SIST EN ISO 16373-2:2014

Textiles - Dyestuffs - Part 2: General method for the determination of extractable dyestuffs including allergenic and carcinogenic substances (ISO 16373-2:2014)

EN ISO 16373-2 specifies the analyses used to detect extractable dyestuffs in textile products, with the extraction performed for all kind of fibres and types of dyestuffs using pyridine/water (1:1). It lists (see Annexes A and B) the allergenic and carcinogenic dyestuffs which can be analysed using this method; the lists of dyestuffs are expandable.

Textilien - Farbstoffe - Teil 2: Allgemeines Verfahren zur Bestimmung von bestimmten extrahierbaren Farbstoffen einschließlich allergener und karzinogener Farbstoffe (ISO 16373-2:2014)

Die in der vorliegenden Norm beschriebenen Analysen werden zum Nachweis extrahierbarer Farbstoffe in
textilen Erzeugnissen angewendet. Die Extraktion erfolgt für alle Faserarten und Farbstofftypen mit
Pyridin/Wasser (1:1).
Allergene und karzinogene Farbstoffe, die mit diesem Verfahren analysiert werden können, sind in den
Anhängen A und B aufgeführt. Diese Farbstofflisten können erweitert werden.
ANMERKUNG In Anhang F wird die mit diesem Verfahren erhaltene prozentuale Wiederfindung für die Klassen (wie in
Teil 1 festgelegt) saurer Farbstoffe, basischer Farbstoffe, direkter Farbstoffe, von Dispersionsfarbstoffen, Flüssigfarbstoffen
und "Fixierfärbemitteln" an unterschiedlichen Textilfasern dargestellt.

Textiles - Colorants - Partie 2: Méthode générale pour la détermination de colorants extractibles dont les substances allergènes et carcinogènes (ISO 16373-2:2014)

L'ISO 16373-2:2014 spécifie les analyses servant à détecter les colorants extractibles dans les produits textiles, l'extraction étant effectuée avec un mélange de pyridine/eau (1:1) pour tous les types de fibres et de colorants. Elle répertorie les colorants allergènes et cancérigènes pouvant être analysés avec cette méthode; ces listes de colorants ne sont pas exhaustives.

Tekstilije - Barvila - 2. del: Splošne metode za ugotavljanje barvil, ki jih je mogoče ekstrahirati, vključno z alergenimi in kancerogenimi snovmi (ISO 16373-2:2014)

Standard EN ISO 16373-2 določa analize za ugotavljanje barvil, ki jih je mogoče ekstrahirati, v tekstilnih izdelkih, pri čemer se ekstrakcija izvaja za vsa vlakna in za tipe barvil, ki uporabljajo piridin/vodo (1:1). Našteva (glejte dodatka A in B) alergene in kancerogena barvila, ki jih je mogoče analizirati s to metodo; seznami barvil se lahko razširijo.

General Information

• Status: Published
• Publication Date: 03-Jul-2014
• ICS: 59.080.01 - Textiles in general
• ICS: 87.060.10 - Pigments and extenders
• Technical Committee: ITEK - Textile and textile products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 12-Jun-2014
• Due Date: 17-Aug-2014
• Completion Date: 04-Jul-2014
• Ref Project: EN ISO 16373-2:2014 - Textiles - Dyestuffs - Part 2: General method for the determination of extractable dyestuffs including allergenic and carcinogenic dyestuffs (method using pyridine-water) (ISO 16373-2:2014)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 16373-2:2014
01-september-2014
7HNVWLOLMH%DUYLODGHO6SORãQHPHWRGH]DXJRWDYOMDQMHEDUYLONLMLKMHPRJRþH
HNVWUDKLUDWLYNOMXþQR]DOHUJHQLPLLQNDQFHURJHQLPLVQRYPL,62
Textiles - Dyestuffs - Part 2: General method for the determination of extractable
dyestuffs including allergenic and carcinogenic substances (ISO 16373-2:2014)
Textilien - Farbstoffe - Teil 2: Allgemeines Verfahren zur Bestimmung von bestimmten
extrahierbaren Farbstoffen einschließlich allergener und karzinogener Farbstoffe (ISO
16373-2:2014)
Textiles - Colorants - Partie 2: Méthode générale pour la détermination de colorants
extractibles dont les substances allergènes et carcinogènes (ISO 16373-2:2014)
Ta slovenski standard je istoveten z: EN ISO 16373-2:2014
ICS:
59.080.01 Tekstilije na splošno Textiles in general
87.060.10 Pigmenti in polnila Pigments and extenders
SIST EN ISO 16373-2:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16373-2:2014

