1、BRITISH STANDARDWater quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 2: Mixed population method using the cell line V79 ICS 13.060.70 BS EN ISO 21427-2:2009 BS 6068-5.45: 2006 Incorporating corrigendum April 2009BS EN ISO 21427-2:2009 This British Standard was
2、 published under the authority of the Standards Policy and Strategy Committee on 29 December 2006 BSI 2009 National foreword The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods. A list of organizations represent
3、ed on this subcommittee 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. Amendment
4、s/corrigenda issued since publication ISBN 978 0 580 65209 7 Date Comments This corrigendum renumbers BS ISO 21427-2:2006 as BS EN ISO 21427-2:2009, and incorporates ISO corrigendum April 2009 which corrects subclause 6.2.15 This British Standard is the UK implementation of EN ISO 21427-2:2009. It i
5、s identical with ISO 21427-2:2006, incorporating corrigendum April 2009. It supersedes BS ISO 21427-2:2006, which is withdrawn. 31 July 2009EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 21427-2 March 2009 ICS 13.060.70 English Version Water quality - Evaluation of genotoxicity by measurem
6、ent of the induction of micronuclei - Part 2: Mixed population method using the cell line V79 (ISO 21427-2:2006) Qualit de leau - valuation de la gnotoxicit par le mesurage de linduction de micronoyaux - Partie 2: Mthode de la population mlange laide de la ligne de cellules V79 (ISO 21427-2:2006) Wa
7、sserbeschaffenheit - Bestimmung der Gentoxizitt mit dem In-vitro-Mikrokerntest - Teil 2: Verwendung einer nicht- synchronisierten V79-Zellkulturlinie (ISO 21427-2:2006) This European Standard was approved by CEN on 1 March 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulatio
8、ns 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 Euro
9、pean 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 na
10、tional 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 an
11、d United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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 ISO 21
12、427-2:2009: EblankContents Page Foreword iv 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 3 5 Interferences . 3 6 Reagents and media. 3 7 Apparatus 7 8 Test facility criteria . 7 9 Procedure 8 10 Evaluation and assessment. 12 11 Precision 14 12 Test report . 14 Annex
13、 A (informative) Bromodeoxyuridine (BrdU) method. 15 Annex B (informative) Evaluation schemes. 17 Annex C (normative) S9 fraction 18 Annex D (informative) Precision data. 19 Bibliography . 20 EN ISO 21427-2:2009 (E) BS EN ISO 21427-2:2009 iiiEN ISO 21427-2:2009 (E) Foreword The text of ISO 21427-2:2
14、006 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 21427-2:2009 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall
15、be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by September 2009, and conflicting national standards shall be withdrawn at the latest by September 2009. Attention is drawn to the possibility that some of the elements of this do
16、cument may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard
17、: 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. End
18、orsement notice The text of ISO 21427-2:2006 has been approved by CEN as a EN ISO 21427-2:2009 without any modification. BS EN ISO 21427-2:2009 iv1 Water quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 2: Mixed population method using the cell line V79 WARNING
19、Persons using this part of ISO 21427 should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure comp
20、liance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted according to this part of ISO 21427 be carried out by suitably trained staff. 1 Scope This part of ISO 21427 specifies a method for the determination of genotoxicity of water and waste water usi
21、ng a mammalian in vitro test which detects damage, induced by water-soluble substances, to the chromosomes or the mitotic apparatus of V79 cells from the Chinese hamster. The micronucleus test allows the identification of substances that cause cytogenetic damage which results in the formation of mic
