SIST EN 71-12:2017

SLOVENSKI STANDARD
01-april-2017
1DGRPHãþD
SIST EN 71-12:2013
9DUQRVWLJUDþGHO1QLWUR]DPLQLLQ1QLWUR]DELOQHVQRYL
Safety of toys - Part 12: N-Nitrosamines and N-nitrosatable substances
Sicherheit von Spielzeug - Teil 12: N-Nitrosamine und N-nitrosierbare Stoffe
Sécurité des jouets - Partie 12 : N-Nitrosamines et substances N-nitrosables
Ta slovenski standard je istoveten z: EN 71-12:2016
ICS:
97.200.50 ,JUDþH Toys
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 71-12
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2016
EUROPÄISCHE NORM
ICS 97.200.50 Supersedes EN 71-12:2013
English Version
Safety of toys - Part 12: N-Nitrosamines and N-nitrosatable
substances
Sécurité des jouets - Partie 12 : N-Nitrosamines et Sicherheit von Spielzeug - Teil 12: N-Nitrosamine und
substances N-nitrosables N-nitrosierbare Stoffe
This European Standard was approved by CEN on 30 October 2016.

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
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 71-12:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Requirements . 7
4.1 Requirements for finger paints . 7
4.2 Requirements for elastomers . 7
5 Principle . 8
6 Reagents and apparatus . 8
6.1 Reagents . 8
6.2 Standards . 9
6.3 Apparatus . 10
7 Sample preparation . 11
7.1 General . 11
7.2 Finger paints . 11
7.2.1 N-nitrosamines. 11
7.2.2 N-nitrosatable substances . 12
7.3 Elastomers . 12
7.3.1 Sample preparation for balloons. 12
7.3.2 Sample preparation for other toys or parts of toys than balloons . 12
7.3.3 Nitrosation . 13
7.3.4 Preparation of solution for determination of N-nitrosamines by HPLC-MS/MS . 13
8 Methods of analysis . 13
8.1 General . 13
8.2 HPLC chromatographic conditions . 14
8.3 MS/MS conditions . 14
8.4 Confirmation of detected N-nitrosamines . 15
9 Calculation of results for N-nitrosamines and N-nitrosatable substances . 16
9.1 Finger paint . 16
9.1.1 General . 16
9.1.2 N-nitrosamines. 16
9.1.3 N-nitrosatable substances . 17
9.2 Elastomers . 17
9.2.1 General . 17
9.2.2 N-nitrosamines. 18
9.2.3 N-nitrosatable substances . 18
10 Method performance . 19
10.1 Finger paints . 19
10.1.1 Repeatability, reproducibility and measurement uncertainty . 19
10.1.2 Limits of detection (LOD) and quantification (LOQ) . 19
10.2 Elastomers . 19
10.2.1 Repeatability, reproducibility and measurement uncertainty . 19
10.2.2 Limits of detection and quantification . 20
11 Test report . 21
Annex A (informative) Background and rationale for this European Standard . 22
A.1 Definition of elastomer . 22
A.2 Requirements for finger paints (see 4.1) . 22
A.3 Requirements for elastomers (see 4.2) . 23
A.4 Avoidance N-nitrosamines in finger paints . 24
A.5 Explanation for decision to do static migration for elastomers and for the specified
migration times . 24
A.6 Reference to other standards and relevant deviations . 25
A.7 Conditions for nitrosation . 25
A.8 NDELA in balloons . 26
A.9 Selection of N-nitrosamines specified in this standard . 26
Annex B (informative) Significant technical changes between this European Standard and
the previous version . 27
Annex C (informative) A–deviations . 29
Annex ZA (informative) Relationship between this European Standard and the essential
requirements of EU Directive 2009/48/EC aimed to be covered . 30
Bibliography . 31
European foreword
This document (EN 71-12:2017) has been prepared by Technical Committee CEN/TC 52 “Safety of
toys”, the secretariat of which is held by DS.
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 2017, and conflicting national standards shall be
withdrawn at the latest by June 2017.
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 71-12:2013.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive 2009/48/EC.
For relationship with EU Directive 2009/48/EC, see informative Annex ZA, which is an integral part of
this document.
