1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 11350:2012W a t e r q u a l i t y D e t e r m i n a t i o n of the genotoxicity of water and waste water Salmonella/microsome fluctuation test (Ames fluctuation test)BS IS
2、O 11350:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 11350:2012. The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained on request to i
3、ts 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 2012. Published by BSI Standards Limited 2012ISBN 978 0 580 66490 8 ICS 13.060.70 Compliance with a British Stand
4、ard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2012.Amendments issued since publicationDate T e x t a f f e c t e dBS ISO 11350:2012 ISO 2012Water quality Determination of the genot
5、oxicity of water and waste water Salmonella/microsome fluctuation test (Ames fluctuation test)Qualit de leau valuation de la gnotoxicit des eaux rsiduaires Essai de Salmonella/microsome (essai dAmes-fluctuation)INTERNATIONAL STANDARDISO11350First edition2012-05-15Reference numberISO 11350:2012(E)BS
6、ISO 11350:2012ISO 11350:2012(E)ii ISO 2012 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2012All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, w
7、ithout permission in writing from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 11350:2012ISO 113
8、50:2012(E) ISO 2012 All rights reserved iiiContents PageForeword iv1 Scope 12 Normative references . 13 Terms and definitions . 24 Interferences . 35 Principle . 46 Apparatus and materials . 47 Reagents, media and dilutions 58 Sampling and samples . 99 Procedure 99.1 Overnight culture . 99.2 Prepara
9、tion of S9 mix .109.3 Testing of water samples 109.4 Measurement of revertant growth .139.5 Calculation of cytotoxicity 1310 Validity criteria .1411 Assessment criteria 1412 Test report .14Annex A (normative) Nutrient broth and agar .15Annex B (normative) Preparation of ampicillin agar plates and st
10、ock cultures16Annex C (normative) Checking of genotype .17Annex D (normative) S9 fraction 18Annex E (informative) Example for application of samples on a 24 well plate 19Annex F (informative) Example for reporting 21Annex G (informative) Testing of chemicals .22Annex H (informative) Precision data25
11、Annex I (informative) Statistical assessment 27Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water A simplified evaluation for testing of waste water .33Annex K (informative) Use of additional tester strains 35Bibliography .36BS ISO 11350:2012ISO 11350:2012(E)
12、ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a te
13、chnical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters
14、of electrotechnical standardization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated t
15、o the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for
16、identifying any or all such patent rights.ISO 11350 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.iv ISO 2012 All rights reservedBS ISO 11350:2012INTERNATIONAL STANDARD ISO 11350:2012(E)Water quality Determination of the genotoxicity of water an
17、d waste water Salmonella/microsome fluctuation test (Ames fluctuation test)WARNING Persons using this International Standard 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 responsibi
18、lity of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions.IMPORTANT It is absolutely essential that tests conducted according to this International Standard be carried out by suitably trained staff.1 ScopeThis International
19、 Standard specifies a method for the determination of the genotoxic potential of water and waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chem
20、icals that induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are involved in the biosynthesis of the amino acid, histidine.NOTE 1 ISO 138298applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents.This method i
21、s applicable to: fresh water; waste water; aqueous extracts and leachates; eluates of sediments (fresh water); pore water; aqueous solutions of single substances or of chemical mixtures; drinking water.NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove ne
22、cessary.2 Normative referencesThe 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 an
23、y amendments) applies.ISO 3696, Water for analytical laboratory use Specification and test methodsISO 7027, Water quality Determination of turbidity ISO 2012 All rights reserved 1BS ISO 11350:2012ISO 11350:2012(E)3 Terms and definitionsFor the purposes of this document, the following terms and defin
24、itions apply.3.1cofactor solutionaqueous solution of chemicals (e.g. NADP, glucose-6-phosphate, and inorganic salts) needed for the activity of the enzymes in the S9 fractionSource: ISO 21427-2:2006,10definition 3.23.2culture mediumnutrients presented in a form and phase (liquid or solidified) which
25、 support microbiological growthSource: ISO 6107-6:2004,6definition 243.3dilution levelDdenominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water with dilution water as integral numberNOTE 1 to entry: For undiluted water or waste water, this coefficient per
26、definition is 11. In this International Standard, the arrow indicates the transition from initial total volume to final total volume. The corresponding and smallest possible value of D is 1.Source: ISO 6107-6:2004,6definition 283.4lowest ineffective dilutionLIDlowest dilution within a test batch whi
27、ch does not show any effect, i.e. no statistically significant increase in the number of revertant wells compared with the negative controlNOTE 1 to entry: LID is determined for each incubation condition (strain, S9 mix). The highest LID value is decisive for the overall assessment.3.5induction rate
28、difference between the mean value of wells with revertant growth counted on the plates treated with a dose of the test sample or with a positive control, and the mean value of the corresponding wells treated with the negative control using the same strain under identical conditionsSource: ISO 6107-6
29、:2004,6definition 43, modified: “wells with revertant growth” replaces “mutant colonies”; “corresponding wells” replaces “corresponding plates”3.6inoculumfraction of a culture of microorganisms used to start a new culture, or an exponentially growing preculture, in fresh mediumSource: ISO 6107-6:200
