SIST EN ISO 11350:2025
(Main)Water quality - Determination of the genotoxicity of water and waste water - Salmonella/microsome fluctuation test (Ames fluctuation test) (ISO 11350:2012)
Water quality - Determination of the genotoxicity of water and waste water - Salmonella/microsome fluctuation test (Ames fluctuation test) (ISO 11350:2012)
This International 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 chemicals 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.
Wasserbeschaffenheit - Bestimmung der Gentoxizität von Wasser und Abwasser - Verfahren mittels Salmonella/Microsomen-Fluktuationstest (Ames-Fluktuationstest) (ISO 11350:2012)
Diese Internationale Norm legt ein Verfahren zur Bestimmung des gentoxischen Potenzials von Wasser und Abwasserproben auf der Basis der Bakterienstämme Salmonella enterica subsp. enterica serotype Typhimurium TA 98 und TA 100 in einem Fluktuationstest fest. Diese Kombination von Bakterienstämmen erlaubt die Messung des gentoxischen Potenzials von Chemikalien, die Punktmutationen (Basenaustauschmutationen und Rasterschubmutationen) in enzymkodierenden Genen auslösen können. Diese Enzyme sind an der Biosynthese der Aminosäure Histidin beteiligt.
ANMERKUNG 1 ISO 13829 [8] dient zur Messung gentoxischer Proben, die DNA-vernetzende Substanzen enthalten.
Das Verfahren ist geeignet für:
Süßwasser/Oberflächenwasser;
Abwasser;
wässrige Extrakte und Eluate;
Sedimenteluate (Süßwasser);
Porenwasser;
wässrige Lösungen von Einzelsubstanzen oder Mischungen von Chemikalien;
Trinkwasser.
ANMERKUNG 2 Für die Testung von Trinkwasser kann eine Extraktion und Aufkonzentrierung der Probe notwendig sein.
Qualité de l'eau - Évaluation de la génotoxicité des eaux résiduaires - Essai de Salmonella/microsome (essai d'Ames-fluctuation) (ISO 11350:2012)
Kakovost vode - Določanje genotoksičnosti za vodo in odpadno vodo - Salmonella/mikrosomski fluktuacijski preskus (Amesov fluktuacijski preskus) (ISO 11350:2012)
Ta mednarodni standard opisuje metodo za določanje genotoksičnega potenciala vode in odpadne vode z uporabo bakterijskih sevov Salmonella enterica, podvrste enterica serotip Typhimurium TA 98 in TA 100 v fluktuacijskem preskusu. Ta kombinacija sevov omogoča merjenje genotoksičnosti kemikalij, ki povzročajo točkovne mutacije (substitucijo baznih parov in mutacije okvirnega položaja) genov, ki kodirajo encime, vključene v biosintezo aminokisline histidin.
OPOMBA 1: Standard ISO 13829[8] se uporablja za meritve genotoksičnosti vzorcev, ki vsebujejo agente za prečno povezovanje DNK.
Ta metoda se uporablja za:
– sladko vodo;
– odpadno vodo;
– vodne ekstrakte in izcedne vode;
– izlužke sedimentov (sladka voda);
– porno vodo;
– vodne raztopine posameznih snovi ali kemičnih mešanic;
– pitno vodo.