Additional information on the rationale for various requirements is given in Annex A. Annex B provides
details of significant technical changes between this European Standard and the previous edition.
This European Standard constitutes the 12th part of the European Standard on safety of toys.
This European Standard for safety of toys consists of the following parts:
— Part 1: Mechanical and physical properties;
— Part 2: Flammability;
— Part 3: Migration of certain elements;
— Part 4: Experimental sets for chemistry and related activities;
— Part 5: Chemical toys (sets) other than experimental sets;
— Part 7: Finger paints — Requirements and test methods;
— Part 8: Activity toys for domestic use;
— Part 9: Organic chemical compounds — Requirements;
— Part 10: Organic chemical compounds — Sample preparation and extraction;
— Part 11: Organic chemical compounds — Methods of analysis;
— Part 12: N-nitrosamines and N-nitrosatable substances;
— Part 13: Olfactory board games, gustative board games, cosmetic kits and gustative kits;
— Part 14: Trampolines for domestic use.
NOTE 1 In addition to the above parts of EN 71, the following guidance documents have been published: CEN
Technical Report CEN/TR 15071, Safety of toys — National translations of warnings and instructions for use in
EN 71, and CEN Technical Report CEN/TR 15371 (all parts), Safety of toys — Interpretations.
NOTE 2 Words in italics are defined in Clause 3 (Terms and definitions).
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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.
1 Scope
This European Standard specifies the requirements and test methods for N-nitrosamines and N-
nitrosatable substances for:
— toys and parts of toys made from elastomers and intended for use by children under 36 months;
— toys and parts of toys made from elastomers and intended to be placed in the mouth;
— finger paints for children under 36 months.
EXAMPLE Examples of toys made from elastomers are balloons and teethers.
NOTE With regard to elastomers, up to now N-nitrosamines and N-nitrosatable substances have mainly been
detected in vulcanized materials.
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.
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
finger paint
aqueous semi-solid or liquid, coloured mixture specially designed for children to apply directly to
suitable surfaces with the fingers and hands
[SOURCE: EN 71-7:2014, definition 3.1]
3.2
elastomer
flexible cross-linked macro-molecular material which returns rapidly to approximately its initial
dimensions and shape after substantial deformation by stress and release of the stress at room
temperature
Note 1 to entry: Not all elastomers are likely to contain N-nitrosamines and N-nitrosatable substances. For
further guidance on possible sources of N-nitrosamines and N-nitrosatable substances in elastomers the term
elastomer and other related aspects see A.1.
[SOURCE: EN ISO 472:2013, 2.327, modified: the phrases “flexible cross-linked”, “approximately” and
“at room temperature” and note 1 to entry added and the phrase “a weak” deleted]
3.3
N-nitrosamine
substance characterised by the -N-N=O functional group, usually formed by the reaction of an amine
with a nitrosating agent at acidic pH
Note 1 to entry: The reacting amines primarily are secondary amines.
Note 2 to entry: An example for a nitrosating agent is nitrite.
3.4
N-nitrosatable substance
substance which when released into the test solution undergoes nitrosation to form a N-nitrosamine
under specified conditions
4 Requirements
4.1 Requirements for finger paints
The concentration of N-nitrosamines in finger paints shall not exceed 0,02 mg/kg when tested in
accordance with 7.2 and Clause 8, calculated as sum of all detected N-nitrosamines according to 9.1.
The migration of N-nitrosatable substances from finger paints shall not exceed 1 mg/kg of toy material,
calculated as sum of all detected N-nitrosamines after nitrosation.
At least the substance in Table 1 shall be determined in finger paint. If there are indications of the
presence of other N-nitrosamines, they shall also be determined.
Table 1 — List of N-nitrosamines in finger paints
Name of the substance CAS number Abbreviation
N-nitrosodiethanolamine 1116–54–7 NDELA
4.2 Requirements for elastomers
The migration of N-nitrosamines (calculated as sum of all detected N-nitrosamines according to 9.2) and
of N-nitrosatable substances (calculated as sum of all detected N-nitrosamines after nitrosation) from
toys or parts of toys shall not exceed the limit values specified in Table 2 when tested in accordance
with 7.3 and Clause 8.
