comparative testing report on the detection and quantification...

Report EUR 25989 EN

20 13

Niccolò Bassani, Adam Niedzwiecki, Marko Maras, Angelo Collotta, Matteo Maretti, Diana Charels

Comparative testing round:

ILC-EURL-GMFF-CT-02/12

Preliminary Report

Comparative Testing Report on the Detection and Quantification of Soybean

40-3-2 and Maize MON 88017 Third Main Title Line Third Line

European Commission

Joint Research Centre

Institute for Health and Consumer Protection

Contact information

Molecular Biology and Genomics Unit – European Union Reference Laboratory for Genetically Modified Food and Feed

(EU-RL GMFF)

Address: Via Enrico Fermi 2749, TP 201, 21027 Ispra (VA), Italy

Functional mailbox: [email protected]

http://ihcp.jrc.ec.europa.eu/

http://www.jrc.ec.europa.eu/

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JRC82214

EUR 25989 EN

ISBN 978-92-79-30349-4 (PDF)

ISSN 1831-9424 (online)

doi: 10.2788/91506

Luxembourg: Publications Office of the European Union, 2013

© European Union, 2013

Reproduction is authorised provided the source is acknowledged.

Printed in Italy

mailto:[email protected]

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http://www.jrc.ec.europa.eu/

EURL-CT-02/12 PreICTR

EUROPEAN COMMISSION JOINT RESEARCH CENTRE

Institute for Health and Consumer Protection Molecular Biology and Genomics Unit

Comparative Testing Report on the Detection and Quantification of Soybean 40-3-2 and

Maize MON 88017 Comparative testing round: ILC-EURL-GMFF-CT-02/12

Date of issue; 23 May 2013

Joint Research Centre Institute for Health and Consumer Protection

Molecular Biology and Genomics Unit

EURI. European Union Referenc· Laboratory

for GM Food & Feed

Report number; EURL-CT-02/12 PrelCTR Status: Preliminary report Confidentiality statement: The laboratory codes assigned to each participant in this comparative testing round are confidential. However, the EU-RL GMFF will disclose details of the National Reference Laboratories that have been appointed under Regulation (EC) No 882/2004 and Regulation (EC) No 1981/2006 to DG SANCO for the purpose of an assessment of their performance.

EU-RL GMFF: Comparative testing report 1/18

Ref. Ares(2013)1393017 - 27/05/2013


Address of Comparative testing provider: European Commission, Joint Research Centre (JRC)

Institute for Health and Consumer Protection (IHCP)

Molecular Biology and Genomics Unit - European Union Reference Laboratory for Genetically

Modified Food and Feed (EU-RL GMFF)

Via E. Fermi 2749,1-21027 Ispra (VA)

Italy

Functional mailbox: mba-comparative-testinatSMrc.ec.euroDa.eu



Executive Summary

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003(1), organised a comparative testing round for National Reference Laboratories nominated under Regulation (EC) No 882/2004(2) and Regulation (EC) No 1981/2006(3), for members of the European Network of GMO Laboratories (ENGL), for official control laboratories and for laboratories from third countries which had volunteered to participate.

In accordance with Article 32 of 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, the EU-RL GMFF shall organise comparative testing and shall ensure an appropriate follow-up of such testing.

The design and execution of the comparative testing round was in accordance with the ISO 17043 Standard'4'. The EU-RL GMFF is accredited according to the ISO 17043 Standard 'General requirements for proficiency testing(4)'.

The test items used in the comparative testing round ILC-EURL-GMFF-CT-02/12 were produced in-house. Monsanto Company provided dried leaves of MON 88017, unique identifier MON-88017-3, and seeds of soybean event 40-3-2 (unique identifier MON-04032-6). A compound feedstuff was spiked with the aforementioned GM events. Participants were required to screen two test items denoted compound feedstuff levels 1 and 2, for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any events detected were then to be quantified. In December 2012, a total of 160 laboratories were invited to participate in ILC-EURL-GMFF-CT-02/12. Test items were shipped at room temperature to participants at the beginning of February 2013 in plastic containers containing approximately 5 g of flour. The EU-RL GMFF managed the on-line registration and submission of results.