22、ronuclei containing lagging chromosome fragments and/or whole chromosomes. The assay is based on the increase in the frequency of micronucleated cells after incubation with and without metabolic activation. 2 Normative references The following referenced documents are indispensable for the applicati
23、on 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. ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples 3 Terms and definitions For the purposes
24、 of this document, the following terms and definitions apply: 3.1 cell lines distinct families of cells grown in culture originated from a single clone 3.2 cofactor solution aqueous solution of chemicals (e.g. NADP, Glucose-6-phosphate and inorganic salts) needed for the activity of the enzymes in t
25、he S9 fraction EN ISO 21427-2:2009 (E) BS EN ISO 21427-2:2009 2 3.3 dilution level D denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water with dilution water as integral number NOTE For undiluted water or waste water, this coefficient is per definition
26、 1:1. The corresponding smallest possible D value is 1. 3.4 D value smallest value of D at which, under the conditions of this part of ISO 21427, no increase in the number of micronuclei per culture is detected NOTE In the case of more than one D value (at maximum two are possible, see 9.2), the hig
27、hest D value is decisive. 3.5 karyotype characteristic of the nucleus of a cell, including its size, form and chromosome number 3.6 micronuclei small particles consisting of acentric fragments of chromosomes and/or entire chromosomes which lag behind at anaphase stage of cell division and form, afte
28、r telophase, single or multiple micronuclei in the cytoplasm 3.7 mitotic index percentage of cells of a cell population under division at a particular time of observation 3.8 plating efficiency measure of the number of colonies originated from single cells 3.9 proliferation index rate at which cells
29、 are dividing within the culture 3.10 proliferation rate rate with which cells replicate, calculated by a formula which takes into account 1, 2, 4 and 8 cell stages of clones 3.11 S9 fraction 9 000 g supernatant of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to 30
30、0 g) pretreated with an appropriate substance or substance combination for enzyme induction 3.12 S9 mix mixture of the S9 fraction and the cofactor solution 3.13 stock culture frozen culture for the preservation of the characteristics of V79 cells 3.14 survival index percentage of surviving cells co
31、mpared to all cells, used as index of toxicity 3.15 test culture culture of cells used for the study EN ISO 21427-2:2009 (E) BS EN ISO 21427-2:2009 3 4 Principle The possible clastogenic and/or aneugenic activity of the test sample is detected by comparing, for the respective activation condition, t
32、he number of micronucleated cells in cultures treated with the negative control and the number in cultures treated with undiluted and diluted test samples, respectively. During cell division, chromatid fragments without centromers will not move to the nuclei of the daughter cells and will stay withi
33、n the cytoplasm. Part of the chromosome aberrations induced by the test item will be chromatid fragments without centromers and will, therefore, not be incorporated in the nuclei of the daughter cells. In addition, spindle disorders may lead to chromosomes which are not incorporated into the nucleus
34、. These particles will form micronuclei in the plasma. V79 cells are exposed for 24 h (4 h with the S9 mix) to a range of concentrations of a test sample. Thereafter, slides are prepared, and cells are stained and evaluated for the presence of micronucleated cells. An increased incidence of these mi
35、cronucleated cells in comparison to the negative control indicates that the test item may cause chromosome breaks or spindle disorders in V79 cells in vitro. 5 Interferences Biologically relevant alterations of the culture conditions may induce chromosome aberration due to secondary mechanisms resul
36、ting in artificial positive and, therefore, irrelevant results 16 . Those factors are, e.g. stronger changes in osmolality or pH, precipitation of test sample and phagocytosis thereof, and strong cytotoxic effects of the test sample. Therefore, test samples should be monitored at least for changes i
