comparative testing report on the detection and...

Niccolo Bassani Adam Niedzwiecki Angelo Collotta Matteo Maretti Marco Mazzara Joachim Kreysa

Comparative testing round:

ILC-EURL-GMFF-CT-01/13

Version b

Comparative Testing Report on the

Detection and Quantification of GM

events in biscuit powder

2014

Report EUR 26823 EN

This document replaces ''Comparative Testing Report on the Detection and Quantification of GM events in biscuit powder'' with ISBN number 978-92-79-37261-2 and PUBSY request number JRC89455. The corrections made in the new document are: Figure 6a corrected, changed grouping of one laboratory and relative figures/tables accordingly and re-calculated z-scores for MON 863 in m/m % by using only results from laboratories not using reference gene adh1 with an amplicon size of 70 bp to estimate the robust mean.

European Commission

Joint Research Centre

Institute for Health and Consumer Protection

Contact information

Molecular Biology and Genomics Unit

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

E-mail: [email protected]

Tel.: +39 0332 78 5165

Fax: +39 0332 78 9333

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

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

This publication is a Science and Policy Report by the Joint Research Centre of the European Commission.

Legal Notice

This publication is a Science and Policy Report by the Joint Research Centre, the European Commission’s in-house science

service. It aims to provide evidence-based scientific support to the European policy-making process. The scientific output

expressed does not imply a policy position of the European Commission. 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.

JRC91449

EUR 26823 EN

ISBN 978-92-79-40073-5 (PDF)

ISSN 1831-9424 (online)

doi: 10.2788/14356

Luxembourg: Publications Office of the European Union, 2014

© European Union, 2014

Reproduction is authorised provided the source is acknowledged.

Printed in Italy

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 GM Events in Biscuit Powder

Comparative testing round: ILC-EURL-GMFF-CT-01/13

Date of Issue Version b: 5 August 2014

Report number: EURL-CT-01/13 CTRb

Status: Recalled final report

Motivation: figure 6a corrected, changed grouping of one laboratory and relative

figures/tables accordingly and re-calculated z-scores for MON 863 in m/m % by using

only results from laboratories not using reference gene adh1 with an amplicon size of 70

bp to estimate the robust mean.

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 to DG SANCO.

ISO 17043 Accreditation Proficiency Test Provider by:

EURL-CT-01/13 CTR Final b

EU-RL GMFF: Comparative testing report 2/89

Corrections from the previous version:

Version b – 05/08/2014

– Overall percentage of laboratories performing satisfactorily changed from 92 % to 89 % according to the newly derived z-scores for MON863 m/m % measurements

Page 7 – Changed numbering of laboratories in different groups: from 6 ENGL members and 3 non-ENGL official EU control laboratories to 7 ENGL members and 2 non-ENGL official EU control laboratories

Page 8 Figure 1 – Changed according to this new laboratory grouping

Page 9 – Section 2.1: Added DNA extraction method used (Nucleospin) and modified description of PCR inhibition test

Page 9 – Section 2.2: Added "taken into account the water content of the base materials" to explain the masses shown in Table 1

Page 10 – Moved the sentence on reporting of measurement uncertainty from Annex 3 to this section

Page 15 – modified text on excluding data from laboratories having used adh1-70 bp for calculation of the robust mean

Page 16 – Added paragraph on new approach for z-scores estimation

Page 16 Table 3 – Changed according to the new robust means estimated on stratified data. Position of asterisk in the table was changed (after µR instead of after N = 55), clarifying that µR was calculated on a subset of the N = 55 only. Footnote explanations were added

Page 18 Table 5 – Text of Section 5.1 modified for clarity and sentence on GMO detection added. New table introduced for comparison between robust means and internal reference value. In Section 5.2, actual numbers of laboratories added (in addition to %).

Page 19 Figure 6a – Changed based on new robust means for MON 863 and corrected numbers for other events

Page 20 Table 6a – Changed underperforming laboratories in accordance with results from newly derived z-scores and used notation "unsatisfactory z-scores" instead of "underperforming z-scores" (also changed elsewhere)

Page 20 – Modified comments on underperformances, with new number of underperforming NRLs and non-NRLs

Page 21 – deleted paragraph on underperforming laboratories (already described in Discussion) and modified text on selection of methods by laboratories and dissemination of new information by EU-RL

Page 22 – Reference list updated

Page 24 – Changed labelling for 1 laboratory from 5 (non-ENGL official control) to 4 (ENGL member)

Page 26 – Order of Annexes changed

Pages 28-29-30 – Table modified with L32 being labelled as 4 (ENGL member) and not 5 (official control in EU)

Page 31 – Notation of acceptance limits for z-scores changed into |z| ≤ 2.0 (also changed in Tables A.4.1-12)

Page 32 – hmgA changed into hmg, which is more commonly used

Page 33 Table A.4.1 – Changed L01 to L44 in legend

Pages 38-40 – New z-scores tables A4.5 – A4.6

Pages 52-53 – New z-scores graphical visualisation in Figures A4.5 – A4.6, in accordance with new z-scores described in Tables A4.5 – A4.6.



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, I-21027 Ispra (VA), Italy


Phone: +39 0332 78 6518 Coordinator

Marco Mazzara

Phone: +39 0332 78 5773




Executive Summary

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF),

accredited under ISO 17043, organised a comparative testing (CT) round for National Reference

Laboratories (NRLs) nominated under Regulation (EC) No 882/2004, with voluntary participation of

other official control laboratories.

The test items consisted of biscuit powder made from conventional maize and wheat grain spiked

with powder of 98140 maize and MON 863 X MON 810 maize in different concentrations (Level 1 and

2). Participants were required to perform species identification and test for the presence of maize

events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603, 1507, MON 88017,

MON 89034, 98140 and MIR162 in the two test items. Any event detected then had to be quantified.

Results could be reported in mass/mass % or copy/copy % and the EU-RL calculated the robust

means (µR) of Level 1 and 2 test items accordingly. The target standard deviation for CT was fixed by

the Advisory Board for Comparative Testing at 0.20 for all events, based on the experience of

previous CT rounds. The robust means and target standard deviation were used to derive z-scores

for the participants’ results.

Sixty-four laboratories from 28 countries registered for this CT round, of which all but one returned

at least qualitative test results.

The EU-RL GMFF issued a final report on this CT round in April 2014. After the final report was

prepared, it was discovered that the distribution of the quantitative data obtained from laboratories

for event MON 863 deviated from normality because some laboratories used the adh1 reference gene

(with target amplicon of 70 bp) for quantification of MON 863 maize, which is known to carry a

nucleotide polymorphism that may affect amplification. Therefore, the robust means for event MON

863 were re-calculated based on the data from laboratories that had used a reference gene different

from adh1-70 bp. The original final report has been re-called; the current version contains the re-

calculated z-scores for maize event MON 863.

When performing species identification, almost all laboratories correctly identified maize species in

each test item. In addition, approximately half of laboratories also identified soybean and a few

oilseed rape.

The majority of laboratories correctly detected the three GM events included in the Level 1 and Level

2 test samples. Results of the quantitative evaluation of the GM content were compromised by the

use of the adh1 gene for MON 863 quantification by a number of laboratories. Forty five laboratories

(71 %) obtained satisfactory z-scores (|z| ≤ 2.0) for the three GM events in both test items. Among

the remaining 18 laboratories, half had unsatisfactory z-scores for MON 863.

Only 46 % of participants provided information on measurement uncertainty in a complete and

consistent manner and further improvement in this crucial area is needed.



