Safety of toys - Part 12: N-Nitrosamines and N-nitrosatable substances

Foreword

This document (EN 71-12:2013) 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of an identical text or by endorsement, at the latest by December 2013, and conflicting national standards shall be withdrawn at the latest by December 2013.

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.

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(s).

For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document.

This European Standard constitutes the 12^th 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 Report, CR 14379, Classification of toys — Guidelines, 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, Safety of toys — Replies to requests for interpretation of EN 71-1, EN 71-2, and EN 71-8.

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.

Introduction

There are ongoing developments in the following area:

• the influence of the mouthing behaviour (mouthing time and sucking/chewing) on the migration out of teethers and other elastomers than balloons.

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.

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.

EXAMPLES Examples of toys made from elastomers are balloons and teethers.

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

paste and/or jelly like, coloured mixture specially designed for children to apply directly to suitable surfaces with the fingers and hands

NOTE 1 to entry: In addition to water, finger paints essentially consist of colorants, binders, preservatives and embittering agent and may additionally contain extenders, humectants and surfactants.

[SOURCE: prEN 71-7:2012, 3.1]

3.2 elastomer

material which undergoes substantial, elastic ((fully) reversible) deformation when put under stress and consisting of three-dimensional networks of cross-linked flexible polymers

NOTE 1 to entry: The cross-links can be chemical bonds in rubbers (like natural rubber, synthetic rubber and silicones) or physical, thermo-reversible fixation points in thermoplastic elastomers (TPE) or the combination of both (TPE-V).

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 tested in finger paint. If there are indications of the presence of other N-nitrosamines, they should also be tested.

4.2 Requirements for elastomers

The migration of N-nitrosamines from toys intended for use by children under 36 months or in other toys or parts of toys intended to be placed into the mouth shall not exceed 0,05 mg/kg of toy material when tested in accordance with 7.3 and Clause 8, calculated as sum of all detected N-nitrosamines according to 9.2.

The migration of N-nitrosatable substances from toys intended for use by children under 36 months or in other toys or parts of toys intended to be placed into the mouth shall not exceed 1 mg/kg of toy material, calculated as sum of all detected N-nitrosamines after nitrosation.

At least the substances in Table 2 shall be tested in elastomers. If there are indications of the presence of other N-nitrosamines they should also be tested.

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.

N-nitrosamines are degraded by ultra-violet light. Exposure of extracts or standards to sources such as sunlight or fluorescent tube light should be avoided. The samples and 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.

6.1.1 Sodium hydrogen carbonate.

6.1.2 Sodium chloride.

6.1.3 Potassium carbonate.

6.1.4 Sodium nitrite.

6.1.7 Saliva test solution.

Dissolve 4,2 g of the sodium hydrogen carbonate (6.1.1), 0,5 g of the sodium chloride (6.1.2), 0,2 g of the potassium carbonate (6.1.3) and 30 mg of the sodium nitrite (6.1.4) in water and dilute to 900 ml with water. Adjust to pH 9,0 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.

The saliva test solution shall be prepared freshly every day to ensure an accurate nitrite concentration. If the solution is stored and not prepared freshly the laboratory shall prove that the nitrite concentration has not decreased during storage.

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

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 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 elastomers and nitrosatable substances in finger paints.

Prepare 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.

6.3 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.

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.

NOTE Using this HPLC column 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.

6.3.7 Membrane filter.

7 Sample preparation

NOTE 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.

7.1 General

N-nitrosamines are degraded by ultra-violet light. 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 HPLCvials used. This could be avoided by prewashing the HPLC-vials with ethanol. In the presence of Nnitrosamines in the blank sample this shall be investigated and removed before analysing samples.

7.2 Finger paints

7.2.1 N-nitrosamines

Finger paints shall be homogenised 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.

7.2.2 N-nitrosatable substances

Finger paints shall be homogenised 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 homogenise 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 Nnitrosamines.

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 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 10. Ensure that the sample is totally immersed, e.g. by adding some glass balls (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 Nnitrosamines. It cannot be reused for the nitrosation step.

7.3.2 Sample preparation for other toys or parts of toys than balloons

If the approximate surface area of the toy or the elastomeric part of the toy is less than 10 cm², test the whole toy or the elastomeric part of the toy uncut. At least 0,2 g of the samples are needed per analyses.

