybean meal 5,0 g Sodium chloride (NaCl) 5,0 g Agar Water (5.2.2.2) 15,0 g to 000,0 ml Sterilize in the autoclave (5.3.2.1 a) After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to the TSA Annex B gives guidance on the neutralizers that may be used BS EN 1656:2009 EN 1656:2009 (E) 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of: Tryptone, pancreatic digest of casein 1,0 g Sodium chloride (NaCl) 8,5 g Water (5.2.2.2) to 000,0 ml Sterilize in the autoclave (5.3.2.1 a) After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2 It shall be sterile NOTE Annex B Information on neutralizers that have been found to be suitable for some categories of products is given in 5.2.2.6 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3 It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3 NOTE Annex B Information on rinsing liquids that have been found to be suitable for some categories of products is given in 5.2.2.7 Hard water for dilution of products For the preparation of 000 ml 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 000 ml Sterilize by membrane filtration (5.3.2.7) or in the autoclave (5.3.2.1 a) Autoclaving – if used – may cause a loss of liquid In this case make up to 000 ml with water (5.2.2.2) under aseptic conditions Store the solution in the refrigerator (5.3.2.8) 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 000 ml Sterilize by membrane filtration (5.3.2.7) Store the solution in the refrigerator (5.3.2.8) for no longer than one week; Place 600 ml to 700 ml of water (5.2.2.2) in a 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9) of solution A, then 8,0 ml of solution B Mix and dilute to 000 ml with water (5.2.2.2) The pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.4) If necessary, adjust the pH by using a solution of approximately 40 g/l (about mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 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 a different final water hardness in each test tube In any case, the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube 5.2.2.8 5.2.2.8.1 Interfering substance General The interfering substance shall be chosen according to the conditions of use laid down for the product BS EN 1656:2009 EN 1656:2009 (E) 2) evaluation of bactericidal activity; 3) number of replicates per test organism; g) special remarks; h) conclusion; i) locality, date and identified signature NOTE 24 An example of a typical test report is given in Annex D BS EN 1656:2009 EN 1656:2009 (E) Annex A (informative) Referenced strains in national collections a) General disinfectant products Enterococcus hirae: Proteus vulgaris7): Pseudomonas aeruginosa: ATCC 10541 CIP 58.55 DSM 3320 NCIMB 8192 ATCC 13315 CIP 58.60 DSM 30118 NCTC 4175 NCIMB 4175 ATCC 15442 CIP DSM 7) Staphylococcus aureus: 103467 939 NCIMB 10421 ATCC 6538 CIP 4.83 DSM 799 NCTC 10788 NCIMB 9518 Also known as Proteus hauserii 25 BS EN 1656:2009 EN 1656:2009 (E) b) Teat disinfectants Escherichia coli: ATCC 10536 CIP 54.127 DSM Staphylococcus aureus: Streptococcus uberis: 682 NCTC 10418 NCIMB 8879 ATCC 6538 CIP 4.83 DSM 799 NCTC 10788 NCIMB 9518 ATCC 19436 CIP 103219 DSM 20569 NCTC 3858 NCIMB 2038 Abbreviations: ATCC CIP DSM NCIMB NCTC 26 American Type Culture Collection Collection de l’Institut Pasteur Deutsche Sammlung von Mikroorganismen und Zellkulturen National Collection of Industrial and Marine Bacteria National Collection of Type Cultures BS EN 1656:2009 EN 1656:2009 (E) Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids Important Neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing fluids shall be validated according to the prescriptions of the standard Antimicrobial agent Quaternary compounds amines ammonium and fatty Amphoteric compounds Biguanides compounds and similar Chemical compounds able to neutralize residual antimicrobial activity Lecithin c), Saponin, Polysorbate 80, Sodium dodecyl sulphate, Ethylene oxide condensate of fatty alcohol (nonionic surfactants) b) Lecithin c), Saponin, Polysorbate 