37、n pH or osmolality of the cultures using the same proportion of test item per culture as will be used later under test conditions. If there is a shift in pH in the culture, the test item should be adjusted to pH 7,0 0,2. If there is a change in osmolality, the highest concentration used in the test
38、has to be reduced so that no relevant alteration of osmolality occurs in the cultures. To avoid artifacts based on phagocytosis or severe cytotoxicity, limitations are given for the highest concentration, which should be used for testing (see 9.1 and 9.2). 6 Reagents and media As far as possible use
39、 “reagent grade” chemicals. If chemicals with different amounts of water of crystallization are used, calculate the needed amounts accordingly. Always perform autoclaving for 20 min at 121 C 2 C. Cover vessels loosely (e.g. with aluminium foil). Never seal air-tight. 6.1 Water. Prepare all aqueous s
40、olutions with water of a conductivity of u 5 S/cm. 6.2 Reagents. 6.2.1 Glucose-6-phosphate dihydrate, C 6 H 11 O 9 PNa 2 2 H 2 O. 6.2.2 Nicotinamide adenine dinucleotide phosphate disodium salt, NADP, C 21 H 26 N 7 Na 2 O 17 P 3 . 6.2.3 Magnesium chloride hexahydrate, MgCl 2 6 H 2 O. 6.2.4 Potassium
41、 dihydrogenphosphate, KH 2 PO 4 . 6.2.5 di-Sodium hydrogenphosphate dihydrate, Na 2 HPO 4 2 H 2 O. 6.2.6 Ethanol (absolute), C 2 H 5 OH. EN ISO 21427-2:2009 (E) BS EN ISO 21427-2:2009 4 6.2.7 Glacial acetic acid, CH 3 COOH. 6.2.8 Formaldehyde, HCHO, 37 % volume fraction. 6.2.9 tri-Sodium citrate dih
42、ydrate, HOC(COONa)(CH 2 COONa) 2 2 H 2 O. 6.2.10 di-Sodium hydrogenphosphate, Na 2 HPO 4 . 6.2.11 Sodium dihydrogenphosphate, NaH 2 PO 4 . 6.2.12 May-Grnwald-solution, modified 1) . 6.2.13 Hydrochloric acid, c(HCl) = 1 mol/l. 6.2.14 Sodium hydroxide solution, c(NaOH) = 1 mol/l. 6.2.15 Dimethyl sulfo
43、xide (DMSO), C 2 H 6 SO. 6.2.16 Positive controls. 6.2.16.1 Cyclophosphamide, monohydrate, C 7 H 15 Cl 2 N 2 O 2 PH 2 O. CAS Registration No: 6055-19-2. 6.2.16.2 Ethyl-methane sulfonate (EMS), CH 3 SO 3 CH 2 CH 3 . CAS Registration No: 62-50-0. 6.2.17 Sodium citrate solution for hypotonic treatment.
44、 Prepare a 1,5 % solution of tri-sodium citrate in water. 6.2.18 Fixation solution. Mix 50 ml of glacial acetic acid with 150 ml of ethanol, add 2,5 ml of a 37 % formaldehyde solution. 6.2.19 Buffer solution according to WEISE (pH 7,2) 1) . This solution is commercially available in ampoules. Dilute
45、 the contents of one ampoule in water and, using a 1 000 ml measuring flask, bring to volume with water. 6.2.20 Giemsa solution 1) . Prepare a 2,6 % Giemsa solution in buffer according to WEISE (pH 7,2) (6.2.19). Filter prior to use. 6.2.21 Phosphate buffer. Dissolve 2,13 g of Na 2 HPO 4in 1 l water
46、. Dissolve 1,8 g of NaH 2 PO 4in 1 l water. Mix both solutions at the ratio of 4:1 and adjust to a final pH of 7,4. 6.2.22 MEM-medium (= Minimal Essential Medium) with stabilized glutamine 1) . 6.2.23 Fetal bovine serum (= FCS) 1) . 1) This reagent is commercially available. This information is give
47、n for the convenience of users of this part of ISO 21427 and does not constitute an endorsement by ISO of these products. EN ISO 21427-2:2009 (E) BS EN ISO 21427-2:2009 5 6.2.24 Penicillin/Streptomycin solution, 10 000 E/10 000 g/ml 1) . 6.2.25 Amphotericin-B solution, 250 g/ml 1) . 6.2.26 Trypsin/E
48、DTA solution, 0,25 % 1) . 6.2.27 Hanks Balanced Salt Solution (= HBSS) 1) . 6.2.28 Hanks Balanced Salt Solution (= HBSS) without Ca 2+and Mg 2+1) . 6.2.29 Potassium chloride solution. Dissolve 4 g of potassium chloride, in 1 l water. 6.3 Preparation of culture media 6.3.1 Culture medium with FCS. Th
49、is medium is used as general culture medium and for treatment of cells without the S9 mix. Mix 500 ml of MEM-medium, 50 ml of FCS, 5 ml of Penicillin/Streptomycin solution and 5 ml of Amphotericin-B solution. The medium is stable for up to 4 weeks if stored in a refrigerator at 4 C 2 C. 6.3.2 Culture medium without FCS. This medium is used only for the treatment period of cells under activation condition (S9 mix). Mix 500 ml of MEM-medium, 5 ml of Penicillin/S