Content

1. Introduction ..................................................................................................................... 7

2. Test items ......................................................................................................................... 9

2.1 Characterisation of base materials ......................................................................................... 9

2.2 Preparation and characterisation of test items ....................................................................... 9

3. Tasks to be performed by participants .......................................................................... 10

4. Results ............................................................................................................................ 11

4.1 Species identification .......................................................................................................... 13

4.2 Qualitative GMO testing results ........................................................................................... 14

4.3 Quantitative GMO testing results ......................................................................................... 15

5. Discussion of laboratories' performance........................................................................ 18

5.1 Overall performance ........................................................................................................... 18

5.2 Measurement uncertainty ................................................................................................... 18

5.3 Underperforming laboratories ............................................................................................. 20

6. Conclusions .................................................................................................................... 21

References ......................................................................................................................... 22

Acknowledgements ............................................................................................................ 23

Annex 1: Homogeneity and stability of test items ............................................................. 25

A1.1 Homogeneity .................................................................................................................. 25

A1.2 Stability of test items ...................................................................................................... 26

Annex 2: Species identification .......................................................................................... 27

A2.1 Soybean .......................................................................................................................... 27

A2.2 Maize .............................................................................................................................. 28

A2.3 Oilseed rape .................................................................................................................... 29

Annex 3: Performance statistics ........................................................................................ 30

Annex 4: Participants' results ............................................................................................ 31

Annex 5: Questionnaire data ............................................................................................. 59

Annex 6: Invitation letter .................................................................................................. 84

Annex 7: Accompanying letter to shipment of samples..................................................... 86

Annex 8: Confirmation of shipment ................................................................................... 88

Annex 9: Acknowledgement of receipt .............................................................................. 89



Drafted by:

N. Bassani (EU-RL GMFF Data analysis officer) _____________________________

Reviewers - Members of the Advisory Board:

B. China _____________________________

P. Corbisier _____________________________

H. Hird _____________________________

L. Hougs _____________________________

M. Sandberg _____________________________

M. Schulze _____________________________

I. Taverniers _____________________________

Scientific and technical approval:

M. Mazzara (Competence group leader) _____________________________

Compliance with EU-RL Quality System:

S. Cordeil (Quality manager) _____________________________

Authorisation to publish:

J. Kreysa (Head of Unit) _____________________________



1. Introduction The Joint Research Centre (JRC) of the European Commission was established as European Union

Reference Laboratory for GM Food and Feed by Regulation (EC) No 1829/2003(1). The EU-RL GMFF is

also mandated by Regulation (EC) No 882/2004(2).

Article 32 of Regulation (EC) No 882/2004 tasks the EU-RL with the organisation of comparative

testing (CT) for National Reference Laboratories (NRLs, nominated under Regulation (EC) No

882/2004) and an appropriate follow-up of such testing. The aim of this activity is ‘to contribute to a

high quality and uniformity of analytical results’(2). Moreover, Article 12 of said Regulation requires

that the nominated NRLs should be accredited under ISO/IEC 17025 on ‘General requirements for the

competence of testing and calibration laboratories’ and 17025-accredited laboratories must prove

their competence, e.g. by taking part in proficiency testing. As the EU-RL GMFF is accredited under

ISO 17043 (‘General requirements for proficiency testing’(3)), successful participation in CT rounds

organised by it, meets this requirement.

Regulations (EC) No 1829/2003 and (EU) No 619/2011 establish a threshold for labelling of food and

feed products (0.9 %) and a minimum required performance limit (0.1 % m/m) for detecting low

level presence of GMO in feed. These values are used by the Member States of the European Union

in the official control of food and feed. Hence, an accurate and harmonised determination of the GM

content is of paramount importance.

The EU-RL GMFF organised a comparative testing round for NRLs nominated under Regulation (EC)

No 882/2004. Participation was open and free of charge for any official control laboratory.

Participation was mandatory for NRLs nominated under Regulation (EC) No 882/2004 and highly

recommended for NRLs nominated under Regulation (EC) No 1981/2006(4). This comparative testing

round met the requirements of ISO 17043.

In August 2013, a total of 161 laboratories were invited to participate in this CT round of the EU-RL

GMFF (ILC-EURL-GMFF-CT-01/13) and 64 laboratories from 28 countries registered for it. Test items

were prepared by the EU-RL and shipped to registered participants at the beginning of September

2013 in plastic containers containing approximately 5 g of flour. The EU-RL GMFF managed the on-

line registration and submission of results and was responsible for their evaluation. It was supported

by the Advisory Board for CT.

Sixty-three laboratories from 28 countries returned at least qualitative results (see Figures 1 and 2).

These laboratories fell into the following groups:

1. 2 were NRLs nominated only under Regulation (EC) No 882/2004 (group 1),

2. 22 were NRLs nominated only under Regulation (EC) No 1981/2006 (group 2),

3. 28 were NRLs nominated under both Regulations (group 3),

4. 7 were ENGL members but did not belong to group 1, 2 or 3 (group 4),

5. 2 were official control laboratories from EU Member States but not ENGL members (group

5),

6. 2 were official control laboratories from a third country (group 6).



Figure 1. Laboratories submitting at least qualitative results, sorted by group.

Figure 2. Overview of laboratories submitting at least qualitative results, sorted by country.



2. Test items

The test items were produced in-house from ground powder of 98140 maize and MON 863 X MON

810 maize, provided by IRMM, Geel (Belgium), and conventional maize and dried wheat grain.

2.1 Characterisation of base materials

Base materials consisted of:

• 3000 g of non-GM maize (kernels)

• 3000 g of non-GM wheat (grain)

• 300 g of GM maize event 98140 powder and 300 g of GM maize event MON 863 X MON 810

powder provided by IRMM, Geel, Belgium

Non-GM materials were ground using an Ultra Centrifugal Mill ZM200 (Retsch GmbH, DE). An oven-

drying method was used for determining the remaining water content in the powders. To determine

the extractability of DNA from the GM and non-GM base materials, DNA was extracted from each of

the powders in 10 independent replicates using the Nucleospin© method(5). Extracted DNA was

quantified with Picogreen in a VersaFluor Fluorometer.

Four DNA extracts, randomly chosen from the 10 replicates, were assessed for the presence of PCR

inhibitors using the validated hmg reference gene system and the maximum amount of DNA validated

for each event-specific method. No inhibition was detected. The DNA extracts were also assessed for

the presence of GM-event(s) or species-specific DNA other than those relevant to the present

comparative testing round, using ABI pre-spotted plates(6). No other species were identified in the

base materials.

2.2 Preparation and characterisation of test items

Two levels of processed material (Levels 1 and 2) test items were gravimetrically prepared and mixed

to obtain nominal concentrations of 0.3 m/m % and 1 m/m % GM of maize 98140 and 1.3 m/m %

and 0.5 m/m % GM of MON 863 X MON 810, respectively.

These test items were prepared by the EU-RL in accordance with ISO Guide 34(7) (‘General

requirements for the competence of reference material producers’), as follows:

• Two different mass fractions (mixtures) of GM materials, representing two different GM

levels, were produced by mixing pure non-GM with pure GM powder base materials, taking

into account the water content of the base materials (see Table 1 for details on mixtures).

Table 1. Mixtures composition (in grams).

Wheat Maize 98140 MON 863 X MON 810 Butter Eggs* Sugar

Level 1 566.93 558.28 1.59 7.06 70.87 311.81 283.46

Level 2 566.93 558.91 5.30 2.72 70.87 311.81 283.46

Non-GM GM Other ingredients

*A medium egg is assumed to weigh 52 g without shell



• Each mixture was manually mixed for 10 minutes, then thoroughly mixed for 60 min in a

Turbula T10B mixer. Finally, each preparation was mixed with the following ingredients: 6

medium eggs (approximately 52 g each), 71 g of butter and 283 g of sugar.

• The mixtures were then formed into biscuits which were baked at 180°C for 10 minutes.

• Once the biscuits had cooled they were milled to a fine powder using an Ultra Centrifugal Mill

ZM200 (Retsch GmbH, Haan, DE).

