SIST EN ISO 17234-1:2025

SLOVENSKI STANDARD
01-februar-2025
Usnje - Kemijski preskusi za določevanje nekaterih azo barvil na barvanem usnju -
1. del: Določevanje nekaterih aromatskih aminov, pridobljenih iz azo barvil (ISO
17234-1:2024)
Leather - Chemical tests for the determination of certain azo colourants in dyed leathers
- Part 1: Determination of certain aromatic amines derived from azo colourants (ISO
17234-1:2024)
Leder - Chemische Prüfungen zur Bestimmung bestimmter Azofarbstoffe in gefärbten
Ledern - Teil 1: Bestimmung bestimmter aromatischer Amine aus Azofarbstoffen (ISO
17234-1:2024)
Cuir - Essais chimiques pour le dosage de certains colorants azoïques dans les cuirs
teints - Partie 1: Dosage de certaines amines aromatiques dérivées des colorants
azoïques (ISO 17234-1:2024)
Ta slovenski standard je istoveten z: EN ISO 17234-1:2024
ICS:
59.140.30 Usnje in krzno Leather and furs
71.040.40 Kemijska analiza Chemical analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 17234-1
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2024
EUROPÄISCHE NORM
ICS 59.140.30 Supersedes EN ISO 17234-1:2020
English Version
Leather - Chemical tests for the determination of certain
azo colourants in dyed leathers - Part 1: Determination of
certain aromatic amines derived from azo colourants (ISO
17234-1:2024)
Cuir - Essais chimiques pour le dosage de certains Leder - Chemische Prüfungen zur Bestimmung
colorants azoïques dans les cuirs teints - Partie 1: bestimmter Azofarbstoffe in gefärbten Ledern - Teil 1:
Dosage de certaines amines aromatiques dérivées des Bestimmung bestimmter aromatischer Amine aus
colorants azoïques (ISO 17234-1:2024) Azofarbstoffen (ISO 17234-1:2024)
This European Standard was approved by CEN on 9 December 2024.

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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17234-1:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 17234-1:2024) has been prepared by Technical Committee ISO/IULTCS
"International Union of Leather Technologists and Chemists Societies" in collaboration with Technical
Committee CEN/TC 289 “Leather” the secretariat of which is held by UNI.
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 June 2025, and conflicting national standards shall be
withdrawn at the latest by June 2025.
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.
This document supersedes EN ISO 17234-1:2020.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. 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 17234-1:2024 has been approved by CEN as EN ISO 17234-1:2024 without any
modification.
International
Standard
ISO
17234-1
IULTCS
IUC 20-1
Fourth edition
Leather — Chemical tests for
2024-12
the determination of certain azo
colourants in dyed leathers —
Part 1:
Determination of certain aromatic
amines derived from azo colourants
Cuir — Essais chimiques pour le dosage de certains colorants
azoïques dans les cuirs teints —
Partie 1: Dosage de certaines amines aromatiques dérivées des
colorants azoïques
Reference numbers
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en) © ISO 2024

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
© ISO 2024
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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General . 1
5 Principle . 3
6 Safety precautions. 3
7 Apparatus . 3
8 Reagents . 4
9 Sampling and preparation of samples . 5
10 Procedure . 5
10.1 Degreasing.5
10.2 Reductive cleavage .5
10.3 Liquid-liquid extraction.5
10.4 Check of the analytical system .6
11 Chromatographic analyses . 6
12 Calibration . 6
13 Evaluation . 6
13.1 Calculation of amine in the sample.6
13.2 Reliability of the method .7
14 Test report . 7
Annex A (informative) Chromatographic analyses. 8
Annex B (informative) Reliability of the method .11
Annex C (informative) Assessment guide — Interpretation of analytical results .12
Annex D (informative) Procedure for liquid/liquid extraction without diatomaceous earth .18
Annex E (normative) Colourants — Method for the determination of certain aromatic amines .21
Annex F (normative) Leather and colourants — Method for the determination of free aromatic
amines .22
Bibliography .25

iii
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of 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 www.iso.org/iso/foreword.html.
IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to further
the advancement of leather science and technology. IULTCS has three Commissions, which are responsible
for establishing international methods for the sampling and testing of leather. ISO recognizes IULTCS as an
international standardizing body for the preparation of test methods for leather.
This document was prepared by the Chemical Test Commission of the International Union of Leather
Technologists and Chemists Societies (IUC Commission, IULTCS) in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 289, Leather, in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
It is based on IUC 20 published in J. Soc. Leather Tech. Chem., 86, pp. 299-305, 2002, and declared an official
method of the IULTCS in June 2003.
This fourth edition cancels and replaces the third edition (ISO 17234-1:2020), which has been technically
revised.
The main changes are as follows:
— normative Annexes E and F have been added.
A list of all parts in the ISO 17234 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 17234-1:2024(en)
International Standard
IULTCS/IUC 20-1:2024(en)
Leather — Chemical tests for the determination of certain azo
colourants in dyed leathers —
Part 1:
Determination of certain aromatic amines derived from azo
colourants
1 Scope
This document specifies a method to determine certain aromatic amines derived from azo colourants.
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 2418, Leather — Chemical, physical, mechanical and fastness tests — Position and preparation of specimens
for testing
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4044, Leather — Chemical tests — Preparation of chemical test samples
ISO 17234-2, Leather — Chemical tests for the determination of certain azo colorants in dyed leathers — Part 2:
Determination of 4-aminoazobenzene
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 General
Certain azo colourants can release, by reductive cleavage of azo group(s), one or more of the aromatic amines
[2] [3]
listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and GB 20400-2006 (see Table 1).

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
[2]
Table 1 — Aromatic amines listed in EU Regulation 1907/2006, Annex XVII, Appendix 8 and
[3]
GB 20400-2006
No. CAS number Index number EC number Substances
1 92–67–1 612–072–00–6 202–177–1 biphenyl-4-ylamine
4-aminobiphenyl
xenylamine
2 92–87–5 612–042–00–2 202–199–1 benzidine
3 95–69–2 612–196–00–0 202–441–6 4-chloro-o-toluidine
4 91–59–8 612–022–00–3 202–080–4 2-naphthylamine
a
5 97–56–3 611–006–00–3 202–591–2 o-aminoazotoluene
4-amino-2',3-dimethylazobenzene
4-o-tolylazo-o-toluidine
a
6 99–55–8 612–210–00–5 202–765–8 5-nitro-o-toluidine
2-amino-4-nitrotoluene
7 106–47–8 612–137–00–9 203–401–0 4-chloroaniline
8 615–05–4 612–200–00–0 210–406–1 4-methoxy-m-phenylenediamine
2,4-diaminoanisole
9 101–77–9 612–051–00–1 202–974–4 4,4'-methylenedianiline
4,4'-diaminodiphenylmethane
10 91–94–1 612–068–00–4 202–109–0 3,3'-dichlorobenzidine
3,3'-dichlorobiphenyl-4,4'-ylenediamine
11 119–90–4 612–036–00-X 204–355–4 3,3'-dimethoxybenzidine
o-dianisidine
12 119–93–7 612–041–00–7 204–358–0 3,3'-dimethylbenzidine
4,4'-bi-o-toluidine
13 838–88–0 612–085–00–7 212–658–8 4,4'-methylenedi-o-toluidine
14 120–71–8 612–209–00–X 204–419–1 6-methoxy-m-toluidine
p-cresidine
15 101–14–4 612–078–00–9 202–918–9 4,4'-methylene-bis-(2-chloro-aniline)
2,2'-dichloro-4,4’-methylene-dianiline
16 101–80–4 612–199–00–7 202–977–0 4,4'-oxydianiline
17 139–65–1 612–198–00–1 205–370–9 4,4'-thiodianiline
18 95–53–4 612–091–00-X 202–429–0 o-toluidine
2-aminotoluene
19 95–80–7 612–099–00–3 202–453–1 4-methyl-m-phenylenediamine
2,4-toluylendiamine
2,4-diaminotoluene
20 137–17–7 612–197–00–6 205–282–0 2,4,5-trimethylaniline
21 90–04–0 612–035–00–4 201–963–1 o-anisidine
2-methoxyaniline
b
22 60–09–3 611–008–00–4 200–453–6 4-aminoazobenzene
c
23 95–68–1 612–027–00–0 202–440–0 2,4-xylidine
2,4-dimethylbenzene-1-amine
c
24 87–62–7 612–161–00-X 201–758–7 2,6-xylidine
2,6-dimethylbenzene-1-amine
a
The CAS-numbers 97–56–3 (no. 5) and 99–55–8 (no. 6) are further reduced to CAS-numbers 95–53–4 (no. 18) and 95–80–7
(no. 19).
