development of a harmonised method for specific migration into … · 2016. 6. 3. · 2012 giorgia...

Report EUR 25680 EN

2 0 1 2

Giorgia Beldì, Natalia Jakubowska, Aurélie Peychès Bach and Catherine Simoneau

Establishment of precision criteria from an EU interlaboratory comparison organised by the EURL- Food Contact Materials for the quantification from and migration into poly(2,6-diphenyl phenylene oxide)

Development of a harmonised method for specific migration into the new simulant for dry foods established in Regulation 10/2011

European Commission Joint Research Centre Institute for Health and Consumer Protection Contact information Catherine Simoneau Address: Joint Research Centre, Via Enrico Fermi 2749, TP 260, 21027 Ispra (VA), Italy E-mail: [email protected] Tel.: +39 0332 78 5889 Fax: +39 0332 78 5707 http://ihcp.jrc.ec.europa.eu/ http://www.jrc.ec.europa.eu/ This publication is a Reference Report by the Joint Research Centre of the European Commission. Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication. Europe Direct is a service to help you find answers to your questions about the European Union Freephone number (*): 00 800 6 7 8 9 10 11 (*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed.

A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server http://europa.eu/. JRC77515 EUR 25680 EN ISBN 978-92-79-28078-8 ISSN 1831-9424 doi:10.2788/76804 Luxembourg: Publications Office of the European Union, 2012 © European Union, 2012 Reproduction is authorised provided the source is acknowledged. Printed in Italy

EC-JRC-IHCP, CAT Unit action programme 15014

2012

EURL Food Contact Materials No SANCO/2011/FOOD SAFETY070-Food Contact Materials

Giorgia Beldì, Natalia Jakubowska, Aurèlie Peychès Bach and Catherine Simoneau

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Table of content

1. Summary........................................................................................ 5

2. Introduction................................................................................... 7

3. Scope ............................................................................................. 7

4. Time frame..................................................................................... 7

5. Test materials ................................................................................ 8

5.1 Preparation ...............................................................................................................................8

5.2 Homogeneity assessment .........................................................................................................8

5.3 Stability test .............................................................................................................................9

5.4 Distribution ............................................................................................................................10

6. Instructions to participants ......................................................... 10

7. Approaches for statistical evaluation of results ........................... 10

7.1. Evaluation of the BHT, BP, DiBP, DEHA and DINCH method performance

characteristics – methods for determination of the consensus value, repeatability ( r ) and

reproducibility ( R ) standard deviation .......................................................................................10

7.2. Identification of modes using Kernel density plotting..........................................................11

7.3. Mandel’s h- and k-statistics ..................................................................................................11

7.4. Evaluation criteria for laboratory performance – z-scores ...................................................12

8. Results and conclusions ............................................................. 12

8.1 Preliminary considerations....................................................................................................12

8.2 Method performance characteristics from the precision experiment....................................13

8.3 z-scores and laboratories performance..................................................................................21

8.4 Final conclusion.....................................................................................................................23

9. Acknowledgements ...................................................................... 44

10. References ................................................................................. 44

11. Annexes ..................................................................................... 45

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1. Summary

Regulation (EU) No 10/2011 establishes (poly (2,6-diphenyl phenylene oxide, PPPO)) as food simulant E for testing specific migration from plastics into dry foodstuffs. Although research data has been available, there was to date no precision or validation data on the ability of a method both for the testing of specific migration and for the subsequent quantification of representative substances from PPPO.

Previous research done in 2010-2011 by the EURL-FCM focused the development and the optimisation of a standard operation procedure for using the new simulant E for testing specific migration into dry foodstuffs. A preliminary part of the research led to the selection of substances that responded to criteria of being able to migrate into PPPO from a spiked film, be amenable to be homogeneously spiked into PPPO and into a plastic film, be stable in the film and in PPPO, and be representative a of a range of substances that are common food contact additives. The outcome of the preliminary research led to the selection of substances chosen as models for the subsequent interlaboratory comparison (ILC) exercise due to their migration properties and stability characteristics in LDPE foil and the PPPO. The models for the exercise were butyl hydroxytoluene (BHT), benzophenone (BP), diisobutylphthalate (DiBP), diethylhexyladipate (DEHA) and 1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH).

An efficient analytical method was developed for the quantification of a range of substances investigated from PPPO including extraction of the PPPO followed by GC-MS analysis. A method for fortifying PPPO and polyethylene film (LDPE) with a range of model substances (surrogates) was also developed. An experimental design was also developed for the migration tests using smaller migration test cells than the larger conventional ones for overall migration.

This work presents the results of the subsequent ILC which involved 17 laboratories at EU level. The general aim of the ILC was to evaluate the laboratory performance and precision criteria of the harmonised method for the extraction and quantification of the models substances from PPPO and also for the migration test from a fortified plastic film into PPPO followed by their subsequent quantification.

The EURL prepared and proposed a SOP describing the exposure of a plastic material or article (intended for use with dry foods) to PPPO and the subsequent extraction of the PPPO and analysis of the extract. The exercise foresaw: 1) one part using a spiked PPPO for which the ILC focused on extraction from PPPO itself and quantification of surrogate substances 2) another part using a film specifically contaminated with a selection of substances for which the ILC consisted of the exposure of a film to PPPO and subsequent extraction and quantification. This allowed to obtain data on precision and laboratory performance for both migration and quantification.

The test materials used in this exercise were spiked PPPO samples prepared by EURL-FCM with three levels of model substances and a spiked LDPE film to perform the migration part of the exercise. The homogeneity and stability studies were performed by the EURL-FCM laboratory.

There were 17 volunteer laboratories participating from 15 countries to whom samples were dispatched and 16 of which submitted results. From the EURL-NRL network 14 laboratories out of 15 reported results. There were 2 guests from Germany that provided results as well. Since the aim of the ILC was evaluation of the laboratory performance and to obtain precision criteria there was a communication to the participants to the use harmonised protocol developed. Participants were invited to report four replicates measurements in repeatability conditions. This was done by

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most of the participants. The results of analyses were received and statistically interpreted. The assigned values were obtained as a consensus values after applying the robust statistics to the results obtained from the participants. Laboratory results were also rated with z-scores in accordance with ISO 13528 [4].

The results showed that the validation ILC was successful with more than 8 valid results even taking into account the complexity of the exercise. The results showed that the method performance could be estimated to obtained 7-9% repeatability standard deviation (sd) for most substances (regardless of concentration), with 12% rsd for the high level of BHT and for DiBP at very low levels. The reproducibility results resulting from the ILC involving the 16 EU laboratories were summarised as performance indicators of the method for the quantification from PPPO (for the 3 levels of concentrations of the 5 substances) as well as from migration experiments from the fortified plastic at 60C for 10 days and subsequent quantification.

Reproducibility S.D. (%) calculated by ISO 5725-5 for the different exercises (LL: legislative limit, i.e. SML) are summarised below.

Considering the lack of the data previously available in the literature, this interlaboratory comparison exercise provided a protocol method description which was tested and evaluated, and most importantly the first successful attempt to derive precision data at EU level for a specific migration experiment. It also represented a challenging scenario and was very extensive with 3 levels of concentrations and 1 film, 5 substances, 4 replicates and results from 16 laboratories, i.e 1360 data points. This exercise consequently provided a great breadth of valuable detailed and traceable precision data, which can be used for a more refined information and proficiency test of official controls planned for 2013.

Substance LL valuemg/kg

R sd % at 1/10 * LL

R sd % at the LL

R sd %At 2 * LL

R sd %From FILM

BHT 2 45% 28% 19% 41%

BP 0.6 (/2) 21% 20% 17% 22%

DiBP ‐‐ 40% 30% 26% 25%

DEHA 18 29% 21% 16% 35%

DINCH 60 24% 21% 16% 16%

Substance LL valuemg/kg

R sd % at 1/10 * LL

R sd % at the LL

R sd %At 2 * LL

R sd %From FILM

BHT 2 45% 28% 19% 41%

BP 0.6 (/2) 21% 20% 17% 22%

DiBP ‐‐ 40% 30% 26% 25%

DEHA 18 29% 21% 16% 35%

DINCH 60 24% 21% 16% 16%

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2. Introduction

ILC studies are an essential element of laboratory quality assurance and allow individual laboratories to compare their analytical results with those from other laboratories while providing them objective standards to perform against.

It is one of the core duties of the European Reference Laboratories to organise interlaboratory comparisons, as is stated in Regulation (EC) No 882/2004 of the European Parliament and of the Council [5].

In accordance with the above requirements the European Reference Laboratory for Food Contact Materials (EURL-FCM) organised in 2011-2012 interlaboratory comparison tests for the network of appointed National Reference Laboratories (NRLs).

3. Scope

The scope of this ILC had 3 objectives:

1. to establish precision criteria of the analytical method (standard Operating protocol, SOP) proposed for the testing specific migration into PPPO for model substances.

2. to test the laboratory performance of the appointed NRLs to analyse butyl hydroxytoluene, benzophenone, diisobutylphthalate, diethylhexyladipate and 1,2-cyclohexanedicarboxylic acid diisononyl ester in PPPO

3. to establish precision criteria and laboratory performance for both migration and quantification using a film specifically contaminated with a selection of substances for which the ILC consisted of the exposure of a film to PPPO and subsequent extraction and quantification.

The concentration levels were chosen in accordance with the existing legislation [6]. The assessment of the measurement results was undertaken on the basis of requirements laid down in international standards and guidelines [4, 7, 8, 9].

4. Time frame

The EURL-FCM prepared a SOP (Annex 1) based on modified and refined CEN 1186-13 and CEN 14338 methods [2, 3].

Invitation letters were sent to the laboratories (Annex 2). Laboratories were invited to fill in a letter of confirmation of their participation (Annex 3).

The samples were dispatched to participants together with a letter accompanying the samples (Annex 4), the SOP of the analytical method to be used for the exercise

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(Annex 1), a format for the compilation of results to be eventually sent in non-electronic format (Annex 7) and electronic files where the result should be inserted.

