EN ISO 19040-3:2022

Water quality - Determination of the estrogenic potential of water and waste water - Part 3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene assay is based on the activation of the human estrogen receptor alpha.
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;
—          the limit of quantification (LOQ) of this method for the direct analysis of water samples is between 0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre concentration of water samples can prove necessary if their estrogenic potential is below the given LOQ.

Wasserbeschaffenheit - Bestimmung des estrogenen Potentials von Wasser und Abwasser - Teil 3: In vitro-Reportergentest mit humanen Zellen (ISO 19040‑3:2018)

Dieses Dokument legt ein Verfahren zur Bestimmung des estrogenen Potentials von Wasser und Abwasser mittels eines Reportergen-Tests unter Verwendung stabil transfizierter, humaner Zellen fest. Dieser Reportergen-Test beruht auf der Aktivierung des humanen Estrogenrezeptors alpha.
Dieses Verfahren ist anwendbar auf:
   Süßwasser;
   Abwasser;
   wässrige Extrakte und Sickerwasser;
   Eluate von Sedimenten (Süßwasser);
   Porenwasser;
   wässrige Lösungen von Einzelsubstanzen oder von chemischen Gemischen;
   Trinkwasser;
Die Bestimmungsgrenze (LOQ) dieser Methode zur direkten Analyse von Wasserproben liegt zwischen 0,3 ng/l und 1 ng/l 17β Estradiol-Äquivalenten (EEQ). Diese Werte wurden in einem internationalen Ringversuch ermittelt (siehe Anhang F). Der obere Arbeitsbereich wurde bis zu einem Gehalt von 75 ng EEQ/l bewertet [basierend auf einem internationalen Ringversuch (siehe Tabelle F.3)]. Proben, die ein estrogenes Potenzial oberhalb dieser Schwelle zeigen, müssen für eine gültige Quantifizierung verdünnt werden. Eine Extraktion und Anreicherung von Wasserproben kann notwendig sein, wenn ihr estrogenes Potential unter der oben angegebenen Bestimmungsgrenze liegt.
WARNUNG —Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortung des Arbeitgebers, angemessene Sicherheits- und Schutzmaßnahmen zu treffen und sicherzustellen.
WICHTIG — Es ist erforderlich, bei den Untersuchungen nach diesem Dokument Fachleute oder Facheinrichtungen einzuschalten.

Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires - Partie 3: Essai in vitro sur cellules humaines avec gène rapporteur (ISO 19040-3:2018)

Le présent document spécifie une méthode permettant de déterminer le potentiel œstrogénique de l'eau et des eaux résiduaires au moyen d’un essai avec gène rapporteur utilisant des cellules humaines transfectées de façon stable. Cet essai avec gène rapporteur se fonde sur l’activation du récepteur des œstrogènes humains alpha.
Cette méthode est applicable:
—    aux eaux douces;
—    aux eaux résiduaires;
—    aux extraits aqueux et lixiviats;
—    aux éluats de sédiments (eau douce);
—    aux eaux interstitielles;
—    aux solutions aqueuses contenant des substances uniques ou des mélanges chimiques;
—    à l’eau potable;
—    la limite de quantification (LDQ) de cette méthode pour l’analyse directe d’échantillons d’eau est comprise entre 0,3 ng/l et 1 ng/l d’équivalents 17β-œstradiol (EEQ) sur la base des résultats de l’essai interlaboratoires international (voir l’Annexe F). Le domaine de mesure supérieur a été évalué [sur la base des résultats de l’essai interlaboratoires international (voir le Tableau F.3)] jusqu’à un niveau de 75 ng d’EEQ/l. Les échantillons présentant un potentiel œstrogénique supérieur à ce seuil doivent être dilués pour une quantification valable. L’extraction et la préconcentration des échantillons d’eau peuvent s’avérer nécessaires, si leur potentiel œstrogénique est inférieur à la LDQ donnée.