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SIST EN ISO 16373-2:2014

EUROPEAN STANDARD
EN ISO 16373-2

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2014
ICS 59.080.01
English Version
Textiles - Dyestuffs - Part 2: General method for the
determination of extractable dyestuffs including allergenic and
carcinogenic dyestuffs (method using pyridine-water) (ISO
16373-2:2014)
Textiles - Colorants - Partie 2: Méthode générale de Textilien - Farbstoffe - Teil 2: Allgemeines Verfahren zur
détermination des colorants extractibles, notamment les Bestimmung von extrahierbaren Farbstoffen einschließlich
colorants allergènes et cancérigènes (méthode utilisant un allergener und karzinogener Farbstoffe (Pyridin-Wasser-
mélange pyridine/eau) (ISO 16373-2:2014) Verfahren) (ISO 16373-2:2014)
This European Standard was approved by CEN on 3 June 2014.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16373-2:2014 E
worldwide for CEN national Members.

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SIST EN ISO 16373-2:2014
EN ISO 16373-2:2014 (E)
Contents Page
Foreword .3
2

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SIST EN ISO 16373-2:2014
EN ISO 16373-2:2014 (E)
Foreword
This document (EN ISO 16373-2:2014) has been prepared by Technical Committee CEN/TC 248 “Textiles
and textile products” the secretariat of which is held by BSI, in collaboration with Technical Committee
ISO/TC 38 "Textiles".
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 December 2014, and conflicting national standards shall be withdrawn
at the latest by December 2014.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16373-2:2014 has been approved by CEN as EN ISO 16373-2:2014 without any modification.


3

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SIST EN ISO 16373-2:2014

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SIST EN ISO 16373-2:2014
INTERNATIONAL ISO
STANDARD 16373-2
First edition
2014-06-01
Textiles — Dyestuffs —
Part 2:
General method for the determination
of extractable dyestuffs including
allergenic and carcinogenic dyestuffs
(method using pyridine-water)
Textiles — Colorants —
Partie 2: Méthode générale de détermination des colorants
extractibles, notamment les colorants allergènes et cancérigènes
(méthode utilisant un mélange pyridine/eau)
Reference number
ISO 16373-2:2014(E)
©
ISO 2014

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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, 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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Safety precautions . 1
6 Reagents . 2
7 Apparatus . 2
7.1 Apparatus and auxiliaries for sample preparation . 2
7.2 Chromatographic equipment (selected from the following). 2
8 Procedure. 3
8.1 Preparation of test specimen . 3
8.2 Extraction . 3
8.3 Detection and quantification of dyestuff . 3
8.4 Calibration . 3
9 Calculation and expression of the results . 4
10 Test report . 4
Annex A (normative) List of carcinogenic dyestuffs . 5
Annex B (normative) List of allergenic and other dyestuffs . 6
Annex C (normative) Calculation . 7
Annex D (informative) Examples of chromatographic methods. 8
Annex E (informative) Reliability of the method .17
Annex F (informative) Multiple-extraction of several textile fibres with pyridine/water .18
Bibliography .20
© ISO 2014 – All rights reserved iii