b
Azo colourants that are able to form 4-aminoazobenzene generate under the condition of this method aniline (CAS-number
62–53–3) and 1,4-phenylenediamine (CAS number 106–50–3). The presence of these colourants shall be tested using ISO 17234-2.
c
Additional aromatic amines in GB 20400–2006.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
5 Principle
After degreasing, the leather sample is treated with sodium dithionite in an aqueous buffer solution (pH 6)
at 70 °C in a closed vessel. The amines released in the process of reductive cleavage are transferred to a
t-butyl methyl ether (8.5) phase by means of liquid-liquid extraction using diatomaceous earth columns. The
t-butyl methyl ether (8.5) extract is then concentrated under mild conditions in a rotary vacuum evaporator
and the residue is dissolved in a suitable solvent, depending on the method used to determine the amines
(see Annex A).
Determination of the amines is performed by means of liquid chromatography (LC) using a diode array
detector (DAD) or mass selective detector (LC-MS), by capillary gas chromatography with a mass selective
detector (GC-MS) or by capillary electrophoresis with a diode array detector (CE-DAD), or qualitatively with
(high-performance) thin layer chromatography (TLC, HPTLC).
The amines shall be identified by means of at least two different chromatographic separation methods in
order to avoid any possible misinterpretations caused by interfering substances (such as position isomers
of the amines to be identified) and hence any incorrect statements. Amine quantification shall be performed
by LC-DAD, LC-MS or GC-MS.
A screening method using liquid-liquid extraction without diatomaceous earth columns is described in
Annex D.
If it is required to analyse the colourant itself, the method in Annex E shall be used.
If it is required to analyse for residual free aromatic amines in the leather or colourant, the method in
Annex F shall be used.
6 Safety precautions
WARNING — The aromatic amines listed in Clause 4 are classified as substances known to be or
suspected to be human carcinogens.
6.1 It is the user's responsibility to use safe and proper techniques when handling materials in this
test method. Consult manufacturers for specific details, such as material safety data sheets and other
recommendations.
6.2 Good laboratory practice should be followed. Wear safety glasses in all laboratory areas and a dust
respirator and single-use gloves while handling powder colourants and aromatic amines.
6.3 National and local safety regulations can apply.
7 Apparatus
The usual laboratory equipment and, in particular, the following is used.
7.1 Suitable reaction vessel, made of temperature-resistant glass with a gas-tight closure.
7.2 Suitable heating system, at (70 ± 2) °C.
7.3 Polypropylene or glass column, inside diameter 25 mm to 30 mm, length 130 mm to 150 mm, packed
with 20 g of diatomaceous earth, fitted with glass fibre filter at the outlet.
The diatomaceous earth columns are either bought pre-packed and used as is, or 20 g of diatomaceous earth
can be packed into a glass or polypropylene column of the dimensions given.
7.4 Vacuum rotary evaporator with vacuum control and water bath.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
7.5 Pipettes, in required sizes or variables pipettes.
7.6 Ultrasonic bath with thermostat.
7.7 Chromatographic equipment, selected from the following.
7.7.1 Liquid chromatography (LC) and DAD or MS.
7.7.2 Capillary gas chromatography (GC), with MS.
7.7.3 Capillary electrophoresis (CE), with DAD.
7.7.4 Thin layer chromatography (TLC) or high-performance thin layer chromatography (HPTLC).
NOTE A description of the chromatographic equipment (7.7) is given in Annex A.