[Not translated]

General Information

• Status: Published
• Publication Date: 20-Sep-2022
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 21-Sep-2022
• Due Date: 16-Oct-2023
• Completion Date: 21-Sep-2022
• Ref Project: SIST EN ISO 19040-3:2023 - Water quality - Determination of the estrogenic potential of water and waste water - Part 3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 19040-3:2022
01-julij-2022
[Not translated]

Water quality - Determination of the estrogenic potential of water and waste water - Part

3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)
Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und

Abwasser - Teil 3: In vitro Reportergentest mit humanen Zellen (ISO 19040-3:2018)

Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires

- Partie 3: Essai in vitro sur cellules humaines avec gène rapporteurns) (ISO 19040-

3:2018)
Ta slovenski standard je istoveten z: prEN ISO 19040-3
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 19040-3:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 19040-3:2022
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oSIST prEN ISO 19040-3:2022
INTERNATIONAL ISO
STANDARD 19040-3
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 3: In vitro human cell-based
reporter gene assay
Qualité de l'eau — Détermination du potentiel oestrogène de l'eau et
des eaux résiduaires —
Partie 3: Essai in vitro sur cellules humaines avec gène rapporteur
Reference number
ISO 19040-3:2018(E)
ISO 2018
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oSIST prEN ISO 19040-3:2022
ISO 19040-3:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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oSIST prEN ISO 19040-3:2022
ISO 19040-3:2018(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Interferences ............................................................................................................................................................................................................ 4

5 Principle ........................................................................................................................................................................................................................ 4

6 Apparatus and materials.............................................................................................................................................................................. 4

7 Reagents, cells and media ........................................................................................................................................................................... 5

8 Sampling and samples .................................................................................................................................................................................... 9

8.1 General ........................................................................................................................................................................................................... 9

8.2 Bottles and material for sampling .......................................................................................................................................... 9

8.3 Bottles and material pre-cleaning .......................................................................................................................................... 9

8.4 Sampling procedure ........................................................................................................................................................................... 9

8.5 Transport of samples ......................................................................................................................................................................... 9

8.6 Pretreatment of sample ................................................................................................................................................................10

8.7 Storage of samples ............................................................................................................................................................................10

9 Procedure..................................................................................................................................................................................................................10

9.1 Cell culture maintenance .............................................................................................................................................................10

9.1.1 Freezing cells ....................................................................................................................................................................10

9.1.2 Starting a new cell culture ....................................................................................................................................10

9.1.3 Culturing cells ..................................................................................................................................................................11

9.2 Human cell reporter gene assay test procedure .....................................................................................................11

9.2.1 Seeding the cells (day 1) ........................................................................................................................................11

9.2.2 Preparation of the E2-reference (day 2) ..................................................................................................11

9.2.3 Preparation of the sample dilutions (day 2) .........................................................................................12

9.2.4 Field blank ..........................................................................................................................................................................12

9.2.5 Exposing the cells (day 2) .....................................................................................................................................12

9.2.6 Harvesting the cells (day 3) .................................................................................................................................13

9.2.7 Measurement of luminescence (day 3) .....................................................................................................13

9.3 Data analysis ..........................................................................................................................................................................................13

9.3.1 Calculation of the reporter gene induction ............................................................................................13

9.3.2 Calculation of the percentage of maximum response ...................................................................14

9.3.3 Calculation of the dose-response curve ....................................................................................................14

10 Validity criteria ...................................................................................................................................................................................................14

10.1 Validity criteria for the assay ...................................................................................................................................................14

10.2 Validity criteria for samples......................................................................................................................................................15

11 Assessment criteria ........................................................................................................................................................................................15

12 Test report ................................................................................................................................................................................................................15

Annex A (informative) Settings of the luminometer .........................................................................................................................16

Annex B (informative) Plate setup .......................................................................................................................................................................17

Annex C (informative) Bioassay characteristics and details .....................................................................................................18

Annex D (informative) Test set up for chemicals and extracts ...............................................................................................20

Annex E (informative) Preparation of dilution series .....................................................................................................................22

Annex F (informative) Performance data .....................................................................................................................................................23

Annex G (informative) Statistical assessment .........................................................................................................................................33

Annex H (informative) Calculation of 17β-estradiol equivalents ........................................................................................34

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Annex I (informative) Measurement of the lowest ineffective dilution (LID) of waste

water — A simplified evaluation for testing of waste water ................................................................................36

Bibliography .............................................................................................................................................................................................................................39

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oSIST prEN ISO 19040-3:2022
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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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following

URL: www .iso .org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.
A list of all parts in the ISO 19040 series can be found on the ISO website.
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oSIST prEN ISO 19040-3:2022
INTERNATIONAL STANDARD ISO 19040-3:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 3: In vitro human cell-based reporter gene assay