The participants were asked to fill in a letter of confirmation of the receipt of the samples (Annex 5). The participants were given 1.5 month to complete the work.

5. Test materials

Samples of spiked LDPE at one concentration level and PPPO at three different concentration levels of five model substances were prepared by the EURL-FCM as presented in Table 1.

To perform the migration tests the 5 Petri dishes, 5 glass o-rings and 50 filters were sent to the laboratories by the EURL. The standard of DINCH (CAS:166412-78-8) was sent by the EURL to ensure the same representative mixture of the isomers.

Table1. Samples for ILC‐2011

Name Sample

TNX1 One bottle with 10 g of the spiked PPPO at level 1



MTNX 4 LDPE discs for migration tests

Blank TNX One bottle with 25 g of clean PPPO

5.1 Preparation

Preparation and homogenization of the test materials were done by the EURL-FCM laboratory according to the procedure described in Annex 6. After spiking and homogenization the PPPO were divided into glass amber bottles and the LDPE foil were wrapped into aluminium foil and hermetically closed together in plastic bag.

5.2 Homogeneity assessment

The samples were tested for homogeneity by the EURL Laboratory. Ten randomly selected test specimens for each sample (TNX1, TNX2, and TNX3 and MTNX) were analysed in duplicate for butyl hydroxytoluene, benzophenone, diisobutylphthalate, diethylhexyladipate and 1,2-cyclohexanedicarboxylic acid diisononyl ester using the method description.

Homogeneity was evaluated by the ProLab Software [10] according to IUPAC International Harmonised Protocol [9], F-test and to the method proposed in the ISO 13528 [4]. The results together with their statistical evaluation are given in Annex 8.

All test materials showed sufficient homogeneity.

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5.3 Stability test

Randomly selected specimens for each sample (TNX1, TNX2, and TNX3) were stored at 3 different temperature conditions (4ºC, 20°C and 40ºC). The test samples were monitored for stability by the EURL laboratory for all model substances from the date of preparation and homogeneity assessment to the closing of the ILC. The stability was evaluated as described in ISO GUIDE 35:2006 [11].

The evaluation of data was carried out by performing a linear regression on the determined concentrations of BHT, BP, DiBP, DEHA and DINCH (mean values) vs time. For a stable material it is expected that the intercept is (within uncertainty) equal to the assigned value, whereas the slope does not differ significantly from zero.

Using the linear regression equation:

Y(BHT, BP, DiBP, DEHA and DINCH mg/kg) = b0 +b1 X (time, weeks)

the slope is not significantly different from zero if the following requirement is respected;

where

b1 is the slope obtained from the linear regression,

t0.95,n-2 is the Student’s t-factor for n-2 degrees of freedom and p = 0.95 (95% level of confidence) and

s(b1) is the uncertainty associated with the slope.

This can be calculated as follows:

The value of s (standard deviation of the time-points) can be obtained from:

where n is the number of points of the linear regression.

The results together with their statistical evaluation are given in Annex 9

It should be pointed out that no significant trend was observed for the test samples at all temperature conditions (4ºC, 20°C and 40ºC) for the time of the ILC. It was concluded that the stability of the samples was adequate for at least two months following their preparation.

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5.4 Distribution

The sample kits were dispatched to the participants by the EURL-FCM. Each participant received a box containing:

• a box containing the test materials: • TNX1, TNX2, TNX3– Three amber glass bottles with 10 g of the spiked PPPO (three

different concentration level for each compound); • MTNX – Amber bottle with 25 g of the clean PPPO for the migration tests and

calibration curves; • 4 LDPE discs – for migration tests; • 5 Petri dishes + 5 glass o-rings - to perform the migration tests (10 days 60°C); • 50 filters – to extract the PPPO ; • 1 vial with Cyclohexane dicarboxylic acid diisononyl ester (DINCH): CAS: 166412-78-8

to use as standard in the exercise;

The letters:

• an accompanying letter with instructions on sample handling (Annex 4); • a form to sent back after receipt of the samples to confirm their arrival (Annex 5); • a form for reporting results when not possible to report in the software form (Annex 7).

6. Instructions to participants

Instructions were given to all participants in the letters that accompanied the samples (Annex 4). Laboratories were asked to report 4 results for each concentration level and 4 results for the migration experiment. Participants were asked to follow the SOP provided and to avoid / report any eventual deviation. The results had to be reported using the unit of measure indicated in the instruction letter.

The results were reported in a special ProLab software [10] form or in a form sent to the participants (Annex 7).

7. Approach for statistical evaluation of results

7.1. Evaluation of the method performance characteristics – consensus value, repeatability ( r ) and reproducibility ( R ) standard deviation

The statistical evaluation of the results was performed using the ProLab software [10] applying different algorithms for the determination of the consensus value, repeatability (r) and reproducibility (R) standard deviation.

ISO 5725-2 [12] is a standard approach for the statistical analysis of method validation interlaboratory studies. It uses specific assumptions as follows:

• all laboratories (apart from only a very few outlier laboratories) must have equal analytical performance in order to guarantee that distribution of the test results is close to the normal distribution (this assumption cannot be made for proficiency testing);

• all laboratories must use the same analytical method; • the method requires replicates.

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The standard ISO 5725-2 applies the Grubbs test for the outlier identification of individual test results and laboratory mean values. Additionally tests for the identification of exceeding intra laboratory standard deviations are applied (Cochran test and F-test, respectively). It is common when analysing data from precision experiments to find data that are borderline between stragglers and outliers, thus requiring judgments that may affect the results.

The robust methods described in ISO 5725-5 [13] allow to assign less weight to extreme results, rather than eliminate them from the data set. In this case it is not required to use decisions potentially affecting the results of the calculations. The algorithm of ISO 5725-5 (Algorithm A+S) is similar to one in ISO 13528 [4]. Where results are normally distributed the statistics in ISO 5725-2 give results that are very similar to robust statistics in 5275-5 or Q/Hampel algorithm in DIN 38402 A45 [14].

7.2. Identification of modes using Kernel density plotting

Kernel density plots were additionally used to identify multi modality in the distributions of the values and identify outlying results. Analytical results from a collaborative study are not always normally distributed or can contain values from different populations giving rise to multiple distribution modes. These modes can be visualised by using Kernel density plots [15, 16]. In case the results are not normally distributed the classical statistics from ISO 5725-2 should not be applied.

Kernel density plots were computed by the ProLab software from the analytical results by representing the individual numeric values each as a normalised Gaussian distribution centred on the respective analytical value. The sum of these normal distributions formed then the Kernel density distribution.

7.3. Mandel’s h- and k-statistics

Mandel's h-statistic and Mandel's k-statistic [12] represent measurements for graphically surveying the consistency of the data. They are helpful for method and laboratory assessment.

Mandel's h-statistic address whether there are differences between the mean values of the laboratories.

Mandel's k-statistic is useful to assess the variance of each laboratory compared to the variances of the other laboratories.

Mandel’s h- and k- values were calculated by ProLab software following ISO 5725. The examination of the plots of Mandel's h- and k-statistics may indicate that specific laboratories exhibit patterns of results that are markedly different from the others. This is indicated by (compared to the other laboratories) consistently high or low variation and/or extreme (high or low) mean values.

Various patterns can appear in the plot of Mandel's h-statistic. All laboratories can have both positive and negative values. Individual laboratories may tend to give either all positive or all negative values.

If one laboratory stands out on the k-statistic as having many large values, the respective laboratory has a poorer repeatability precision than the other laboratories. A laboratory could give rise to consistently small k-values because of such factors as excessive rounding of its data or an insensitive measurement scale.

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7.4. Evaluation criteria for laboratory performance – z-scores

Individual laboratory performance was expressed in terms of z and z’-scores in accordance with ISO 13528 and the International Harmonised Protocol:

p

assignedlab Xxz

σ)( −

=

22

)('

assignedp

lab

u

Xxz

+

−=

σ

Where:

xlab is the measurement result reported by a participant;

Xassigned is the assigned value;

σp is the target standard deviation for proficiency assessment;

uassigned is the standard uncertainty of the assigned value.

The z- and z’-scores can be interpreted as follow:

|z|≤2 satisfactory result;

2<|z|≤3 questionable result;

|z|>3 unsatisfactory result.

The z-scores compared the participant's deviation from the assigned value with target standard deviation accepted for the interlaboratory comparison σp.

z’-scores can be used when the assigned value is not calculated using the results reported by the participants. z’-score takes in consideration the uncertainty of the assigned values. In case the guidelines for limiting the uncertainty of the assigned value uassigned < 0.3 σp [4] are met, then z-scores will be similar to z’-scores.

The present ILC uses the mean values of all results reported by the participant as an assigned value. Therefore the z’-scores could not be used for assessment of the laboratory performance. For results reported as "smaller than" (<-values), the reported value was not used in any calculations and no evaluation of the measurement results was made. No scores were given.

8. Results and conclusions

8.1 Preliminary considerations

There were 17 participants from 15 countries to whom samples were dispatched. The ILC was closed permanently in the end of January 2012 for statistical interpretation.

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Sixteen laboratories submitted results. From the EURL-NRL network 14 laboratories out of 15 reported results. There were 2 guests from Germany that provided results as well. As requested, most of the laboratories reported four replicate results under repeatability conditions.

The participation of the laboratories was regarded as satisfactory for the aim of the precision experiment with regards of the numbers of received results. Absolute minimum of participating laboratories for conducting a precision experiment is 8. Since the aim of the ILC was evaluate the laboratory performance and precision criteria of the harmonised method there was a communication to the participants that different techniques were not fit to the scope of this ILC. Fifteen participants followed strictly the SOP and one laboratory used a minor modification which did not influence the results, so all the data were evaluated together.

8.2 Method performance characteristics from the precision experiment

The mean values, reproducibility and repeatability standard deviations were calculated according to 3 different algorithms: DIN 38402 A45 Q-Hampel algorithm, robust ISO 5725-5 and classical ISO 5725-2. There are shown in Tables 2-4. In DIN 38402 A45 Q-Hampel algorithm and robust ISO 5725-5 all the data are taken for the calculations as foreseen. In the ISO 5725-2 algorithm, the Grubs and Cochran test showed a few outliers and stragglers. The outliers were taken out from further statistical elaboration performed with that method. Their number and type per samples are shown in Table 5.