8 Reagents
Unless otherwise specified, analytical grade chemicals shall be used.
8.1 n-hexane.
8.2 Citrate buffer solution, 0,06 mol/l, pH = 6, preheated to (70 ± 2) °C.
1)
8.3 Aqueous sodium dithionite solution, ρ = 200 mg/ml , freshly prepared, to be used immediately
after resting for 1 h in a closed vessel.
8.4 Sodium hydroxide aqueous solution, a mass fraction of 40 %.
8.5 t-butyl methyl ether.
8.6 Methanol.
8.7 Acetonitrile.
8.8 Amines, listed in Table 1 (highest available purity standard).
8.9 Standard solutions.
8.9.1 Stock solution of the amines (8.8), 400 µg/ml in ethyl acetate for TLC.
8.9.2 Stock solution of the amines (8.8), 200 µg/ml of each amine in an appropriate solvent.
NOTE Acetonitrile is an appropriate solvent for this stock solution, resulting in good stability of amines.
8.9.3 Standard solution for amine process control, 30 µg amine per millilitre solvent, freshly prepared
from stock solutions (8.9.1 or 8.9.2) depending on the analytical method.
1) ρ = mass concentration.
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
8.9.4 Internal standard in solution (IS), ρ = 10 μg of IS/ml of t-butyl methyl ether (8.5).
In the case of GC-MS analysis, one of the following internal standards can be used:
— IS1: naphthalene-d8, CAS no. 1146-65-2;
— IS2: 2,4,5-trichloroaniline (TCA), CAS no. 636-30-6;
— IS3: anthracene-d10, CAS no. 1719-06-8.
8.10 Water, Grade 3 according to ISO 3696.
9 Sampling and preparation of samples
The leather shall be sampled in accordance with ISO 2418 and prepared in accordance with ISO 4044. If
sampling in accordance with ISO 2418 is not possible (e.g. in the case of leathers from finished products such
as shoes or garments), details about sampling shall be given in the test report. Any traces of adhesives shall
be removed mechanically.
In the case of leather patchwork fabrics with varicoloured patterns, the various colours shall be taken into
account separately as far as possible. For commodities consisting of various leather qualities, specimens of
the various qualities shall be analysed separately.
10 Procedure
10.1 Degreasing
Weigh a representative specimen of (1,0 ± 0,1) g to the nearest 0,01 g of the leather sample in the reaction
vessel (7.1), and add 40 ml n-hexane (8.1). Close the vessel (7.1) and put it in an ultrasonic bath (7.6) at
(40 ± 2) °C for (40 ± 5) min.
Decant the n-hexane layer from the leather specimen. Any loss of leather particles during decanting shall be
avoided. Evaporate the residual n-hexane at least overnight in the open vessel.
10.2 Reductive cleavage
Add 15 ml buffer solution (8.9) preheated to (70 ± 2) °C to the sample.
Close the reaction vessel tightly and treat for (30 ± 1) min at (70 ± 2) °C.
Subsequently, add 3 ml aqueous sodium dithionite solution (8.3) for the reductive cleavage of the azo groups
to the reaction vessel, then shake vigorously and immediately keep at (70 ± 2) °C for another (30 ± 1) min.
Then cool to room temperature (20 °C to 25 °C) within 2 min with a cooling mixture of ice, water and salt.
10.3 Liquid-liquid extraction
Add 1,5 ml of the NaOH solution (8.4) to the reaction solution and shake vigorously. Transfer the reaction
solution to the diatomaceous earth column (7.3) and allow it to be absorbed by the column for 15 min.
Meanwhile, add 10 ml t-butyl methyl ether (8.5) to the reaction vessel and shake vigorously. After the 15 min
period decant the t-butyl methyl ether (8.5) onto the top of the column and collect the eluate in a 250 ml
round-bottom flask.
Rinse the reaction vessel with 10 ml t-butyl methyl ether (8.5) and transfer the solvent to the column.