Participants could report the results in either mass/mass % or copy/copy %. The EU-RL GMFF calculated the robust means (/¿д) of the maize powder levels 1 and 2 test items in mass/mass % and in copy/copy %. All data were log-transformed and then robust statistics were applied to obtain a robust mean(5'6'7). In addition, values {μ) were assigned by the EU-RL GMFF on the basis of the data from the homogeneity study(8) (mass/mass % data only).

ll) European Commission (2003). Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. Off. J. Eur. Union L 268: 1-23 (2) European Commission (2004). 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. Off. J. Eur. Unioni. 191: 1-52 (3) European Commission (2006). Regulation (EC) No 1981/2006 of 22 December 2006 on detailed rules for the implementation of Article 32 of Regulation (EC) No 1829/2003 of the European Parliament and of the Council as regards the Community reference laboratory for genetically modified organisms. Off. J. Eur. Union L 368: 99-109 W ISO/IEC 17043:2010 Conformity assessment - General requirements for proficiency testing {5)Analytical Methods Committee (2004). GMO Proficiency Testing: Interpreting z-scores derived from log-transformed data, RSC. AMC Technical Brief, No. 18, December 2004



On average, 94.37 % of the participating labs returning results in mass/mass % reported a value with a Z-score estimated on the basis of the robust mean lying within the working range of -2 to +2, whereas this percentage decreased to 78.76 % for laboratories returning results in copy/copy %. This difference was larger for maize event MON88017 (mean 96.08 % versus 71.57 % for m/m % and cp/cp %, respectively) than for soybean event 40-3-2 (mean 92.66 % versus 85.95 % for m/m % and cp/cp %, respectively). Moreover, a relevant difference between the assigned value and the robust mean for level 2 concentration of maize event MON 88017 was observed. Such a difference occurred because of a substantial under-estimation of the actual concentration level by 43 out of 51 laboratories (i.e. 84.31 %) reporting results in m/m % format.

(6) Thompson, M., Ellison, SLR., Owen, L., Mathieson, K., Powell, J., Key, P., Wood, R., Damant, ΑΡ. (2006). Scoring in Genetically Modified Organism Proficiency Tests Based on Log-Transformed Results. J. AOAC Int. 89; 232-239 !7) Analytical Methods Committee (1989). Robust statistics - How not to reject outliers Part 1. Basic Concepts. Analyst 114: 1359-1364 <" GP41GP40_EURL_4_Assessment of the homogeneity and stability of test items for comparative testing (2012). EU-RL GMFF internal quality document.



Content

1. Introduction 6 2. Approach 6

2.1 Assigned values determined by the test item producer 7 2.2 Robust mean from participants 7 2.3 Statistical data and summaries 7

3. Participants'results 7 4. Interpretation of z-scores 16 5. Conclusions 17 6. References 17

Drafted by:

N. Bassani (Scientific and technical support officer) / A<c J,

Scientifìc and technical approval:

M. Mazzara (Competence group leader) ·. <?. m Jmoia

Compliance with EU-RL Quality System:

S. Cordeil (Quality manager) Í4'vv

Authorisation to publish:

J. Kreysa (Head of Unit)



1. Introduction

The aim of this preliminary report is to inform participants about the outcome of this comparative testing round. Participants having obtained a z-score(s) outside the working range of -2 to +2 will be informed by e-mail and requested to submit their raw data. Participants that will be asked to repeat the experimental work will receive advice regarding the approach to be followed for the experimental analyses. Those participants are not informed about the exact value of their z-score(s). A final report will be issued not later than two months after this preliminary report, taking account of the repeated experiments carried out and reported on in time. For those laboratories that did not repeat their experiments in time or that had again unacceptable Z-scores, further follow-up, possibly involving an on-site visit by EURL experts, may be planned to help them improving their performance. Confirmation of that improvement can then only be provided by the next CT-round.

2. Approach

The assignment of a laboratory number to each participant and the submission of results were managed by the EU-RL GMFF. Results had to be reported using an on-line form for which each participant received an individual access code. A questionnaire was attached to the on-line reporting form to provide details of the analytical methods used.

Participants had to screen two test items denoted compound feedstuff levels 1 and 2, for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any events detected were then to be quantified. Participants could report the quantitative results in either mass/mass % (m/m %) or DNA copy/copy % (cp/cp %). The expression of measurement results in cp/cp % follows the Recommendation (EC) No 2004/787(9), where it is recommended that the results of quantitative analyses are expressed as GM DNA copy numbers in relation to target taxon-specific copy numbers calculated in terms of haploid genomes.