80 Examples of suitable neutralizers and of rinsing liquids (for membrane filtration methods) a) Ethylene oxide condensate of fatty alcohol, g/l + lecithin c), 20g/l + polysorbate 80, 5g/l Rinsing liquid : tryptone, 1g/l + NaCl, g/l; polysorbate 80, g/l Polysorbate 80, 30 g/l + saponin, 30 g/l + lecithin c), Oxidizing compounds Sodium thiosulphate d) (Chlorine, iodine, hydrogen peroxide, peracetic acid, hypochlorites, etc.) Catalase [for hydrogen peroxide or products releasing hydrogen peroxide] Aldehydes L-histidine Glycine Phenolic and related compounds: orthophenylphenol, phenoxyethanol, triclosan, phenylethanol, etc Lecithin c) Polysorbate 80 3g/l Rinsing liquid: tryptone, 1g/l + NaCl, g/l; polysorbate 80, g/l Sodium thiosulphae, g/l to 20 g/l + polysorbate 80, 30 g/l + lecithin c), g/l Polysorbate 80, 50 g/l + catalase 0,25g/l + lecithin c) 10 g/l Rinsing liquid : sodium thiosulphate, g/l Polysorbate 80, 30 g/l + lecithin c), 3g/l + L-histidine, 1g/l (or + glycine, g/l) Polysorbate 80, 30 g/l + saponin, 30 g/l + L-histidine, g/l (or + glycine, g/l) Rinsing liquid: polysorbate 80, g/l + L-histidine, 0,5 g/l (or + glycine, g/l) Polysorbate 80, 30 g/l + lecithin c), 3g/l Ethylene oxide condensate of fatty alcohol, g/l + lecithin c), 20 g/l + polysorbate 80, g/l Ethylene oxide condensate of fatty alcohol b) Rinsing liquid: tryptone, g/l + NaCl, g/l; polysorbate 80, g/l Lecithin c), Saponin, Polysorbate 80 e) Polysorbate 80, 30 g/l + saponin, 30 g/l +lecithin c), g/l Rinsing liquid: tryptone, g/l + NaCl, g/l; polysorbate 80, g/l Anilides Alcohols Polysorbate 80, 30 g/l + saponin, 30 g/l + lecithin c), 3g/l Polysorbate 80, 30 g/l + sodium dodecyl sulphate, g/l + lecithin c), 3g/l a) According to the pH of the tested product, the pH of the neutralizer or the rinsing liquid may be adjusted at a suitable value or prepared in phosphate buffer (ex.: phosphate buffer 0,25 mol/l: potassium dihydrogen phosphate (KH2PO4) 34 g; distilled water (500 ml); adjusted to pH 7,2 ± 0,2 with sodium hydroxide (NaOH) mol/l; distilled water up to 000 ml) b) The carbon chain-length varies from C12 to C18 carbon atoms c) Egg and soya; egg is preferable d) The toxic effect of sodium thiosulphate differs from one microorganism to another e) For the neutralization of short chain alcohols (less than C5), simple dilution may be appropriate Care should be taken if the alcoholbased products contain additional antimicrobial agents NOTE Other neutralizer mixtures may be required for products containing more than one antimicrobial agent NOTE The concentration of the various neutralizing compounds or of the neutralizer as such may not be adequate to neutralize high concentrations of the products 27 BS EN 1656:2009 EN 1656:2009 (E) Annex C (informative) Graphical representations of dilution-neutralization method and membrane filtration method C.1 Dilution-neutralization method Key Figure C.1 – Dilution-neutralization method – Test (Na) 28 BS EN 1656:2009 EN 1656:2009 (E) Key Figure C.2 – Dilution-neutralization method - Validation 29 BS EN 1656:2009 EN 1656:2009 (E) C.2 Membrane filtration method Key Figure C.3 – Membrane filtration method – Test (Na) 30 BS EN 1656:2009 EN 1656:2009 (E) Key Figure C.4 – Membrane filtration method - Validation 31 BS EN 1656:2009 EN 1656:2009 (E) Annex D (informative) Example of a typical test report NOTE All names and examples in Annex D are fictitious apart from those used in the standard NOTE Test reports for bactericidal activity should be entitled “EN 1656, BACTERICIDAL ACTIVITY”, and be presented in the same format NOTE Only the test results of one replicate for Pseudomonas aeruginosa are given as an example - HHQ Laboratories Antiseptville/Euroland Tel ++011.57 83 62-0 Fax ++011.57 83 62-19 e-mail: h.h.Q.lab@net.com TEST REPORT EN 1656, BACTERICIDAL ACTIVITY (obligatory and additional conditions) Client: Mult Formulations Inc., Mannheim / Euroland Disinfectant-sample Name of the product : W Batch number: 26-01-48 Manufacturer or – if not known – supplier: Centipede Formulations Inc (manufacturer) Storage conditions (temp and other): Room temperature, darkness Appearance of the product: Liquid, clear, yellowish Active substance(s) and their concentration(s): Not indicated Product diluent recommended by the manufacturer for use: Potable water Period of testing Date of delivery of the product: 2008-04-01 32 Dates of tests: see “Test results” (attached) BS EN 1656:2009 EN 1656:2009 (E) Experimental conditions Product diluent: hard water Concentrations of the product tested: see “Test results” (attached) Obligatory conditions: test-organisms: Enterococcus hirae ATCC 10541, Proteus vulgaris ATCC 13315, Pseudomononas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538 test temperature: 10 °C contact time: 30 interfering substance: 3,0 g/l bovine albumin = low-level soiling Incubation temperature: 36 °C Additional conditions: test organism: Pseudomononas aeruginosa ATCC 15442 Test temperature: 40 °C contact time: 60 Interfering substance: 10,0 g/l bovine albumin + 10,0 g/l yeast extract = high level soiling Incubation temperature: 36 °C Test results: see attached sheets test organism: Staphylococcus aureus ATCC 6538 Test temperature: °C; contact time: 15 Interfering substance: 10,0 g/l skimmed milk Incubation temperature: 36 °C Test results: see attached sheets Special remarks regarding the results: All controls and validation were within the basic limits At least one concentration of the product demonstrated an lg reduction of less than lg No precipitate during the test procedure (test mixtures were homogeneous) 33 BS EN 1656:2009 EN 1656:2009 (E) Conclusion: For the product W (batch 26-01-48), the bactericidal concentration for general purposes determined according to the EN 1656 standard (obligatory conditions) under low-soiling conditions is: 1,0 % (v/v) (The mean reduction of six replicates with the limiting test organism Pseudomononas aeruginosa was 1,2 x 10 Staphylococcus aureus, Proteus vulgaris and Enterococcus hirae were tested once and showed a lg reduction or more at a lower concentration than Pseudomononas aeruginosa.) For the product W (batch 26-01-48), the bactericidal concentration for specific purposes determined according to the EN 1656 standard at 40 °C, with 60 contact time under high-level soiling conditions, using Pseudomononas aeruginosa ATCC 15442 as test organism is: 0,25 % (v/v) For the product W (batch 26-01-48) the bactericidal concentration for specific purposes determined according to the EN 1656 standard at °C, with 15 contact time with skimmed milk as interfering substance, using Staphylococcus aureus ATCC 6538 as test organism is: 2,5 % (v/v) Antiseptville, 2008-06-01 Alexandra May, MD, PhD, Scientific Director 34 BS EN 1656:2009 EN 1656:2009 (E) Test results (bactericidal suspension test) EN 1656… .(Phase 2, step1) Product-name: W Batch No 26-01-48 Remarks: X Dilution neutralization method Pour plate Spread plate x Number of plates .2 / ml Neutralizer: Lecithin 3,0 g/l in diluent Membrane filtration method Test temperature: 10 °C Rinsing liquid: interfering substance:….bovine albumin: 3,0 g/l ……………………………… Test organism: Pseudomonas aeruginosa ATCC 15442 Incubation temperature: 36 °C Internal lab No : QS 68/00 Date of test:.2008-05-06 Responsible person: Fang Signature: Fang Diluent used for product test solutions: hard.water Appearance of the product test solutions: clear Validation and controls Validation Experimental suspension (Nv0) Conditions control (A) Vc1 x 86 = Vc1 (40 + 46) Vc2 92 x yes 89 of Nv0 ≤ 160 ? no x = Vc1 (43 + 36) Vc2 (47 + 45) 30 ≤ 79 Neutralizer control (B) 84 81,5 of A is ≥ 0,5 x x of Nv0 ? yes no filtration Method validation (C) Product conc.: 10 ml/l 86 x Vc1 = (42 + 44) Vc2 (39 + 45) x or 91 Vc2 88,5 of B is ≥ 0,5 x x of Nv0 yes no x = (35 + 40) (43 + 48) x 75 87 81 (41 + 46) x of C is ≥ 0,5 x x of Nv0 ? yes no 35 BS EN 1656:2009 EN 1656:2009 (E) Test suspension and Test Test-suspension (N and N0): Conc of product % N Vc1 Vc2 x wm = 193,64 10-6 168 213 N0 = N/10 (≡ lg 7,29) 10-7 20 25 7,17 ≤ N0 ≤ 7,70? Vc1 the Vc2 Na = x x 10 lgNa x 106 (≡ lg 8,29) yes no lgR Contact- (No=7,29) time (min) 0,50 > 660 > 630 > 450 > 3,81 < 3,48 30 0,75 122 154 380 3,14 4,15 30 1,00 < 140 < 2,15 > 5,14 30 Remarks: Counting per plate for N –6 10 : 80 + 88; 105 + 108 –7 10 : + 11; 15 + 10 Explanations: Vc = count per ml (one plate or more) x = average of Vc1 and Vc2 (1 + duplicate) x wm = weighted mean of x R = reduction (lg R = lgN0 – lgNa) 36 Na 0,75 % : 66 + 56; 71 + 83 1,00 % Vc1: + BS EN 1656:2009 EN 1656:2009 (E) Bibliography [1] European Pharmacopoeia (EP), Edition 1997 supplement 2000, Water for injections 37 BS EN 1656:2009 BSI - British Standards Institution BSI is the independent national body responsible for preparing British Standards It presents the UK view on standards in Europe and at the international level It is incorporated by Royal Charter Revisions British Standards are updated by amendment or revision Users of British Standards should make sure that they possess the latest amendments or editions It is the constant aim of BSI to improve the quality of our products and services We would be grateful if anyone finding an inaccuracy or ambiguity while using this British Standard would inform the Secretary of the technical committee responsible, the identity of which can be found on the inside front cover Tel: +44 (0)20 8996 9000 Fax: +44 (0)20 8996 7400 BSI offers members an individual updating service called PLUS which ensures that subscribers automatically receive the latest editions of standards Buying standards Orders for all BSI, international and foreign standards publications should be addressed to Customer Services Tel: +44 (0)20 8996 9001 Fax: +44 (0)20 8996 7001 Email: orders@bsigroup.com You may also buy directly using a debit/credit card from the BSI Shop on the Website http://www.bsigroup.com/shop In response to orders for international standards, it is BSI policy to supply the BSI implementation of those that have been published as British Standards, unless otherwise requested Information on standards BSI provides a wide range of information on national, European and international standards through its Library and its Technical Help to Exporters Service Various BSI electronic information services are also available which give details on all its products and services Contact Information Centre Tel: +44 (0)20 8996 7111 Fax: +44 (0)20 8996 7048 Email: info@bsigroup.com Subscribing members of BSI are kept up to date with standards developments and receive substantial discounts on the purchase price of standards For details of these and other benefits contact Membership Administration Tel: +44 (0)20 8996 7002 Fax: +44 (0)20 8996 7001 Email: membership@bsigroup.com Information regarding online access to British Standards via British Standards Online can be found at http://www.bsigroup.com/BSOL Further information about BSI is available on the BSI website at http:// www.bsigroup.com Copyright BSI Group Headquarters 389 Chiswick High Road, London, W4 4AL, UK Tel +44 (0)20 8996 9001 Fax +44 (0)20 8996 7001 www.bsigroup.com/ standards Copyright subsists in all BSI publications BSI also holds the copyright, in the UK, of the publications of the international standardization bodies Except as permitted under the Copyright, Designs and Patents Act 1988 no extract may be reproduced, stored in a retrieval system or transmitted in any form or by any means – electronic, photocopying, recording or otherwise – without prior written permission from BSI This does not preclude the free use, in the course of implementing the standard, of necessary details such as symbols, and size, type or grade designations If these details are to be used for any other purpose than implementation then the prior written permission of BSI must be obtained Details and advice can be obtained from the Copyright and Licensing Manager Tel: +44 (0)20 8996 7070 Email: copyright@bsigroup.com