In other cases, select the most appropriate part of the elastomeric part of the toy, from which to obtain a test portion of (10 ± 1) cm², in such a way as to minimise the inaccessible and internal surfaces. Where possible, choose a test portion from a thin part of the toy. Remove a disc, or other shaped piece if these reduces the amount of cut edges, with a surface area of (10 ± 1) cm² using a suitable cutting instrument. Measure the approximate surface area of the test portion taking into account the thickness when greater than 1 mm. The edges of the test portion should be smooth in appearance.

In cases where the preparation of the elastomeric part of the toy leads to unrealistic results because of the cutting process, the whole uncut toy or elastomeric part of the toy may be tested.

The toy or test portion is weighed and saliva test solution is added. Thereby, the ratio of sample weight to volume of saliva test solution of 10 is maintained as described in 7.3.1 for balloons. Ensure that the sample is totally immersed and wetted, e.g. by adding some glass balls (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 ± 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 12 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 Nnitrosamines. It cannot be reused for the nitrosation step.

NOTE In a future revision of this standard the migration conditions specified in this clause may change depending on further studies expected to be undertaken in the near future.

7.3.3 Nitrosation

Transfer immediately 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 Nnitrosamines.

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 has 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.

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.5.

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: 20 µl

Gradient: See Table 3.

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 (6.3.6). 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 4.

NOTE MRM-transitions can vary between different instruments. Suitable transitions need to be chosen for quantification and identification of N-nitrosamines.

8.4 Confirmation of detected N-nitrosamines

Using HPLC-MS/MS detected N-nitrosamines are confirmed via their specific ratio of 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 5.

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 (366 nm, 3 h) 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 the sample peak is not substantially reduced after irradiation, the initial peak was a false positive and no further investigation for the presence of N-nitrosamines is required,
• 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).

9 Calculation of results for N-nitrosamines and N-nitrosatable substances

9.1 Finger paint

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.

9.1.1 General

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:

$M=w\times \left[\raisebox{1ex}{$\left(m+19\right)$}\!\left/ \!\raisebox{-1ex}{$m$}\right.\right]$(1)

where

M content of NDELA in sample, in mg/kg;

w mass fraction of NDELA in sample solution, in mg/kg;

m 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/cm³ 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 NDLEA after nitrosation in the sample solution directly from the calibration curve. For the concentration C_nitrosatable in mg/l of N-nitrosatable substances, a dilution factor of 1,3 l/kg applied to the calculation:

$w_{\mathrm{nitrosatable}}=C·\mathrm{1,3}$(2)

where

w_nitrosatable is the mass fraction of N-nitrosatable substances in the sample solution, in mg/kg;

C is the concentration of NDLEA 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_nitrosatable) in mg/kg in the analysed finger paint:

$M_{\mathrm{nitrosatable}}=\left(w_{\mathrm{nitrosatable}}·\left(\raisebox{1ex}{$m_{\mathrm{total}}$}\!\left/ \!\raisebox{-1ex}{$m$}\right.\right)\right)-M$(3)

where

M_nitrosatable is the content of N-nitrosatable substances in finger paint, in mg/kg;

w_nitrosatable is the mass fraction of N-nitrosatable substances in the sample solution, in mg/kg;

m_total total mass of sample and saliva test solution, in g;

m mass of sample, in g;

M is the content of NDELA in the finger paint, in mg/kg.

NOTE 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/cm³ at 20 °C. Therefore the difference to the density of water of 1 g/cm³ 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.

9.2 Elastomers

9.2.1 General

For NDELA and NDMA 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 and NDMA calibration solutions against the corresponding R value for these N-nitrosamines.

For all other N-nitrosamines the external standard calibration method is used. Prepare a calibration curve by plotting the concentration of the nitrosamine calibration solutions in mg/l against the peak area.

9.2.2 N-nitrosamines

Determine the concentration C_NA in mg/l of N-nitrosamines in the sample solution directly from the calibration curve.

Calculation of the content of N-nitrosamines (MNA) in mg/kg in the analysed toy sample:

$M_{\mathrm{NA}}=C_{\mathrm{NA}}·V/m$(4)

where

M_NA is the content of N-nitrosamines in the toy, in mg/kg;

C_NA is the concentration of N-nitrosamines in the sample solution, in mg/l;

V volume of saliva test solution, in ml;

m mass of sample, in g.