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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(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.
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. 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. 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 meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
ISO 16373-2 was prepared by the European Committee for Standardization (CEN) Technical Committee
CEN/TC 248, Textiles and textile products, in collaboration with ISO Technical Committee TC 38, Textiles,
in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
ISO 16373 consists of the following parts, under the general title Textiles — Dyestuffs:
— Part 1: General principles of testing coloured textiles for dyestuff identification
— Part 2: General method for the determination of extractable dyestuffs including allergenic and
carcinogenic (method using pyridine-water)
— Part 3: Method for determination of carcinogenic extractable dyestuffs (method using
triethylamine/methanol)
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Introduction
The ISO 16373 series deals with dyestuffs used in textile for qualification and quantification.
1)
ISO 16373-1 includes the definition of the dyestuff classes and the description of some procedures to
identify qualitatively the dyestuff class used in textile material.
The other parts of ISO 16373 are related to the quantification of some dyestuffs.
In this part of ISO 16373, the principle of the test method is based on the extraction using pyridine-water
solution, which has been found to be the most efficient solution to extract a large range of dyestuffs,
including allergenic and carcinogenic dyestuffs.
In ISO 16373-3, the principle of the test method is based on extraction using triethylamine/methanol
solution. This solution has been found efficient to extract some dyestuffs in some cases.
Additional information related to the recovery rate (to characterize the extraction efficiency) obtained
from the application of ISO 16373-3 and this part of ISO 16373 will be summarized in ISO 16373-1.
It is important to note that other test methods exist related to azo dyes, for which a reduction of those
extracted azo dyes leads to the release of some aromatic amines, to be detected and determined using
chromatography.
The percentage of recovery using the method of this part of ISO 16373 is shown in Annex F for the
dyestuff classes (to be defined in ISO 16373-1) acid, basic, direct, disperse, solvent dyestuffs and
“mordant dyestuffs” on different textile fibres.
1) To be published.
© ISO 2014 – All rights reserved v

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SIST EN ISO 16373-2:2014

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SIST EN ISO 16373-2:2014
INTERNATIONAL STANDARD ISO 16373-2:2014(E)
Textiles — Dyestuffs —
Part 2:
General method for the determination of extractable
dyestuffs including allergenic and carcinogenic dyestuffs
(method using pyridine-water)
1 Scope
This part of ISO 16373 specifies the analyses used to detect extractable dyestuffs in textile products,
with the extraction performed for all kind of fibres and types of dyestuffs using pyridine/water (1:1). It
lists (see Annexes A and B) the allergenic and carcinogenic dyestuffs which can be analysed using this
method; the lists of dyestuffs are expandable.
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
allergenic dyestuff
dyestuff which may cause an allergic skin reaction
3.2
carcinogenic dyestuff
dyestuff which is classified as carcinogenic substance
Note 1 to entry: Harmonized classification according to the Globally harmonized system of classification and
[2] [3]
labelling of chemicals (GHS), incorporated in EU Regulation 1272/2008 (CLP) .
4 Principle
A coloured test specimen is selected from the textile article and extracted with pyridine/water at
100 °C. The extract is analysed by liquid chromatography/diode array detection (LC/DAD) and/or by
liquid chromatography/mass spectrometry (LC/MS).
5 Safety precautions
WARNING —The substances listed in Tables A.1, B.1 and B.2 are classified as substances known
to be or suspected to be human allergens or carcinogens.
Ensure that any handling and disposal of these substances is in strict accordance with the
appropriate national health and safety regulations.
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

It is the user’s responsibility to use safe and proper techniques in handling materials in this test
method. Consult manufacturers for specific details such as material safety data sheets and other
recommendations.
Good laboratory practice should be followed. Wear safety glasses in all laboratory areas and a
single use dust respirator while handling powder dyestuffs.
Attention is drawn to any national and local safety regulations.
6 Reagents
Unless otherwise specified, analytical grade chemicals shall be used.
6.1 Pyridine.
6.2 Acetonitrile, chromatographic grade.
6.3 Ammonium acetate.
6.4 Tetrabutylammonium dihydrogen phosphate.
6.5 Deionized water, grade 3 in accordance with ISO 3696.
6.6 Pyridine/water (1:1), prepared by mixing 500 ml pyridine (6.1) and 500 ml water (6.5).
Keep the solution in a brown glass bottle.
6.7 Individual stock solutions, prepared in pyridine-water 1:1, of all reference substances listed in
Annexes A and B
It is recommended that reference substances (including those listed in Annexes A and B) of the highest
purity grade available on the market be used. The given purity has to be considered for the calculation
(see Clause 9).
7 Apparatus
7.1 Apparatus and auxiliaries for sample preparation
7.1.1 Standard laboratory equipment.
7.1.2 Analytical balance, resolution at 0,01 g.
7.1.3 Glass vials (20 ml to 40 ml), with tight closure.
7.1.4 Heating source that generates heat at a temperature of (100 ± 2)°C (thermal block or laboratory
sand-bath, controllable).
7.1.5 Autosampler glass vials, with tight closure.
7.1.6 Thermo-sensing device, e.g. thermocouple, to measure at 100 °C with a resolution of 0,1 °C.
7.2 Chromatographic equipment (selected from the following)
7.2.1 Equipment for LC/DAD
— High performance liquid chromatograph (HPLC),
— DAD detector,
— separating column,
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