Participants were instructed to apply the formulas described below when reporting their results.

mass GM event [g] m/m % = x 100 % (1)

Total mass species [g]

GM event DNA copy numbers [cp] cp/cp % = x 100 % (2)

Target taxon-specific DNA copy numbers [cp]

9 European Commission (2004). Recommendation (EC) No 2004/787 of 4 October 2004 on technical guidance for sampling and detection of genetically modified organisms and material produced from genetically modified organisms as or in products in the context of Regulation (EC) No 1830/2003. Off. J. Eur. Union L 348; 18-26

EU-RL GMFF; Comparative testing report 6/18


2.1 Assigned values determined by the test item producer

Following evaluation of the data it was decided to include in this preliminary report assigned values determined by the EU-RL GMFF. The assigned value in m/m % {μ) is derived from the homogeneity datat8). For this study the assigned values in cp/cp % were not determined.

2.2 Robust mean from participants

The robust mean (/^) from participants in the comparative testing round was calculated using robust statistics'10'. This approach minimises the influence of outlying values. All results were log-transformed prior to the calculation of the robust mean to establish a near-normal distribution allowing the interpretation of results on the basis of a normal distribution'6'. Robust means {μα) were calculated on the basis of the results reported in m/m % and cp/cp %, respectively.

2.3 Statistical data and summaries

The aim of a performance statistic is to provide participants with a meaningful result that can be easily interpreted. The procedure followed for the evaluation of participants' performance was agreed by the Members of the Advisory Board and relies on the calculation of z-scores on the assigned values'8' {μ) and the robust means'10' {μη) of the participants' results. In this context, the z-score is a measure indicating to which degree a laboratory deviates from the "true" value (assigned value μ or robust mean μ«) of the GM concentration, relatively to the defined standard deviation for proficiency testing. The larger the absolute value of the z-score, the more likely it is that a laboratory is underperforming by over- (if z-score is too high) or under-estimating (if z-score is too low) the actual level of GMO-concentration of a specific sample.

3. Participants' results

As for soybean event 40-3-2, 63 labs returned results in m/m % and 15 in cp/cp % for Level 1 and 62 returned results in m/m % and 15 in cp/cp % for Level 2. For maize event MON 88017, 53 labs returned results in m/m % and 17 in cp/cp % for Level 1 and 53 in m/m % and 18 in cp/cp % for Level 2. Tables 1, 2, 5 and 6 show the z-scores gained by participants who had reported the results in m/m % whereas the z-scores on the basis of the results reported in cp/cp % are displayed in Tables 3, 4, 7 and 8. Additionally, Table 9 reports the percentages of laboratories presenting a z-score (calculated both on the basis of a robust mean and of an assigned value) within and outside the working range of (-2,+2) for each concentration level of the two GM events.

10 Analytical Methods Committee (2001). Robust statistics: a method for coping with outliers. AMC Technical Brief, No. 6, April 2001



Table 1. z-scores for soybean event 40-3-2 compound feedstuff level 1 for results reported in m/m %. 1 z-score calculated on the basis of the robust mean,2 z-score calculated on the basis of the assigned value, * z-score outside the working range of -2 to +2

Soybean event 40-3-2 (level 1, m/m %) Laboratory number z-score1 z-score2

L02 * *

L03 -0.12 0.00 L04 -0.37 -0.26 L05 0.29 0.41 LOG -0.50 -0.39 L07 0.63 0,74 L08 0.43 0.55 Lll 0.28 0.39 L12 0.06 0.17 L13 0.07 0.19 L14 no z-score attributed no z-score attributed L16 -0.22 -0.10 LIS 0.15 0.26 L20 -0.08 0.03 L21 -0.43 -0.31 L22 -0.15 -0.04 L23 0.06 0.17 L24 -0.23 -0.12 L26 1.70 1.81 L28 0.88 1.00 L29 1.01 1.12 L32 0.49 0.60 L33 -0.22 -0.10 L34 0.39 0.51 L35 0.10 0.22 L36 0.51 0.63 L37 0.05 0.17 L39 -0.02 0.09 L40 0.59 0.71 L41 -0.46 -0.34 L42 -0.23 -0.12 L46 -0.67 -0.56 L48 -0.84 -0.72 L49 -0.22 -0.10 L50 -1.17 -1.06 LSI -0.69 -0.58 L53 0.27 0.38 L54 -0.02 0.09 L58 0.09 0.20 L59 -0.29 -0.17 L61 0.09 0.20 L62 -0.50 -0.39 L64 -0.60 -0.48 L65 -0.12 0.00 L66 -0.22 -0.10 L68 0.01 0.13 L69 -lš01 -0.90 L70 * »