OPOMBA 2: Pri preskušanju pitne vode je lahko potrebna ekstrakcija in predkoncentracija vodnih vzorcev.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-november-2025
Kakovost vode - Določanje genotoksičnosti za vodo in odpadno vodo -
Salmonella/mikrosomski fluktuacijski preskus (Amesov fluktuacijski preskus) (ISO
11350:2012)
Water quality - Determination of the genotoxicity of water and waste water -
Salmonella/microsome fluctuation test (Ames fluctuation test) (ISO 11350:2012)
Wasserbeschaffenheit - Bestimmung der Gentoxizität von Wasser und Abwasser -
Verfahren mittels Salmonella/Microsomen-Fluktuationstest (Ames-Fluktuationstest) (ISO
11350:2012)
Qualité de l'eau - Évaluation de la génotoxicité des eaux résiduaires - Essai de
Salmonella/microsome (essai d'Ames-fluctuation) (ISO 11350:2012)
Ta slovenski standard je istoveten z: EN ISO 11350:2025
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 11350
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2025
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Determination of the genotoxicity of water
and waste water - Salmonella/microsome fluctuation test
(Ames fluctuation test) (ISO 11350:2012)
Qualité de l'eau - Évaluation de la génotoxicité des eaux Wasserbeschaffenheit - Bestimmung der Gentoxizität
résiduaires - Essai de Salmonella/microsome (essai von Wasser und Abwasser - Verfahren mittels
d'Ames-fluctuation) (ISO 11350:2012) Salmonella/Microsomen-Fluktuationstest (Ames-
Fluktuationstest) (ISO 11350:2012)
This European Standard was approved by CEN on 4 August 2025.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11350:2025 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
The text of ISO 11350:2012 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 11350:2025
by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
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 February 2026, and conflicting national standards
shall be withdrawn at the latest by February 2026.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 11350:2012 has been approved by CEN as EN ISO 11350:2025 without any modification.
INTERNATIONAL ISO
STANDARD 11350
First edition
2012-05-15
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome fluctuation test
(Ames fluctuation test)
Qualité de l’eau — Évaluation de la génotoxicité des eaux résiduaires —
Essai de Salmonella/microsome (essai d’Ames-fluctuation)
Reference number
ISO 11350:2012(E)
©
ISO 2012
ISO 11350:2012(E)
All 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, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
ISO 11350:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Interferences . 3
5 Principle . 4
6 Apparatus and materials . 4
7 Reagents, media and dilutions . 5
8 Sampling and samples . 9
9 Procedure . 9
9.1 Overnight culture . 9
9.2 Preparation of S9 mix .10
9.3 Testing of water samples .10
9.4 Measurement of revertant growth .13
9.5 Calculation of cytotoxicity .13
10 Validity criteria .14
11 Assessment criteria .14
12 Test report .14
Annex A (normative) Nutrient broth and agar .15
Annex B (normative) Preparation of ampicillin agar plates and stock cultures.16
Annex C (normative) Checking of genotype .17
Annex D (normative) S9 fraction .18
Annex E (informative) Example for application of samples on a 24 well plate .19
Annex F (informative) Example for reporting .21
Annex G (informative) Testing of chemicals .22
Annex H (informative) Precision data.25
Annex I (informative) Statistical assessment .27
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water —
A simplified evaluation for testing of waste water .33
Annex K (informative) Use of additional tester strains .35
Bibliography .36
ISO 11350:2012(E)
Foreword
ISO (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 technical 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 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 to 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 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 reserved
INTERNATIONAL STANDARD ISO 11350:2012(E)
Water quality — Determination of the genotoxicity of water and
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 responsibility 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 Scope
This International 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 chemicals 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.
[8]
NOTE 1 ISO 13829 applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents.
This method is 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 necessary.
2 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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 7027, Water quality — Determination of turbidity
ISO 11350:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
cofactor solution
aqueous solution of chemicals (e.g. NADP, glucose-6-phosphate, and inorganic salts) needed for the activity
of the enzymes in the S9 fraction
[10]
[Source: ISO 21427-2:2006, definition 3.2]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[6]
[Source: ISO 6107-6:2004, definition 24]
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 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. [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.
[6]
[Source: ISO 6107-6:2004, definition 28]
3.4
lowest ineffective dilution
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant increase in the
number of revertant wells compared with the negative control
NOTE 1 to entry: LID is determined for each incubation condition (strain, ±S9 mix). The highest LID value is decisive for
the overall assessment.