• The powdered biscuits were then further mixed for 60 minutes in a Turbula T10B mixer to

obtain homogeneous mixtures.

From each of these two powder test materials, 300 test items of approximately 5 g were prepared in

30 ml-bottles using a sample divider (Retsch GmbH, Haan, DE). Bottles were labelled according to the

GM level of the test items and stored at 4°C.

The homogeneity and stability of test items was performed in-house. Test items were found to be

homogeneous for all GM events tested for both concentration levels (p-value > 0.05). Additionally the

test items were found to be stable over both the short and long term at the 5 % significance level.

Detailed methods and results are described in Annex 1.

3. Tasks to be performed by participants

Participants in this CT round were required to screen the two test items (Level 1 and 2), i.e. to

perform species identification and test for the presence of maize events 3272, Bt11, Bt176, 59122,

GA21, MIR604, MON 810, MON 863, NK603, 1507, MON 88017, MON 89034, 98140 and MIR162. Any

event detected had to be quantified. Participants could report the quantitative results in m/m % or

DNA cp/cp %. Additionally, laboratories were asked to report the estimated measurement uncertainty

as an absolute value, and the practical LOD and LOQ in the appropriate measurement unit.

Participants were instructed to apply the following formulas when reporting their results:

Mass GM event [g]

m/m % = x 100 % (1)

Total mass species [g]

GM event DNA copy number [cp]

cp/cp % = x 100 % (2)

Target taxon-specific DNA copy number [cp]



4. Results

A total of 63 laboratories from 28 countries submitted at least qualitative results. One laboratory

submitted only qualitative results for all events, and one laboratory provided qualitative results for

maize event 98140 only. Table 2 lists additional deviations from the requested method of reporting

quantitative results.

Table 2. Observed deviations in the reporting of quantitative results

LabCode Issue

L63 Results were reported in both m/m % and cp/cp % for maize events MON 810 and MON 863

L05, L12, L18, L29, L33, L42, L63 No results reported for maize event 98140

Most laboratories (approximately 90 %) reported the GM content of test items in m/m %, whereas

the remaining laboratories expressed their results only, or additionally, in cp/cp % (Figures 3a-c).

Figure 3a. Overview of quantitative results for maize event 98140, grouped by measurement unit. L1 = Level 1, L2 = Level 2.



Figure 3b. Overview of quantitative results for maize event MON 863, grouped by measurement unit. L1 =

Level 1, L2 = Level 2.

Figure 3c. Overview of quantitative results for maize event MON 810, grouped by measurement unit. L1 =

Level 1, L2 = Level 2.



To facilitate the comparison between the laboratory groups defined on page 7, the qualitative and

quantitative results reported hereunder were stratified according to the following three categories:

• Category (a)

o NRLs appointed only under Regulation (EC) No 882/2004 or appointed under both

relevant Regulations (groups 1 and 3),

• Category (b)

o NRLs appointed only under Regulation (EC) No 1981/2006 (group 2),

• Category (c)

o ENGL members (not in group 1, 2, or 3), non-ENGL EU laboratories and third

countries official control laboratories (groups 4, 5, and 6, respectively).

4.1 Species identification

Overall, all but one laboratory correctly detected the presence of maize, and that laboratory, that

stated that it did not test for maize, actually performed quantification of the maize events and

therefore has de facto tested for the presence of maize. In addition, approximately half of the

laboratories also identified the presence of soybean and a minority the presence of oilseed rape. NRLs

appointed under either Regulation performed similarly to non-NRLs with similar percentages of

species identification, for all the species considered. Results for all species, regardless of the

laboratory category, are summarised in Figures 4a-b.

Figure 4a. Overview of species identification data for Level 1 test item. D = Detected, ND = Not Detected, NT =

Not Tested.



Figure 4b. Overview of species identification data for Level 2 test item. D = Detected, ND = Not Detected, NT =

Not Tested.

4.2 Qualitative GMO testing results

A summary of results for the GMO screening in both test items is reported in Figure 5. Approximately

90 % of laboratories returned results that agreed with the consensus, i.e. did not detect any GM

event other than 98140, MON 863 and MON 810. Amongst the laboratories reporting results

deviating from the consensus for at least one event, 3 were NRLs appointed under Regulations

882/2004 and 1981/2006, while 5 were NRLs appointed under Regulation 1981/2006 only. Of these 8

laboratories, only one (NRL appointed under Regulation 1981/2006) also quantified the additional

events detected (59122, GA21, MIR604 and NK603).

Figure 5. Overview of screening results for both test items, excluding the spiked events. D = Detected, ND =

Not Detected.



4.3 Quantitative GMO testing results

For each event, the consensus value (µR) from participants in the CT round was calculated using

robust statistics(8,9). This approach minimises the influence of outlying values. Robust means (µR)

were calculated separately for measurements results reported in m/m % and cp/cp %.

The expanded uncertainty (U) comprises standard uncertainty (u) contributions from the

characterisation of the material (uchar) and the between-test item homogeneity (ubb)(10), and is

estimated according to:

22bbchar uukU += (3)

A coverage factor (k) of 2 was used to calculate the expanded uncertainty corresponding to a 95 %

level of confidence(11). The standard uncertainty (uchar) on the characterisation was calculated using

the formula:

Nuchar

σ= (4)

where: σ = robust Relative Standard Deviation of the robust mean expressed in m/m %

N = number of data points

The robust means (µR) for data on the non-transformed scale, as determined by the EU-RL GMFF,

are reported in Table 3. For maize event MON 863, the robust mean of results submitted in m/m %

was calculated using data from all laboratories except those who have used adh1 with amplicon size

of 70 bp (adh1-70 bp), because this is known to carry a polymorphism that can lead to unstable and

severely biased (up-ward or down-ward) results(12). Analysing all data submitted for MON 863, it was

found that the distribution of the quantitative laboratory results was indeed deviating from normality,

therefore, µR was determined based on a subset of the results obtained. One laboratory did not

provide any information on the amplicon size of the adh1 reference gene used and the data provided

were therefore not included in the calculation of the robust mean.



Table 3. Overview of the robust means (µR) and expanded uncertainties for Level 1 and 2 test items.

U abs [m/m %] U rel [%] ( u char, rel )1

( u bb, rel)2

Level 1 0.33 (N = 49) 0.03 8.18 3.09 2.68

Level 2 1.06 (N = 49) 0.12 11.10 3.14 4.57

Level 1 1.36* (N = 55) 0.08 6.14 2.43 1.88

Level 2 0.53* (N = 55) 0.05 8.92 3.85 2.25

Level 1 0.91 (N = 53) 0.09 10.05 4.74 1.68

Level 2 0.36 (N = 53) 0.06 17.65 6.18 6.30

U abs [cp/cp %] U rel [%] ( u char, rel )1

( u bb, rel)2

Level 1 0.18 (N = 5) 0.06 31.77 15.66 2.68

Level 2 0.69 (N = 5) 0.08 11.16 3.19 4.57

Level 1 0.68 (N = 8) 0.16 23.80 11.75 1.88

Level 2 0.26 (N = 8) 0.06 24.08 11.83 2.25

Level 1 0.35 (N = 10) 0.04 11.44 5.47 1.68

Level 2 0.13 (N = 10) 0.02 17.89 6.35 6.30

μ R [m/m %]

Expanded uncertainty Relative standard uncertainty

contribution [ % ](U = 2 * u c )

Maize 98140

Maize 98140

Maize MON 863

Maize MON 810

Maize MON 863

Maize MON 810

μ R [cp/cp %]

Expanded uncertainty Relative standard uncertainty

contribution [ % ](U = 2 * u c )

* Robust mean was estimated only using the data from laboratories quantifying the event using a reference gene different from adh1-70 bp 1 Relative standard uncertainty relating to the characterisation 2 Relative standard uncertainty resulting from the homogeneity assessment

The z-scores were calculated for all three events and for both Level 1 and 2 test items on the basis of

the robust means for both m/m % and cp/cp % data. For consistency, all decimal numbers have

been rounded to two digits. The information is given, when the group size allowed it, by laboratory

category and, for indicative purposes, by laboratory group (see page 7).

