BS EN 14204:2012 BSI Standards Publication Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants and antiseptics used in the veterinary area — Test method and requirements (phase 2, step 1) BS EN 14204:2012 BRITISH STANDARD National foreword This British Standard is the UK implementation of EN 14204:2012 It supersedes BS EN 14204:2004 which is withdrawn The UK participation in its preparation was entrusted to Technical Committee CH/216, Chemical disinfectants and antiseptics A list of organizations represented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 © The British Standards Institution 2013 Published by BSI Standards Limited 2013 ISBN 978 580 74073 ICS 71.100.35 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 28 February 2013 Amendments issued since publication Date Text affected BS EN 14204:2012 EN 14204 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM November 2012 ICS 71.100.35 Supersedes EN 14204:2004 English Version Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal 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 mycobactéricide 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 mykobakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 22 September 2012 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Ref No EN 14204:2012: E BS EN 14204:2012 EN 14204:2012 (E) Contents Page Foreword 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Terms and definitions 5 4 Requirements 5 5 5.1 5.2 5.2.1 5.2.2 5.3 5.3.1 5.3.2 5.4 5.4.1 5.4.2 5.5 5.5.1 5.5.2 5.5.3 5.6 5.6.1 5.6.2 5.7 5.7.1 5.7.2 5.7.3 5.8 5.8.1 5.8.2 5.8.3 5.9 5.9.1 5.9.2 5.9.3 5.10 Test method 6 Principle 6 Materials and reagents 7 Test organisms 7 Culture media and reagents 7 Apparatus and glassware 10 General 10 Usual microbiological laboratory equipment and, in particular, the following: 10 Preparation of mycobacterial test suspension and product test solutions 11 Mycobacterial test suspensions (test and validation suspension) 11 Product test solutions 13 Procedure for assessing the mycobactericidal activity of the product 14 General 14 Test procedure - Dilution-neutralization method 15 Test procedure - Membrane filtration method 17 Experimental data and calculation 19 Explanation of terms and abbreviations 19 Calculation 19 Verification of methodology 23 General 23 Control of weighted mean counts 23 Basic limits 23 Expression of results 24 Reduction 24 Control of active and non-active product test solution (5.4.2) 24 Mycobactericidal concentration 24 Interpretation of results - conclusion 24 General 24 Mycobactericidal activity for general purposes 24 Qualification for certain fields of application 24 Test report 25 Annex A (informative) Referenced strain in national collections 27 Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids 28 Annex C (informative) Graphical representations of dilution-neutralization method 30 Annex D (informative) Example of a typical test report 32 Bibliography 36 BS EN 14204:2012 EN 14204:2012 (E) Foreword This document (EN 14204:2012) 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 2013, and conflicting national standards shall be withdrawn at the latest by May 2013 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 14204:2004 Data obtained using the former version of EN 14204 may still be used This document was revised 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 The following technical changes have been made: Membrane filtration (5.5.3) method and associated media and equipment have been added 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom BS EN 14204:2012 EN 14204:2012 (E) Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has mycobactericidal activity in the area and 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 substances, 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 the chosen experimental conditions However, for some applications the instructions of use of a product may differ and therefore additional test conditions need to be used BS EN 14204:2012 EN 14204:2012 (E) Scope This European Standard specifies a test method and the minimum requirements for