30、4,6definition 443.7negative controldilution water without test sampleSource: ISO 6107-6:2004,6definition 512 ISO 2012 All rights reservedBS ISO 11350:2012ISO 11350:2012(E)3.8revertant growthvisible mutant colonies on the microplate at the end of the respective test3.9overnight cultureculture started
31、 late in the afternoon and incubated overnight (usually about 16 h) to be ready during the following morning for purposes such as the inoculation of a precultureSource: ISO 6107-6:2004,6definition 54NOTE 1 to entry For specification, see 9.1.3.10positive controlany well characterized material and/or
32、 substance, which, when evaluated by a specific test method, demonstrates the suitability of the test system to yield a reproducible, appropriate positive or negative response in the test systemSource: ISO 10993-12:,7definition 3.12NOTE 1 to entry The positive controls mentioned in this Internationa
33、l Standard are dissolved in dimethyl sulfoxide (DMSO) prior to use. For the purposes of this International Standard, the positive controls are known mutagens which are suitable for the verification of the sensitivity of the method and/or the activity of the S9 mix.3.11S9 fractionsupernatant at 9 000
34、g of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to 300 g) pretreated with a substance or substance combination appropriate for enzyme inductionSource: ISO 6107-6:2004,6definition 743.12S9 mixmixture of S9 fraction and cofactor solutionSource: ISO 6107-6:2004,6def
35、inition 753.13stock cultureculture of a strain of organisms maintained under conditions to preserve original features such as nucleotide sequencesSource: ISO 6107-6:2004,6definition 873.14test sampleundiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all pr
36、eparation steps such as centrifugation, filtration, homogenization, pH adjustment and determination of ionic strengthSource: ISO 6107-6:2004,6definition 924 InterferencesBacteriotoxic effects of the test sample can lead to a reduction of viable bacteria and to a reduction of wells with revertants du
37、e to a repression of revertant growth.This method includes sterile filtration of water and waste water prior to the test. Due to this filtration, solid particles are separated from the test sample. Thus, there is a possibility that genotoxic substances adsorbed on particles are not detected. ISO 201
38、2 All rights reserved 3BS ISO 11350:2012ISO 11350:2012(E)5 PrincipleThe bacteria are exposed under defined conditions to various concentrations of the test sample and incubated for 100 min at 37 C 1 C in 24 well plates. Due to this exposure, genotoxic agents enclosed in the test sample can induce mu
39、tations in one or both marker genes of the bacterial strains used (hisG46 for TA 100 and hisD3052 for TA 98) in correlation with the applied concentrations. Induction of mutations causes a concentration-related increase in the number of mutant colonies.After exposure of the bacteria, reversion indic
40、ator medium (7.40), containing the pH indicator dye bromocresol purple (7.7), is added to the wells. Subsequently, the batches are distributed to 384 well plates (48 wells for each parallel) and incubated for 48 h to 72 h (9.3.2, 9.3.3).Mutagenic activity of the test sample is determined by counting
41、 the number of purple to yellow shifted wells (per 48 wells of each parallel), treated with the undiluted or the diluted test sample, compared to the negative control.The lowest dilution (1N) of the test sample which induces no mutagenic effect under all experimental conditions (if any mutagenic eff
42、ect is induced by the test sample) is the criterion for evaluating the mutagenic potential. Sample dilutions above this (1A, A N) shall induce a mutagenic effect according to the criteria of this International Standard in at least one strain under at least one activation condition (with or without a
43、ddition of S9 mix). The respective LID value is N. If no mutagenic effect is observed under all experimental conditions, this dilution is 11 and the respective LID value is 1.6 Apparatus and materials6.1 Temperature- and time-controlled incubator, 37 C 1 C.6.2 pH meter.6.3 Analytical balance.6.4 Ste
44、am sterilizer.6.5 Dry sterilizer.6.6 Magnetic stirrer.6.7 Rotary mixer.6.8 Freezer, capable of being maintained at 18 C and at 70 C.6.9 Pipettes, 0,1 ml, 0,5 ml, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, of glass or plastics.6.10 Storage bottles, 250 ml and 1 000 ml.6.11 Measuring cylinders, 100 ml and 200
45、 ml.6.12 Volumetric flasks, 20 ml, 200 ml and 500 ml.6.13 Sterile filters, 0,2 m and 0,45 m.6.14 Erlenmeyer flasks, 50 ml, 100 ml and 250 ml.6.15 Inoculating loops.4 ISO 2012 All rights reservedBS ISO 11350:2012ISO 11350:2012(E)6.16 Eight-channel multistepper pipette (repeater pipette).6.17 Eight-ch
46、annel pipettes, 5 l to 50 l and 50 l to 300 l.6.18 Spectrophotometer.6.19 Transparent sterile polystyrene 24 well and 384 well plates with flat bottom and lid.6.20 Microplate photometer for 24 well plates and optionally for 384 well plates, filters: 420 nm 15 nm and 595 nm 10 nm.6.21 Clean bench.6.2
47、2 Petri dishes with venting ribs, diameter approximately 94 mm, height approximately 16 mm.6.23 Cryogenic vials, sterile, 1 ml, 10 ml.7 Reagents, media and dilutions7.1 General. As far as possible, use “reagent grade” chemicals. If hydrates of anhydrous compounds or hydrates different from those spe
48、cified are used, ensure that the appropriate mass of the main compound is employed.When necessary, autoclave for 20 min at 121 C 2 C. Cover vessels loosely (e.g. with aluminium foil). Never seal air-tight.7.2 Water, grade 1, as defined in ISO 3696, or water with a conductivity of 5 S/cm.If sterile w
49、ater is needed, sterilize by sterile filtration (0,2 m) or autoclaving. Water as specified here is also used for the stepwise dilution of the test sample.7.3 Tester strains. Use mutant strains of Salmonella Typhimurium LT2, which enable detection of point mutations, to determine the mutagenic potential of a test sample. Since point mutations can be subdivided into two classes (frameshift mutations and base pair substitutions), the tw
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