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
1 Scope

This document specifies a method for the determination of the estrogenic potential of water and waste

water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene

assay is based on the activation of the human estrogen receptor alpha.
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;

— the limit of quantification (LOQ) of this method for the direct analysis of water samples is between

0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international

interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of

the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing

estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction

and pre concentration of water samples can prove necessary if their estrogenic potential is below

the given LOQ.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org ./obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
culture medium

nutrients presented in a form and phase (liquid or solidified) which support cellular growth

[SOURCE: ISO 6107-6:2004, 24, modified — “cellular” replaces “microbiological”]
3.2
dilution level

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. The corresponding and

smallest possible value of D is 1. In this document, the arrow indicates the transition from initial total volume to

final total volume.
[SOURCE: ISO 6107-6:2004, 28]
3.3
dilution water
sterile water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.4
effective concentration of a compound which causes 50 % of an effect

Note 1 to entry: In the sense of the present document the EC is the effective concentration of a compound which

induces 50 % of the maximal reporter gene activity which can be achieved by this compound.

3.5
extract
test sample after extraction and possible removal of extraction vehicle
3.6
field blank

container prepared in the laboratory, using reagent water or other blank matrix, and sent with the

sampling personnel for exposure to the sampling environment to verify possible contamination during

sampling
[SOURCE: ISO 11074:2015, 4.5.3]
3.7
induction rate

quotient of the mean value of wells with enhanced reporter gene activity measured 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 cells under identical conditions

[SOURCE: ISO 6107-6:2004, 43, modified — “wells with enhanced reporter gene activity measured”

replaces “mutant colonies”; “corresponding wells” replaces “corresponding plates”, “quotient” replaces

“difference”; “cells” replaces “strain”.]
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3.8
limit of quantification
LOQ

lowest value that can be determined with an acceptable level of accuracy and precision

[SOURCE: ISO 15839:2003, 3.18]
3.9
lowest ineffective-dilution value
LID

lowest dilution within a test batch which does not show any effect, i.e. no statistically significant

increase in the reporter gene activity compared with the negative control

[SOURCE: ISO 11350:2012, 3.4, modified — “increase in the reporter gene activity” replaces “increase

in the number of revertant wells”]
3.10
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.11
passage number

the number of subcultures from cells in a new culture vessel (cell culture flask or micro titer plate)

3.12
reference compound

compound with one or more property values that are sufficiently reproducible and well established to

enable the calibration of the measurement method

[SOURCE: ISO 7405:2008, 3.6, modified — “compound” replaces “material”; “the calibration of the

measurement method” replaces “use of the material or substance for the calibration of an apparatus,

the assessment of a measurement method or for the assignment of values to materials”.]

3.13
relative light units
RLU

amount of reporter gene activity as measured by light produces using a luminometer, expressed as

relative light units
3.14
reporter gene activity

quantitative activity of a gene attached to the promoter sequence of another gene

3.15
stock culture

frozen culture of cells for the preservation of the characteristics of the cell line

[SOURCE: ISO 21427-2:2006, 13, modified — “the cell line” replaces “V79 cells”]
3.16
subculturing

transfer of part of a cell culture into a new cell culture vessel during cell culture

3.17
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
[SOURCE: ISO 6107-6:2004, 92]
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4 Interferences

Toxic effects present in the test samples may lead to a reduction of cell viability and hence to a reduction

of the measured cellular response. Consequently, estrogenic effects of a sample may be masked by acute

toxic effects leading to false negative test results (see Clause 9 for further information). In this case, the

sample should be diluted further until no cytotoxicity is observed (see manual of cytotoxicity test used).

The use of inappropriate sampling devices and/or sampling flask may influence the test result because

of the possible adsorption of active compounds on surfaces leading to false negative results. On the

other hand, active compounds could be released into the sample from sampling flasks, especially if

plastic ware is used, and false positive results might be generated. See Clause 7 for more information.

High salinity can cause toxic effects due to the resulting osmotic pressure. The ER(α) CALUX cells

(References [10] to [16]) tolerates a conductivity of the sample up to 34,000 µS/cm (1,0 % w/w salinity).