Table 2.: Assigned values and repeatability/reproducibility S.D. calculated by DIN 38402 A45 algorithm

Sample Compound Assigned

value [mg/kg]

Reprod. S.D. [mg/kg]

Rel. reprod. S.D. [%]

Repeat.S.D. [mg/kg]

Rel.repeat. S.D. [%]

DIN 38402 A45 BHT 0.2046 0.1158 56.58 0.0158 7.74 BP 0.2521 0.0591 23.43 0.0174 6.92 DiBP 0.7928 0.3483 43.93 0.1049 13.23 DEHA 2.8681 0.6930 24.16 0.2043 7.12

TNX1

DINCH 8.9760 2.4395 27.18 0.6185 6.89 BHT 1.6312 0.4352 26.68 0.1273 7.80 BP 0.4867 0.1054 21.67 0.0352 7.24 DiBP 1.6463 0.4623 28.08 0.1213 7.37 DEHA 17.4297 4.1591 23.86 0.7570 4.34

TNX2


TNX3


MTNX

DINCH 68.7739 22.0725 32.09 10.8243 15.74

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Table 3: Assigned values and repeatability/reproducibility S.D. calculated by ISO 5725‐5 (A+S) algorithm


value [mg/kg]


Rel. reprod.S.D. [%]

Repeat.S.D. [mg/kg]


ISO 5725-5 (Algorithm A+S) BHT 0.2204 0.0983 44.60 0.0148 6.71 BP 0.2513 0.0528 21.01 0.0188 7.50 DiBP 0.8293 0.3277 39.51 0.1007 12.14 DEHA 2.8676 0.8309 28.98 0.2149 7.49

TNX1


TNX2


TNX3


MTNX

DINCH 70.2446 18.7338 26.67 11.0554 15.74 Table 4: Assigned values and repeatability/reproducibility S.D. calculated by ISO 5725‐2 algorithm


value [mg/kg]


Rel. reprod. S.D. [%]

Repeat.S.D. [mg/kg]


ISO 5725-2 BHT 0.2024 0.0976 48.22 0.0139 6.89 BP 0.2436 0.0603 24.76 0.0238 9.76 DiBP 0.7872 0.3294 41.84 0.1138 14.45 DEHA 2.8246 0.8064 28.55 0.2095 7.42

TNX1


TNX2


TNX3


MTNX

DINCH 65.5067 15.0684 23.00 8.9718 13.70

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Table 5: Number of Grubs and Cochran outliers for all the samples

Mandel's h- and Mandel's k-statistics are shown in the following pages as calculated according to ISO 5725 (Figures 1-5). Values differing statistically significantly from values of other laboratories are marked in a different colour. For a 1% significant level the indicative Mandel’s h value is 2.33 and k-value (for n=4 replicates) is 1.88 Laboratories with higher values are marked in red. For a 5% significant level the indicative Mandel’s h value is 1.86 and k-value (for n=4 replicates) is 1.59. Laboratories with higher values are marked in yellow.

The outcome of the Mandel's h- and Mandel's k-statistics were similar to and in correspondence with the Grubs and Cochran outliers’ tests according to ISO 5725-2.

The calculated mean/assigned values from the three different algorithms gave similar results. Slight differences could be observed in the estimation of the repeatability/ reproducibility standard deviations.

To allocate less weight to extreme results rather than eliminate them from the data set, the robust method ISO 5725-5 was chosen for further calculations. The experiments conducted with BHT, BP, DiBP, DEHA and DINCH in PPPO allowed to derive precision parameters (Table 3). Factors that were relevant in the values obtained for repeatability and reproducibility standard deviation were the sample preparation procedure, which included two extraction steps with filtration and evaporation which could influence the reproducibility of the method. Another factor could be the difficulty to deal with the model analytes (e.g. DiBP could cause ‘blank’ problems when analysed at low concentrations, while DINCH was a mixture of isomers). It should be also pointed out that it was the first exercise organised using PPPO as simulant which is a complex porous polymer difficult to manage.

Sample Compound Grubs outliers (mean value)

Cochran outliers (standard deviation)

Grubs + Cochran outliers

BHT - LC0056, LC0092 - BP - LC0034 - DiBP LC0028 - - DEHA - LC0041 -

TNX1

DINCH LC0005 - - BHT - LC0056, LC0092 - BP - - - DiBP LC0028 - - DEHA LC0005 - -

TNX2

DINCH LC0056, LC0005 - - BHT LC0005 LC0092 - BP - - - DiBP LC0028 - - DEHA - - -

TNX3

DINCH LC0005 - - BHT - LC0013 - BP - LC0013 - DiBP - LC0044 - DEHA - - - MTNX

DINCH - LC0005, LC0013, LC0044 -

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Figure 1: Mandel’s h and k statistics for BHT

Bar 1: TNX1Bar 2: TNX2Bar 3: TNX3Bar 4: MTNX1

Mandel's h statistics for BHT-butylated hyrdotoluene

Laboratory

LC00

05

LC00

10

LC00

13

LC00

16

LC00

25

LC00

28

LC00

31

LC00

34

LC00

38

LC00

41

LC00

44

LC00

48

LC00

49

LC00

50

LC00

55

LC00

56

LC00

92

3.6

3.4

3.2

3

2.8

2.6

2.4

2.2

2

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

-0.2

-0.4

-0.6

-0.8

-1

-1.2

-1.4

-1.6


Mandel's k statistics for BHT-butylated hyrdotoluene

Laboratory

LC00

05

LC00

10

LC00

13

LC00

16

LC00

25

LC00

28

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31

LC00

34

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44

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48

LC00

49

LC00

50

LC00

55

LC00

56

LC00

923.83.73.63.53.43.33.23.1

32.92.82.72.62.52.42.32.22.1

21.91.81.71.61.51.41.31.21.1

10.90.80.70.60.50.40.30.20.1

0

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Mandel's h statistics for benzophenone

Laboratory

LC00

05

LC00

10

LC00

13

LC00

16

LC00

25

LC00

28

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LC00

92

2.2

2

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

-0.2

-0.4

-0.6

-0.8

-1

-1.2

-1.4

-1.6

-1.8

-2

-2.2

-2.4

-2.6


Mandel's k statistics for benzophenone

Laboratory

LC00

05

LC00

10

LC00

13

LC00

16

LC00

25

LC00

28

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31

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55

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56

LC00

92

3.1

3

2.9

2.8

2.7

2.6

2.5

2.4

2.3

2.2

2.1

2

1.9

1.8

1.7

1.6

1.5

1.4

1.3

1.2

1.1

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Figure 2: Mandel’s h and k statistics for BP

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Mandel's h statistics for diisobuthylphthalate

Laboratory

LC00

05

LC00

10

LC00

13

LC00

16

LC00

25

LC00

28

LC00

31

LC00

34

LC00

38

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41

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44

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Mandel's h statistics for diethylhexyladipate

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- 20 -


Mandel's h statistics for DINCH

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Figure 5: Mandel’s h and k statistics for DINCH

- 21 -

8.3 z-scores and laboratories performance

For calculation of the z-score, the most important decisions are the choice of the assigned value and that of the target standard deviation (SD) against which the performance will be assessed. For the assigned values a robust mean was chosen as a consensus assigned value. For the target SD the choice impacts the correct assignment of “satisfactory”, “questionable” and “unsatisfactory”. While Horwitz SD can be a good compromise, it does not reflect various levels of complexity of analytical methods. Thus in this case it was more adequate to use the reproducibility SD as target SD. In Table 6-10 z-scores calculated against two different target SDs are presented. The z-score calculated by reproducibility SD as target SD were more realistic and were used to assess the laboratories’ performance.

Table 6. Summary of the z‐scores against different target S.D. for BHT

Table 7. Summary of the z‐scores against different target S.D. for BP z-scores for BP

classical Horwitz ILC Reproducibility SD

Laboratory TNX1 TNX2 TNX3 MTNX TNX1 TNX2 TNX3 MTNX LC0005 -3.007 -2.938 -3.922 -6.474 -2.818 -2.673 -3.817 -3.143 LC0010 -0.531 0.598 -0.185 -0.587 -0.498 0.545 -0.181 -0.285 LC0013 -0.228 -0.149 0.852 5.560 -0.214 -0.136 0.829 2.743 LC0016 - - - - - - - - LC0025 -0.127 0.483 0.621 -1.235 -0.119 0.440 0.604 -0.599 LC0028 -0.632 -0.896 -0.926 0.123 -0.593 -0.816 -0.901 0.060 LC0031 0.125 0.570 1.098 -0.537 0.117 0.518 1.069 -0.260 LC0034 2.062 -0.168 0.786 1.340 1.932 -0.153 0.765 0.651 LC0038 -0.228 0.512 0.226 0.300 -0.214 0.466 0.220 0.145 LC0041 1.893 2.180 -1.733 -1.067 1.774 1.983 -1.686 -0.518 LC0044 -0.026 -0.092 0.094 2.230 -0.025 -0.083 0.092 1.083 LC0048 -0.430 0.330 0.391 1.555 -0.403 0.300 0.380 0.755 LC0049 0.468 -2.444 -4.402 -2.042 0.439 -2.224 -4.284 -0.992 LC0050 -1.299 -0.804 0.396 1.210 -1.218 -0.732 0.385 0.587 LC0055 1.035 1.633 0.522 -2.020 0.970 1.486 0.508 -0.981 LC0056 -0.683 -0.523 0.687 -1.054 -0.640 -0.476 0.669 -0.511 LC0092 0.984 0.138 -0.048 1.685 0.922 0.126 -0.047 0.867

z-scores for BHT

classical Horwitz ILC Reproducibility SD Laboratory TNX1 TNX2 TNX3 MTNX TNX1 TNX2 TNX3 MTNX