3.5
induction rate
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 conditions
[6]
[Source: ISO 6107-6:2004, definition 43, modified: “wells with revertant growth” replaces “mutant colonies”;
“corresponding wells” replaces “corresponding plates”]
3.6
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing preculture,
in fresh medium
[6]
[Source: ISO 6107-6:2004, definition 44]
3.7
negative control
dilution water without test sample
[6]
[Source: ISO 6107-6:2004, definition 51]
2 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
3.8
revertant growth
visible mutant colonies on the microplate at the end of the respective test
3.9
overnight culture
culture started 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 preculture
[6]
[Source: ISO 6107-6:2004, definition 54]
NOTE 1 to entry For specification, see 9.1.
3.10
positive control
any well characterized material and/or 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 system
[7]
[Source: ISO 10993-12:—, definition 3.12]
NOTE 1 to entry The positive controls mentioned in this International 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.
3.11
S9 fraction
supernatant at 9 000g 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 induction
[6]
[Source: ISO 6107-6:2004, definition 74]
3.12
S9 mix
mixture of S9 fraction and cofactor solution
[6]
[Source: ISO 6107-6:2004, definition 75]
3.13
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[6]
[Source: ISO 6107-6:2004, definition 87]
3.14
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all preparation
steps such as centrifugation, filtration, homogenization, pH adjustment and determination of ionic strength
[6]
[Source: ISO 6107-6:2004, definition 92]
4 Interferences
Bacteriotoxic effects of the test sample can lead to a reduction of viable bacteria and to a reduction of wells with
revertants due 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 11350:2012(E)
5 Principle
The 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 mutations 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 indicator 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 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 (1→N) of the test sample which induces no mutagenic effect under all experimental
conditions (if any mutagenic effect is induced by the test sample) is the criterion for evaluating the mutagenic
potential. Sample dilutions above this (1→A, 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 addition
of S9 mix). The respective LID value is N. If no mutagenic effect is observed under all experimental conditions,
this dilution is 1→1 and the respective LID value is 1.
6 Apparatus and materials
6.1 Temperature- and time-controlled incubator, 37 °C ± 1 °C.
6.2 pH meter.
6.3 Analytical balance.
6.4 Steam 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 ml.
6.12 Volumetric flasks, 20 ml, 200 ml and 500 ml.
6.13 Sterile filters, 0,2 µm and 0,45 µm.
6.14 Erlenmeyer flasks, 50 ml, 100 ml and 250 ml.
6.15 Inoculating loops.
4 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
6.16 Eight-channel multistepper pipette (repeater pipette).
6.17 Eight-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
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.
6.22 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 dilutions
7.1 General. As far as possible, use “reagent grade” chemicals. If hydrates of anhydrous compounds or hydrates
different from those specified 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 µS/cm.
If sterile water is needed, sterilize by sterile filtration (0,2 µm) 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 two tester strains TA 98 and TA 100 are used.
TA 98 contains as a marker the frameshift mutation (+2 type) hisD3052, whereas TA 100 bears the base pair
substitution hisG46.
In addition, both strains shall have the following genetic properties:
— they contain the plasmid pKM101, coding for ampicillin resistance;
— they are all deep rough, e.g. partly deficient in lipopolysaccharide side chains, enabling also larger
molecules to penetrate the bacterial cell wall and to cause mutations;
— due to a mutation in uvrB, the capability of the tester strains to repair DNA-damage is limited and the
likelihood that DNA-damage results in mutations is increased.
NOTE The use of additional tester strains is described in Annex K.
7.4 2-Aminoanthracene (2-AA), C H N, CAS No: 613-13-8.
14 11
7.5 Ampicillin sodium salt, C H N NaO S, CAS No: 69-52-3.
16 18 3 4
7.6 d-Biotin, C H N O S, CAS No: 58-85-5.
10 16 2 3
7.7 Bromocresol purple, sodium salt, CAS No: 62625-30-3.
ISO 11350:2012(E)
7.8 Citric acid monohydrate, C H O •H O, CAS No: 5949-29-1.
6 8 7 2
7.9 Dimethylsulfoxide, DMSO, C H SO, CAS No: 67-68-5.
2 6
7.10 d-Glucose, anhydrous, C H O , CAS No: 50-99-7.