— guard column.
7.2.2 Equipment for LC/MS
— High performance liquid chromatograph (HPLC),
— electrospray ion source,
— MS detector,
— separating column,
— guard column.
8 Procedure
8.1 Preparation of test specimen
The test specimen shall be selected based on the following criteria:
— parts of the textile article;
— nature of the fibre component (fibre composition);
— colours.
Prepare a test specimen of 1,0 g max. by cutting the laboratory sample up into small pieces no larger
2
than 1 cm . Determine the mass of the test specimen to the nearest 0,01 g and record it as m (see 8.2).
E
8.2 Extraction
Add 7,5 ml of pyridine/water (1:1) (6.6) to the test specimen and close the vial tightly. Heat the vial in
the heating source until the temperature of the solvent reaches (100 ± 2) °C, and hold this temperature
for (35 ± 5) min.
Check the time taken for the temperature of the solvent to reach the required temperature using a glass
vial (see 8.1) containing 7,5 ml of pyridine/water (1:1)(6.6) with a thermo-sensing element (7.1.6), which
is plunged in the solvent and sealed with a septum.
Allow the vial to cool down to 40 °C or below before opening it.
Transfer approximately 1 ml of (test) liquid from the vial to a smaller vial for further analysis.
NOTE This step could be done with a syringe through the closed septum to minimize contact with pyridine.
8.3 Detection and quantification of dyestuff
Dyestuff detection can be performed using the above-specified chromatographic techniques (7.2). If
other analytical techniques are used it shall be reported.
Dyestuff quantification is performed by means of HPLC/DAD/MS.
NOTE Some dyes can be quantified with HPLC/DAD.
8.4 Calibration
For the calibration, the mixed standards of the reference substances listed in Table A.1 (and respectively
in Tables B.1 and B.2) are prepared in pyridine/water (1:1). The stock solutions are used to prepare
mixed standards with concentrations levels of 1 mg/l, 5 mg/l, 10 mg/l and 20 mg/l related to dyestuff
content.
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

9 Calculation and expression of the results
Amounts of dyestuff are usually calculated by means of a software program. The calculation can be
carried out manually as described in Annex C.
Amounts of dyestuff are expressed in mg dyestuff per kg of textile (mg/kg).
If the detected amount of a dye is over 100 mg/kg, it is certain that this dye is used – such as one of those
listed in Table A.1, B.1 or B.2.
10 Test report
The test report shall refer to this method and shall include at least the following:
a) reference to this part of ISO 16373, i.e. ISO 16373-2;
b) all information necessary for the identification of the test specimen;
c) date of sample receipt and date of analysis;
d) sampling procedure;
e) detection method and quantification method;
f) results reported as level and detection limit per dyestuff, in mg/kg;
g) any deviation from the given procedure.
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Annex A
(normative)