LC0005 -4.979 -4.583 -5.469 -6.559 -2.242 -2.384 -3.739 -1.689 LC0010 - - - - - - - - LC0013 1.572 0.176 -0.344 9.305 0.708 0.092 -0.235 2.396 LC0016 - - - - - - - - LC0025 - 0.076 0.518 0.941 - 0.040 0.354 0.242 LC0028 -2.777 -2.112 -1.650 1.147 -1.250 -1.099 -1.128 0.295 LC0031 -1.026 0.246 1.602 0.274 -0.462 0.128 1.095 0.071 LC0034 0.141 -1.181 0.740 -4.512 0.064 -0.614 0.506 -1.162 LC0038 0.612 -0.572 0.259 1.355 0.275 -0.298 0.177 0.349 LC0041 0.499 2.082 1.269 0.724 0.225 1.083 0.868 0.186 LC0044 1.459 -0.183 0.201 -0.822 0.657 -0.095 0.137 -0.212 LC0048 2.475 -0.103 -0.064 2.206 1.115 -0.054 -0.044 0.568 LC0049 - - - 0.692 - - - -0.178 LC0050 -1.636 -1.163 -0.574 -0.521 -0.737 -0.605 -0.392 -0.134 LC0055 -0.574 -0.103 -0.418 0.454 -0.259 -0.054 -0.286 -0.117 LC0056 -0.744 3.199 -1.539 -5.380 -0.335 1.664 -1.052 -1.385 LC0092 21.373 10.792 45.456 8.172 9.626 5.615 31.078 2.105

- 22 -

Table 8. Summary of the z‐scores against different target S.D. for DiBP

z-scores for DiBP


Laboratory TNX1 TNX2 TNX3 MTNX TNX1 TNX2 TNX3 MTNX LC0005 -5.308 -4.176 -5.332 -4.178 -2.210 -2.348 -2.849 -1.759 LC0010 -0.819 0.022 -1.089 -1.239 -0.341 0.012 -0.582 -0.522 LC0013 -1.735 -1.384 0.397 2.503 -0.723 -0.778 0.212 1.054 LC0016 - - - - - - - - LC0025 -0.874 -0.367 -1.058 -0.867 -0.364 -0.206 -0.565 -0.365 LC0028 10.596 14.771 10.618 -0.294 4.412 8.306 5.673 -0.124 LC0031 -1.369 -0.935 -1.120 -2.492 -0.570 -0.526 -0.598 -1.049 LC0034 2.400 -0.201 2.741 0.285 0.999 -0.113 1.464 0.120 LC0038 0.500 0.411 0.243 -0.309 0.208 0.231 0.130 -0.130 LC0041 1.471 1.538 2.302 -1.782 0.613 0.865 1.230 -0.751 LC0044 1.746 -0.387 -1.182 2.021 0.727 -0.217 -0.632 0.851 LC0048 -4.868 -1.570 -0.423 2.995 -2.027 -0.883 -0.226 1.261 LC0049 -0.611 -1.350 -1.329 -1.549 -0.254 -0.759 -0.710 -0.652 LC0050 -0.324 -0.790 -0.708 1.477 -0.135 -0.444 -0.378 0.622 LC0055 2.845 2.106 0.645 -0.189 1.185 1.184 0.345 -0.080 LC0056 1.032 2.934 1.481 -0.559 0.430 1.650 0.792 -0.235 LC0092 -0.703 0.278 -0.893 3.903 -0.293 0.156 -0.477 1.644

Table 9.Summary of the z‐scores against different target S.D. for DEHA

z-scores for DEHA


Laboratory TNX1 TNX2 TNX3 MTNX TNX1 TNX2 TNX3 MTNX LC0005 4.189 7.418 7.553 9.952 1.974 3.667 4.369 2.921 LC0010 1.219 2.025 2.704 -0.898 0.575 1.001 1.564 -0.264 LC0013 0.485 -0.108 -0.323 -4.371 0.229 -0.054 -0.187 -1.283 LC0016 - - - - - - - - LC0025 0.210 -0.683 -0.657 -2.124 0.099 -0.338 -0.380 -0.623 LC0028 -2.152 1.324 2.113 4.130 -1.014 0.654 1.222 1.212 LC0031 -3.072 -1.877 -1.040 -3.047 -1.447 -0.928 -0.602 -0.894 LC0034 -3.860 -3.353 -1.379 1.366 -1.818 -1.658 -0.798 0.401 LC0038 -0.109 0.592 0.292 0.744 -0.051 0.293 0.169 0.218 LC0041 3.059 0.368 -4.925 -2.787 1.441 0.182 -2.849 -0.818 LC0044 -0.237 -0.683 -0.898 2.854 -0.111 -0.338 -0.520 0.838 LC0048 - -2.341 0.223 2.391 - -1.157 0.129 0.702 LC0049 -0.117 0.880 -0.571 -2.441 -0.055 0.435 -0.331 -0.716 LC0050 -1.203 -2.690 -1.535 -0.127 -0.567 -1.330 -0.888 -0.037 LC0055 1.117 0.634 0.-371 0.232 0.526 0.313 -0.215 0.068 LC0056 0.376 1.823 1.474 -3.650 0.177 0.901 0.852 -1.071 LC0092 0.423 0.737 0.147 2.791 0.199 0.364 0.085 0.819

- 23 -

Table 10. Summary of the z‐scores against different target S.D. for DINCH

z-scores for DINCH


Laboratory TNX1 TNX2 TNX3 MTNX TNX1 TNX2 TNX3 MTNX LC0005 21.917 29.137 29.313 27.173 10.263 12.630 14.882 8.595 LC0010 0.962 4.074 3.614 1.748 0.451 1.766 1.835 0.553 LC0013 1.477 -1.705 -2.477 1.113 0.692 -0.739 -1.258 0.352 LC0016 - - - - - - - - LC0025 0.709 -0.962 -0.646 -2.386 0.332 -0.417 -0.328 -0.755 LC0028 -2.893 -0.458 0.100 0.077 -1.355 -0.198 0.051 0.024 LC0031 -1.693 -0.311 0.058 -3.965 -0.793 -0.135 0.030 -1.254 LC0034 -2.365 -4.151 -2.463 1.005 -1.107 -1.799 -1.251 0.318 LC0038 0.125 0.516 0.490 -0.954 0.058 0.224 0.249 -0.302 LC0041 2.815 0.007 -0.987 -2.860 1.318 0.003 -0.501 -0.905 LC0044 -0.592 -2.014 -1.104 7.047 -0.277 -0.873 -0.561 2.229 LC0048 0.346 0.131 0.386 1.541 0.162 0.057 0.196 0.487 LC0049 0.483 0.310 0.098 -0.208 0.226 0.134 0.050 -0.066 LC0050 -2.719 -2.245 -0.285 0.524 -1.273 -0.973 -0.145 0.166 LC0055 -0.473 -0.460 -1.622 -2.108 -0.222 -0.199 -0.824 -0.667 LC0056 1.914 4.360 4.082 -4.383 0.896 1.890 2.072 -1.386 LC0092 -1.264 0.346 -0.149 1.794 -0.592 0.150 -0.076 0.567

Five sets of figures for each of the four samples are presented in Figures 6-25. Each set included: individual laboratories values and their mean and standard deviation (a), Kernel density plot (b) and z- scores (c).

8.4 Final conclusion

The participation in the ILC was satisfactory for the purpose of the study with 16 out of 17 laboratories which reported results; The ILC on method performance was successful with more than 8 valid results, even taking into account the complexity of the exercise. Considering the lack of the data previously available in the literature, this ILC exercise provided the first successful attempt to derive precision data at EU level for a specific migration experiment. It also represented a worst case and was very extensive with 3 levels of concentrations and 1 film, 5 substances, 4 replicates and 16 laboratories, i.e 1360 data points. This exercise consequently provided a great breadth of valuable detailed and traceable precision data, which can be used for a more refined validation of the method and proficiency test of official controls planned for 2013.

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Sample: TNX1Measurand: BHT-butylated hyrdotolueneMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 13

Assigned value: 0.220 mg/kg (Empirical value)Rel. target s.d.: 44.60% (Empirical value)Rel. repeatability s.d.: 6.71%Limits of tolerance: 0.024 - 0.417 mg/kg (|Z score| < 2.00)

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Figure 6. Summary graphs of the laboratories test results for BHT with their repeatability SD (a), Kernel density plot (b) and z‐scores (c)

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Figure 7. Summary graphs of the laboratories test results for BP with their repeatability SD (a), Kernel density plot (b) and z‐scores (c)

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Figure 8. Summary graphs of the laboratories test results for DiBP with their repeatability SD (a), Kernel density plot (b) and z‐scores (c)

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Figure 9. Summary graphs of the laboratories test results for DEHA with their repeatability SD (a), Kernel density plot (b) and z‐scores (c)

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Figure 10. Summary graphs of the laboratories test results for DINCH with their repeatability SD (a), Kernel density plot (b) and z‐scores (c)

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Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