Table 2 — Limit values for elastomers
Product type N-nitrosamines N-nitrosatable substances
mg/kg mg/kg
a) toys intended for use by
children under 36 months and
0,01 0,1
intended or likely to be placed
into the mouth
b) toys intended for use by
children under 36 months not 0,05 1
covered by a)
c) toys intended for use by
children of 36 months and over
0,05 1
and intended to be placed into
the mouth
d) balloons 0,05 1
At least the substances in Table 3 shall be determined in elastomers. If there are indications of the
presence of other N-nitrosamines they shall also be determined.
Table 3 — List of N-nitrosamines in elastomers
Name of the substance CAS number Abbreviation
N-nitrosodiethanolamine 1116–54–7 NDELA
N-nitrosodimethylamine 62–75–9 NDMA
N-nitrosodiethylamine 55–18–5 NDEA
N-nitrosodipropylamine 621–64–7 NDPA
N-nitrosodiisopropylamine 601–77–4 NDiPA
N-nitrosodibutylamine 924–16–3 NDBA
N-nitrosodiisobutylamine 997–95–5 NDiBA
N-nitrosodiisononylamine 1207995–62–7 NDiNA
(N-Nitroso-N,N-di(3,5,5-
trimethylhexyl)amine))
N-nitrosomorpholine 59–89–2 NMOR
N-nitrosopiperidine 100–75–4 NPIP
N-nitrosodibenzylamine 5336–53–8 NDBzA
N-nitroso-N-methyl-N- 614–00–6 NMPhA
phenylamine
N-nitroso-N-ethyl-N-phenylamine 612–64–6 NEPhA
5 Principle
N-nitrosamines and N-nitrosatable substances migrate into a test solution. N-nitrosatable substances are
converted to N-nitrosamines by acidification. The final test solutions are analysed for N-nitrosamines by
a suitable validated analytical technique. The N-nitrosamines and N-nitrosatable substances are
expressed in mg/kg of sample.
The test solution for N-nitrosamines in finger paints is water. The test solution for N-nitrosatable
substances in finger paints and for N-nitrosamines and N-nitrosatable substances in elastomers is saliva
test solution.
6 Reagents and apparatus
6.1 Reagents
Unless otherwise specified, all reagents shall be of analytical grade and distilled water, or water of
equivalent purity conforming to at least grade 3 of EN ISO 3696.
6.1.1 Sodium hydrogen carbonate (CAS 144-55-8)
6.1.2 Sodium chloride (CAS 7647-14-5)
6.1.3 Potassium carbonate (CAS 584-08-7)
6.1.4 Sodium nitrite (CAS 7632-00-0)
6.1.5 Hydrochloric acid (CAS 7647-01-0), aqueous solution, c(HCL) = 0,1 mol/l
6.1.6 Sodium hydroxide (CAS 1310-73-2), aqueous solution, c(NaOH) = 0,1 mol/l
6.1.7 Saliva test solution
Dissolve (4,2 ± 0,021) g of the sodium hydrogen carbonate (6.1.1), (0,5 ± 0,002 5) g of the sodium
chloride (6.1.2), (0,2 ± 0,001 0) g of the potassium carbonate (6.1.3) and (0,03 ± 0,000 15) g of the
sodium nitrite (6.1.4) in water and dilute to (950 ± 5) ml with water. Adjust to pH 9,0 ± 0,1 if necessary
by adding the hydrochloric acid solution (6.1.5) or sodium hydroxide solution (6.1.6) drop by drop.
Transfer into a 1 l volumetric flask and dilute to the mark with water.
This solution has a limited stability. Saliva test solution kept for more than five days shall not be used.
6.1.8 Hydrochloric acid solution, c(HCL) = 1 mol/l.
6.1.9 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
6.1.10 Methanol, ≥ 99,9 %, HPLC-grade.
6.1.11 Formic acid, ≥ 99,5 %, LC-MS grade.
6.1.12 Eluent A: 0,1 % (volume fraction) formic acid in water.
6.1.13 Eluent B: 0,1 % (volume fraction) formic acid in methanol.
6.2 Standards
N-nitrosamines are degraded by ultraviolet light. Exposure of extracts or standards to sources such as
sunlight or fluorescent tube light should be avoided. The standards shall be protected by wrapping in
aluminium foil or using amber glassware and stored in the dark at a temperature of (5 ± 3) °C.