L71 0.25 0.37 L72 0.01 0.13 L74 -0.69 -0.58 L75 -0.22 -0.10 L76 1.41 1.53 L77 0.10 0.22 L78 -0.07 0.05 L79 -0.36 -0.24 L82 • *

L83 0.21 0.32 L84 -0.23 -0.12 L87 0.57 0.68 L88 0.43 0.55 L89 0.07 0.19 L90 0.25 0.37



Table 2. z-scores for soybean event 40-3-2 compound feedstuff level 2 for results reported in m/m %.1 z-score calculated on the basis of the robust mean,2 z-score calculated on the basis of the assigned value, * z-score outside the working range of -2 to +2

Soybean event 40-3-2 (level 2, m/m 0/o) Laboratory number z-score1 z-score2

L02 * *

L03 0.61 1.07 L04 -0.29 0.17 L05 1.06 1.52 L06 -0.16 0.30 L07 1.45 1.91 L08 0.08 0.55 Lil -0.29 0.17 L12 -0.29 0.17 L13 -0.16 0.30 L14 no z-score attributed no z-score attributed L16 0.20 0.66 LIS -0.29 0.17 L20 -0.71 -0.25 L21 -1.59 -1.13 L22 1.06 1.52 L23 0.20 0.66 L24 0.61 1.07 L26 -0.42 0.04 L28 -0.68 -0.22 L29 no results reported no results reported L32 -0.03 0.43 L33 1.14 1.60 L34 -0.56 -0.10 L35 -0.71 -0.25 L36 -0.56 -0.10 L37 -1.36 -0.90 L39 0.51 0.97 L40 * *

L41 * *

L42 -1.07 -0.61 L46 0.89 1.35 L48 # *

L49 0.20 0.66 L50 -1.39 -0.93 LSI 0.61 1.07 L53 -0.71 -0.25 L54 1.06 1.52 L58 0.71 1.17 L59 0.08 0.55 L61 -0.56 -0.10 L62 -0.56 -0.10 L64 -0.56 -0.10 L65 1.85 *

L66 0.08 0.55 L68 -0.42 0.04 L69 -0.16 0.30 L70 * *

L71 0.51 0.97 L72 -0.03 0.43 L74 -1.21 -0.75 L75 -0.87 -0.41 L76 0.61 1.07 L77 0.20 0.66 L78 -0.56 -0.10 L79 0.41 0.87 L82 * *

L83 -1.03 -0.57 L84 -0.56 -0.10 L87 -0.42 0.04 L88 0.41 0.87 L89 -1.81 -1.35 L90 -0.29 0.17



Table 3. z-scores for soybean event 40-3-2 compound feedstuff level 1 for results reported in cp/cp %. 1 z-score calculated on the basis of the robust mean, * z-score outside the working range of -2 to +2

Soybean event 40-3-2 (level 1, cp/cp в/о)

Laboratory number z-score1

LOI -0.24 L09 0.27 LIS -0.29 H7 -0.27 L25 -0.35 L26 1.12 L30 0,24 L31 *

L43 -0.33 L45 1.43 L52 0.37 L60 0.22 L63 0.16 L85 0.08 L86 -0.58



Table 4. z-scores for soybean event 40-3-2 compound feedstuff level 2 for results reported in cp/cp %. 1 z-score calculated on the basis of the robust mean, * z-score outside the working range of -2 to +2

Soybean event 40-3-2 (level 2, cp/cp %)


LOI 1.97 L09 -0.48 LIS -1.27 L17 -0.62 L2S no z-score attrbuted L26 -0.48 L30 0.74 LSI »

L43 -1.66 L4S *

LS2 •

L60 -0.48 L63 0.14 L8S 0.35 L86 -0.22



Table 5. z-scores for maize event MON 88017 compound feedstuff level 1 for results reported in m/m %. 1 z-score calculated on the basis of the robust mean, 2 z-score calculated on the basis of the assigned value, * z-score outside the working range of -2 to +2