48

LC00

13

LC00

49

LC00

31

LC00

50

LC00

44

LC00

25

LC00

34

LC00

10

LC00

92

LC00

38

LC00

41

LC00

55

LC00

56

LC00

28

Z sc

ore

98.5

87.5

76.5

65.5

54.5

43.5

32.5

21.5

10.5

0-0.5

-1-1.5

-2-2.5

-3

E

E

Limit of tolerance

Limit of tolerance



- 32 -

ProLab 2010

Laboratory

LC00

34

LC00

50

LC00

48

LC00

31

LC00

25

LC00

44

LC00

13

LC00

41

LC00

38

LC00

55

LC00

92

LC00

49

LC00

28

LC00

56

LC00

10

LC00

05

mg/

kg

32.5

30

27.5

25

22.5

20

17.5

15

12.5

10

7.5

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

34

LC00

50

LC00

48

LC00

31

LC00

25

LC00

44

LC00

13

LC00

41

LC00

38

LC00

55

LC00

92

LC00

49

LC00

28

LC00

56

LC00

10

LC00

05

Z sc

ore

4

3.5

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

E

Limit of tolerance

Limit of tolerance



- 33 -

ProLab 2010

Laboratory

LC00

34

LC00

50

LC00

44

LC00

13

LC00

25

LC00

55

LC00

28

LC00

31

LC00

41

LC00

48

LC00

49

LC00

92

LC00

38

LC00

10

LC00

56

LC00

05

mg/

kg

200

180

160

140

120

100

80

60

40

20

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

34

LC00

50

LC00

44

LC00

13

LC00

25

LC00

55

LC00

28

LC00

31

LC00

41

LC00

48

LC00

49

LC00

92

LC00

38

LC00

10

LC00

56

LC00

05

Z sc

ore

13

12

11

10

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

E

Limit of tolerance

Limit of tolerance



- 34 -

ProLab 2010

Laboratory

LC00

05

LC00

28

LC00

56

LC00

50

LC00

55

LC00

13

LC00

48

LC00

44

LC00

38

LC00

25

LC00

34

LC00

41

LC00

31

LC00

92

mg/

kg

27.5

25

22.5

20

17.5

15

12.5

10

7.5

5

2.5

0

E

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

28

LC00

56

LC00

50

LC00

55

LC00

13

LC00

48

LC00

44

LC00

38

LC00

25

LC00

34

LC00

41

LC00

31

LC00

92

Z sc

ore

32302826

242220181614

1210

8642

0-2-4

E

E

Limit of tolerance

Limit of tolerance



- 35 -

ProLab 2010

Laboratory

LC00

49

LC00

05

LC00

41

LC00

28

LC00

10

LC00

92

LC00

44

LC00

38

LC00

48

LC00

50

LC00

55

LC00

25

LC00

56

LC00

34

LC00

13

LC00

31

mg/

kg

2

1.5

1

0.5

0

EE



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance


ProLab 2010

Laboratory

LC00

49

LC00

05

LC00

41

LC00

28

LC00

10

LC00

92

LC00

44

LC00

38

LC00

48

LC00

50

LC00

55

LC00

25

LC00

56

LC00

34

LC00

13

LC00

31

Z sc

ore

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

-3.5

-4

-4.5

-5

E E

Limit of tolerance

Limit of tolerance



- 36 -

ProLab 2010

Laboratory

LC00

05

LC00

49

LC00

44

LC00

31

LC00

10

LC00

25

LC00

92

LC00

50

LC00

48

LC00

38

LC00

13

LC00

55

LC00

56

LC00

41

LC00

34

LC00

28

mg/

kg

3.5

3

2.5

2

1.5

1

0.5

0

E

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

49

LC00

44

LC00

31

LC00

10

LC00

25

LC00

92

LC00

50

LC00

48

LC00

38

LC00

13

LC00

55

LC00

56

LC00

41

LC00

34

LC00

28

Z sc

ore

76.5

65.5

54.5

43.5

32.5

21.5

10.5

0-0.5

-1-1.5

-2-2.5

-3-3.5

-4-4.5

-5

E

E

Limit of tolerance

Limit of tolerance



- 37 -

ProLab 2010

Laboratory

LC00

41

LC00

50

LC00

34

LC00

31

LC00

44

LC00

25

LC00

49

LC00

55

LC00

13

LC00

92

LC00

48

LC00

38

LC00

56

LC00

28

LC00

10

LC00

05

mg/

kg

60

55

50

45

40

35

30

25

20

15

E

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

41

LC00

50

LC00

34

LC00

31

LC00

44

LC00

25

LC00

49

LC00

55

LC00

13

LC00

92

LC00

48

LC00

38

LC00

56

LC00

28

LC00

10

LC00

05

Z sc

ore

54.5

43.5

32.5

21.5

10.5

0-0.5

-1-1.5

-2-2.5

-3-3.5

-4-4.5

-5

E

E

Limit of tolerance

Limit of tolerance



- 38 -

ProLab 2010

Laboratory

LC00

13

LC00

34

LC00

55

LC00

44

LC00

41

LC00

25

LC00

50

LC00

92

LC00

31

LC00

49

LC00

28

LC00

48

LC00

38

LC00

10

LC00

56

LC00

05

mg/

kg

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

E

E



Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance


ProLab 2010

Laboratory

LC00

13

LC00

34

LC00

55

LC00

44

LC00

41

LC00

25

LC00

50

LC00

92

LC00

31

LC00

49

LC00

28

LC00

48

LC00

38

LC00

10

LC00

56

LC00

05

Z sc

ore

16151413121110

9876543210

-1-2-3-4-5

E

E

Limit of tolerance

Limit of tolerance



- 39 -

ProLab 2010

Laboratory

LC00

05

LC00

56

LC00

34

LC00

44

LC00

49

LC00

50

LC00

55

LC00

31

LC00

41

LC00

25

LC00

28

LC00

38

LC00

48

LC00

92

LC00

13

mg/

kg

45

40

35

30

25

20

15

10

5

0

E

E

Sample: MTNX1Measurand: BHT-butylated hyrdotolueneMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 15


Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

56

LC00

34

LC00

44

LC00

49

LC00

50

LC00

55

LC00

31

LC00

41

LC00

25

LC00

28

LC00

38

LC00

48

LC00

92

LC00

13

Z sc

ore

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

EE

Limit of tolerance

Limit of tolerance



- 40 -

ProLab 2010

Laboratory

LC00

05

LC00

49

LC00

55

LC00

25

LC00

41

LC00

56

LC00

10

LC00

31

LC00

28

LC00

38

LC00

50

LC00

34

LC00

48

LC00

92

LC00

44

LC00

13

mg/

kg

40

35

30

25

20

15

10

5

0

E

E

Sample: MTNX1Measurand: benzophenoneMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 16


Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

49

LC00

55

LC00

25

LC00

41

LC00

56

LC00

10

LC00

31

LC00

28

LC00

38

LC00

50

LC00

34

LC00

48

LC00

92

LC00

44

LC00

13

Z sc

ore

4

3.5

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

-3.5

-4

E

E

Limit of tolerance

Limit of tolerance



- 41 -

ProLab 2010

Laboratory

LC00

05

LC00

31

LC00

41

LC00

49

LC00

10

LC00

25

LC00

56

LC00

38

LC00

28

LC00

55

LC00

34

LC00

50

LC00

44

LC00

13

LC00

48

LC00

92

mg/

kg26

24

22

20

18

16

14

12

10

8

6

Sample: MTNX1Measurand: diisobuthylphthalateMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 16


Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

05

LC00

31

LC00

41

LC00

49

LC00

10

LC00

25

LC00

56

LC00

38

LC00

28

LC00

55

LC00

34

LC00

50

LC00

44

LC00

13

LC00

48

LC00

92

Z sc

ore

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

Limit of tolerance

Limit of tolerance



- 42 -

ProLab 2010

Laboratory

LC00

13

LC00

56

LC00

31

LC00

41

LC00

49

LC00

25

LC00

10

LC00

50

LC00

55

LC00

38

LC00

34

LC00

48

LC00

92

LC00

44

LC00

28

LC00

05

mg/

kg

50

45

40

35

30

25

20

15

10

5

0

E

Sample: MTNX1Measurand: diethylhexyladipateMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 16


Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

13

LC00

56

LC00

31

LC00

41

LC00

49

LC00

25

LC00

10

LC00

50

LC00

55

LC00

38

LC00

34

LC00

48

LC00

92

LC00

44

LC00

28

LC00

05

Z sc

ore

4

3.5

3

2.5

2

1.5

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

E

Limit of tolerance

Limit of tolerance



- 43 -

ProLab 2010

Laboratory

LC00

56

LC00

31

LC00

41

LC00

25

LC00

55

LC00

38

LC00

49

LC00

28

LC00

50

LC00

34

LC00

13

LC00

48

LC00

10

LC00

92

LC00

44

LC00

05

mg/

kg

300

275

250

225

200

175

150

125

100

75

50

25

E

E

Sample: MTNX1Measurand: DINCHMethod: ISO 5725-5 (Alg. A+S)No. of laboratories: 16


Assign

ed va

lue

Mean

Limit of tolerance

Limit of tolerance

ProLab 2010

Laboratory

LC00

56

LC00

31

LC00

41

LC00

25

LC00

55

LC00

38

LC00

49

LC00

28

LC00

50

LC00

34

LC00

13

LC00

48

LC00

10

LC00

92

LC00

44

LC00

05

Z sc

ore

10

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

E

E

Limit of tolerance

Limit of tolerance



- 44 -

9. Acknowledgements

The laboratories who participated in this exercise - listed below - are kindly acknowledged. NRLs

CZECH NIPH- NRL for Food Contact Materials and for Articles for children under 3 years old,

National Institute of Public Health (SZU’)

DENMARK Department of Food Chemistry, National Food Institute Technical University of

Denmark

ESTONIA Health Protection Inspectorate - Central Laboratory of Chemistry

FINLAND Finnish Customs Laboratory

FRANCE SCL Laboratoire de Bordeaux-Pessac

GERMANY Bundesinstitut für Risikobewertung (BFR) (Federal Institute for Risk Assessment)

IRELAND Public Analyst Laboratory - Sir Patrick Duns Hospital

LUXEMBOURG Laboratoire National de Santé, Division du Controle des Denrées Alimentaires

POLAND Laboratory of Department of Food and Consumer Articles Research, National Institute

of Hygene

PORTUGAL ESB-SE (Portuguese Catholic University - Biotechnology College – Packaging

Department)

SLOVENIA National Institute of Public Health of Republic of Slovenia, Department of Sanitary

Chemistry

SPAIN Centro Nacional de Alimentación, Agencia Espanola de Seguridad Alimentaria y

Nutrición (AESAN)

THE NETHERLANDS Food and Consumer Product Safety Authority (VWA), Ministry of Economic Affairs,

Agriculture and Innovation

UNITED KINGDOM Food Environment Research Agency (FERA)

GUESTs

GERMANY LAV Sachsen-Anhalt, Fachbereich Lebensmittelsicherheit, Halle

GERMANY Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen

10. References

[1] Regulation EC10/2011 of the European Parliament and the Council of 14 January 2011 on plastic materials and articles intended to come into contact with food.