6 12 6
7.11 d-Glucose-6-phosphate disodium salt hydrate, G-6-P-Na , C H Na O P•2H O CAS No: 3671-99-6.
2 6 11 2 9 2
7.12 Hydrochloric acid solution, HCl, c(HCl) = 1 mol/l.
7.13 Magnesium chloride hexahydrate, MgCl •6H O, CAS No: 7791-18-6.
2 2
7.14 Magnesium sulfate heptahydrate, MgSO •7H O, CAS No: 10034-99-8.
4 2
7.15 Potassium chloride, KCl, CAS No: 7447-40-7.
7.16 Dipotassium hydrogenphosphate, K HPO , CAS No: 7758-11-4.
2 4
7.17 Sodium ammonium hydrogenphosphate tetrahydrate, NaNH HPO •4H O, CAS No: 7583-13-3.
4 4 2
7.18 Sodium chloride, NaCl, CAS No: 7647-14-5.
7.19 Sodium dihydrogenphosphate, anhydrous, NaH PO , CAS No: 7558-80-7.
2 4
7.20 Disodium hydrogenphosphate, anhydrous, Na HPO , CAS No: 7558-79-4.
2 4
7.21 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
7.22 β-Nicotinamide adenine dinucleotide phosphate sodium salt, NADP·H O, C H N NaO P ·H O,
2 21 27 7 17 3 2
CAS No: 698999-85-8.
7.23 Nitrofurantoin (NF), CAS No: 67-20-9.
7.24 4-Nitro-o-phenylenediamine (4-NOPD), CAS No: 99-56-9.
1)
7.25 Nutrient broth powder.
1)
7.26 S9 fraction (liver homogenate; induced by phenobarbital/β-naphthoflavone).
7.27 l-Histidine, C H N O , CAS No: 71-00-1.
6 9 3 2
7.28 Phosphate buffer.
7.28.1 Sodium dihydrogenphosphate buffer, c(NaH PO ) = 0,2 mol/l.
2 4
Dissolve 14,39 g NaH PO (or 16,55 g NaH PO •H O) in 600 ml of water (7.2).
2 4 2 4 2
1) This reagent is commercially available. This information is given for the convenience of users of this International
Standard and does not constitute an endorsement by ISO of these products.
6 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
7.28.2 Disodium hydrogenphosphate buffer, c(Na HPO ) = 0,2 mol/l. Dissolve 28,39 g Na HPO in 1 000 ml
2 4 2 4
of water (7.2).
Add sodium dihydrogenphosphate buffer (7.28.1) to disodium hydrogenphosphate buffer (7.28.2) until a pH value
of 7,4 is reached and autoclave. Store at room temperature in the dark. The solution is stable for at least 1 year.
7.29 d-Biotin solution. Dissolve 12,2 mg d-biotin (7.6) in 100 ml of water (7.2) by boiling up. After cooling,
sterilize by filtration (0,2 µm filter). Store 10 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.30 l-Histidine solution. Dissolve 50 mg of l-histidine (7.27) in 50 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 1,5 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23). Aqueous solutions stored
as frozen aliquots are stable for at least 1 year.
7.31 Glucose-6-phosphate solution. Dissolve 0,68 g of d-glucose-6-phosphate (7.11) in 10 ml of water (7.2)
and sterilize by filtration (0,2 µm). Store aliquots (e.g. 200 µl) at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.32 NADP solution, c(NADP) = 0,04 mol/l. Dissolve the appropriate mass of NADP in 10 ml of water (7.2) to
obtain a final concentration of 0,04 mol/l and sterilize by filtration (0,2 µm). Store aliquots (e.g. 700 µl) at −18 °C
or below in sterile cryogenic vials (6.23). Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
Various hydrates of NADP are available. The actual molecular weight is specified in the product data sheet.
Calculate the amount of NADP required according to the molecular weight given.