WARNING — N-nitrosamines can endanger human health owing to their toxicity. Persons using this
European Standard should be familiar with normal laboratory practice. This European Standard does
not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of
the user to establish appropriate safety and health practices and to ensure compliance with any
national regulatory conditions.
6.2.1 N-nitrosamines or their solutions, with known purities.
6.2.2 Internal standard, d8-N-nitrosodiethanolamine (d8-NDELA), with known purity.
6.2.3 Internal standard, d6-N-nitrosodimethylamine (d6-NDMA), with known purity.
6.2.4 Stock solution of N-nitrosamines
Prepare a stock solution containing 10 mg/l of each N-nitrosamine (6.2.1) in methanol (6.1.10). This
solution shall be stored in the absence of light at (−18 ± 3) °C.
NOTE Certified N-nitrosamines standards can be purchased from several suppliers including storage and
stability information.
6.2.5 Solutions of internal standards
6.2.5.1 Stock solution of internal standards for elastomers
Prepare a stock solution containing 1 mg/l d8-NDELA (6.2.2) and 1 mg/l d6-NDMA (6.2.3) in methanol
(6.1.10). This solution shall be stored in the absence of light at (−18 ± 3) °C.
6.2.5.2 Working solution of internal standards for finger paints
Prepare an internal standard working solution containing 1 mg/l d8-NDELA (6.2.2) in water. This
solution has a stability of one day.
6.2.6 Calibration solutions
6.2.6.1 Calibration solutions for N-nitrosamines in finger paints
Prepare five calibration solutions by diluting the stock solution of the N-nitrosamines (6.2.4) and the
working solution of d8-NDELA (6.2.5.2) with water. The concentrations of the calibration solutions shall
be 0,001 mg/l, 0,002 mg/l, 0,005 mg/l, 0,010 mg/l and 0,020 mg/l. Each solution shall contain
0,010 mg/l of d8-NDELA. These solutions have a stability of one day.
6.2.6.2 Calibration solutions for N-nitrosatable substances in finger paints
Prepare at least five calibration solutions by diluting the stock solutions of the N-nitrosamines (6.2.4)
with saliva test solution (6.1.7). The concentration range of the calibration solutions shall be 0,001 mg/l
to 0,05 mg/l. To 1 ml of each solution, add 0,020 ml of the internal standards stock solution containing
d8-NDELA and d6-NDMA (6.2.5.1). Shake to mix the solutions. These solutions have a stability of one
day.
6.2.6.3 Calibration solutions for elastomers
Prepare at least five calibration solutions by diluting the stock solutions of the N-nitrosamines (6.2.4)
with saliva test solution (6.1.7). The concentration range of the calibration solutions shall be
0,000 2 mg/l to 0,05 mg/l. To 1 ml of each solution, add 0,020 ml of the internal standards stock
solution containing d8-NDELA and d6-NDMA (6.2.5.1). Shake to mix the solutions. These solutions have
a stability of one day.
6.3 Apparatus
Laboratory equipment that can come into contact with the sample shall be free from N-nitrosamines.
Standard laboratory equipment and equipment appropriate for applying the test method shall be used.
The following equipment could be used when applying the test method specified in this standard.