Bacterial and fungal contaminations can negatively influence the response of the cells. Therefore,

antibiotics are added to the cell culture medium. Contamination of the cells is assessed by visual

observation (microscope) when testing the sample. See Clause 9 for further information.

If filtered samples are tested in order to remove bacteria from the sample solid particles are separated

from the sample also. Thus, substances with estrogenic activity which are adsorbed on particles might

not be detected.

Anti-estrogenic compounds and other non-toxic inhibitory compounds might mask estrogenic effects.

The presence of interfering compounds can be assessed by samples which are spiked with a defined

amount of an estrogenic compound with defined properties (e.g. 17β-estradiol) leading to a known

induction of the test system.

Compounds with estrogenic properties might be present as inactive conjugates. A chemical de-

conjugation can be necessary in order to quantify the overall estrogenic potential of a sample.

5 Principle

Estrogen receptor (ER) - mediated signaling is essential in estrogen action and the mechanism of

estrogen receptor signaling is well established. Upon estrogen binding the estrogen receptor becomes

activated, and binds to recognition sequences in promoter regions of target genes, the so-called

estrogen responsive elements (EREs). These EREs have been linked to a promoter element and a gene

transcribing for the easily measurable protein luciferase. In these cells, the ligand-activated receptor

will activate luciferase transcription, and the transcribed luciferase protein will emit light when a

substrate is added. The signal dose-dependently increases as a result of increasing concentrations of

ligand. The luciferase activity in cellular lysates is measured with a luminometer, allowing reliable,

sensitive and quantitative measurements.
6 Apparatus and materials

Beside the equipment which is usually present in a laboratory for cell culture the following apparatus

and materials are needed. For suitable sampling devices see Clause 8.
6.1 Laminar air flow cabinet, standard: “biological hazard”.
6.2 Water bath, 37 °C.
6.3 CO incubator, 5 % CO , 37 °C, humidity 100 %.
2 2
6.4 Inverted phase-contrast microscope.
6.5 Freezer, at least ≤−18 °C and at ≤−70 °C.
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6.6 Shaking apparatus for micro-test plates.
6.7 Centrifuge.
6.8 Laboratory balance.
6.9 Sterile pipettes, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, glassware or plastics.
6.10 Pipette controller.
6.11 Cell culture flasks, 75 cm with filter lids.
6.12 Sterile plastic containers, 12 ml and 50 ml with sterile cap.
6.13 Sterile plates with 12 wells.

6.14 Multi-channel multi-stepper pipette (repeater pipette), including 5 ml and 10 ml tips.

6.15 Pipettes, 1 μl, 50 μl, 200 μl and 1 000 μl, with sterile tips.
6.16 Multi-channel pipettes, up to 50 μl and up to 300 μl.

6.17 Sterile polystyrene 96-well plates, with flat transparent bottom and lid, appropriate for cell

culture, volume 300 μl per well.

6.18 Microplate luminometer with two injectors, for addition of substrate and stop reagent.

6.19 Cell counter or hemacytometer.
6.20 pH meter.
6.21 Cryovials, sterile, 2 ml.
6.22 Liquid nitrogen container for long term cell storage.
6.23 Filter, cellulose acetate, 0,45 µm pore size.
7 Reagents, cells and media
7.1 Reagents

As far as possible, use "reagent grade" chemicals. If (different) hydrates are used that differ from the

compounds specified, ensure that the appropriate mass of the main compound is employed.

7.1.1 Dimethyl sulfoxide (DMSO).

7.1.2 Glycerol for molecular biology, ≥99 %, molecular weight: 92,09 g/mol, CAS: 56-81-5.

7.1.3 17β-estradiol, ≥98 %, (C H O ), molecular weight: 272,38 g/mol, CAS: 50-28-2.

18 24 2
7.1.4 Ethylene-diamine-tetra-acetate (EDTA), CAS: 6381-92-6.
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7.1.5 Trypsine, CAS: 9002-07-7.
7.1.6 Fetal calf serum (FCS).
7.1.7 DMEM/F12 medium with phenol red as pH indicator.
7.1.8 Non-essential amino acids (100x).

7.1.9 Penicillin-streptomycin (100x), concentration used 5 000 units penicillin per ml / 5 000 μg

streptomycin per ml).
7.1.10 Phosphate buffered saline pH 7,2 (PBS), without calcium and magnesium.
7.1.11 Adenosine-5'-triphosphate (ATP), CAS: 34369-07-8.