9.2.3 N-nitrosatable substances

Determine the concentration C in mg/l of N-nitrosamines after nitrosation in the sample solution directly from the calibration curve. For the concentration Cnitrosatable in mg/l of N-nitrosatable substances, a dilution factor of 1,3 applied to the calculation:

$C_{\mathrm{nitrosatable}}=C·\mathrm{1,3}$(5)

where

C_nitrosatable is the concentration of N-nitrosatable substances in the sample solution, in mg/l;

C is the concentration of N-nitrosamines in the sample solution after nitrosation, in mg/l.

For getting the N nitrosatable substances the concentration of N-nitrosamines (MNA) has to be subtracted from the content of the N-nitrosamines determined after the nitrosation. Calculation of the content of N-nitrosatable substances (M_nitrosatable) in mg/kg in the analysed toy sample:

$M_{\mathrm{nitrosatable}}=\left(C_{\mathrm{nitrosatable}}·\raisebox{1ex}{$V$}\!\left/ \!\raisebox{-1ex}{$m$}\right.\right)-M_{\mathrm{NA}}$(6)

where

M_nitrosatable is the content of N-nitrosatable substances in the toy, in mg/kg;

C_nitrosatable is the concentration of N-nitrosatable substances in the sample solution, in mg/l;

V volume of saliva test solution, in ml;

m mass of sample, in g;

M_NA is the content of N-nitrosamines in the toy, in mg/kg.

10 Quality

10.1 Finger paints

10.1.1 Repeatability, reproducibility and measurement uncertainty

Table 6 shows the results from a round robin test on a finger paint containing NDELA.

NOTE An Eurachem/CITAC Guide [9] gives guidance on how to use measurement uncertainty for interpretation of analytical results in relation to compliance assessment.

10.1.2 Limits of detection (LOD) and quantification (LOQ)

For a solution containing 1 µg/l NDELA in water, the signal to noise ratios of the ions measured was calculated to be around 80. The relative standard deviation of the peak areas at this concentration is about 5 % (n = 6). Therefore an acceptable limit of quantitation (LOQ) has also been set at 1 µg/l for NDELA in water. Recovery of NDELA in finger paints is ensured by the use of d8-NDELA as an internal standard. The internal standard minimises the impact of certain matrix effects (e.g. ion suppression) on the recovery.

10.2 Elastomers

10.2.1 Repeatability, reproducibility and measurement uncertainty

Table 7 shows the results from a round robin test on N-nitrosamines and N-nitrosatable substances in elastomers.

NOTE An Eurachem/CITAC Guide [9] gives guidance on how to use measurement uncertainty for interpretation of analytical results in relation to compliance assessment.

10.2.2 Limits of detection and quantification

For solutions of N-nitrosamines in saliva test solution containing 1 µg/l of each N nitrosamine the calculated signal to noise ratio (S/N) of the ions was not lower than 20 (NDMA, MRM-transition 75 - 58). The relative standard deviation (n=6) of the peak areas at this concentration were lower than 6 % for each ion. Therefore an acceptable limit of quantitation (LOQ) has also been set at 1 µg/l for each N-nitrosamine in saliva test solution included in this standard.

11 Test report

The test report shall at least contain:

1. a title, e.g. Test report, Test Certificate;
2. the name and address of the laboratory and the name and address of the client;
3. a serial number or similar unique identification for the report which should appear on each page together with pagination in the form Page .. of ..; 20
4. the client’s identification details for the samples and the identifiers used by the laboratory, for example sample numbers;
5. the date of receipt of the items and the date(s) of testing;
6. identification of the analytical technique used;
7. a reference to this European Standard (i.e. EN 71-12:2013);
8. a note of any deviations from the standard and any environmental conditions which may bear upon the results;
9. the test results themselves with units and the measurement uncertainty if necessary;
10. the name, position and signature or other identification of the person accepting responsibility for the report and the report’s date of issue;
11. a statement that the results only apply to the items tested.