6.3.1 Vortex mixer
6.3.2 Shaking machine
6.3.3 Centrifuge, capable of achieving not less than 20 000 g
6.3.4 Glass balls
6.3.5 Conical flask, made of amber glass
6.3.6 Oven, capable of maintaining a temperature of (40 ± 2) °C
6.3.7 High performance liquid chromatography apparatus coupled with tandem mass
spectrometry equipped with atmospheric pressure ionization (APCI)
6.3.8 Analytical reversed phase HPLC column, C18, for example Macherey-Nagel, Nucleodur C18
1)
Sphinx®, column length 50 mm, internal diameter 3 mm, particle size 1,8 μm. Another phase that
)
proved to be suitable is porous graphitic carbon, PGC, for example Thermo Scientific HyperCarb®
NOTE Using these HPLC columns and the conditions given in 8.2 NDELA was not eluted within the void
volume. When using another column it needs to be ensured that NDELA has sufficient retention and does not elute
within the void volume of the column used. Due to the fact that the two given columns exhibit a different retention
mechanism they can be used complementary to solve possible chromatographic problems, e.g. poor separation of
NDBA/NDiBA or certain interferences with matrix peaks (such matrix peaks are known for example for
NDBA/NDiBA or NDBzA when using low resolution mass spectrometry).
6.3.9 Membrane filter, membrane made of polyester
7 Sample preparation
7.1 General
Other determination methods than HPLC-MS/MS might require modifications of this procedure after
the migration. For nitrosation, it is mandatory to use the given volume ratios of sample solution,
hydrochloric acid and sodium hydroxide as well as the given time and temperature.
N-nitrosamines are degraded by ultraviolet light. If samples are stored prior to analysis, they shall be
sealed airtight (e.g. in a suitable sized container or wrapped in aluminium foil) and stored in the dark at
temperature below 25 °C. The contamination of samples shall be avoided, e.g. avoiding the cross-
contamination between samples.
NOTE Similar conditions are appropriate for sampling and transport.
Exposure of samples, extracts, migrates or standard solutions to sources such as sunlight or fluorescent
tube light shall be avoided. All extracts, migrates and standard solutions shall be protected by wrapping
in aluminium foil or using amber glassware. If not analysed directly after preparing extracts, migrates
and standard solutions shall be stored in the dark at a temperature of (5 ± 3) °C.
For analysis of finger paints and elastomers a method blank should be prepared and analysed to check if
there are any blank values. For NDELA, blank values have been reported which originated from the
HPLC-vials used. This could be avoided by prewashing the HPLC-vials with ethanol or by irradiating the
vials with UV light (8.4). In the case of N-nitrosamines being detected in the blank sample their source
shall be investigated and removed before analysing samples.
7.2 Finger paints
7.2.1 N-nitrosamines
Finger paints shall be homogenized by mixing (for example using a glass rod).
Weigh about 1,0 g of the sample (note the exact mass) into a sample tube, add 0,20 ml of d8-NDELA
working solution (6.2.5.2) and add 18,8 ml water. After vortexing for about 1 min, shake continuously
for approximately 15 min on a shaking machine (6.3.2). Centrifuge the sample solution at
approximately 4 000 g for about 15 min. Transfer a portion of the supernatant into a second sample
tube and centrifuge at approximately 20 000 g for about 15 min. If necessary, filter the supernatant
through a membrane filter which will not retain analytes.

1)
This information is given for the convenience of users of this European Standard and does not constitute an
endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same
results.
7.2.2 N-nitrosatable substances
Finger paints shall be homogenized by mixing (for example using a glass rod).
Weigh about 0,4 g of the sample (note the exact mass) into a sample tube and add saliva test solution
until the total weight is 20 g (note the exact mass). After vortexing for about 1 min, shake continuously
for approximately 15 min on a shaking machine (6.3.2) to homogenize the sample solution.
Transfer 5,0 ml of the test solution in an amber vial and add 0,5 ml of 1 mol/l hydrochloric acid solution
(6.1.8). Cap the vial, shake to mix and allow the solution to stand for (30 ± 1) min at (40 ± 2) °C. Add
1,0 ml of 1 mol/l sodium hydroxide solution (6.1.9) and shake to mix. Transfer the solution in a
centrifugation tube and centrifuge the sample solution at 20 000 g for 15 min. If necessary, filter the
supernatant through a membrane filter.
This test solution should be stored in a refrigerator at (5 ± 3) °C and may be used to reanalyse for N-
nitrosamines.
Transfer immediately 1,0 ml of the test solution into an amber HPLC-Vial and add 0,020 ml of the
internal standards stock solution containing d8-NDELA and d6-NDMA (6.2.5.1). Shake to mix the
solution. The solution is now ready for measurement of N-nitrosamines after nitrosation.