mycobactericidal 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 – i.e in the breeding, husbandry, production, 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 2 This method corresponds to a phase step test Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application 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, 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 apply Requirements The product shall demonstrate at least a decimal log (lg) reduction when tested in accordance with Table and Clause under simulated low level (3,0 g/l bovine albumin) or high level soiling (10 g/l yeast extract and 10 g/l bovine albumin) BS EN 14204:2012 EN 14204:2012 (E) Table - Obligatory and additional test conditions Test conditions Mycobactericidal activity Test organism a) obligatory Mycobacterium avium b) additional any relevant test organism Test temperature a) obligatory 10°C ± 1°C b) additional 4°C ± 1°C; 20°C ± 1°C; 40°C ± 1°C Contact time a) obligatory 60 ± 10 s b) additional a) Interfering substance obligatory low level soiling a ± s; ± 10 s; 10 ± 10 s, 15 ± 10 s, 30 ± 10 s, 120 ± 10 s 3,0 g/l bovine albumin high level soiling 10 g/l yeast extract plus 10 g/l bovine albumin b) additional any relevant substance NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions a The obligatory contact time for disinfectants stated in Table was chosen to enable comparison of standard conditions The referenced test conditions are by no means intended as requirements for the use of a product The recommended contact time for the use of the product is within the responsibility of the manufacturer Any additional specific mycobactericidal activity shall be determined in accordance with 5.2.1 and 5.5.1.1 in order to take into account intended specific use conditions 5.1 Test method Principle A sample of the product as delivered and/or diluted in hard water (or water for ready to use products) is added to a test suspension of mycobacteria in a solution of an interfering substance The mixture is maintained at (10 ± 1) °C for 60 ± 10 s At the end of this contact time, an aliquot is taken and the mycobactericidal action in this portion is immediately neutralized or suppressed by a validated method The number of surviving mycobacteria in each sample is determined and the reduction in viable counts calculated BS EN 14204:2012 EN 14204:2012 (E) The test is performed using Mycobacterium avium as the test organism (Clause 4, Table 1) Additional and optional contact times and temperatures are specified (Clause 4, Table 1) Additional interfering substances and test organisms may be used 5.2 Materials and reagents 5.2.1 5.2.1.1 Test organisms The mycobactericidal activity shall be evaluated using the following strain : Mycobacterium avium ATCC 157691) NOTE See Annex A for corresponding strain numbers in some other culture collections The required incubation temperature for this test organism is (36 ± 1) °C or (37 ± 1) °C 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 5.2.2.1 Culture media and reagents 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 If additional strains not grow on the media (5.2.2.3) or cannot be used with diluent (5.2.2.4) additional media shall be used and shall be reported as well as additional incubation conditions To improve the 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 Ready-to-use media may be used if it complies with the required specification For each culture medium and reagent, a limitation for use should be fixed 5.2.2.2 Water The water shall be freshly glass distilled and not demineralised water If distilled water of adequate quality is not available, water for injectable preparations (see bibliographic reference [1] Pharmacopoeia) can be used 1) ATCC 15769 is the collection number of 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 BS EN 14204:2012 EN 14204:2012 (E) 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 NOTE See 5.2.2.6 for the procedure to prepare hard water 5.2.2.3 Middlebrook and Cohn 7H10 medium + 10 % oleic acid albumin dextrose complex (OADC) (reported as 7H10 in the text) For