"Value" refers to the reported value and uncertainty as calculated and reported by the laboratory.

Also practical “LOD” (limit of detection) and “LOQ” (limit of quantification) are values calculated and

provided by the laboratories and refer to the methods they used. The z-score, measurement

uncertainty (MU; % of incorrectly reported MU is estimated using data from laboratories which

reported a value only), mean LOD (µLOD) and mean LOQ (µLOQ) as well as their standard deviation are

calculated by the EU-RL. Detailed results are reported in Annex 4, Tables A4.1 to A4.12 and Figures

A4.1 to A4.12.

In Table 4 a summary of results with respect to the indicators described above (% of incorrectly

reported MU, LOD, LOQ and % of unsatisfactory z-scores) is presented for each event and test item,

for the two measurement units separately.



Table 4. Summary of quantitative GMO testing results reported in m/m % and cp/cp %.

μ2

σ3 μ σ

Maize 98140

Level 1

Level 2

Maize MON 863

Level 1 0.08 4/55 (7%)

Level 2 0.07 9/55 (16%)

Maize MON 810

Level 1 3/53 (6 %)

Level 2 5/53 (9 %)

μ σ μ σ

Maize 98140

Level 1 0/5 (0 %)

Level 2 1/5 (20 %)

Maize MON 863

Level 1

Level 2

Maize MON 810

Level 1 0/10 (0 %)

Level 2 1/10 (10 %)4/9 (44 %)

0.05

0.11

0.04 0.05 0.12 0.09

0.06

LOQ

(cp/cp %)

LOD

(cp/cp %) Unsatisfactory

z-scores (%)

20/44 (45%)

24/50 (48 %)

4/8 (50 %)

3/49 (6 %)

0.04 0.05 0.07

1/8 (12 %)

Results

reported in

m/m%

Results

reported in

cp/cp%

0.06 0.06 0.15

0.05

0.12

0.05

0.09

1/5 (20 %)

LOD

(m/m %)

LOQ

(m/m %)

Incorrectly

Reported MU (%)

Unsatisfactory

z-scores (%)

Incorrectly

reported MU1

(%)

0.04 0.11 0.06

24/52 (46%) 0.130.04

0.13

1 MU = Measurement Uncertainty 2 µ = Sample mean of reported LOD/LOQ values 3 σ = Sample standard deviation of reported LOD/LOQ values



5. Discussion of laboratories' performance

5.1 Overall performance

In this seventh CT round most laboratories correctly identified maize in the test items. In addition, of

the laboratories that performed species identification, more than half also detected soybean and two

participants detected oilseed rape.

The majority of laboratories also correctly detected the three GM events included in the Level 1 and

Level 2 test samples and provided quantitative results for these events. With respect to maize event

MON 863, it should be noted that the underperformance of the adh1-70 bp as a reference gene has

been reported in the literature already several years ago(12). Thus, as laboratories are free to use any

method in a CT round, provided that there is a scientific ground for this choice, they should have

been aware of the issue and should have taken it into account when quantifying the event, i.e. they

should have used a different maize reference gene.

There were differences in the mean MON 863 content in both test items between laboratories using

adh1-70 bp and all the other laboratories (see Table 5). Similarly, the MON 863 content measured by

laboratories that used adh1-70 bp was approximately two-fold higher than the reference value

estimated by the EU-RL GMFF on the basis of the homogeneity study (see Table 5). As a result, 8 out

of 9 laboratories with an underperforming z-score for at least one test item had used the adh1-70 bp

reference gene method.

Table 5. Robust means (by reference gene group) and internal reference value for maize event MON 863.

adh1-70 bp Others

Level 1 1.30 2.88 1.36 1.50

Level 2 0.50 1.22 0.53 0.58

Robust mean (%) Reference value

(%)

Nominal %

MON 863Test item

5.2 Measurement uncertainty

In Figures 6a-b the robust means (µR) for all events and both reporting units (m/m % and cp/cp %)

are presented, and the expanded measurement uncertainties (see Table 3) are indicated with vertical

bars.

It should be noted that 28 out of 63 laboratories (approximately 44 %) reported a complete and

consistent estimate of measurement uncertainty (MU), a trend in reporting MU which is consistent

with that obtained in the previous CT round. In particular:

- 15 out of 63 laboratories (approximately 24 %) answered the questions relating to MU on the

questionnaire inconsistently, since these laboratories answered yes to both Q31.1 and Q31.2,

which are mutually exclusive questions;

- 9 out of 63 laboratories (approximately 14 %) did not answer all the questions relating to MU;

- 7 out of 63 laboratories (approximately 11 %) did not provide an estimate of the MU;

- 5 out of 63 laboratories (approximately 8 %) reported a relative estimate, even though on the

questionnaire it was explicitly stated that an absolute value had to be reported for the MU.



Figure 6a. Comparison of robust means (µR) of GM events in Level 1 and 2 test items in m/m %.

Figure 6b. Comparison of robust means (µR) of GM events in Level 1 and 2 test items in cp/cp %.



5.3 Underperforming laboratories

An overview of the laboratories which obtained unsatisfactory z-scores is provided in Table 6a (m/m

%) and Table 6b (cp/cp %).

Table 6a. Laboratories with unsatisfactory z-scores on the basis of the robust mean for Level 1 and 2 test items in m/m %.

Level 1 Level 2 Level 1 Level 2 Level 1 Level 2

L44 (a) 1 X X

L23 (a) 3 X

L24 (a) 3 X

L30 (a) 3 X X

L38 (a) 3 X X

L48 (a) 3 X

L53 (a) 3 X

L63 (a) 3 X

L64 (a) 3 X X

L09 (b) 2 X X X

L15 (b) 2 X X

L61 (b) 2 X X

L62 (b) 2 X X

L33 (c) 4 X

L42 (c) 5 X

L03 (c) 6 X X

MON 810Unsatisfactory z-scores [m/m %]

Category Group98140 MON 863Laboratory

number

Table 6b. Laboratories with unsatisfactory z-scores on the basis of the robust mean for Level 1 and 2 test items in cp/cp %.

Level 1 Level 2 Level 1 Level 2 Level 1 Level 2

L41 (a) 3 X

L63 (a) 3 X X

L29 (b) 2 X

Laboratory number

Category Group

Unsatisfactory z-scores [cp/cp %]98140 MON 863 MON 810

Out of 30 unsatisfactory z-scores, 16 (i.e. 53 %) were obtained by 11 NRLs appointed under

Regulation 882/2004, 10 (i.e. 33 %) were obtained by 5 NRLs appointed under Regulation 1981/2006

and the remaining 4 by 3 non-NRLs. It is relevant to note that the 11 underperforming NRLs from

category (a) represented the 37 % of all laboratories in this category, whereas the 4 from category

(b) represented only the 18 % of the related category.



6. Conclusions

In this seventh comparative testing round participants were asked to screen two test items (biscuit

powder Levels 1 and 2), i.e. to perform species identification and then test for the presence of maize

events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603, 1507, MON 88017,

MON 89034, 98140 and MIR162, and quantify any event detected.

All laboratories correctly identified maize during the species identification task. Almost half of the

laboratories also identified traces of soybean, of these ≈50 % were NRLs appointed under Regulation

882/2004. Oilseed rape was identified by two laboratories. Thus, with regards to species identification

the performance of laboratories was considered satisfactory.