In the event the measured N-nitrosamine concentrations are above the calibration range the test
solutions stored in the refrigerator may be diluted with saliva test solution for reanalysing.
7.3 Elastomers
7.3.1 Sample preparation for balloons
Cut the balloons longitudinal in two halves to obtain nearly symmetrical pieces. Depending on the
weight of the sample the halves shall be cut again longitudinal to get again two nearly symmetrical
pieces. Weigh the balloon halves and/or quarters in a 50 ml conical flask (6.3.5) until the sample weight
is between 3,5 g and 4,5 g. Add a corresponding amount of saliva test solution ((6.1.7), preheated to
(40 ± 2) °C) between 35,0 ml and 45,0 ml to get a weight to volume ratio of 1:10. Ensure that the sample
is totally immersed, e.g. by adding some glass balls (6.3.4, preheated to (40 ± 2) °C). Close the flask with
a ground stopper and allow the solution to stand at (40 ± 2) °C for (60 ± 3) min.
Immediately decant the test solution into a suitable glass vessel to stop the migration process. The test
solution is used for both, the determination of N-nitrosamines according to 7.3.4 and for the nitrosation
according to 7.3.3. It is important to commence the nitrosation step immediately after decanting the test
solution.
This test solution should be stored in a refrigerator at (5 ± 3) °C and may be used to reanalyse for N-
nitrosamines. It cannot be reused for the nitrosation step.
7.3.2 Sample preparation for other toys or parts of toys than balloons
If possible, separate the elastomeric part of the toy from other materials, e.g. associated textiles.
The toy or part of toy should be tested uncut. If not possible (i.e. if the weight of the toy or part of toy is
more than 4 g or if it does not fit into a 50 ml conical flask), the elastomeric part of the toy shall be cut in
such a way as to minimize the inaccessible and internal surfaces. The surface area resulting from the
cutting edges should be as low as possible. Weigh the sample in a 50 ml conical flask (6.3.5) until the
sample weight is between 2,0 g and 4,0 g. Add a corresponding amount of saliva test solution ((6.1.7),
preheated to (40 ± 2) °C) between 20,0 ml and 40,0 ml to get a weight to volume ratio of 1:10. Ensure
that the sample is totally immersed and wetted, e.g. by adding some glass balls ((6.3.4), pre-heated to
(40 ± 2) °C) in such a way to prevent the sample lying directly on the bottom of the glass vessel and the
test portions sticking together. Close the flask with a ground stopper and allow the solution to stand at
(40 ± 2) °C for (240 ± 5) min.
NOTE For deviating migration conditions (migration time) applicable for Germany for certain toys see
Annex C.
Immediately decant the test solution into a suitable glass vessel to stop the migration process. The test
solution is used for both, the determination of N-nitrosamines according to 7.3.4 and for the nitrosation
according to 7.3.3. It is important to commence the nitrosation step immediately after decanting the test
solution.
This test solution should be stored in a refrigerator at (5 ± 3) °C and may be used to reanalyse for N-
nitrosamines. It cannot be reused for the nitrosation step.
7.3.3 Nitrosation
Immediately after migration, transfer 5,0 ml of the test solution in an amber vial and add 0,5 ml of
1 mol/l hydrochloric acid solution (6.1.8). Cap the vial, shake to mix and allow the solution to stand for
(30 ± 1) min at (40 ± 2) °C. Add 1,0 ml of 1 mol/l sodium hydroxide solution (6.1.9) and shake to mix.
The test solution is used for the determination of N-nitrosamines according to 7.3.4.
This test solution should be stored in a refrigerator at (5 ± 3) °C and may be used to reanalyse for N-
nitrosamines.
7.3.4 Preparation of solution for determination of N-nitrosamines by HPLC-MS/MS
Transfer immediately 1,0 ml of the test solution into an amber HPLC-Vial and add 0,020 ml of the
internal standards stock solution containing d8-NDELA and d6-NDMA (6.2.5.1). Shake to mix the
solution. The solution is now ready for measurement of N-nitrosamines.