Annex A (informative) Background and rationale for this European Standard

A.1 Requirements for finger paints (see 4.1)

The migration limits value set in Directive 2009/48/EC [6] for N-nitrosamines and N-nitrosatables substances in toys intended for use by children under 36 months or in toys intended to be placed in the mouth are focused on the oral exposure to these compounds.

NDELA is a genotoxic carcinogen without a safe threshold value. It is well known from cosmetics, that NDELA has a high skin penetration rate. A risk assessment for NDELA in finger paints has clearly demonstrated that the dermal exposure may exceed the oral exposure by two orders of magnitude. Therefore, the dermal exposure is considered the dominant exposure route of the total systemic exposure, while oral intake plays a minor role. Hence, embittering agents, which were usually added to reduce the risk of ingestion, cannot protect from dermal exposure to NDELA by finger paints. Due to the dermal exposure and the analytical procedure it has been decided to specify a limit value of 0,02 mg/kg for N-nitrosamines.

Therefore, the test solution for N-nitrosamines in finger paints is water (dermal exposure) and the test solution for N-nitrosatable substances in finger paints is saliva test solution (oral intake).

A.2 Requirements for elastomers (see 4.2)

The threshold values for elastomers are adopted from Directive 2009/48/EC, Annex II, Chapter III, number 8 [6].

A.3 Avoidance N-nitrosamines in finger paints

The formation of N-nitrosamines is possible when secondary amines and nitrosating agents are both present in the finger paint under certain conditions. It is known from studies on cosmetic products that NDELA can be formed by the reaction of diethanolamine with a nitrite source.

To avoid the possible formation of N-nitrosamines avoid using formulations that combine diethanolamine and triethanolamine (which is known to be often contaminated with diethanolamine or which can be decomposed to diethanolamine) with possible sources of nitrite that may be present in certain preservatives (e.g bronopol) and other raw materials.

A.4 Explanation for decision to do static migration for elastomers

The legislative requirements for N-nitrosamines contained in Directive 2009/48/EC [6] are based on information provided by the German and Dutch Authorities which was reviewed by the EU Scientific Committee SCCP/1132/07 [8]. The information provided by the German and Dutch Authorities was based on data for balloons using the method described in EN 12868:1999 [2] using a static migration method on a sample of specified area into a known volume of nitrite containing saliva test solution, but using a migration time of 1 h. EN 12868:1999 [2] implements Commission Directive 93/11/EEC [7] which regulates the release of N-nitrosamine and N-nitrosatable substances from elastomer or rubber teats and soothers.

To ensure comparison with existing data it has been decided to use the conditions described in EN 12868:1999 [2] which is a static migration test at 40 °C. According to SCCP/1132/07 [8] the migration time of 1 h is used for balloons.

A.5 Reference to other standards and relevant deviations

There are other standards that describe alternative detection systems for the determination of N nitrosamines. EN 12868 [2] describes a system using gas-chromatography coupled to a thermal energy analyser (GC-TEA) to determine volatile nitrosamines. Also ISO 10130 [3] describes a detection system for determining NDELA.

These alternative detection systems may be used after the migration and nitrosation specified in 7.3.1 to 7.3.3 on the toy material has been completed but will require validation of the relevant system to ensure that any results are comparable to the results obtained using the LC-MS/MS detection system described in this European Standard.

It should be noted that laboratories using GC-TEA as described in EN 12868 [2] or other GC techniques like GC-MS will require an additional preconcentration step that requires extraction into an organic solvent which is amenable to GC (e.g. by the procedure specified in EN 12868 [2]). Furthermore this will need a derivatisation step to enable the determination of non-volatile N-nitrosamines, e.g. NDELA. These additionally steps shall be carried out only after the migration and nitrosation steps has been applied.

For laboratories using an HPLC with post-column photolysis and derivatisation as described in ISO 10130 [3] to determine NDELA there may be matrix problems using this detection method which hinders the reliable evaluation of chromatograms obtained for finger paint samples.

A.6 Conditions for nitrosation

N-nitrosatable substances are converted into N-nitrosamines due to acidification of the nitrite containing saliva test solution after migration of the sample. Experiments showed that conversion rate depends on a lot of influences like temperature, time and nitrite concentration and differs also for different precursors. Therefore it is crucial to specify each of these parameters as it was done in this European Standard to get comparable results. The conditions were taken from EN 12868:1999 [2] where each of these critical parameters is specified. For the temperature used during nitrosation this European Standard specifies a more accurate temperature of (40 ± 2) °C contrary to EN 12868 [2] where only room temperature is given.