performance of viable counts: Middlebrook 7H10 agar 19 g ; Glycerol ml ; Water (see 5.2.2.2) to 895 ml Heat to boiling to dissolve completely Sterilize for 10 in the autoclave and cool to 50 °C to 55 °C Add 100 ml Middlebrook OADC enrichment under aseptic conditions Final pH = 6,6 ± 0,2 at 25 °C In case of encountering problems with neutralization (5.5.1.2), it may be necessary to add neutralizer to the 7H10 Annex B gives guidance on the neutralizers that may be used 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 (see 5.2.2.2) to 000 ml Sterilize in the autoclave [see 5.3.2.1a)] After sterilization the pH of the medium 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 and 5.5.2 The neutralizer shall be sterile NOTE Annex B Information on neutralizers that have been found to be suitable for some categories of products is given in Sterilize in the autoclave 5.2.2.6 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 [5.3.2.1a)] Store the solution at °C to 8°C for no longer than one month NOTE In the case of loss of volume during sterilization by autoclave, make up to 000 ml with water (5.2.2.2) under aseptic conditions before storage BS EN 14204:2012 EN 14204:2012 (E) d) B is equal to or greater than 0,5 x NV0 e) C is equal to or greater than 0,5 x NV0 f) control of weighted mean counts (5.7.2): quotient is not lower than and not higher than 15 5.8 5.8.1 Expression of results Reduction The reduction (R = N0/Na) is expressed in logarithm Record the number of cfu/ml in the test suspension N (5.6.2.3) and the test Na (5.6.2.4) Calculate N0 (5.6.2.3) For each product concentration and each experimental condition, calculate and record the decimal log reduction (lg) separately using the formula: lg R = lg N0 − lg Na 5.8.2 Control of active and non-active product test solution (5.4.2) At least one concentration per test (5.5.2) shall demonstrate a lg or more reduction and at least one concentration shall demonstrate a lg reduction of less than 5.8.3 Mycobactericidal concentration Record the lowest concentration of the product which passes the test (lg R ≥ 4) 5.9 5.9.1 Interpretation of results - conclusion General According to the chosen experimental conditions (obligatory or obligatory and additional) the mycobactericidal concentrations determined according to this standard may differ (Clause 4) A product can only pass the test if the requirements of 5.8.2 are fulfilled 5.9.2 Mycobactericidal activity for general purposes The product shall be deemed to have passed the EN 14204 standard if it demonstrates in a valid test at least a lg reduction within 60 or less at 10 °C with the chosen interfering substance simulating low- or highlevel soiling conditions defined by this standard when the test organism is Mycobacterium avium 5.9.3 Qualification for certain fields of application The mycobactericidal concentration for specific purpose is the concentration of the tested product for which at least a lg reduction is demonstrated in a valid test under the additional chosen test conditions The product shall have passed the EN standard under the obligatory test conditions The mycobactericidal concentration for specific purposes may be lower than the one determined for general purposes For more details see EN 14885 24 BS EN 14204:2012 EN 14204:2012 (E) 5.10 Test report ) The test report7 shall refer to this European Standard (EN 14204) Any operation not included in the standard as well as any incidents which may have affected the results shall be reported The test report shall state, at least, the following information: a) identification of the laboratory; b) identification of the client; c) identification of the sample: d) 1) name of the product; 2) batch number; 3) manufacturer; 4) date of delivery; 5) storage conditions; 6) product diluent recommended by the manufacturer for use; 7) active substance(s) and its (their) concentration(s) (optional); 8) appearance of the product; test method and its validation: Full details of the tests for validation of the neutralizer shall be given e) experimental conditions: 1) date(s) of test (period of