A group of 14 laboratories used adh1 with an amplicon size of 70 bp, a reference gene that has been

shown previously to provide biased results for quantification of maize event MON 863(12). Their results

appeared to confirm this bias. Therefore, as the choice of the method is to be made by the

laboratory, laboratories should ensure that the method chosen is fit for the purpose and updated to

the latest scientific knowledge. In addition, when new information becomes available with regards to

the performance of previously validated methods, it should be disseminated by the EU-RL GMFF.

The reporting of measurement uncertainty (MU) was, on average across GM events and concentration

levels, reported in a complete and consistent manner by only approximately 46 % of laboratories, a

result which is worse than the one obtained in the previous CT round (57 %, p-value > 0.05 with

Normal approximation test). Thus, given the importance of a correct estimation of measurement

uncertainty, there is still a need to provide laboratories with guidance and training on the calculation

and reporting of MU.



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 verification of

compliance with feed and food law, animal health and animal welfare rules. Off. J. Eur. Union

L 191: 1-52

3. ISO/IEC 17043:2010 Conformity assessment – General requirements for proficiency testing

European Commission (2006).

4. 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

5. JRC.I.3 S11B6/EURL [DNA Extraction – Nucleospin Food]. EU-RL GMFF internal quality

document

6. Querci, M., Foti, N., Bogni, B., Kluga, L., Broll, H. & Van den Eede, G. (2009). Real-time PCR-

based ready-to-use multi-target analytical system for GMO detection. Food Anal. Methods

2(4): 325-336

7. ISO Guide 34:2009 General requirements for the competence of reference material producers

8. Analytical Methods Committee (1989). Robust statistics – How not to reject outliers Part 1.

Basic Concepts. Analyst 114: 1359-1364

9. Analytical Methods Committee (2001). Robust statistics: a method for coping with outliers.

AMC Technical Brief. No. 6. April 2001

10. JCGM 100:2008 Evaluation of measurement data - Guide to the Expression of Uncertainty in

Measurement

11. EURACHEM/CITAC Guide CG4 (2000). Quantifying Uncertainty in Analytical Measurement, 2nd

edition

12. Broothaerts, W., Corbisier, P., Schimmel, H., Trapmann, S., Vincent, S., Emons, H. (2008). A

Single Nucleotide Polymorphism (SNP839) in the adh1 Reference Gene Affects the

Quantitation of Genetically Modified Maize (Zea mays L.). J. Agric. Food. Chem. 56: 8825-8831

13. ISO 13528:2005 Statistical methods for use in proficiency testing by interlaboratory

comparisons

14. Thompson, M., Wood, R. (1993). The international harmonized protocol for the proficiency

testing of (chemical) analytical laboratories. J. AOAC Int. 76: 926-940

15. Linsinger, TPJ., van der Veen, AMH., Gawlik, BM., Pauwels, J., Lamberty, A. (2004). Planning

and combining of isochronous stability studies of CRMs. Accred. Qual. Assur. 9: 464-472

16. Thompson, M., Ellison, SLR., Owen, L., Mathieson, K., Powell, J., Key, P., Wood, R., Damant,

AP. (2006). Scoring in Genetically Modified Organism Proficiency Tests Based on Log-

Transformed Results. J. AOAC Int. 89: 232-239

17. Analytical Methods Committee (2004). GMO Proficiency Testing: Interpreting z-scores derived

from log-transformed data. RSC. AMC Technical Brief. No. 18. December 2004

18. Powell, J., Owen, L. (2002). Reliability of Food Measurements: The Application of Proficiency

Testing to GMO Analysis. Accred. Qual. Ass. 7: 392-402



Acknowledgements With respect to the raw material(s) used in this study we kindly acknowledge IRMM, JRC-Geel for

providing the maize events 98140 and MON 863 X MON 810. We sincerely thank Adam Niedzwiecki,

Matteo Maretti, Angelo Collotta, Fabrizia Scabini, Roberta Brustio, Stéphane Cordeil, Steven Price and

Lorella Vidmar of the MBG Unit and EU-RL GMFF for their invaluable contributions to this seventh

comparative testing round and Wim Broothaerts for reviewing the final report.

The CT-Advisory Board members (Bernard China, Philippe Corbisier, Hez Hird, Lotte Hougs, Martin

Sandberg, Manuela Schulze and Isabel Taverniers) have provided invaluable input for the planning

and analysis of the CT round and intensively revised and agreed to this report. Their constructive

contribution is highly appreciated.

The laboratories listed below are kindly acknowledged for their participation in this exercise.

Organisation Department Country Group 1

AGES - Institute for Food Safety Vienna AU 3

Agricultural Institute of Slovenia Crop Science Department SI 2

Agroscope Liebefeld-Posieux Research Station ALP Analytics CH 6

Bavarian Health and Food Safety Authority DE 2

Central Control and Testing Institute in Agriculture Dptm. of Molecular Biology SK 3

Centre for Research in Agrigenomics (CRAG) Molecular Genetics ES 4

Centre Wallon de Recherches Agronomiques Valorisation des productions BE 3

Centro Nacional de Alimentacion (Agencia Espanola de Seguridad Alimentaria y Nutricion) Biotechnology Unit ES 3

Chemisches und Veterinäruntersuchungsamt Ostwestfalen-Lippe DE 2

Croatian National Institute of Public Health GMO and Risk Assessment Unit HR 4

Crop Research Institute CZ 3

CVUA Freiburg DE 2

Danish Vererinary and Food Administration Section for Plant Diagnostics DK 3

Dutch Food and Consumer Product Authority Micro 3 NL 2

Federal Agency For the Safety of the Food Chain FLSFC Melle BE 5

Federal Office of Consumer Protection and Food Safety DE 1

Federal Office of Public Health FOPH Microbiol. & Biotechnol. Riscs CH 4

Fera UK 3

Finnish Customs Laboratory ET2 FI 3

Finnish Food Safety Authority Evira Research and Laboratory FI 4

Hessian State Laboratory DE 2

IDAH BMOMG RO 1

INIAV UEIS-SAFSV PT 2

INRAN - ENSE Laboratorio analisi sementi IT 2

Institute for Agricultural and Fisheries Research Unit Technolgy and Food - PI BE 3

Institute of Biochemistry and Biophysics PAS Lab.Gen.Mod.Anal. PL 2

Institute of Food Safety, Animal Health and Environment „BIOR” Virology Department LV 3

Instytut Zootechniki PIB KLP Pracownia w Szczecinie PL 3

Istituto Superiore di Sanità, National Institute of Health DSVSA IT 2

Istituto Zooprofilattico Sperimentale Lazio è Toscana Biotechnology Unit IT 3

Laboratoire National de Santé Food Control LU 3

Laboratorio Arbitral Agroalimentario - MAGRAMA OGM ES 3

Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei M-V

DE 2



Organisation Department Country Group 1

Landesamt für Verbraucherschutz Sachsen-Anhalt DE 2

Landeslabor Berlin-Brandenburg Fb I-6 DE 2

Landeslabor Schleswig-Holstein DE 2

Landesuntersuchungsamt Rheinland-Pfalz Institut f. Lebensmittelchemie DE 2

Landesuntersuchungsanstalt für das Gesundheits- und Veterinärwesen Sachsen (LUA)

Amtliche Lebensmitteluntersuch DE 2

LAVES LVI-Braunschweig/Hannover DE 2

LGC UK 3

LTZ Augustenberg Referat 24 DE 2

Ministry of Finance, General Chemical State Laboratory Food Directorate GR 3

National Center of Public Health and Analyses GMO Unit BG 3

National Food Agency Science Department SE 3

National Food and Veterinary Risk Assessment Institute Molecular Biology and GMO LT 3

National Food Chain Safety Office HU 3

National Institute of Biology SI 3

National Veterinary Research Institute Feed Hygiene PL 3

Norwegian Veterinary Institute Bacteriology-Feed and GMO NO 4

Plant Breeding and Acclimatization Institute – National Research Institute GMO Controlling Laboratory PL 2