Maize event MON 88017 (level 1, m/m °/o) Laboratory number z-score1 z-score2

L02 0.03 -0.31 L03 -0.08 -0.43 L04 -0.34 -0.68 L05 -0.95 -1.29 L06 -0.29 -0.64 L07 -0.47 -0.82 L08 -0.29 -0.64 L12 -0.29 -0.64 L13 0.67 0.33 L14 no z-score attributed no z-score attributed L16 -0.83 -1.18 L18 -0.29 -0.64 L20 -0.52 -0.86 L21 -1.19 -1.54 L23 -0.38 -0.72 L24 no z-score attributed no z-score attributed L28 -0.01 -0.36 L33 0.73 0.38 L35 -0.12 -0.47 L36 -0Ś29 -0.64 L37 1.00 0.66 L39 0.31 -0.03 L40 * 1.89 L42 -0.10 -0.44 L46 -0.47 -0.82 L49 -0.62 -0.96 L51 1.19 0.85 L53 -0.17 -0.51 L54 0.81 0.46 L5S -0.08 -0.43 L59 -0.83 -1.18 L61 0.98 0.64 L62 • #

L64 0.44 0.09 L65 1.26 0.91 L66 -0.17 -0.51 L68 -0.21 -0.55 L69 -1.26 -1.60 L70 0.96 0.62 L71 0.18 -0.17 L72 1.44 1.10 L74 0.53 0.19 L76 0.10 -0.24 L77 -0.67 -1.01 L78 -0.29 -0.64 L79 1.63 1.28 L82 0.70 0.36 L83 0.14 -0.20 L84 -0.95 -1.29 L86 -0.38 -0.72 L87 -0.29 -0.64 L88 0.07 -0.28 L90 0.53 0.19



Table 6. z-scores for maize event MON 88017 compound feedstuff level 2 for results reported in m/m %. 1 z-score calculated on the basis of the robust mean,2 z-score calculated on the basis of the assigned value, * z-score outside the working range of -2 to +2

Maize event MON 88017 (level 2, m/m %)

Laboratory number z-score1 z-score2

L02 -0.22 -0.90 L03 -0.39 -1.07 L04 0.22 -0.46 L05 -0.94 -1.62 L06 0.08 -0.60 L07 -0.78 -1.46 L08 -0.75 -1.43 L12 -0.29 -0.98 L13 0.73 0.05 L14 no z-score attributed no z-score attributed L16 -0.44 -1.12 LIS -0.25 -0.93 L20 -0.34 -1.02 L21 -1.57 *

L23 -0.58 -1.26 L24 no z-score attributed no z-score attributed L28 -0.02 -0.70 L33 0.71 0.03 L35 0.12 -0.56 L36 0.14 -0.54 L37 0.35 -0.33 L39 0.50 -0.18 L40 0.94 0.26 L42 0.29 -0.39 L46 -0.15 -0.84 L49 -0.29 -0.98 LSI 0.95 0.27 LS3 -0.11 -0.79 LS4 0.52 -0.16 LSS 0.31 -0.37 LS9 0.35 -0.33 L61 0.95 0.27 L62 * *

L64 0.50 -0.18 L65 0.74 0.06 L66 -0.52 -1.20 L68 0.24 -0.44 L69 -1.04 -1.72 L70 * »

L71 0.00 -0.68 L72 0.94 0.26 L74 0.42 -0.26 L76 -0.55 -1.23 L77 -0.44 -1.12 L7S -0.07 -0.75 L79 1.30 0.62 L82 0.47 -0.21 L83 0.22 -0.46 L84 -0.61 -1.29 L86 0.10 -0.58 LS7 -0.34 -1.02 L8S 0.18 -0.50 L90 -0.02 -0.70



Table 7. z-scores for maize event MON 88017 compound feedstuff level 1 for results reported in cp/cp %. 1 z-score calculated on the basis of the robust mean, * z-score outside the working range of -2 to +2

Maize event MON 88017 (level cp/cp %)


LOI 1.62 L09 -1.29 LIS -0.91 L17 -0.51 L26 1.13 L30 0.17 L31 1.67 L32 0.88 L34 -0.82 L43 no results reported L48 *