analysis); 2) diluent used for product test solutions (hard water or distilled water); 3) product test concentrations (= desired test concentrations according to 5.4.2) 4) appearance of product dilutions; 5) contact time(s); 6) test temperature(s); 7) interfering substance; 8) stability and appearance of the mixture during the procedure (note the formation of any precipitate or flocculant); 9) temperature of incubation; 7) An example of a typical test report is given in Annex D 25 BS EN 14204:2012 EN 14204:2012 (E) 10) neutralizer; 11) identification of the mycobacterial strain; f) test results: 1) controls and validation; 2) evaluation of mycobactericidal activity; 3) number of replicates per test organism; g) special remarks; h) conclusion; i) locality, date and identified signature NOTE 26 An example of a typical test report is given in Annex D BS EN 14204:2012 EN 14204:2012 (E) Annex A (informative) Referenced strain in national collections Mycobacterium avium - ATCC 15769 CIP 105415 Abbreviations: ATCC American Type Culture Collection CIP Collection de l'Institut Pasteur 27 BS EN 14204:2012 EN 14204:2012 (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 shall be validated according to the prescriptions of the standard Table B.1 - Neutralizers and rinsing liquids (1 of 2) Antimicrobial agent Chemical neutralize activity Quaternary ammonium compounds and fatty amines Lecithin , Saponin, Polysorbate 80, Sodium dodecyl sulphate, Ethylene oxide condensate of fatty alcohol b) (non-ionic surfactants) Amphoteric compounds compounds able to residual antimicrobial c) Examples of suitable neutralizers and of rinsing a) liquids (for membrane filtration methods) - 30 g/l Polysorbate 80 + 30 g/l saponin + 3g/l lecithin c) - 30 g/l Polysorbate 80 + g/l sodium dodecyl sulphate c) + 3g/l.lecithin - g/l Ethylene oxide condensate of fatty alcohol + c) 20g/l lecithin + 5g/l polysorbate 80 - Rinsing liquid: 1g/l tryptone + g/l NaCl + g/l polysorbate 80 c) Biguanides and similar compounds Lecithin , Saponin, Polysorbate 80 Oxidizing compounds Sodium thiosulphate (Chlorine, iodine, hydrogen peroxide, peracetic acid, hypochlorites, etc…) Catalase [for hydrogen peroxide or products releasing hydrogen peroxide] Aldehydes L-histidine c) - 30 g/l Polysorbate 80 + 30 g/l saponin + 3g/l lecithin - Rinsing liquid: 1g/l tryptone + g/l NaCl + g/l polysorbate 80 d) - g/l to 20 g/l Sodium thiosulphae + 30 g/l polysorbate c) 80 + g/l lecithin - 50 g/l Polysorbate 80 + 0,25g/l catalase + 10 g/l c) lecithin - Rinsing liquid: g/l sodium thiosulphate Glycine c) - 30 g/l Polysorbate 80 + 3g/l lecithin + 1g/l L-histidine (or + g/l glycine) - 30 g/l Polysorbate 80 + 30 g/l saponin + g/l Lhistidine (or + g/l glycine) - Rinsing liquid: g/l polysorbate 80 + 0,5 g/l L-histidine (or + g/l glycine) 28 BS EN 14204:2012 EN 14204:2012 (E) Table B.1 - Neutralizers and rinsing liquids (2 of 2) Antimicrobial agent Chemical neutralize activity Phenolic and related compounds: orthophenylphenol, phenoxyethanol, triclosan, phenylethanol, etc Lecithin compounds able to residual antimicrobial c) Examples of suitable neutralizers a) c) - 30 g/l Polysorbate 80 + 3g/l lecithin Polysorbate 80 Ethylene oxide condensate of fatty b) alcohol - g/l Ethylene oxide condensate of fatty alcohol + 20 c) g/l lecithin + g/l polysorbate 80 - Rinsing liquid: g/l tryptone + g/l NaCl + g/l polysorbate 80 Anilides Alcohols c) Lecithin , Saponin, Polysorbate 80 e) c) - 30 g/l Polysorbate 80 + 30 g/l saponin + g/l lecithin - Rinsing liquid: g/l tryptone + g/l NaCl + g/l polysorbate 80 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 1000 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 29 BS EN 14204:2012 EN 14204:2012 (E) Annex C (informative) Graphical representations of dilution-neutralization method Figure C.1 30 BS EN 14204:2012 EN 14204:2012 (E) Figure C.2 31 BS EN 14204:2012 EN 14204:2012 (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 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 14204, MYCOBACTERICIDAL ACTIVITY (obligatory and additional conditions) Client: Centipede Formulations