Subsequently, pour 60 ml t-butyl methyl ether (8.5) directly onto the column.
For amine detection and quantification, the t-butyl methyl ether extract is concentrated to a volume less than
5 ml (not to dryness) with a vacuum rotary at a temperature less than 50 °C and a pressure of approximately

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
2)
450 mbar . If it is necessary to change to another solvent, remove the remainder of the solvent very carefully
by means of a weak flow of inert gas.
NOTE 1 Removal of the solvent (concentration in the rotary vacuum evaporator, evaporation to dryness) can lead
to substantial amine losses if performed under uncontrolled conditions.
Make up the extract or residue to 2,0 ml with an appropriate solvent for detection and determination of
the amines using chromatography [acetonitrile (8.7), t-butyl methyl ether (8.5) or methanol (8.6)] without
delay. If the complete analysis cannot be performed within (24 ± 1) h, keep the extract at (−18 ± 3) °C and
warm carefully to room temperature before analysis.
NOTE 2 Owing to the matrix, individual amines such as 2,4-diaminotoluene and 2,4-diaminoanisole are likely to
exhibit a very poor stability, especially in methanol. Where delays occur in the work routine, it is possible that amines
are no longer detectable by the time of instrumental measurement.
10.4 Check of the analytical system
Amine recovery rates shall conform with the following minimum requirements:
— amines nos 1 to 4, 7, 9 to 17 and 20 to 21: recovery rate 70 %;
— amine no. 8: recovery rate 20 %;
— amines nos 18, 19, 23 and 24: recovery rate 50 %;
— amines nos 5, 6 and 22, see footnotes to Table 1.
If an amine recovery does not comply with the appropriate minimum requirement, then check the procedure
and perform a new test.
11 Chromatographic analyses
The detection of the aromatic amines can be performed using the chromatographic techniques listed in 7.7
and examples described in Annex A. Other validated methods can be used. The quantification of the aromatic
amines is performed by means of LC-DAD, LC-MS or GC-MS. Where gas chromatography is used, appropriate
internal standards as described in 8.9.4 shall be employed.
If any amine is detected by one chromatographic method, then confirmation shall be made using one or
more alternative methods. The result is positive only if both methods give a positive result.
12 Calibration
Use the standard solution (8.9.2) to prepare at least three calibration solutions in a range of 2 µg/ml to
30 µg/ml.
13 Evaluation
13.1 Calculation of amine in the sample
Calculate the amine concentration based on the peak areas of the individual amine components. Calculate
the content of the amine as a mass fraction, w, in milligrams of the individual component per kilogram of
leather material (mg/kg) according to Formula (1):
AV×
s
w=×ρ (1)
c
Am×
cE
5 2
2) 1 bar = 0,1 MPa = 10 Pa; 1 MPa = 1 N/mm

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
where
ρ is the concentration of the amine in the calibration solution, in micrograms per millilitre (μg/ml);
c
A is the peak area of the amine in the sample solution, in area units;
s
A is the peak area of the amine in the calibration solution, in area units;
c
V is the volume of the specimen according to 10.3 (final sample volume), in millilitres (ml) (here 2 ml);
m is the mass of the leather specimen, in grams (g).
E
13.2 Reliability of the method
For the reliability of the method, see Annex B.
14 Test report
The test report shall refer to this official method and give information on at least the following aspects:
a) a reference to this document, i.e. ISO 17234-1:2024;
b) identification of the sample;
c) sampling procedure;
d) any deviations from the analytical procedure, particularly any additional steps performed;
e) declaration of analytical techniques used for detection and confirmation;
f) the date of the test;
g) the analytical results for the amines in milligrams per kilogram (see Clause 13), individually listed and
reported according to the identification threshold values as follows:
— In the case of levels per amine component ≤ 30 mg/kg:
According to the analysis as carried out, azo colourants which release the listed aromatic amines
were not detected.
— In the case of levels per amine component > 30 mg/kg:
The analysis result suggests that the leather submitted has been manufactured or treated using azo
colourants which release one or more of the listed amines.