7.33 Potassium chloride solution. Dissolve 74,56 g of KCl (7.15) in 1 000 ml of water (7.2) and autoclave.
Store at room temperature. The solution is stable for at least 1 year.
7.34 MgCl •6H O solution. Dissolve 50,83 g of MgCl •6H O (7.13) in 1 000 ml of water (7.2) and autoclave
2 2 2 2
the solution. Store at room temperature. The solution is stable for at least 1 year.
7.35 Bromocresol purple solution. Dissolve 51 mg of bromocresol purple sodium salt (7.7) in 30 ml of
water (7.2). Prepare this solution freshly before addition to the reversion indicator medium (7.40).
7.36 Ampicillin solution. Dissolve 500 mg of ampicillin (7.5) in 10 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 500 µl aliquots at −18 °C or below in sterile cryogenic vials (6.23). The solution is stable for
at least 6 months.
2)
7.37 Growth medium. Dissolve 4,7 g of nutrient broth powder and 0,31 g of sodium chloride (7.18) in 200 ml
of water (7.2). Adjust the pH to 7,5 ± 0,1. Add water (7.2) to 250 ml and autoclave the solution.
The following final concentrations in the growth medium shall result:
— 7,5 g/l meat extract;
— 7,5 g/l peptone;
— 5,0 g/l sodium chloride.
Solutions stored under sterile conditions as frozen aliquots are stable for at least 1 year.
2) Use nutrient broth powder containing 40 % meat extract, 40 % peptone, and 20 % sodium chloride.
ISO 11350:2012(E)
7.38 Exposure medium. Prepare a medium for incubation of bacteria with the sample containing a low amount
of l-histidine to support a few cell divisions.
Dissolve consecutively the following ingredients in 900 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 1 000 ml, adjust the pH to 7,0 ± 0,2, if necessary, and sterilize by filtration (0,2 µm filter).
Store the medium at 2 °C to 8 °C.
Add, per 100 ml, 0,6 ml of d-biotin solution (7.29) and 0,1 ml of l-histidine solution (7.30) under sterile conditions.
Prepare only the amount of medium necessary for the next 2 weeks. Store the medium at 2 °C to 8 °C.
7.39 Exposure medium concentrate. Dissolve consecutively the following ingredients in 70 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 93 ml, adjust the pH, if necessary, and sterilize by filtration (0,2 µm filter). Store the medium
concentrate at 2 °C to 8 °C.
Add 6 ml of d-biotin solution (7.29) and 1 ml of l-histidine solution (7.30) under sterile conditions. Prepare only
the amount of medium necessary for the next 2 weeks. Store the medium concentrate at 2 °C to 8 °C.
7.40 Reversion indicator medium. Prepare a pH indicator medium without l-histidine.
7.40.1 Solution I. Dissolve the following ingredients in 950 ml water in the given order:
— 0,4 g magnesium sulfate heptahydrate (7.14);
— 4,0 g citric acid (7.8);
— 20,0 g dipotassium hydrogenphosphate (7.16);
— 7,0 g sodium ammonium hydrogenphosphate tetrahydrate (7.17).
Add water (7.2) to 1 000 ml and add 30,0 ml of bromocresol purple solution (7.35). Adjust the pH to 7,3 ± 0,1.
Transfer the solution one half each into two 1 000 ml flasks and autoclave.
7.40.2 Solution II. Dissolve 8,0 g of d-glucose (7.10) in 800 ml of water (7.2). Adjust the pH to 7,3 ± 0,1.
Transfer both halves of the solution into two 1 000 ml flasks and autoclave.
7.40.3 Mixing and storage. After cooling to ambient temperature, mix 515 ml of solution I (7.40.1) with 400 ml of
solution II (7.40.2) under sterile conditions. Add 20 ml of d-biotin solution (7.29) under sterile conditions to each flask.
Store the medium at room temperature in the dark. The medium is stable for at least 1 month.