[2] CEN 1186‐13: 1999‐05, Materials and articles in contact with foodstuffs – Plastics, Part 13‐Test methods for overall migration at high temperature, December 2002

[3] CEN 14338:2003, Paper and board intended to come into contact with foodstuffs Conditions for determination of migration from paper and board using modified polyphenylene oxide (MPPO) as simulant, March 2004

[4] ISO 13528:2005; Statistical Methods for Use in Proficiency Testing by Interlaboratory Comparisons

[5] Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules

[6] 2002/72/EC Commission Directive of 6 August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs. (Plastics: Unofficial consolidated version

- 45 -

including 2002/72/EC, 2004/1/EC, 2004/19/EC, 2005/79/EC, 2007/19/EC, 2008/39/EC)

[7] M. Thompson, Analyst, (2000), 125, 385‐386

[8] T. Linsinger et al., Accreditation and Quality Assurance in Analytical Chemistry (2001), 6, 20‐25

[9] The International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories by M. Thompson et al., Pure and Applied Chemistry (2006), 78, 145–196

[10] ProLab Software – QuoData, Drezden – www.quodata.de

[11] ISO GUIDE 35:2006; Reference materials–General and statistical principles for certification.

[12] ISO 5725‐2:1994 (E) Accuracy (trueness and precision) of measurement methods and results – Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method

[13] ISO 5725 ‐5 Accuracy (trueness and precision) of measurement method and results. Part 5: Alternative methods for the determination of the precision of a standard measurement method.

[14] DIN 38402 A45 Ringversuche zur externen Qualitätskontrolle von Laboratorien.

[15] AMC, Representing data distributions with kernel density estimates. AMC Technical Brief, 2006, http://www.rsc.org/images/brief4_tcm18‐25925.pdf.

[16] P.J. Lowthian and M. Thompson, Bump‐Hunting for the proficiency tester ‐ searching for multimodality. The Analyst, 2002. 127: p. 1359, https://www.swetswise.com/eAccess/viewAbstract.do?articleID=14625081

11. Annexes

Annex 1. Standard Operating Procedure: Determination of buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl ester in PPPO

Annex 2. Invitation letter to laboratories ILC01 2011

Annex 3. Format for confirmation of participation to ILC01 2011

Annex 4. Letters accompanying the sample ILC01 2011

Annex 5. Letter of confirmation of receipt of ILC01 2011

Annex 6. Procedure for the preparation of the spiked PPPO and LDPE foil

Annex 7. Form for the compilation of the results in non-electronic format

Annex 8. Results of the homogeneity study

Annex 9. Results of the stability study

- 46 -

Annex 1. Standard Operating procedure ILC

DETERMINATION OF BUTYL HYDROXYTOLUENE, BENZOPHENONE,

BIS(2-ETHYLHEXYL)ADIPADE, DIISOBUTYLPHTALATE

AND 1,2-CYCLOHEXANEDICARBOXYLIC ACID DIISONONYL ESTER

IN PPPO

0 INTRODUCTION Butyl hydroxytoluene (BHT) is an antioxidant, benzophenone (BP) is a light stabilizer (UV absorber), bis(-ethylhexyl)adipade (DEHA), diisobutylphtalate (DiBP) and 1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) are plasticizers for the manufacture of plastic articles in application area such as food packaging.

This standard operation procedure (SOP) represents an analytical method for the determination of buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl ester in PPPO a simulant for fatty and dry foods.

1 SCOPE This protocol describes the methods for the cleaning of the PPPO, preparation of migration tests and the extraction procedure with following quantification of buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl ester from the PPPO.

The method is appropriate for the quantitative determination of BHT, BP, DEHA, DiBP and DINCH in PPPO in approximate analyte concentration range of 0.25 to 75 mg/kg, 0.05 to 50 mg/kg, 1 to 50mg/kg, 0.05 to 50 and from 5 to 100 mg/kg respectively.

2 PRINCIPLES Prior to each using, the PPPO is cleaned with acetone in a Soxhlet apparatus for 6h and than dried in the oven at 160°C for 6h.

Migration tests are performed using 1g of the PPPO, which is in one side contact with the spiked LDPE foil inside closed Petri dish and incubated for 10 days in 60°C.

BHT, BP, DEHA, DiBP and DINCH are extracted from the PPPO manually with hexane (2 x 20 ml) at room temperature. The extracts are filtrated and dried under nitrogen to around 5 mL. Determination is carried out by means of gas chromatography-mass spectrometry (GC-MS). Quantification is achieved using benzyl butyl phthalate (BBP) as an internal standard.

- 47 -

3 REAGENTS

3.1 Reference material, reagents and solvents

3.1.1. 2,6-Di-tert-butyl-4-methylphenol (BHT): Sigma-Aldrich, Cat no. 24.002-8, purity 99%, CAS no. 128-37-0.

3.1.2. Benzyl butyl phthalate (BBP): Sigma, Cat. no. P-6699, Purity 98%, CAS: 85-68-7;

3.1.3. Benzophenone (BP): Sigma-Aldrich, Cat no. B-3634, CAS no. 119-61-9;

3.1.4. Bis(2-ethylhexyl)adipate (DEHA): Fluka, Cat. no. 02138, Purity ≥ 99%, CAS: 103-23-1;

3.1.5. Bis(2-methylpropyl)benzene-1,2-dicarboxylate (DiBP): Fluka, Cat. no. 80130, Purity ≥ 98%, CAS: 84-69-5;

3.1.6. Cyclohexane dicarboxylic acid diisononyl ester (DINCH): Industrial supplier, CAS: 166412-78-8;

3.1.7. Acetone for HPLC, 99.8%

3.1.8. Hexane for HPLC, 97%

3.2 Solutions

3.2.1 Standard stock solutions of each compound: buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl ester in hexane with an accurately known concentration of approximately 1000 µg·mL-1 of each compound.

Weight accurately about 100 mg of each compound (3.1.1, 3.1.3 – 3.1.6) into a 100 mL volumetric flasks. Fill volumetric flask up to the mark with hexane (3.1.8) and mix.

Calculate the exact concentration of the substances in µg/mL.

3.2.2 Intermediate standard solutions of buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl ester in hexane (100 µg/mL)

Pipette 1.0 mL of the standard stock solutions (3.2.1) into a 10 mL volumetric flasks and fill the flasks up to the mark with hexane.


3.2.3 Intermediate standard solutions of buthyl hydroxytoluene, benzophenone, bis(-

ethylhexyl)adipade, diisobutylphtalate in hexane (10 µg/mL)

Pipette 1.0 mL of the standard stock solutions (3.2.2) into a 10 mL volumetric flasks

and fill the flasks up to the mark with hexane.


3.2.4 Internal standard solution of benzyl butyl phthalate (BBP) in hexane with an accurately known concentration of approximately 1000 µg·mL-1

- 48 -

Weight accurately about 100 mg of BBP (3.1.2) into a 100 mL volumetric flask. Complete the volume with hexane (3.1.8).

Calculate the exact concentration of BBP in µg·mL-1. 3.2.5 PPPO extraction solution of buthyl phthalate (BBP) in hexane with accurately know

concentration of approximately 2.5 µg·mL-1.

Pipette 2.5 mL of the standard stock solution (3.2.4) into a 1000 mL volumetric flask and fill the flask up to the mark with hexane.

Calculate the exact concentration of BBP in µg·mL-1.

4 LABORATORY EQUIPMENT Cleaning of the PPPO :

4.1 Extraction thimbles;

4.2 Apparatus for Soxhlet extraction;

Migration tests:

4.3 Calibrated balance accurate to 0.1 mg;

4.4 Oven or incubator;

4.5 Migration cells: Petri dishes (Duran® 11.28; 60 x 20 mm) and glass O-rings;

Calibration curve and extraction of the PPPO:

4.6 Syringes or digital pipettes, 5-250 µL;

4.7 Funnels with the frit or Buckner (Ø 45mm);

4.8 Glass microfiber filters (Whatman™, GF/A, 47mm Ø circles);

4.9 Nitrogen evaporator;

4.10 Normal laboratory glassware and apparatus.

5 GC-MS apparatus 5.1 Gas chromatograph equipped with an autosampler and in connection with mass

selective detector.

5.2 GC column, capable of delivering reproducible peaks of buthyl hydroxytoluene, benzophenone, bis(-ethylhexyl)adipade, diisobutylphtalate and 1,2-cyclohexanedicarboxylic acid diisononyl and benzyl butyl phthalate (BBP) as an internal standard, and capable to separate this peaks from interference peaks

NOTE 1: All the solution shall be storage in refrigerator (app. 4ºC) and protected from light (till 3 months).

- 49 -

originated from samples used.

6 PROCEDURE

6.1 Cleaning of the PPPO procedure

− Put PPPO into the Soxhlet thimble;

− Add acetone (3.1.7) into the boiling flask (250 mL of Acetone for 20g of PPPO);

− Turn on the heater and clean (extract continuously) the PPPO for 6h;

− After 6h turn off the heating system take out the PPPO from the cartridge into the backer or big Petri dish;

− Place the covered Petri dish under the fumehood to evaporate the solvent while mixing;

− Put Petri dish into the oven at 160°C for 6h;

− After heating, store the PPPO into closed Erlenmeyer flask.

6.2 Migration test sample preparation

− Place LDPE foil into the Petri dish (4.5);

− Place the glass O-ring in the middle of the LDPE foil;

− Weight 1.0 g of the clean PPPO and put inside the O-ring;

1 – Petri dish; 2 – glass O-ring; 3 – LDPE foil; 4 – PPPO

− Close the Petri dish;

− Shake gently the closed Petri dish to cover all the LDPE foil by the PPPO

− Wrap the Petri dish by aluminum foil carefully to keep the LDPE foil surface covered by PPPO;

− Put the Petri dish inside the oven at 60°C for 10 days;

− After migration test take out the Petri dish from the oven;

NOTE 4: LDPE foil, Petri dish, O‐ring and cleaned PPPO are provided by JRC.