A.7 NDELA in balloons

NDELA has been shown to migrate from balloons in a number of studies, although there is no known accelerator precursor to NDELA that is used in the manufacturing process when making balloons. Current thinking (based on a limited number of samples tested) is that the NDELA is not produced during manufacture of the latex material but arises from a subsequent surface treatment on the balloon using an ‘anti-tack’ solution where ethanolamines may be present.

A.8 Selection of N-nitrosamines specified in this standard

N-nitrosamines and N-nitrosatable substances given in Table 1 and Table 2 have been chosen because these N-nitrosamines either have been found in toys or these are known to be produced by pre-cursors, namely some accelerators used during rubber production. The selection reflects the current state of knowledge for producing toys or parts of toys made from elastomers and finger paints.

If there is new knowledge or if there are indications that also other N-nitrosamines are relevant for a specific sample they have to be analysed.

Annex B (informative)
Sample storage conditions

The degradation of N-nitrosamines shall be avoided, e.g. by avoiding the exposure of samples to UV light.

The contamination of samples shall be avoided, e.g. avoiding the cross-contamination between samples.

Annex C (informative)
A-deviations

A-deviation: National deviation due to regulations, the alteration of which is for the time being outside the competence of the CEN/ CENELEC member.

This European Standard falls under Directive 2009/48/EC.

NOTE (From CEN/CENELEC Internal Regulations Part 2: 2011, 2.17): Where standards fall under EU Directives, it is the view of the Commission of the European Communities (OJ No C 59, 9.3.1982) that the effect of the decision of the Court of Justice in case 815/ 79 Cremonini/ Vrankovich (European Court Reports 1980, p. 3583) is that compliance with Adeviations is no longer mandatory and that the free movement of products complying with such a standard should not be restricted within the EU except under the safeguard procedure provided for in the relevant Directive.

A-deviations in an EFTA-country are valid instead of the relevant provisions of the European Standard in that country until they have been removed.

Annex ZA (informative)
Relationship between this European Standard and the Essential Requirements of EU Directive 2009/48/EC

This European Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association to provide a means of conforming to Essential Requirements of the New Approach Directive 2009/48/EC.

Once this European Standard is cited in the Official Journal of the European Union under that Directive and has been implemented as a national standard in at least one Member State, compliance with the clauses of this European Standard given in Table ZA.1 confers, within the limits of the scope of this European Standard, a presumption of conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.

WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.

Bibliography

[1]   prEN 71-7:2012, Safety of toys — Part 7: Finger paints — Requirements and test methods

[2]   EN 12868:1999, Child use and care articles — Methods for determining the release of N-Nitrosamines and N­nitrosatable substances from elastomer or rubber teats and soothers

[3]   ISO 10130, Cosmetics — Analytical methods — Nitrosamines: Detection and determination of Nnitrosodiethanolamine (NDELA) in cosmetics by HPLC, post-column photolysis and derivatization

[4]   ISO 15819, Cosmetics — Analytical methods — Nitrosamines: Detection and determination of Nnitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS

[5]   ISO 29941, Condoms — Determination of nitrosamines migrating from natural rubber latex condoms

[6]   Directive 2009/48/EC of the European Parliament and of the Council of 18 June 2009 on the safety of toys, OJ L 170, 30.6.2009, p. 1-37, available from http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:170:0001:0037:EN:PDF

[7]   Commission Directive 93/11/EEC of 15 March 1993 concerning the release of the N-nitrosamines and Nnitrosatable substances from elastomer or rubber teats and soothers, OJ L 93, 17.4.1993, p. 37-38, available from http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:1993:093:0037:0038:EN:PDF

[8]   Scientific Committee on Consumer Products (SCCP): Opinion on the Presence and release of Nitrosamines and Nitrosatable Compounds from Rubber Balloons; adopted at the 14th plenary of 18 December 2007, available from http://ec.europa.eu/health/archive/ph_risk/committees/04_sccp/docs/sccp_o_121.pdf

[9]   Eurachem/CITAC Guide, Use of uncertainty information in compliance assessment, First edition 2007