Regional Laboratory of Genetically Modified Food PL 3

RIKILT Wageningen UR NFA NL 3

Scientific Institute of Public Health Platform Biotech & mol Biol BE 3

Service Commun des Laboratoires du MINEFI - Laboratoire de Strasbourg

FR 3

Staatliche Betriebsgesellschaft für Umwelt und Landwirtschaft Geschäftsbereich 6, FB 63 DE 2

Staatliches Gewerbeaufsichtsamt Hildesheim Dezernat 33 Gentechnik DE 4

State Veterinary and Food Institute Dolny Kubin SK 3

Tallinn University of Technology Department of Gene Technology EE 2

Thüringer Landesamt für Verbraucherschutz Lab for detection of GMO/foods DE 2

Thüringer Landesanstalt für Landwirtschaft Untersuchungswesen DE 4

Umweltbundesamt GmbH AU 3

USDA- GIPSA Technical Services Division US 6

Wojewodzki Inspektorat Weterynarii w Opolu Zaklad Higieny Weterynaryjnej PL 5

1 See the description of laboratory groups on page 7



Annex 1: Homogeneity and stability of test items

A1.1 Homogeneity

The assessment of the homogeneity(13) was performed by the EU-RL GMFF after the test items had

been packed in their final form and before distribution to participants in line with the following

consideration:

Samples are considered to be adequately homogeneous if:

∧≤ σ3.0ss (A1.1)

Where ss is the between-test item standard deviation as determined by a 1-way random effects

ANOVA(14) and ∧σ is the standard deviation for comparative testing.

If this criterion is met, the between-test item standard deviation contributes no more than about

10 % to the standard deviation for comparative testing.

The repeatability of the test method is the square root of the mean sum of squares within-test item

MSwithin. The relative between-test item standard deviation ss,rel is given by

%100, ×

−

=yn

MSMS

s

withinbetween

rels (A1.2)

where: MSbetween is the mean sum of squares between test items

MSwithin is the mean sum of squares within test items

n is the number of replicates for each sample

y is the mean of the homogeneity data

If MSwithin > MSbetween, then:

( )%100

1

24

*, ×

−==

y

nNn

ityrepeatabil

us bbrels (A1.3)

where: u*bb is the maximum uncertainty contribution that can be obtained by the hidden

heterogeneity of the material.

For each group of test items 10 bottles (N = 10) were randomly selected and analysed in five-fold

replicates (n = 5). The criterion described in formula (A1.1) was in all cases fulfilled, indicating that all

groups of test items were homogeneous. The data from the homogeneity study were also used for

the estimation of the uncertainty contribution related to the level of homogeneity of test items.



A1.2 Stability of test items

An isochronous short term stability study involving two Level 1 test items with three replicates each

(N = 2, n = 3), was conducted over one, two and four weeks at +4°C and +18°C (15). The results did

not reveal an influence of time or temperature on the stability of test items, which were thus shipped

at ambient temperature.

An isochronous long term stability study involving two Level 1 test items with three replicates each

(N = 2, n = 3) was conducted over two, four and six months at +4°C(15). No trends were detected in

the GM concentration level for any of the test items analysed at a 5 % significance level, thus

indicating that test items can be stored over a prolonged period at +4°C.



Annex 2: Species identification

A2.1 Soybean

Overall, 52 % and 44 % of laboratories that tested for the presence of soybean species, for Level 1

and 2 test items respectively, identified samples as containing soybean, with similar detection

percentages across different laboratory categories. These results may indicate some soybean

contamination, as no soy flour was intentionally added to either test item. Additionally, 14 % of

laboratories (i.e. 9 out of 63), all of which were NRLs, did not perform species identification for

soybean, although this was an element of the CT round.

Table A2.1. Results of species identification analysis for soybean species by laboratory category; D = Detected, ND = Not Detected. When calculating %, only laboratories that actually tested for soybean were considered in the denominator.

Lab Code Group Level 1 Level 2 Lab Code Group Level 1 Leve l 2 Lab Code Group Level 1 Level 2

L39 1 Detected Detected L06 2 Not Detected Not Detected L05 4 Detected Not Detected

L44 1 Detected Detected L08 2 Not Detected Not Detected L14 4 Detected Detected

L01 3 Detected Detected L09 2 Not Tested Not Tested L17 4 Detected Not Detected

L02 3 Detected Not Detected L11 2 Not Detected Not Detected L25 4 Detected Detected

L07 3 Detected Not Detected L13 2 Detected Detected L32 4 Not Detected Not Detected

L10 3 Not Detected Not Detected L15 2 Detected Detected L33 4 Not Detected Not Detected

L12 3 Detected Detected L19 2 Not Detected Not Detected L51 4 Not Detected Not Detected

L16 3 Not Detected Not Detected L20 2 Not Tested Not Tested L18 5 Not Detected Detected

L21 3 Not Tested Not Tested L22 2 Detected Detected L42 5 Not Detected Not Detected

L23 3 Not Detected Not Detected L26 2 Not Detected Not Detected L03 6 Not Detected Not Detected

L24 3 Detected Detected L27 2 Detected Detected L46 6 Detected Detected

L30 3 Not Detected Not Detected L28 2 Detected Detected % D 45 36

L35 3 Detected Detected L29 2 Not Detected Not Detected % ND 55 64

L37 3 Not Detected Not Detected L31 2 Detected Detected



L43 3 Detected Detected L40 2 Not Detected Not Detected

L47 3 Not Detected Not Detected L45 2 Detected Not Detected

L48 3 Detected Detected L52 2 Detected Detected

L49 3 Not Detected Not Detected L60 2 Not Tested Not Tested

L50 3 Not Tested Not Tested L61 2 Not Detected Not Detected

L53 3 Detected Detected L62 2 Not Detected Not Detected

L54 3 Not Tested Not Tested % D 53 47

L55 3 Not Detected Not Detected % ND 47 53

L56 3 Detected Detected


L58 3 Not Detected Not Detected

L59 3 Not Tested Not Tested



% D 54 46

% ND 46 54

(a) (b) (c)



A2.2 Maize

Of the 63 laboratories that submitted results for this CT round, 62 correctly identified the presence of

maize in the test items. One NRL appointed under both regulations did not perform the species

identification test for maize in either test item, yet performed quantification of the three maize events

involved in the CT round.

Table A2.2. Results of species identification analysis for maize species by laboratory category; D = Detected, ND = Not Detected. When calculating %, only laboratories that actually tested for maize were considered in the denominator.













L30 3 Detected Detected L28 2 Detected Detected % D 100 100

L35 3 Detected Detected L29 2 Detected Detected % ND 0 0










L54 3 Detected Detected % D 100 100

L55 3 Detected Detected % ND 0 0







% D 100 100

% ND 0 0

(a) (b) (c)



A2.3 Oilseed rape

The majority of laboratories that tested for the presence of oilseed rape did not identify oilseed rape

in either of the test items (96 % for both Level 1 and 2 test items). Of the 62 laboratories submitting

at least qualitative results, 14 % (i.e. 9 out of 62) did not test for it at all. Notably, two laboratories

did not agree with consensus: one laboratory was an NRL appointed under Regulation 1981/2006

whilst the other was a member of the ENGL.

Table A2.3. Results of species identification analysis for oilseed rape species by laboratory category; D = Detected, ND = Not Detected. When calculating %, only laboratories that actually tested for oilseed rape were considered in the denominator.