8 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
7.41 Control solutions.
7.41.1 Negative controls. For preparation of the negative controls, always use the same solvent as for the samples
to be tested. This is usually water (7.2) when testing water samples and DMSO (7.9) when testing chemicals.
7.41.2 Positive controls. In general, dissolve 10 mg of each positive control substance in 10 ml of
DMSO (7.9). Prepare 50 µl aliquots as stock solutions in sterile cryogenic vials and store them at −18 °C or below.
Under these conditions stock solutions are stable for at least 1 year. On the day of the test, unfreeze one aliquot.
7.41.3 Strain TA 98 without S9 mix. Use 4-nitro-o-phenylenediamine (4-NOPD) (7.24) as positive control
substance for strain TA 98 without S9 mix.
Dilute the stock solution 1→2 with DMSO (7.9). This dilution is used in the test.
7.41.4 Strain TA 100 without S9 mix. Use nitrofurantoin (NF) (7.23) as a positive control substance for strain
TA 100 without S9 mix.
Dilute the stock solution 1→80 with DMSO. This dilution is used in the test.
7.41.5 Strain TA 98 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as a positive control substance for
strain TA 98 with S9 mix.
Dissolve the stock solution 1→200 with DMSO. This dilution is used in the test.
7.41.6 Strain TA 100 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as positive control substance for
strain TA 100 with S9 mix.
Dissolve the stock solution 1→50 with DMSO. This dilution is used in the test.
8 Sampling and samples
Test the samples immediately after sampling. If this is not possible, keep water samples at 0 °C to 5 °C (for
<48 h) or below −18 °C (for up to 2 months). For multiple testing divide larger samples in advance into appropriate
portions, since thawed samples can only be used on the same day.
Samples containing solids should be centrifuged to separate them. In this case, only the supernatant is
processed further.
Sterilize all samples using sterile filters (0,45 µm). Homogenize test samples by thoroughly shaking before use.
Adjust the sample to a pH of 7,2 ± 0,2 using either HCl (7.12) or NaOH solution (7.21). Select the acid or alkali
concentrations such that the added volumes are as small as possible. Avoid overtitration. Take into account the
[5]
change in the sample’s pH and resulting effects (see ISO 5667-16 ).
Perform dilutions of the test sample as specified in Tables 2 and 3 with sterilized water (7.2).
9 Procedure
9.1 Overnight culture
Under sterile conditions, pipette 20 ml of growth medium (7.37) supplemented with 20 µl of ampicillin
solution (7.36) into a 100 ml Erlenmeyer flask (6.14) hermetically sealed with caps or aluminium foil and mix by
gentle agitation.
Add 20 µl of the respective test strain (TA 98 or TA 100) immediately after thawing. Incubate the culture at
37 °C ± 1 °C for 10 h. If the required cell density (9.3.1, G.1.1) is not reached, extend incubation time to 12 h.
If the required cell density is still not reached after 12 h, inoculate a fresh overnight culture. A clock timer may
ISO 11350:2012(E)
be used. Use a shaking frequency of about 150 r/min. From the inoculation of test bacteria to the beginning of
incubation at 37 °C, ensure that the temperature of the incubation bath is 19 °C ± 4 °C.
9.2 Preparation of S9 mix
Treatment for enzyme induction and preparation of the S9 fraction are described in Annex D. If the S9 fraction
is purchased commercially, it shall also be prepared in accordance with Annex D.
Prepare the S9 mix freshly on the day of testing. Mix:
— 66 µl KCl solution (7.33);
— 64 µl MgCl •6H O solution (7.34);
2 2
— 50 µl d-glucose-6-phosphate solution (7.31);
— 200 µl NADP solution (7.32);
— 1 000 µl phosphate buffer (7.28);
— 20 µl water (sterile);
— 600 µl S9 fraction (Annex D).
This mixture is sufficient for two exposure plates. If more than two plates are required, increase the amount of
S9 mix proportionally.
Keep the S9 mix permanently on ice for not more than 1 h and use it on the same day. Discard remaining S9
mix at the end of this day.