— In the case of levels of 4-aminodiphenyl and/or 2-naphthylamine and the 4-methoxy-m-
phenylenediamine > 30 mg/kg:
Use of this analytical method has detected 4-aminodiphenyl and/or 2-naphthylamine. According to
the current state of knowledge it cannot be unequivocally confirmed without additional information
that azo colourants which release amines were used.
Care should be taken in the interpretation of less than 30 mg/kg of amines as these can be due to false-
positive results. For the interpretation of results, see Annex C.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Annex A
(informative)
Chromatographic analyses
A.1 Preliminary remark
As the chromatographic equipment (7.7) of the laboratories can vary, no generally applicable instructions can
be provided for chromatographic analyses. The following parameters have been successfully tested and used.
A.2 Liquid chromatography (LC)
A.2.1 Liquid chromatography/diode array detector (LC-DAD)
Eluent 1: methanol;
Eluent 2: 0,575 g of ammonium dihydrogen phosphate + 0,7 g of disodium hydrogen
phosphate in 1 000 ml of water, pH 6,9;
Stationary phase: LiChrospher 60 RP-select B (5 µm); length: 250 mm; inside diameter: 4,6 mm;
Flow rate: (0,7 to 1,0) ml/min;
Gradient: start: 15 % eluent 1, linear increase to 80 % eluent 1 within 45 min;
Column temperature: 40 °C;
Injection volume: 10,0 μl;
Detection: DAD, spectrograph;
Quantification: at 240 nm, 280 nm and 305 nm.
NOTE LiChrospher 60 RP-select B is an example of a suitable product available commercially. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.
Equivalent products can be used if they can be shown to lead to the same results.
A.2.2 liquid chromatography/mass selective detector (LC-MS)
Eluent 1: acetonitrile;
Eluent 2: ammonium acetate in 1 000 ml of water, 5 mmol, pH 3,0;
Stationary phase: C18 (3,5 μm); length: 50 mm; inside diameter: 2,1 mm;
Flow rate: 300 μl/min;
Gradient: see Table A.1;
Column temperature 40 °C;
Injection volume: 2,0 μl;
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Detection: quadrupole-trap and/or ion-trap mass detector, scanning mode and/or MS
daughter ion MS detection; DAD: for wavelengths, see A.2.1;
Spray gas: nitrogen (bottled/generator);
Ionization: API electrospray positive, fragmentor 120 V.
Table A.1 — Gradient programme
Time Eluent 1 Eluent 2
min % %
0 10 90
1,5 20 80
7,5 90 10
A.3 Capillary gas chromatography (GC-MS)
Capillary column: medium polarity, e.g. SE 54 or DB 5, length: 50 m, inside diameter: 0,32 mm,
film thickness: 0,5 μm;
Injector system: split/splitless;
Injector temperature: 250 °C;
Carrier gas: helium;
Temperature programme: 70 °C (2 min), 70 °C to 280 °C (at 10 °C/min), 280 °C (5 min);
Injection volume: 1,0 μl, splitless 2 min;
Detection: MS, scan 45 amu to 300 amu.
A.4 Capillary electrophoresis (CE-DAD)
250 μl of the sample solution (10.3) is mixed with 50 μl HCl (c = 0,01 mol/l) and passed through a membrane
filter (0,2 μm). This solution is analysed by means of capillary zone electrophoresis.
Capillary 1: 56 cm, uncoated, inside diameter 50 μm, with extended light path;
Capillary 2: 56 cm, coated with polyvinyl alcohol (PVA), inside diameter 50 μm, with
extended light path;
Buffer solution: phosphate buffer solution (c = 50 mmol/l), pH 2,5;
Column temperature: 25 °C;
Voltage: 30 kV;
Injection time: 4 s;
Flushing time: 5 s;
Detection: DAD spectrograph at 214 nm, 240 nm, 280 nm, 305 nm.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
A.5 Thin-layer chromatography (TLC); HPTLC or TLC only for semiquantitative
confirmation
A.5.1 General
Plates (HPTLC): silica gel 60 with fluorescence indicator F254, (20 × 10) cm;
Applied volume: 5 μl, applied as a line with automatic applicator;
Mobile solvent 1: chloroform/acetic acid (90 + 10) parts per volume.