In the event the measured N-nitrosamine concentrations are above the calibration range the test
solutions stored in the refrigerator may be diluted with saliva test solution for reanalysing.
Experiments have shown that the response in the MS for the non-polar NDiNA in saliva test solution
declines over time. This can be avoided by diluting the sample and calibration solutions with methanol
(1:1) before measurement of NDiNA. This should be done if there is an indication for the presence of
NDiNA in the sample. Another possibility is to homogenize the solution by shaking just before
measurement.
8 Methods of analysis
8.1 General
The analysis of N-nitrosamines and N-nitrosatable substances shall be performed in accordance with the
methods of analysis described in this European Standard. Alternative methods of analysis or
modifications to the procedures described are acceptable only if they are capable of achieving at least
the accuracy and precision of the methods described in this European Standard, an adequate sensitivity
and have been validated to show that the results are equivalent to those of these standard methods.
NOTE The experience and the results of the round robin test (see Clause 10) have shown that the detection
using GC techniques, e.g. GC-TEA as specified in EN 12868, is suitable. For further information see also A.6.
The high amount of salts in the migration solution might clog the inlet of certain mass spectrometers.
This can be overcome by adding a switching valve between column outlet and mass spectrometer and
switching the eluent to waste until the void peak is eluted. Care should be taken that the eluent is
switched back to the mass spectrometer in time for the early eluting NDELA.
8.2 HPLC chromatographic conditions
Flow rate: 0,3 ml/min
Column temperature: 30 °C
Injection volume: injection volume that enables a LOQ of 0,000 2 mg/l (this is depending on the
sensitivity of the instrument used; typical injection volumes are up to 100 µl
but the volume may be even higher if such technical resources are available)
Gradient: See Table 4.
Table 4 — Gradient for HPLC conditions
Time Eluent A Eluent B
min % %
0 90 10
10 10 90
12 10 90
12,2 90 10
14,5 90 10
Flow rate, injection volume and gradient depend on the analytical column used. These conditions might
be modified when working with other columns or column dimensions (6.3.8). Chromatographic
conditions have to be suitable for achieving sufficient retention for NDELA to elute not within the void
volume of the column used.
8.3 MS/MS conditions
Ion source: APCI
Polarity: positive
Minimum resolution 0,7 Da
Suitable MRM-transitions: See Table 5.
Table 5 — Suitable MRM-transitions for MS/MS conditions
Compound Q1 mass Q3 masses
m/z m/z
NDMA 75,0 42,9 58,0
NDEA 103,1 75,0 -
NPIP 115,1 69,1 40,9
NMOR 117,0 86,9 85,9
NDPA 131,1 89,1 43,1
NDiPA
NDELA 135,0 104,0 74,0
d8-NDELA 143,0 111,0 80,2
NDBA 159,1 57,1 103,0
NDiBA
d6-NDMA 81,0 46,1 -
NDBzA 226,9 91,1 65,1
NEPhA 150,9 77,1 -
NMPhA 136,9 66,0 107,1
NDiNA 299,0 57,1 71,1
NOTE MRM-transitions can vary between different instruments. Suitable transitions need to be chosen for
quantification and identification of N-nitrosamines. The given transitions were obtained using nitrogen as collision
gas.
8.4 Confirmation of detected N-nitrosamines
Using HPLC-MS/MS detected N-nitrosamines are confirmed via their specific ratio of the intensities of
the detected ions. Therefore, relative intensities of two or more detected ions from sample
preparations, expressed as a percentage of the intensity of the most intense ion, shall correspond to
those of the calibration standard solutions, at comparable concentrations, measured under the same
conditions, within the tolerances listed in Table 6.