L44 1 Not Detected Not Detected L08 2 Not Detected Not Detected L14 4 Detected Detected

L01 3 Not Detected Not Detected L09 2 Not Tested Not Tested L17 4 Not Detected Not Detected



L10 3 Not Detected Not Detected L15 2 Detected Detected L33 4 Not Detected Not Detected


L16 3 Not Detected Not Detected L20 2 Not Tested Not Tested L18 5 Not Tested Not Tested

L21 3 Not Tested Not Tested L22 2 Not Detected Not Detected L42 5 Not Detected Not Detected



L30 3 Not Detected Not Detected L28 2 Not Detected Not Detected % D 10 10

L35 3 Not Detected Not Detected L29 2 Not Detected Not Detected % ND 90 90

L37 3 Not Detected Not Detected L31 2 Not Detected Not Detected







L50 3 Not Tested Not Tested L61 2 Not Detected Not Detected


L54 3 Not Tested Not Tested % D 5 5

L55 3 Not Detected Not Detected % ND 95 95







% D 0 0

% ND 100 100

(a) (b) (c)



Annex 3: Performance statistics

The aim of performance statistics 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 from log10-transformed

data(16,17) based on the robust means(8,9) (µR) of the participants’ results.

The EU-RL GMFF calculated the consensus values from participants taking the robust means (µR) for

Level 1 and 2 test items in m/m % and cp/cp % on both original and log10-transformed scale.

The value of

∧σ , the target standard deviation for comparative testing, was defined by the Members

of the Advisory Board on the basis of the experience acquired with previous CT rounds, and set to 0.2

for all events(18).

The z-scores (zi) for participant i reporting measurement result xi were calculated in comparison to

the robust mean as follows:

( ) σµ ˆ/loglog 1010 Rii xz −= (A3.1)

A z-score zi was considered satisfactory if |zi| ≤ 2.0.



Annex 4: Participants' results

For consistency, all decimal numbers have been rounded to two digits. The information is given, when

the sample size allowed it, by laboratory category and, for indicative purposes, by laboratory group

(see page 7). Results for maize event MON 863 were seen to be largely dependent on the choice of

the reference gene used (adh1-70 bp versus hmg or another reference gene), so that data

distribution deviated from normality and two sub-populations of laboratories could be devised. For

such reason robust means and z-scores for this event were calculated on two distinct sub-groups: the

first composed of the 21 laboratories that used adh1-70 bp as reference gene and the second

composed of the 34 laboratories that used hmg or another gene as reference. One laboratory used

adh1 as reference gene but did not provide any quantitative result.

"Value" refers to the reported value and uncertainty as calculated and reported by the laboratory.

Also “LOD” (limit of detection) and “LOQ” (limit of quantification) are values calculated and provided

by the laboratories and refer to the methods they used. The z-scores, measurement uncertainty (MU;

% of incorrectly reported MU is estimated only using data from laboratories which reported a value),

mean LOD (µLOD) and mean LOQ (µLOQ) as well as their standard deviation are calculated by the EU-

RL. As an indicator for the overall performance, the fraction of laboratories outside the acceptable

range of the z-score (|z| ≤ 2.0) is given and corresponding data are highlighted in bold.



Table A4.1a-c. z-scores for maize event 98140 Level 1 test item for results reported in m/m %, laboratory category: (a), (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported (e.g. Table A4.1a, data from laboratory L44 not considered because it was not clear what the % symbol referred to).

(a)

Laboratory Number

Group Value Uncertainty LOD m/m LOQ m/mz-score based on

µR = 0.33L44 1 0.31 0.13 L1: 0.012% L1:0.024% -0.10L01 3 0.30 (1) 0.05 - 0.10 -0.16L02 3 0.30 (2) 0.10 - - -0.16L07 3 0.36 (2) 0.11 - - 0.24L16 3 0.30 0.10 0.01 0.07 -0.16L21 3 0.27 - <0.04 - -0.39L23 3 0.28 (1) 0.08 0.02 0.08 -0.31L24 3 0.32 0.20 <=0.04 <=0.08 -0.02L30 3 0.38 - - - 0.35L35 3 0.26 (1) (3) 30.00 - - -0.47L37 3 0.33 0.10 0.05 0.5 0.05L38 3 0.37 0.12 0.02 0.10 0.30L43 3 0.27 (2) 0.04 - - -0.39L47 3 0.47 0.08 0.50 0.50 0.81L48 3 0.30 (1) 0.14 - - -0.16L49 3 0.32 - 0.05 0.10 -0.02L53 3 0.40 (1) 0.09 0.10 0.10 0.47L54 3 0.36 (1) 0.09 0.05 0.10 0.24L55 3 0.25 (1) 0.05 0.02 0.10 -0.56L56 3 0.24 (3) 47.10 - - -0.64L57 3 0.18 0.08 0.02 0.20 -1.27L58 3 0.70 (2) (3) 16 0.90/1.60 - 1.68L59 3 0.44 0.28 0.15 0.30 0.67L64 3 1.40 (1) 0.42 0.01 0.10 3.19

% Incorrect MU μLOD = 0.08 μLOQ = 0.17 % |zμR| ≥ 2.0

65% σLOD = 0.14 σLOQ = 0.15 4%

(b)

Laboratory Number


µR = 0.33L06 2 0.32 (1) 0.12 0.01 0.10 -0.02L08 2 0.33 0.06 - - 0.03L09 2 0.09 (3) 71.64 0.01 0.04 -2.77L11 2 0.33 0.06 0.03 0.10 0.05L13 2 0.39 0.14 0.03 0.10 0.41L15 2 0.03 0.01 - - -5.16L19 2 0.30 0.10 0.10 0.10 -0.16L20 2 0.50 0.04 <0.1 0.10 0.95L22 2 0.37 0.10 0.01 0.05 0.30L26 2 0.33 (3) 48.10 0.02 0.07 0.05L27 2 0.38 0.05 0.09 0.18 0.35L28 2 0.20 0.05 - - -1.04L31 2 0.36 (2) 0.08 0.01 0.10 0.24L34 2 0.29 0.11 - - -0.23L36 2 0.29 0.05 0.02 0.10 -0.23L40 2 > 0 - - - *L45 2 > 0 - - - *L52 2 0.32 0.07 0.05 0.10 -0.02L60 2 0.40 0.09 - - 0.47L61 2 0.27 - - - -0.39L62 2 0.26 0.02 0.10 0.10 -0.47


18% σLOD = 0.04 σLOQ = 0.03 11%



(c)

Laboratory Number


µR = 0.33L14 4 0.37 (2) (3) 20.20 0.04 0.08 0.30L17 4 0.40 - 0.10 0.10 0.47L51 4 0.36 0.11 0.07 0.28 0.24L32 5 0.28 (1) 0.13 0.03 0.10 -0.31L03 6 0.40 (1) 0.12 0.05 0.05 0.47L46 6 0.28 0.01 0.01 0.10 -0.31


60% σLOD = 0.03 σLOQ = 0.08 0%



Table A4.2a-c. z-scores for maize event 98140 Level 2 test item for results reported in m/m %, laboratory category: (a), (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported.