List of carcinogenic dyestuffs
See Table A.1.
Table A.1 — Reference carcinogenic dyestuffs
a b c
Number Carcinogenic dyestuff C. I. Number CAS Number Molecular formula
1 Disperse Blue 1 64500 2475–45–8 C H N O
14 12 4 2
2 Solvent Yellow 1 11000 60–09–4 C H N
12 11 3
4-aminoazobenzene
3 Solvent Yellow 2 11020 60–11–7 C H N
14 15 3
4 Solvent Yellow 3 11160 97–56–3 C H N
14 15 3
o-aminoazotoluene
5 Basic Red 9 42500 569–61–9 C H N HCl
19 17 3
6 Basic Violet 14 42500 632–99–5 C H N HCl
20 19 3
7 Disperse Yellow 3 11855 2832–40–8 C H O N
15 15 2 3
8 Acid Red 26 16150 3761–53–3 C H N Na O S
18 14 2 2 7 2
9 Direct Black 38 30235 1937–37–1 C H N Na O S
34 25 9 2 7 2
10 Direct Blue 6 22610 2602–46–2 C H N O S Na
32 24 6 14 4 4
11 Direct Red 28 22120 573–58–0 C H N Na O S
32 22 6 2 6 2
12 Disperse Orange 11 60700 82–28–0 C H NO
15 11 2
13 Acid Red 114 23635 6459–9-5 C H N Na O S
37 28 4 2 10 3
a
Numbering used in Tables D.1, D.4, D.5.
b [2] [3]
Classified according to (GHS) (and to CLP).
c [4]
Colour index number.
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Annex B
(normative)

List of allergenic and other dyestuffs
See Tables B.1 and B.2.
NOTE Not all dyestuffs in Table B.1 are clinically verified as allergenic.
Table B.1 — Reference disperse dyestuffs
a
Number Allergenic dyestuff C.I. Number CAS Number Molecular formula
A1 Disperse Blue 1 64500 2475–45–8 C H N O
14 12 4 2
A2 Disperse Blue 3 61505 2475–46–9 C H N O
17 16 2 3
A3 Disperse Blue 7 62500 3179–90–6 C H N O
18 18 2 6
A4 Disperse Blue 26 63305 3860–63–7 C H N O
16 14 2 4
A5 — 56524–77–7 C H N O
15 12 2 4
Disperse Blue 35
A6 — 56524–76–6 C H N O
16 14 2 4
A7 Disperse Blue 102 11945 12222–97–8 C H N O S
15 19 5 4
A8 Disperse Blue 106 111935 12223–01–7 C H N O S
14 17 5 3
A9 Disperse Blue 124 111938 61951–51–7 C H N O S
16 19 5 4
A10 Disperse Brown 1 11152 23355–64–8 C H N O Cl
16 15 4 4 3
A11 Disperse Orange 1 11080 2581–69–3 C H N O
18 14 4 2
A12 Disperse Orange 3 11005 730–40–5 C H N O
12 10 4 2
A13 Disperse Orange 37/76/59 11132 13301–61–6 C H N O Cl
17 15 5 2 2
A14 Disperse Red 1 11110 2872–52–8 C H N O
16 18 4 3
A15 Disperse Red 11 62015 2872–48–2 C H N O
15 12 2 3
A16 Disperse Red 17 11210 3179–89–3 C H N O
17 20 4 4
A17 Disperse Yellow 1 10345 119–15–3 C H N O
12 9 3 5
A18 Disperse Yellow 3 11855 2832–40–8 C H N O
15 15 3 2
A19 Disperse Yellow 9 10375 6373–73–5 C H N O
12 10 4 4
A20 Disperse Yellow 39 480095 12236–29–2 C H N O
17 16 2
A21 Disperse Yellow 49 — 54824–37–2 C H N O
22 22 4 2
a
Numbering used in Tables D.2 and D.3.
Table B.2 — Other reference dyestuffs
a
Number Other dyestuff CI Number CAS Number Molecular formula
O1 Disperse Yellow 23 26070 6250–22–3 C H N O
18 14 4
O Disperse Orange 149 — 85136–74–9 C H N O
2 25 26 6 3
— 118685–33–9 C H ClCrN O S Na
39 23 7 12 2
O3 Navy Blue 018112
— — C H CrN O S Na
46 30 10 20 2 3
O4 Disperse Orange 61 111355 55281–26–0 C H Br N O
17 15 2 5 2
a
Numbering used in Tables D.2 and D.3.
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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Annex C
(normative)