9.3 Testing of water samples
9.3.1 Preparation of tester strains
3)
In the test cultures a cell density of 180 FAU for TA 98 and 45 FAU for TA 100 is recommended. Laboratory-
specific adaptation of tester strain density may be necessary to achieve the number of negative control revertant
wells as defined in Clause 10.
In the test culture, the tester strain inoculum is diluted 10-fold (Table 2 and Table 3). Therefore, adjust the
cell density in the overnight culture inoculum to 1 800 FAU for TA 98 and 450 FAU for TA 100. Calculate the
required dilution factor and the volume of 1× exposure medium that must be added to the overnight culture in
order to adjust the cell density according to Formulae (1), (2), and (3).
For the determination of the actual cell density of the overnight culture, dilute 100 µl of the overnight culture
with 900 µl growth medium (7.37), otherwise the FAU is out of range. Measure cell densities (Χ ) of this
595 nm
dilution of the tester strains TA 98 and TA 100 immediately before exposure and calculate FAU values of this
dilution using a FAU calibration curve in accordance with ISO 7027. Set up the calibration curve using Χ .
595 nm
Calculate the dilution factor according to Formulae (1) and (2):
In the case of strain TA 98:
X [FAU]
595nm
d = (1)
where
3) Formazine attenuation units (see ISO 7027).
10 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
d is the dilution factor;
X [FAU] is the FAU value of the 10-fold diluted overnight culture of strain TA 98 that is determined
595 nm
as described above.
In the case of strain TA 100:
X [FAU]
595nm
d = (2)
where
d is the dilution factor;
X [FAU] is the FAU value of the 10-fold diluted overnight culture of strain TA 100 that is determined
595 nm
as described above.
Use the calculated dilution factor to determine the required volume of 1× exposure medium that shall be added
to the overnight culture in order to adjust the cell density according to Formula (3).
VV=−()dV (3)
add cultureculture
where
d is the dilution factor according to Formula (1) or (2);
V is the volume of 1× exposure medium to be added to the overnight culture, in millilitres (ml);
add
V is the volume of the undiluted overnight culture, in millilitres (ml).
culture
Table 1 shows a typical example for the adjustment of the cell density for a test with the strain TA 98.
Table 1 — Dilution of the overnight culture (TA 98) as an example
Volume of the
Dilution factor Volume of the
Volume of the
exposure medium
FAU of the 10-fold
[according to overnight culture
adjusted overnight
to be added to the
diluted overnight
Formula (1)] (undiluted)
culture for exposure
overnight culture
culture
(1 800 FAU)
d V V
culture add
240 1,33 20 ml 6,6 ml 26,6 ml
9.3.2 Test culture without S9 mix
Prepare test cultures according to Table 2 using sterile 24-well plates (plate A) (6.19). For each culture,
incubate at least three replicates. Perform under sterile conditions. An example for the configuration of plate A
is given in Annex E.
ISO 11350:2012(E)
Table 2 — Preparation of test culture plate A without S9 mix
Positive
Negative Dilution Dilution Dilution
control batch
a
control batch level 1 level 2 level 4
Component
(see 7.41.2)
µl µl µl µl µl
Exposure medium concentrate (7.39) 100 100 100 100 100
Dilution water (7.2) 800 0 300 550 780
b
Sample 0 800 500 250 20
Adjusted overnight culture of tester
100 100 100 100 100
strain TA 98 or TA 100 (9.3.1)
Total volume 1 000 1 000 1 000 1 000 1 000
a
Dilution level 1: sample concentration 80 %; dilution level 2: sample concentration 50 %; dilution level 3: sample concentration
25 %. If more than three dilution levels are required, extend the dilution series by means of a graduated dilution (e.g. D = 8, 16, 32)
b
As positive control substances, use 4-NOPD for strain TA 98 and NF for strain TA 100.