Plates (TLC): silica gel 60, (20 × 10) cm, saturated chamber;
Applied volume: 10,0 μl, applied as a dot with an automatic applicator;
Mobile solvent 2: chloroform/ethyl acetate/acetic acid (60 + 30 + 10) parts per volume;
Mobile solvent 3: chloroform/methanol (95 + 5) parts per volume;
Mobile solvents 2 and 3: successively without drying of the plates.

Detection: 1) ultraviolet (UV) lamp;
2) after successive treatment with reagents 2 and 3, reaction time
approximately 5 min;
Reagent 1: 0,1 % NaNO in KOH (c = 1 mol/l);
Reagent 2: 0,2 % α-naphthol in KOH (c = 1 mol/l);
Reagent 3: 0,5 % to 1,0 % of ammonium sulphamate in methanol.
A.5.2 Derivatization procedure
After developing the TLC plate, it is dried in air or by a hand-held hot air drier (e.g. hair dryer) for 1 min or
2 min. Next the plate is immersed in reagent 1 for 30 s to 1 min, then immersed in reagent 3 for 30 s to 1 min.
The plate is dried like earlier and then immersed in reagent 2 for 1 min. The plate is then dried by a hot air
drier. Instead of immersion, spraying the reagents using an atomizer is also possible.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Annex B
(informative)
Reliability of the method
The data indicated in Table B.1 were obtained in an interlaboratory collaborative trial on different kinds of
leathers. The data were obtained by using LC with DAD. The leather specimens were ground according to
TM ®
ISO 4044. For liquid-liquid extraction Merck columns, type EXtrelut NT201, were used.
Table B.1 — Interlaboratory trial — Precision data
Repeatability Reproducibility
Mean
Leather
Detected amines r R
sample
mg/kg
mg/kg mg/kg
A Benzidine 13,5 5,4 8,4
3,3’-Dimethoxybenzidine 15,4 4,4 6,4
3,3’-Dimethylbenzidine 20,5 7,1 9,5
B Benzidine 12,9 3,8 8,9
2-Toluidine 37,5 15,4 38,5
C 3,3’-Dimethylbenzidine 25,6 8,0 17,0
2-Toluidine 50,1 20,2 42,1
D Benzidine 16,5 3,0 7,1
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Annex C
(informative)
3)
Assessment guide — Interpretation of analytical results
C.1 General
C.1.1 General
This annex gives complementary technical guidance but does not question the results obtained following
the procedure described in this document.
As the occurrence of the amines in very small amounts can lead to false-positive results, the Regulation
[2]
REACH 1907/2006/Annex XVII defines a limit value of 30 mg/kg of sample material. This value only
applies to a single test specimen.
If the detected amount of amine is over 30 mg/kg, it shall be assumed that an azo colourant which release
amines (see Table 1) was used. Below 30 mg/kg, it is at present not possible to make a reliable statement on
the use of certain azo colourants which release amines (see Table 1) without further information, such as the
type and/or purity of the used colourants or the other raw material used.
Assign a specimen with reduced mass as a minor component and give the advice of a greater uncertainty
due to lower material homogeneity.
Due to the existence of isomers for some targeted amines (see Table C.1), the laboratory should ensure that
the chromatographic and spectral characteristics of the detected analytes are equivalent to the standard
amine substances.
C.1.2 Determination of 4-aminoazobenzene
Azo colourants that are able to form 4-aminoazobenzene generate, under the condition of this method,
aniline and 1,4-phenylenediamine. Due to detection limits and recovery of 1,4-phenylenediamine, only
aniline can be detected. If aniline is detected above 5 mg/kg in a combined test specimen of three parts,
then the presence of 4-aminoazobenzene-releasing colourants shall be tested according to ISO 17234-2.