NOTE 2: For guidance, the instrument parameters which are found suitable for the analysis, using the selected column are given in Annex 2.

NOTE 3: Sent PPPO is already cleaned.

1

1

2

3 4

- 50 -

− Transfer carefully the PPPO into 40 mL vial or the Erlenmeyer flask using funnel;

6.3 Extraction of the PPPO

− Transfer 1.0 g of the PPPO (or all the PPPO from the Petri dish after migration) into the 40 mL vial;

− Add 20 mL of extract solution (3.2.5);

− Shake manually for 1 min;

− Leave it for 5 min without shaking;

− Insert the funnel with the filter into a new 40mL vial;

− Decant the hexane from the PPPO through the filter into the new vial;

− Repeat this extraction procedure using once again 20mL of the extract solution (3.2.5);

− Collect the extracts in the same 40mL vial;

− Evaporate the hexane extract under the nitrogen to around 5mL;

− Inject the concentrated extract into GC-MS.

6.4 Preparation of the calibration curve in PPPO for GC-MS analysis Calibration curve for spiked PPPO samples (TNX1, TNX2, TNX3):

− Weight 1.0 g of clean PPPO in 40 mL vials or Erlenmeyer flasks;

− Spike the PPPO using the syringe or digital pipette to obtained concentrations of BHT from 0.25 to 10 µg/g, BP and DiBP from 0.05 to 2.5 µg/g, DEHA from 1 to 50 µg/g and from 5 to 120 µg/g for DINCH (see table below);

− Extract the PPPO as in 6.3;

− Use the PPPO without the spiking as a blank and extract it as in 6.3.

Concentration level in PPPO

[mg/kg]

Intermediate stock solution of:

[µg/mL]

Spiking volume

[µL]

BHT

BP

and

DiBP

DEHA DINCH

PPPO

weight

[g] BHT

BP

and

DiBP

DEHA DINCH BHT

BP

and

DiBP

DEHA DINCH

0 0 0 0 1 10 10 10 100 0 0 0 0

0.25 0.05 1 5 1 10 10 10 100 25 5 100 50

0.5 0.1 2.5 10 1 10 10 10 100 50 10 250 100

1 0.25 5 20 1 10 10 100 100 100 25 50 200

1.5 0.5 10 40 1 10 10 100 1000 150 50 100 40

2.5 1 25 60 1 10 10 100 1000 250 100 250 60

5 2 35 90 1 100 10 1000 1000 50 200 35 90

10 2.5 50 120 1 100 10 1000 1000 100 250 50 120

- 51 -

Calibration curve for PPPO samples after migration tests (MTNX):

Weight 1.0 g of clean PPPO in 40 mL vials or Erlenmeyer flasks;

Spike the PPPO using the syringe or digital pipette to obtained concentrations of BHT from 10 to 75 µg/g, BP and DiBP from 2.5 to 50 µg/g, DEHA from 1 to 50 µg/g and from 5 to 120 µg/g for DINCH (see table below);

Extract the PPPO as in 6.3;

Use the PPPO without the spiking as a blank and extract it as in 6.3.

Concentration level in PPPO

[mg/kg]

Intermediate stock solution of:

[µg/mL]

Spiking volume

[µL]

BHT

BP

and

DiBP

DEHA DINCH

PPPO

weight

[g] BHT

BP

and

DiBP

DEHA DINCH BHT

BP

and

DiBP

DEHA DINCH

0 0 0 0 1 100 10 10 100 0 0 0 0

10 2.5 1 10 1 100 10 10 100 100 250 100 100

15 5 5 20 1 100 100 100 100 150 50 50 200

25 10 10 40 1 100 100 100 1000 250 100 100 40

50 25 25 60 1 1000 100 100 1000 50 250 250 60

75 50 50 120 1 1000 1000 1000 1000 75 50 50 120

6.5 GC-MS analysis

Before starting measurements, examine the base line stability.

Maintain the same operating conditions of the GC-MS system throughout the measurements of all samples and calibration solutions.

7. CALIBRATION

7.1. Analysis of calibration standard solutions

Inject the relevant calibration solutions (6.4). Integrate peaks and measure peak area for BHT, BP, DEHA, DiBP, DINCH and BBP as an internal standard. Construct a calibration function by plotting the analytes concentration against the ratio of the peak area of the analytes and the peak area internal standards in the calibration solutions. Calculate the regression parameters and correlation coefficient.

The calibration curve shall be linear and the correlation coefficient shall be 0,99 or better. If either of the two requirements is not met, fresh standard solutions shall be prepared from the original standard solutions. Analysis of the solutions and construction of the calibration graph have to be repeated.

NOTE 5: For guidance, the instrument parameters suitable for analysis using the selected column are given in Annex 2.

- 52 -

7.2 Analysis of samples

Inject the sample solutions prepared in 6.3 under the same conditions used for the calibration solutions.

Measure the area of the peaks for calculation of the substance concentrations against established in 7.1 regression. Internal standard is used to compensate for the losses of analytes caused by sample handling, adsorption effects etc.

7.3 Evaluation of data

Calculation of the BHT, BP, DEHA, DiBP and DINCH in the PPPO sample in mg/kg.

Concentration in PPPO [mg/kg] = C/W

Where:

C is the substance mass in µg derived from the calibration curve, and

W is the weight of sample taken in g.

8 CONFIRMATION For confirmation of peak identity the retention time and a ratio between target and qualifier ions shall be taken into account.

9 VALIDATION RESULTS To be included after performing interlaboratory comparison study FCM ILC -02-2011

- 53 -

BHT chemical information

Common name: Butylhydroxytoluene, 2,6-di-tert-butyl-p-cresol

IUPAC name: 2,6-bis(1,1-dimethylethyl)-4-methylphenol

PM reference number:

46640

CAS no.: 128-37-0

Molecular formula: C15H24O

Molecular weight: 220.35

Boiling point (°C): 265

Melting point (°C): 70-73

SML value: 3 mg/kg

Water solubility: 1.1 mg/L (20°C)

Log P: 5.31

2D structure:

- 54 -

BP chemical information

Common name: Benzophenone

IUPAC name: Methanone, diphenyl


38240

CAS no.: 119-61-9

Molecular formula: C13 H10 O


Boiling point(°C): 305.4 °C

Melting point(°C): 47.9 °C

SML value: 0.6 mg/kg

Water solubility: Insoluble, 137 mg/L (estimated)

Log P: 3.18 (estimated)

2D structure:

- 55 -

DiBP chemical information

Common name: DiBP, Diisobutylphtalate

IUPAC name: Bis(2-methylpropyl)benzene-1,2-dicarboxylate


CAS no.: 84-69-5

Molecular formula: C16H22O4



Melting point (°C): -37

SML value: 0.5 mg/kg

Water solubility: 20 mg/L

Log P: 4.11

2D structure:

- 56 -

DEHA chemical information

Common name: DEHA, diethyladipate

IUPAC name: Bis(2-ethylhexyl)adipate

PM reference number: 31920

CAS no.: 103-23-1




Melting point (°C): - 67.8

SML value: 18 mg/kg

Water solubility: <0.005 mg/L

Log P: >6-8.39

2D structure:

- 57 -

DINCH chemical information

Common name: 1,2-cyclohexanedicarboxylic acid, diisononyl

IUPAC name: 1,2-bis(3,5,5-trimethylhexyl) cyclohexane-1,2-dicarboxylate


45705

CAS no.: 166412-78-8



Boiling point (°C): 240-250°C

Melting point (°C): -54°C

SML value: 60 mg/kg

Water solubility: < 0.02 mg/L (25°C)

Log P: 10 (25°C)

2D structure:

- 58 -

GC-MS Conditions (for information)

GC conditions

System GC Agilent 7890A

Analytical Column: DB-17 HT, 30 m, 0.25 mm fused silica provided with a 0.15 μm thick layer of (50%-Phenyl)-methylpolysiloxane

Oven temperature program: 70°C 1 min; → 20°C/min → 250°C;

→ 10°C/min → 320°C 5min

Carrier gas Helium

Flow rate 1.3 mL/min

Injector temperature: 280°C

Injection mode: splitless

Injection volume: 1µL

MS conditions

Mass-selective detector MSD Agilent technologies, inert XL EI/CI MSD 5975C

Transfer line temperature 320°C

Source temperature 230°C

Solvent delay 4 minutes

Detection: SIM mode

Compound Target ion Qualifier ion RT (minutes)

BHT 205.1 220.2 7.2

BP 104.9 182 8.7

DEHA 129 147 11.2

DiBP 148.9 223 9.5

DINCH 155 281 12.5-13.7

BBP 206 149 12.3

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Annex 2. Invitation letter to laboratories

- 60 -

Annex 3. Format for confirmation of participation to the ILC

- 61 -

Annex 4. Letters accompanying the samples

- 62 -

Annex 5. Letter of confirmation of receipt

- 63 -

Annex 6. Procedure for the preparation of the spiked PPPO and LDPE foil PPPO:

1. Washing of the 10 L round bottom flask (spiking PPPO): - 10 L mixing round bottom flask was washed in a dish washer, then filled with the deionised water (resistivity 18

MΩ.cm @ 25 °C) and mixed; - The water was removed; - The mixing bottle was filled with 0.5 L of pentane (Aldrich, HPLC grade + 99%) and shaked; - Pentane was removed.