L50 -0.12 L52 -0.24 L55 *

L57 *

L60 -0.59 L63 -1.00 L85 *



Table 8. z-scores for maize event MON 88017 compound feedstuff level 2 for results reported in cp/cp %. 1 z-score calculated on the basis of the robust mean, * z-score outside the working range of -2 to +2

Maize event MON 88017 (level 1, cp/cp %)


LOI 1.41 L09 *

LIS -0.74 L17 -0.47 L26 1.21 L30 -0.05 L31 *

L32 1.31 L34 -0.58 L43 -0.17 L48 *

L50 -0.14 L52 -0.87 L55 *

L57 *

L60 -0.08 L63 -1.46 L85 *



Table 9. % of laboratories providing a quantitative result for the GM events with a Z score within and outside the working range of -2 to +2 for both GM events and concentration levels, according to Z scores based both on robust mean and assigned value for m/m % data and only on robust mean for cp/cp % data.

Level Unit z-score % in (-2, +2) % out (-2, +2)

Level 1 m/m

Robust

Assigned

95.16

95.16

4.84

4.84

Soybean 40-3-2 cp/cp Robust 93.33 6.67

Soybean 40-3-2

Level 2 m/m

Robust

Assigned

90.16

88.52

9.84

11.48

cp/cp Robust 78.57 21.43

Level 1 m/m

Robust

Assigned

96.08

98.04

3.92

1.96

Maize MON 88017 cp/cp Robust 76.47 23.53

Maize MON 88017

Level 2 m/m

Robust

Assigned

96.08

94.12

3.92

5.88

cp/cp Robust 66.67 33.33

4. Interpretation of z-scores

In general one assumes a normal distribution when calculating z-scores. In which case there is a 5 % probability that some z-scores will fall outside the working range of -2 to +2 and a 0.3 % probability that some z-scores will fall outside the working range of -3 to +3. A z-score outside the working range of -2 to +2 indicates that a participant is probably not performing according to specifications although this cannot be stated with 100 % certainty. The higher the value of the

л target standard deviation for proficiency assessment σ, the more likely participants with a z-

л score outside the working range of -2 to +2 are underperforming. However, a higher σ will also increase the probability of accepting unsatisfactory measurement results. Thus, the choice of the

л Advisory Board for Comparative Testing concerning the values of σ for the different GM events reflects the need to take into account both these aspects, i.e. maximizing the number of truly under-performing labs lying outside the working range of -2 to +2 while in the meantime minimizing the number of truly underperforming labs being declared as performing.



5. Conclusions In this sixth comparative testing round participants were asked to screen two test items denoted compound feedstuff levels 1 and 2, for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. A compound feedstuff was spiked with soybean event 40-3-2 and maize event MON 88017 at different concentration levels. Any events detected were then to be quantified.

Overall, results expressed in m/m % were characterised by a higher percentage of z-scores lying within the working range of -2 to +2 with respect to results reported in cp/cp % (mean 94.37 % versus 78.76 %, estimated only on z-scores calculated on the basis of the robust means). The differences in the percentages of z-scores within the working range between m/m % and cp/cp % results for maize event MON88017 (mean 96.08 % versus 71.57 % for m/m % and cp/cp % respectively, estimated only on Z calculated on the basis of the robust means) appear more relevant than what can be seen for soybean event 40-3-2 (mean 92.66 % versus 85.95 % for m/m % and cp/cp % respectively, estimated only on z-scores calculated on the basis of the robust means).

Some differences between robust means and assigned values for m/m % data were observed, in particular for Level 2 of maize event MON 88017 (see Table 6). In this case, robust mean is relevantly lower than the corresponding assigned value, thus reflecting a substantial underestimation of the actual concentration levels. As a matter of fact, 43 out of 51 laboratories (i.e. 84.31 %) reported a value significantly lower than the nominal concentration level. As for the remaining GM events and levels to be quantified, some mild differences were observed for Level 1 of maize MON 88017 and for Level 2 of Soybean 40-3-2, with the former concentration being slighty under-estimated (i.e. robust mean < assigned value) and the latter over-estimated (i.e. robust mean > assigned value). Also in this case, however, no relevant differences were observed in terms of Z-scores lying within and outside the working range.

It can thus be concluded that the results of this CT-round confirm findings from previous rounds that large majority of the laboratories possess the required skills tested. Identified issues will be addressed with the concerned laboratories and a repetition of the experiments or the result of the next CT will be used to verify that any underlying problems have been resolved.