C.1.3 False-positive results
Table C.1 shows substances which can generate false-positive results (including interferences by isomers).
3) Adapted from ISO 14362-1:2017, Annex C.

ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
Table C.1 — Listing of possible reasons for false-positive results
List of aromatic amines Reasons and substances for false-positive test results
No. CAS no. Remarks
Chemical name/
Chemical name Chemical structure Chemical structure
number of isomers
Solvent Yellow 7
(SY7) = 4-phenylazo-
Only aminobiphe-
phenol = 4-hydroxya-
nyl findings are
zobenzene
unusual.
Such findings can
originate from
biphe- dyestuffs which
Acid Red1 (AR1)
nyl-4-ylamine form 4-amino-
1 92–67–1
4-aminobiphenyl biphenyl during
p-xenylamine the procedure by
molecular rear-
rangement.
Three dyestuffs
Direct Black 168
of this kind are
listed.
No further action
2 92–87–5 benzidine
needed.
Take care of the
4-chloro-o-tolu-
3 95–69–2 10 isomers in all separation of
idine
isomers.
Desulfonation
possible — low
Reactive Red 174
commercial rele-
vance.
2-naphthyl-
Take care of
4 91–59–8
Dyes based on Tobias
amine
impurities of
Acid
2-naphthylamine.
Take care of the
Two isomers in all separation of
isomers.
o-aminoazotol-
uene
4-amino-2',3-di- Detected as
5 97–56–3 methylazoben- o-toluidine, look at
zene no. 18.
4-o-tolyla-
zo-o-toluidine
5-nitro-o-tolu- Detected as
idine 2,4-toluylene-
6 99–55–8
2-amino-4- diamine, look at
nitrotoluene no. 19.
Take care of the
7 106–47–8 4-chloroaniline Three isomers in all separation of
isomers.
Two reduction
steps:
1)  step to 2-meth-
Pigment Red 23
oxy-5-nitroani-
line;
2)  step to
4-methoxy-m-phe-
4-meth-
oxy-m-phenylen- nylene-diamine.
ediamine
8 615–05–4
Two dyes with
2-methoxy-5-
2,4-diaminoan-
Pigment Orange 3
nitroaniline azo
isole
bounded are listed
beside (see C.2.1.1
and C.2.2.2).
Take care of the
Six isomers in all separation of
isomers.
ISO 17234-1:2024(en)
IULTCS/IUC 20-1:2024(en)
TTabablele C C.11 ((ccoonnttiinnueuedd))
List of aromatic amines Reasons and substances for false-positive test results
No. CAS no. Remarks
Chemical name/
Chemical name Chemical structure Chemical structure
number of isomers
Foams and print
Polyurethane
fixing, prepoly-
polymers of
mers, high tem-
4,4'-methylenedi-phe-
perature cleavage
4,4'-methylene-
nyl-diisocyanate
confirm GC result
dianiline
(MDI)
9 101–77–9
by LC technique.
4,4'-diaminodi-
phenyl-methane
N,N'-(methylene-
Cross-linking
di-p-pheny-lene) bis
auxiliary for print
(aziridine-1-carbox-
applications.
amide)
No further action
needed.
For information,
3,3'-dichloro-
combinations of
benzidine
Pigment Black
10 91–94–1 3,3'-dichloro-
7 with Pigment
biphenyl-4,4'-
Orange 13 or Pig-
ylene-diamine
ment Orange 34
have been known
to release the con-
cerned amine.
3,3'-dimethoxy-
No further action
11 119–90–4 benzidine
needed.
o-dianisidine
High tempera-
ture cleavage of
CI Azoic Coupling
amides, confirm
Component 5
GC result by LC
technique.
3,3'-dimethyl-
Dyes on base of
benzidine
12 119–93–7
CI Azoic Coupling
4,4'-bi-o-tolui-
Component 5,
dine
Dyes on base of CI
high tempera-
Azoic Coupling Compo-
ture cleavage of
nent 5
amides, confirm
GC result by LC
technique.
No further action
needed (note that
4,4'-methyl- a compound with
...