7.1.12 Trans-1, 2-diaminocyclohexane-N, N, N', N'-tetraacetic acid monohydrate (CDTA),

CAS: 125572-95-4.
7.1.13 Dithiothreitol (DTT), CAS: 3483-12-3.
7.1.14 2-amino-2-(hydroxyl-methyl)-1,3-propanediol (TRIS), CAS: 77-86-1.
7.1.15 Dextran T500.
7.1.16 Activated charcoal.
7.1.17 Coenzyme A, free acid grade, Add CAS: 85-61-0.
7.1.18 D-Luciferin sodium salt, CAS: 103404-75-7.
7.1.19 Magnesium hydroxide carbonate pentahydrate, C H Mg O , CAS: 56378-72-4.
4 2 5 14
7.1.20 Magnesium sulfate, CAS: 7487-88-9.
7.1.21 Sodium bicarbonate, CAS: 144-55-8.

7.1.22 Cell culture medium powder, Sigma, D2902, without phenol red and sodium bicarbonate.

7.1.23 Tricine, CAS: 5704-04-1.
7.1.24 Acetone, (purity p.a.), CAS: 67-64-1.
7.1.25 Sodium hydroxide, molecular weight 40,00 g/mol, CAS: 1310-73-2.
7.1.26 Triton X-100, CAS: 9002-93-1.

7.1.27 Hydrochloric acid solution, 1 M (HCl), molecular weight 36,46 g/mol, CAS: 7647-01-0.

7.2 Water, grade 3, as defined in ISO 3696; water with a conductivity up to 5 µS/cm is acceptable.

If sterile water is needed, autoclave or sterilize by filtration (cellulose acetate, 0,2 µm). Water as

specified here is also used for the stepwise dilution of the test sample.
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7.3 Cell line.

A genetically modified human cell line, expressing the human estrogen receptor (hERα and/or hERβ)

and containing a reporter plasmid for luciferase. More detailed descriptions of the individual cell lines

are given in Annex C.
7.4 Media.

Always use sterile solutions, glassware, etc. Carry out all procedures under aseptic conditions and in the

sterile environment of a laminar flow cabinet (biological hazard standard). If autoclaving is necessary,

always autoclave for 20 min at (121 ± 2) °C. Cover vessels loosely, never seal air tight.

7.4.1 Cell culture medium.

Add 5,4 ml non-essential amino acids (7.1.8), 42 ml FCS (7.1.6) and 1 ml of penicillin-streptomycin

(7.1.9) to a bottle containing 500 ml DMEM/F12 medium with phenol red. Complete cell culture medium

should be kept at 4 °C and stored for no longer than eight weeks.
7.4.2 Freezing medium.

Add 1 ml non-essential amino acids, 10 ml DMSO, 20 ml FCS and 1 ml penicillin-streptomycin (7.1.9) to

68 ml of DMEM/F12 medium with phenol red. Distribute 20 ml batches in a sterile manner over sterile

tubes. Complete freezing medium should be kept at (−20 ± 1) °C until use and kept on ice on use.

7.4.3 Concentrated assay medium.
7.4.3.1 3x concentrated assay medium.

Dissolve the contents of one vial of cell culture medium powder (7.1.22) in 250 ml of water (at room

temperature) and then dissolve 3,7 g sodium bicarbonate (7.1.21) in the same solution. Adjust the

pH to 7,4 using a 1 mol/l HCl or 1 mol/l NaOH solution. Add water to a final volume of 300 ml and

filter sterilize. Add 12 ml of non-essential amino acids (7.1.8), 55 ml stripped serum (7.4.8) and 37 ml

penicillin-streptomycin (7.1.9). Complete media should be kept at (4 ± 1) °C and stored for no longer

than eight weeks.
7.4.3.2 1x concentrated assay medium.

Dilute 100 ml of 3x concentrated assay medium with 200 ml of water and filter sterilize. Complete

media should be kept at (4 ± 1) °C and stored for no longer than eight weeks.
7.4.4 Trypsin solution.

Add 20 ml of trypsin (7.1.5) to 980 ml PBS (7.1.10) and add 0,2 g EDTA (7.1.4). Sterile filter the trypsin

solution and aliquot into 50
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