6. References

1. European Commission (2003). Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. Off. J. Eur. Union L 268: 1-23

2. European Commission (2004). Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the



vérification of compliance with feed and food law, animal health and animal welfare rules. Off. J. Eur. Union L 191: 1-52

3. European Commission (2006). Regulation (EC) No 1981/2006 of 22 December 2006 on detailed rules for the implementation of Article 32 of Regulation (EC) No 1829/2003 of the European Parliament and of the Council as regards the Community reference laboratory for genetically modified organisms. Off. J. Eur. Union L 368: 99-109

4. ISO/IEC 17043:2010 Conformity assessment - General requirements for proficiency testing

5. Analytical Methods Committee (2004). GMO Proficiency Testing: Interpreting z-scores derived from log-transformed data, RSC. AMC Technical Brief, No. 18, December 2004

6. Thompson, M., Ellison, SLR., Owen, L, Mathieson, K., Powell, J., Key, P., Wood, R., Damant, ΑΡ. (2006). Scoring in Genetically Modified Organism Proficiency Tests Based on Log-Transformed Results. J. AOAC Int. 89: 232-239

7. Analytical Methods Committee (1989). Robust statistics - How not to reject outliers Part 1. Basic Concepts. Analyst\\Ą\ 1359-1364

8. GP41GP40_EURL_4_Assessment of the homogeneity and stability of test items for comparative testing (2012). EU-RL GMFF internal quality document.

9. European Commission (2004). Recommendation (EC) No 2004/787 of 4 October 2004 on technical guidance for sampling and detection of genetically modified organisms and material produced from genetically modified organisms as or in products in the context of Regulation (EC) No 1830/2003. Off. J. Eur. Union L 348: 18-26

10. Analytical Methods Committee (2001). Robust statistics: a method for coping with outliers. AMC Technical Brief, No. 6, April 2001


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European Commission

EUR 25989 EN – Joint Research Centre – Institute for Institute for Health and Consumer Protection

Title: Comparative Testing Report on the Detection and Quantification of Soybean 40-3-2 and Maize MON 88017

Author(s): Niccolò Bassani, Adam Niedzwiecki, Marko Maras, Angelo Collotta, Matteo Maretti, Diana Charels

Luxembourg: Publications Office of the European Union

2013 – 18 pp. – 21.0 x 29.7 cm

EUR – Scientific and Technical Research series – ISSN 1831-9424 (online)

ISBN 978-92-79-30349-4 (PDF)

doi: 10.2788/91506

Abstract

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food

and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these

activities. This preliminary report describes the outcome of the sixth comparative testing round ILC-EURL-GMFF-CT-

02/12. Participants were required to screen two test items denoted compound feedstuff levels 1 and 2, for the presence

of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any events

detected were then to be quantified.

The test items were produced in-house. The EU-RL GMFF managed the on-line registration and submission of results.

A total of 160 laboratories were invited to participate in ILC-EURL-GMFF-CT-02/12. Participants could report the results

of the exercise either in mass/mass % or in copy/copy %.

Overall, in this sixth comparative testing round results expressed in m/m % were characterised by a higher percentage

of z-scores lying within the working range of -2 to +2 with respect to results reported in cp/cp % (mean 94.37 % versus

78.76 %, estimated only on z-scores calculated on the basis of the robust means). However, The differences in the

percentages of z-scores within the working range between m/m % and cp/cp % results for maize event MON88017

(mean 96.08 % versus 71.57 % for m/m % and cp/cp % respectively, estimated only on Z calculated on the basis of the

robust means) appear more relevant than what can be seen for soybean event 40-3-2 (mean 92.66 % versus 85.95 %

for m/m % and cp/cp % respectively, estimated only on z-scores calculated on the basis of the robust means).

Moreover, a relevant difference between the assigned value and the robust mean for level 2 concentration of maize

event MON 88017 was observed. Such a difference occurred because of a substantial under-estimation of the actual

concentration level by 43 out of 51 laboratories (i.e. 84.31 %) reporting results in m/m % format.

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 methods, tools and standards,

and sharing its know-how with the Member States, the scientific community and international

partners.

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.

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-25989-E

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doi: 10.2788/91506

comparative testing report on the detection and quantification...

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