Calculation
Dyestuff levels are calculated from the peak areas of the individual dyestuff components.
The dyestuff level is calculated as a mass fraction, w, in mg/kg, of the extractable dyestuff in the test
solution, using Formula (1):
ρ ×V
c
w= (C.1)
m
E
where
w is the concentration of dyestuff, in mg/kg;
ρ is the concentration of the dyestuff in the test solution, in mg/l (from the calibration curve);
c
V is the volume (final specimen volume), in ml;
m is the mass of the textile specimen, in g.
E
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ISO 16373-2:2014(E)

Annex D
(informative)

Examples of chromatographic methods
D.1 High performance liquid chromatography (HPLC/DAD) method for disperse
and carcinogenic dyestuffs
2)
D.1.1 UHPLC system with pressure over 400 bar
Eluent 1: 2 % Tetrabutylammonium dihydrogen phosphate with 10 % acetonitrile
Eluent 2: Acetonitrile
Stationary phase: HALO-C18 2,7 µm; 150 × 2,1 mm with guard column
Flow rate: 0,5 ml/min
Column temperature: 40 °C
Injection volume: 5 µl
Pressure: 470 bar/6 800 psi
Detection: DAD, spectrograph
Quantification: at 400 nm, 500 nm, 600 nm, (700 nm)
Gradient: Time [min]: Eluent 2 [%]: Flow [ml]:
1,0 0 0,5
15,0 47 0,5
25,0 55 0,5
26,0 100 0,666
28,0 100 0,8
30,0 100 0,8
35 0 0,5
D.1.2 Standard HPLC system with maximum pressure of 400 bar
Conditions as in D.1.1, with the exception of the following parameters:
Stationary phase: HALO-C18 2,7 µm; 100 × 2,1 mm with guard column
Column temperature: 50 °C
2 5 2
2) 1 bar = 0,1 MPa = 0,1 N/mm = 10 N/m .
8 © ISO 2014 – All rights reserved

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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Pressure: 250 bar/3 600 psi
Gradient: Time [min]: Eluent 2 [%]: Flow [ml]:
1,0 0 0,5
15,0 47 0,5
30,0 80 0,5
35,0 80 0,5 start flow gradient
40,0 80 flow gradient from
0,5 to 1,2 ml/min
45,0 100 1,2 end flow gradient
46,0 100 1,2
46,1 0 0,5
53,0 0 0,5
Table D.1 — Reference substances (carcinogenic dyestuffs) and HPLC/DAD retention times
Number Carcinogenic dyestuff CAS Number Retention time Quantification wavelength
[min] with
400 nm 500 nm 600 nm
UHPLC
(see D.1.1)
1 Disperse Blue 1 2475–45–8 8,8 X
2 Solvent Yellow 1 4-ami- 60–09–4 14,6 X
noazobenzene
3 Solvent Yellow 2 60–11–7 21,2 X
4 Solvent Yellow 3 o-ami- 97–56–3 18,2 X
noazotoluene
5 Basic Red 9 569–61–9 1,9 X
6 Basic Violet 14 632–99–5 6,5 X
7 Disperse Yellow 3 2832–40–8 15,8 X
8 Acid Red 26 3761–53–3 15,1 X
9 Direct Black 38 1937–37–1 17,7 X
18,7
10 Direct Blue 6 2602–46–2 16,8 X
11 Direct Red 28 573–58–0 16,0 X
12 Disperse Orange 11 82–28–0 15,6 X
13 Acid Red 114 6459–9-5 24,6 X
NOTE All standards of Tables D.1 and D.2 were diluted in methanol and as well in pyridine/water 1:1. Comparing the two
solutions with HPLC/DAD, no differences in retention time as well in DAD spectra occurred.
© ISO 2014 – All rights reserved 9

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SIST EN ISO 16373-2:2014
ISO 16373-2:2014(E)

Table D.2 — Reference substances (allergenic and other dyestuffs) and HPLC/DAD retention
times
Number Allergenic
...