If calculation of cytotoxicity is performed (9.5), measure initial Χ (t = 0 min) and final Χ (t = 100 min)
595 nm 595 nm
of plate A using a microplate photometer (6.20). It is recommended that only tester strain TA 98 be used for
measurement of growth since cell density of TA 100 remains low.
Incubate plate A in the dark at 37 °C ± 1 °C for 100 min while shaking (150 r/min).
Pour 2,5 ml of reversion indicator medium (7.40) into each well of another 24 well plate (plate B) (6.19).
Immediately thereafter, transfer 500 µl of test culture from plate A into plate B by using a multistepper
pipette (6.16). Mix thoroughly.
Subsequently, transfer the content of one well of plate B to 48 wells of a 384 well plate (plate C) (6.19) in 50 µl
aliquots using a multistepper pipette (6.16). An example of this procedure is given in Annex E.
Incubate the 384 well plate in the dark at 37 °C ± 1 °C for 48 h without shaking. Avoid evaporation-promoting
conditions (e.g. ventilation). For a low number of wells with revertant growth in the positive controls, extend the
incubation time in 384 well plates to 72 h.
If calculation of cytotoxicity is not performed, fill plate A with half the volume of each ingredient (Table 2). After
100 min of incubation, directly add the reversion indicator medium (7.40) to plate A and mix thoroughly. Transfer
the cultures to the 384-well plate and incubate as specified in the previous paragraph.
9.3.3 Test culture with S9 mix
Prepare test cultures according to Table 3 using sterile 24 well microplates (plate A) (6.19). For each culture,
incubate at least three replicates. Perform under sterile conditions. An example for the configuration of plate A
is given in Annex E.
12 © ISO 2012 – All rights reserved
ISO 11350:2012(E)
Table 3 — Preparation of test culture plate A with S9 mix
Positive
Negative Dilution Dilution Dilution
control batch
a
control batch level 1 level 2 level 4
Component
(7.41.2)
µl µl µl µl µl
Exposure medium concentrate (7.39) 100 100 100 100 100
Dilution water (7.2) 800 0 300 550 780
b
Sample 0 800 500 250 20
Adjusted overnight culture of tester 100 100 100 100 100
strain TA 98 or TA 100 (9.3.1)
S9 mix (9.2) 34 34 34 34 34
Total volume 1 034 1 034 1 034 1 034 1 034
a
Dilution level 1: sample concentration 80 %; dilution level 2: sample concentration 50 %; dilution level 3: sample concentration
25 %. If more than three dilution levels are required extend the dilution series by means of a graduated dilution (e.g. D = 8, 16, 32)
b
As positive control substances, use 2-AA for strain TA 98 and TA 100.
If calculation of cytotoxicity is performed (9.5), measure initial Χ (t = 0 min) and final Χ (t = 100 min)
595 nm 595 nm
of plate A using a microplate photometer (6.20). It is recommended that only tester strain TA 98 be used for
measurement of growth since cell density of TA 100 remains low.
Incubate the plates in the dark at 37 °C ± 1 °C for 100 min while shaking (approximately 150 r/min).
Pour 2,5 ml reversion indicator medium (7.40) into each well of another 24 well plate (plate B) (6.19).
Immediately thereafter, transfer 500 µl of test culture from plate A into plate B by using a multistepper pipette
(6.16). Mix thoroughly.
Then, transfer the content of one well of plate B to 48 wells of a 384 well plate (plate C) (6.19) in 50 µl aliquots
using a multistepper pipette (6.16). An example of this procedure is given in Annex E.
Incubate the 384 well plate in the dark at 37 °C ± 1 °C for 48 h without shaking. Avoid evaporation-promoting
conditions (e.g. ventilation). For a low number of wells with revertant growth in the positive controls, extend the
incubation time in 384 well plates to 72 h.
If calculation of cytotoxicity is not performed, fill plate A with half of the volume of each ingredient (Table 3).
After 100 min of incubation, directly add reversion indicator medium (7.40) to plate A and mix thoroughly.
Transfer to the 384 well p
...
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