Inc., Mannheim / Euroland Disinfectant sample Name of the product: W Batch number: Manufacturer or - if not known - supplier 11-47-013 Centipede Formulations Inc (manufacturer) Storage conditions (temp and other): Room temperature, darkness Appearance of the product: Liquid, clear, yellowish Active substance(s) and its/their concentration(s): Not indicated Product diluent recommended by the manufacturer for use: Potable water Period of testing Date of delivery of the product: 2010-10-06 Dates of tests: see "Test results" (attached) Experimental conditions Product diluent: hard water Concentrations of the product tested: see "Test results" (attached) Obligatory conditions: Test organism: Mycobacterioum avium ATCC 15769 Test temperature: 10 °C Contact time: 60 Interfering substance: 3,0 g/l bovine albumin = low-level soiling; 32 BS EN 14204:2012 EN 14204:2012 (E) Incubation temperature: 36 °C Neutralizer: 30 g/l lecithin, sterilized in the autoclave Additional conditions Test temperature: 40 °C Contact time: 60 Interfering substance: 10,0 g/l bovine albumin + 10,0 g/l yeast extract = high level soiling Temperature of incubation 36 °C Bacterial strain used: Mycobacterioum avium ATCC 15769 Additional conditions Test temperature: °C Contact time: 60 Interfering substance: 10,0 g/l bovine albumin + 10,0 g/l yeast extract = high level soiling Temperature of incubation: 36 °C Bacterial strain used: Mycobacterioum avium ATCC 15769 Special remarks regarding the results: All controls and validation were within the basic limits At least one concentration of the product demonstrated a lg reduction of less than lg No precipitate during the test procedure (test mixtures were homogeneous) Conclusion: According to EN 14204 (date of edition) the batch 11-47-013 of the product W possesses mycobactericidal activity for the reference strain of Mycobacterium avium at a concentration of 1,00 % (v/v) at 10 °C, with a 60 contact time under high-level soiling conditions According to EN 14204 (date of edition) the batch 11-47-013 of the product W possesses mycobactericidal activity at a concentration of 0,25 % (v/v) at 40 °C, with a 60 contact time under high-level soiling conditions According to EN 14204 (date of edition) the batch 11-47-013 of the product W possesses mycobactericidal activity at a concentration of 2,5 % (v/v) at °C, with a 60 contact time under high-level soiling conditions Antiseptville, 2010-11-07 Alexandra May, MD, PhD, Scientific Director 33 BS EN 14204:2012 EN 14204:2012 (E) Test results (mycobactericidal quantitative suspension test) EN 14204 (Phase step.1) Product-name: Manufacturer: W Batch No: Centipede Formulation Inc Storage conditions (temp and other): Appearance of the product: 10-47-013 liquid, clear, yellowish room temperature, darkness Dilution neutralization method Diluent used for product test solutions: hard water Appearance of the product dilutions: clear, transparent Pour plate Spread plate Test temperature: 10 °C Number of plates: / ml Interfering substances: Neutralizer: 30 g/l lecithin 10,0 g/l bovine albumin + 10,0 g/l yeast extract Test organism: Mycobacterium avium ATCC 15769 Incubation temp: 36 °C Internal lab no: Date of test: 2010-11-06 Responsible person: Fang Signature: Fang Validation and controls Validation (NV0) VC suspension Experimental control (A) VC 86 (40 + 46), Conditions Neutralizer control (B) Method validation (C) Product conc.: 10 ml/l VC1 79 (43 + 36), 86 (42 + 44), VC1 75 (35 + 40), 78 (35 +43) 84 (44 + 40) 94 (48 + 46) 80 (35 + 45) = 82 = 81,5 = 90 = 77,5 14 ≤ VC of NV0 ≤ 330 ? yes no VC of A is ≥ 0,5 x x of VC of B is ≥ 0,5 x x of NV0? VC of C is ≥ 0,5 x x of NV0? NV0? yes no yes no yes no Test suspension and Test (N and N0): -6 (150 + 150) = (150 300 =298 -7 (15, 15) =30 10 10 Conc of product % 0,50 the Dilution N a−1 -1 VC2 VC1 Test-suspension VC1 N = 301,36 x 10 (lg N = 8,48) + 148) N0 = N/10 (lg N0 = 7,48) (20, 15) =35 VC2 7,48 ≤ N0 ≤ 7,90? Na = x x10 lg Na yes no lgR Contact (N0=7,48) time (min) (>330 +, >330) = >330 (>330 +, 330) = >330 >3300 x10 >4,52 330 +, >330) = >330 (150 + 148) =298 >3140 >3,50