Table 6 — Maximum permitted tolerances for relative ion intensities
Relative intensity Relative range of the response
(% of base ion intensity)
> 50 % ±20 %
> 20 % to 50 % ±25 %
> 10 % to 20 % ±30 %
≤ 10 % ±50 %
Other techniques, or N-nitrosamines which are only detectable via one ion or if the ratio of the detected
ions does not meet the permitted tolerances, require other confirmation methods which could be:
— placing an aliquot of the remaining test solution in a clear, UV transparent glass vial and subjecting
it to UV radiation for (180 ± 5) min at a wavelength of 366 nm along with a similarly prepared
standard solution in a separate vial. On analysis any peaks due to the presence of N-nitrosamines
will disappear or be substantially reduced due to decomposition. However, if any of the peaks are
not substantially reduced after irradiation, this indicates that the initial peak did not correspond to
a N-nitrosamine;
— by the use of at least one alternative column with different selectivity;
— by the use of another detector (for example GC-TEA or HPLC-DAD after post-column
derivatisation);
— by the use of high resolution mass spectrometry (hrMS).
NOTE In high resolution mass spectrometry (HRMS), the resolution typically needs to be greater than 10 000
for the entire mass range at 10 % valley (equal 20 000 FWHM). To use this technique for confirmation of the
identity of a detected substance, it is important that the resolution is at least 35 000 FWHM and that two HRMS
ions with their specific ion ratio are monitored.
9 Calculation of results for N-nitrosamines and N-nitrosatable substances
9.1 Finger paint
9.1.1 General
NOTE The calculations of results for finger paint in the following subclauses only consider NDELA as this is
the only N-Nitrosamine specified in Table 1. If other N-Nitrosamines are detected it is important to adapt the
calculation of the following subclauses accordingly.
For NDELA determination, the internal standard calibration method is used. An R value is determined,
which is the ratio between the intensity from the ion used for quantification and the internal standard
ion. Prepare a calibration curve by plotting the concentration of NDELA calibration solutions against the
corresponding R value.
9.1.2 N-nitrosamines
Determine the concentration c of NDELA in mg/l in the sample solution directly from the calibration
curve. This determined value, in mg/l, of the concentration corresponds to the mass fraction, w, in
mg/kg of NDELA in the sample solution.
Calculation of the content M in mg/kg of NDELA in the finger paint:
Mw=×+m 19 /m (1)
( )

where
M is the content of NDELA in sample, in mg/kg;
w is the mass fraction of NDELA in sample solution, in mg/kg;
m is the mass of sample, in g.
NOTE Pure water is used for preparing the calibration solutions and is also used as the extracting agent for
the samples. Due to the density of water of 1 g/cm the measured concentration c in mg/l is equal to the mass
fraction w in mg/kg and the factor for calculating the content of NDELA is the ratio of the total weight of sample
plus extraction agent (18,8 ml water + 0,2 ml d8-NDELA working solution = 19 g) and the sample weight.
9.1.3 N-nitrosatable substances
Determine the concentration C in mg/l of NDELA after nitrosation in the sample solution directly from
the calibration curve. For the concentration C in mg/l of N-nitrosatable substances, a dilution
nitrosatable
factor of 1,3 l/kg is applied to the calculation:
wC×1, 3 l / kg (2)
nitrosatable
where
w is the mass fraction of N-nitrosatable substances in the sample solution, in mg/kg;
nitrosatable
C is the concentration of NDELA in the sample solution after nitrosation, in mg/l.
For getting the N-nitrosatable substances the concentration of NDELA (M) has to be subtracted from the
content of the NDELA determined after the nitrosation. Calculation of the content of N-nitrosatable
substances (M ) in mg/kg in the analysed finger paint:
nitrosatable
M =w × m /mM− (3)
( ( ))
nitrosatable nitrosatable total
where
M is the content of N-nitrosatable substances in finger paint, in mg/kg;
nitrosatable
w is the mass fraction of N-nitrosatable substances in the sample solution, in mg/kg;
nitrosatable
m total mass of sample and saliva test solution, in g;
total
m is the mass of sample, in g;
M is the content of NDELA in the finger paint, in mg/kg.
NOTE 1 Saliva test solution is used for preparing the calibration solutions and is also used as the extracting
agent for the samples. It has a density of 1,001 g/cm at 20 °C. Therefore, the difference to the density of water of
1 g/cm is negligible and the measured concentration c in mg/l is equal to the mass fraction w in mg/kg. The
factor for calculating the content of N-nitrosatable substances is the ratio of the total weight of the sample plus
saliva test solution and the sample weight.
NOTE 2 The dilution factor
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