2. Preparation of the spiking solutions:

a) Stock solution of BHT, BP, DiBP, DEHA and DINCH in hexane (approx. 1000 mg/L) - 100 mg of each compound was weighted separately into a 100 ml class A volumetric flask and dissolved in hexane; - the flask was filled up to the mark by hexane.

b) spiking solution of BHT, BP, DiBP, DEHA and DINCH in pentane (1 level) - 210µl of BHT, 105 µl of BP, 87.5 µl of DiBP, 1260 µl of DEHA and 4200 µl of DINCH (stock solutions) were pipetting

into the 1L volumetric flask; - the flask was filled up to the mark by pentane;

c) spike solution of BHT, BP, DiBP, DEHA and DINCH in pentane (2 level) - 1050 µl of BHT, 210 µl of BP, 175 µl of DiBP, 6300 µl of DEHA and 17500 µl of DINCH (stock solutions) were

pipetting into the 1 L volumetric flask; - the flask was filled up to the mark by pentane;

d) stock solution of BHT, BP, DiBP, DEHA and DINCH in pentane (3 level) - 2100 µl of BHT, 420 µl of BP, 350 µl of DiBP, 12600 µl of DEHA and 35000 µl of DINCH (stock solutions) were

pipetting into the 1 L volumetric flask; - the flask was filled up to the mark by pentane.

3. Preparation of the spiking PPPO: - 350 g of the cleaned PPPO was weighted and transferred into the 10 L round bottom flask; - 1 L of the spiking solution in pentane was add to the PPPO; - The flask was rinsed by pentane and all liquid was collected in the round bottom flask; - The flask was closed and the PPPO was mixed continuously for 2 hours; - The flask was open and the PPPO was mixed till the complete evaporation of the pentane. - The same procedure was repeated for the other two spiking levels.

LDPE foil:

1. Washing of the 10L reaction chamber (spiking the LDPE) - 10 L reaction chamber was washed in a dish washer, then filled with the deionised water (resistivity 18 MΩ.cm @ 25

°C) and mixed; - the water was removed; - the mixing bottle was filled with 0.5 L of ethanol (Aldrich, + 99.8%) and shaked; - ethanol was removed.

2. Preparation of the spiking solution:

a) spiking solution of BHT, BP, DiBP, DEHA and DINCH in ethanol: - 1.5 g of BHT, 1.5 g of BP,2.5 g of DiBP,2.5 g of DEHA and 10 g of DINCH were weighted and transferred into the 10

L volumetric flask; - the flask was filled up to the mark by ethanol.

b) spiking of the LDPE foil: - the LDPE foil (size: 30x100 cm) was placed into the reaction chamber; - the foil was rolled and did not touch the chamber walls; - to avoid the contact between the foil itself and the chamber walls the glass bars were used; - the chamber was filled by the ethanol spiking solution to cover up the LDPE foil; - the reaction chamber was placed into the oven at 60°C for 2 days; - after the exposure time the foil was taken out from the chamber and rinsed carefully with the fresh ethanol; - the foil was dried using paper and cut into the discs.

- 64 -

Annex 7. Form for the compilation of the results in non-electronic format

- 65 -

Annex 8. Results of the homogeneity study

Sample Measurand Unit Mean s(analytical) [%]

Mode s(target) HORRAT s(target)

[%]

F-test Check for significant

heterogeneity

ISO 13528 Check for sufficient

homogeneity

Harmonised Protocol - test on significant heterogeneity

BHT mg/kg 0.4440 2.4672 HORWITZ 1 18.0763 OK OK OK

BP mg/kg 0.3760 2.5231 HORWITZ 1 18.5342 OK OK OK

DiBP mg/kg 0.7660 23.2655 HORWITZ 1 16.6519 OK OK OK

DEHA mg/kg 3.2065 1.6982 HORWITZ 1 13.4240 OK OK OK

TNX1

DINCH mg/kg 15.4140 4.4754 HORWITZ 1 10.5988 OK OK OK





TNX2






TNX3






MTNX1


- 66 -

Annex 9. Results of the stability study

A) BHT

- 67 -

B) BP

- 68 -

C) DiBP

- 69 -

D) DEHA

- 70 -

E) DINCH

- 71 -

F) Stability spiked PPPO ® level 1 (TNX1)

Sample Compound Test condition

Intercept (b0)

Slope (b1) s(b1) t(α=0.95,n-2)·s(b1) |b1|< t(α=0.95,n-

2)·s(b1)

40ºC 0.3269 0.0054 0.0037 0.0119 OK

20ºC 0.3508 0.0052 0.0037 0.0117 OK BHT

4ºC 0.3451 0.0056 0.0036 0.0116 OK

40ºC 0.2926 0.0030 0.0020 0.0064 OK

20ºC 0.3249 0.0023 0.0020 0.0065 OK BP

4ºC 0.3058 0.0033 0.0020 0.0064 OK

40ºC 1.0001 0.0017 0.0135 0.0430 OK

20ºC 1.3282 -0.0018 0.0166 0.0529 OK DiBP

4ºC 1.1291 0.0029 0.0159 0.0505 OK

40ºC 3.2312 -0.0014 0.0142 0.0452 OK

20ºC 3.3213 -0.0014 0.0163 0.0519 OK DEHA

4ºC 3.2289 0.0024 0.0157 0.0501 OK

40ºC 13.0878 -0.0507 0.0670 0.2132 OK

20ºC 13.6816 -0.0531 0.0725 0.2308 OK

TNX1

DINCH

4ºC 13.1026 -0.0315 0.0736 0.2341 OK

G) Stability spiked PPPO ® level 2 (TNX2)


Intercept (b0)

Slope (b1) s(b1) t(α=0.95,n-2)·s(b1) |b1|< t(α=0.95,n-

2)·s(b1)

40ºC 1.8563 0.0057 0.0124 0.0395 OK

20ºC 1.8402 0.0093 0.0136 0.0432 OK BHT

4ºC 1.8775 0.0091 0.0125 0.0399 OK

40ºC 0.5632 0.0063 0.0036 0.0115 OK

20ºC 0.5670 0.0065 0.0037 0.0117 OK BP

4ºC 0.5686 0.0063 0.0036 0.0113 OK

40ºC 1.4003 -0.0117 0.0083 0.0265 OK

20ºC 1.3489 -0.0094 0.0092 0.0292 OK DiBP

4ºC 1.2993 -0.0099 0.0097 0.0309 OK

40ºC 22.8427 -0.0368 0.1068 0.3398 OK

20ºC 21.3140 -0.0251 0.0954 0.3035 OK DEHA

4ºC 22.6420 -0.0527 0.0987 0.3142 OK

40ºC 66.0950 -0.1327 0.2277 0.7247 OK

20ºC 63.5977 -0.1214 0.2243 0.7139 OK

TNX2

DINCH

4ºC 66.5340 -0.1843 0.1789 0.5693 OK

- 72 -

H) Stability spiked PPPO ® level 3 (TNX3)


Intercept (b0)

Slope (b1) s(b1) t(α=0.95,n-

2)·s(b1)

|b1|< t(α=0.95,n-2)·s(b1)

40ºC 3.7986 -0.0004 0.0169 0.0538 OK

20ºC 3.8582 0.0031 0.0156 0.0496 OK BHT

4ºC 3.8816 0.0074 0.0149 0.0474 OK

40ºC 0.9550 0.0131 0.0053 0.0169 OK

20ºC 0.9435 0.0133 0.0060 0.0191 OK BP

4ºC 0.9101 0.0147 0.0066 0.0210 OK

40ºC 1.9289 0.0027 0.0192 0.0609 OK

20ºC 1.8649 0.0048 0.0215 0.0684 OK DiBP

4ºC 1.9175 0.0061 0.0195 0.0620 OK

40ºC 39.3880 -0.0374 0.0453 0.1442 OK

20ºC 38.7364 -0.0002 0.0468 0.1489 OK DEHA

4ºC 38.9665 -0.0080 0.0457 0.1453 OK

40ºC 103.0942 -0.0771 0.1778 0.5658 OK

20ºC 102.5737 -0.0311 0.1886 0.6002 OK

TNX3

DINCH

4ºC 103.0735 -0.0093 0.1806 0.5749 OK

- 73 -

European Commission EUR 25680 – Joint Research Centre – Institute for Health and Consumer Protection Title: Development of a harmonised method for specific migration into the new simulant for dry foods established in Regulation 10/2011; Establishment of precision criteria from an EU interlaboratory comparison organised by the EURL- Food Contact Materials for the quantification from and migration into poly(2,6-diphenyl phenylene oxide) Author(s): Giorgia Beldì, Natalia Jakubowska, Aurèlie Peychès Bach and Catherine Simoneau Luxembourg: Publications Office of the European Union 2012 – 72 pp. – 21.0 x 29.7 cm EUR – Scientific and Technical Research series – ISSN 1831-9424 (online) ISBN 978-92-79-28078-8 (pdf) doi:10.2788/76804 Abstract

This report presents the results of the EU Interlaboratory comparison (ILC) of the EURL-FCM towards validation criteria for the using the new simulant for specific migration into dry foods, poly(2,6-diphenyl phenylene oxide), PPPO. Regulation EU 10/2011 has established PPPO as new simulant for testing compliance of plastics in contact with foods. The enforceabilityof the Regulation required the validation of a method effectively to measure the quantification of a range of substances to use as models into PPPO as well as the validation of the migration test for long term storage which is typical for dry foods.This was accomplished under the work programme of the EURL with 16 volunteer National Reference Laboratories (NRLs). The laboratories performance were successful with more than 8 valid results, even taking into account the complexity of the exercise. Considering the lack of the data previously available in the literature, this interlaboratory comparison exerciseprovided the first successful attempt to derive precision data at EU level for a specific migration experiment. It alsorepresented a worst case and was very extensive with 3 levels of concentrations and 1 film, 5 substances, 4 replicates and17 labs, i.e 1360 data points. This exercise consequently provided a great breadth of valuable detailed and traceableprecision data, which could be used for a more refined validation of the method and proficiency test of official controls planned for 2013.

z

As the Commission’s in-house science service, the Joint Research Centre’s mission is to provide EU policies with independent, evidence-based scientific and technical support throughout the whole policy cycle. Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges while stimulating innovation through developing new standards, methods and tools, and sharing and transferring its know-how to the Member States and international community. Key policy areas include: environment and climate change; energy and transport; agriculture and food security; health and consumer protection; information society and digital agenda; safety and security including nuclear; all supported through a cross-cutting and multi-disciplinary approach.

LB-N

A-25680-E

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development of a harmonised method for specific migration into … · 2016. 6. 3. · 2012 giorgia...

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