(a)

Laboratory Number


µR = 1.06L44 1 1.02 0.08 - - -0.06

L01 3 0.98 (1) 0.15 - 0.10 -0.14L02 3 1.00 (2) 0.30 - - -0.10L07 3 0.93 (2) 0.28 - - -0.25L16 3 1.20 0.30 0.01 0.07 0.30L21 3 0.73 - <0.04 - -0.78L23 3 0.85 (1) 0.30 0.02 0.08 -0.45L24 3 1.25 0.42 <=0.04 <=0.08 0.39L30 3 0.99 - - - -0.12L35 3 1.04 (1) (3) 30 - - -0.01L37 3 0.81 0.24 0.05 0.50 -0.55

L38 3 0.35 0.11 0.02 0.10 -2.38L43 3 1.01 (2) 0.09 - - -0.08L47 3 1.31 0.54 0.50 0.50 0.49L48 3 1.22 (1) 0.59 - - 0.34L49 3 1.08 - 0.05 0.10 0.07L53 3 1.31 (1) 0.29 0.10 0.10 0.49L54 3 1.34 (1) 0.34 0.05 0.10 0.54L55 3 0.90 (1) 0.28 0.02 0.10 -0.33L56 3 1.03 (3) 34.64 - - -0.03L57 3 0.75 0.28 0.02 0.20 -0.72L58 3 1.90 (2) (3) 16 0.9/1.6 - 1.30L59 3 1.01 0.22 0.15 0.30 -0.08L64 3 4.80 (1) 1.40 0.01 0.10 3.31


65% σLOD = 0.14 σLOQ = 0.16 8%

(b)

Laboratory Number


µR = 1.06L06 2 1.10 (1) 0.34 0.01 0.10 0.11L08 2 1.03 0.02 - - -0.02L09 2 0.43 (3) 70.19 0.01 0.04 -1.93L11 2 1.12 0.13 0.03 0.10 0.15L13 2 1.39 0.27 0.03 0.10 0.62L15 2 0.17 0.07 - - -3.94L19 2 0.90 0.30 0.10 0.10 -0.33L20 2 1.30 0.50 <0.1 0.10 0.47L22 2 1.20 0.33 0.01 0.05 0.30L26 2 1.26 (3) 33.20 0.02 0.07 0.41L27 2 1.07 0.07 0.09 0.18 0.05L28 2 0.80 0.14 - - -0.58L31 2 1.00 (2) 0.11 0.01 0.10 -0.10L34 2 1.08 0.46 - - 0.07L36 2 0.97 0.26 0.02 0.10 -0.16L40 2 0.00 - - - *L45 2 0.00 - - - *L52 2 0.96 0.14 0.05 0.10 -0.19L60 2 1.10 0.24 - - 0.11L61 2 1.10 - - - 0.11L62 2 0.85 0.03 0.10 0.10 -0.45


18% σLOD = 0.04 σLOQ = 0.03 5%



(c)

Laboratory Number


µR = 1.06L14 4 1.34 (2) (3) 23.5 0.04 0.08 0.52L17 4 1.28 - 0.10 0.10 0.42L51 4 1.28 0.30 0.07 0.28 0.42L32 5 0.74 (1) 0.34 0.03 0.10 -0.77L03 6 1.29 (1) 0.3 0.05 0.05 0.44L46 6 0.93 0.05 0.01 0.10 -0.25


60% σLOD = 0.03 σLOQ = 0.08 0%



Table A4.3. z-scores for maize event 98140 Level 1 test item for results reported in cp/cp %, all laboratory categories. - = not reported, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner.

Laboratory Number

Group Value Uncertainty LOD cp/cp LOQ cp/cpz-score based on

µR = 0.18

L39 1 0.25 0.009 - - 0.71

L25 4 0.21 (1) 0.07 0.001 0.01 0.33

L50 3 0.2 0.03 0.02 0.1 0.22

L10 3 0.16 0.04 0.1 0.1 -0.26

L41 3 0.1 0.05 - - -1.28


20% σLOD = 0.05 σLOQ = 0.05 0%

Table A4.4. z-scores for maize event 98140 Level 2 test item for results reported in cp/cp %, all laboratory categories. - = not reported, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner.

Laboratory Number

Group Value Uncertainty LOD cp/cp LOQ cp/cpz-score based on µR = 0.69

L10 3 0.73 0.18 0.10 0.10 0.13

L39 1 0.72 0.03 - - 0.10

L25 4 0.70 (1) 0.16 0.00 0.01 0.04

L50 3 0.67 0.06 0.02 0.10 -0.06

L41 3 0.27 0.20 - - -2.03


20% σLOD = 0.05 σLOQ = 0.05 20%



Table A4.5a-c. z-scores for maize event MON 863 Level 1 test item, laboratory category: (a), (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported.

(a)

Laboratory Number

Reference Gene


µR = 1.36L44 Adh1 (70 bp) 1 3.03 1.80 L1: 0.026% L1: 0.259% 3.57L01 Adh1 (70 bp) 3 2.70 (1) 0.40 - 0.10 1.49L02 Hmg 3 1.40 (2) 0.40 - - 0.07L07 - 3 1.16 (2) 0.35 - - -0.34L10 Adh1 (136 bp) 3 2.09 0.39 0.03 0.10 0.94L16 Hmg 3 1.50 0.50 0.05 0.10 0.22L21 Adh1 3 0.89 (1) 0.28 <0.04 <0.09 -0.92L23 Adh1 (136 bp) 3 3.35 (1) 1.00 0.02 0.09 1.96L24 Adh1 (70 bp) 3 3.22 1.29 <0.05 <=0.1 1.88L30 Adh1 (70 bp) 3 5.09 - - - 2.87L35 Hmg 3 1.17 (1) (3) 30.00 - - -0.32L37 Adh1 (70 bp) 3 2.44 (2) 0.73 0.03 0.10 1.27L38 Hmg 3 1.31 0.50 0.02 0.10 -0.08L43 Hmg 3 1.52 (2) 0.02 - - 0.25L47 Hmg 3 1.46 1.24 0.01 0.10 0.16L48 Adh1 (70 bp) 3 2.89 (1) 1.24 - - 1.64L49 Adh1 (134 bp) 3 1.58 0.39 0.05 0.10 0.33L53 Adh1 (70 bp) 3 3.30 (1) 0.73 0.10 0.10 1.93L54 ZSSIIb 3 1.20 (1) 0.30 0.05 0.10 -0.27L55 Hmg 3 1.28 (1) 0.30 0.02 0.10 -0.13L56 Adh1 (70 bp) 3 1.84 (3) 26.70 - - 0.66L57 Hmg 3 1.19 0.45 0.03 0.30 -0.28L58 Hmg 3 1.50 (2) (3) 48.00 0.7/1.7 - 0.22L59 Hmg 3 1.3 0.54 0.15 0.30 -0.09

L63 Adh1 (70 bp) 3 6.57 (1) 1.19 - - 3.43L64 - 3 3.2 (1) 0.96 0.01 0.10 1.86


64% σLOD = 0.04 σLOQ = 0.08 12%

(b)

Laboratory Number

Reference Gene


µR = 1.36L06 Hmg 2 1.24 (1) 0.38 0.01 0.10 -0.19L08 Hmg 2 1.55 0.10 - - 0.30L09 Hmg 2 0.64 (3) 48.12 0.02 0.07 -1.63L11 Adh1 (70 bp) 2 2.27 0.23 0.03 0.10 1.12L13 Hmg 2 1.41 0.36 0.03 0.10 0.08L15 Adh1 (70 bp) 2 1.26 0.21 - - -0.16L19 Hmg 2 1.40 0.20 0.10 0.10 0.07L20 Hmg 2 1.40 0.15 <0.1 0.10 0.07L22 Hmg 2 1.41 0.21 0.01 0.13 0.08L26 Hmg 2 1.33 (3) 28.90 0.02 0.15 -0.04L27 Hmg 2 1.34 0.08 0.05 0.20 -0.03L28 Hmg 2 1.00 0.09 - 0.10 -0.66L31 Hmg 2 1.49 (2) 0.21 0.01 0.10 0.20L34 Hmg 2 1.36 0.28 - - 0.01L36 Hmg 2 1.52 0.24 0.02 0.18 0.25L40 Invertase 2 1.40 0.08 0.05 0.10 0.07L45 Adh1 2 > 0.00 - 0.10 0.10 *L52 Hmg 2 1.52 0.18 0.05 0.20 0.25L60 Hmg 2 1.3 0.31 - - -0.09L61 Adh1 (134 bp) 2 1.26 0.17 - - -0.16L62 Adh1 (70 bp) 2 3.87 0.93 0.10 0.10 2.28


20% σLOD = 0.03 σLOQ = 0.04 5%



(c)

Laboratory Number

Reference Gene


µR = 1.36L14 Hmg 4 1.44 (2) 22.40 0.05 0.15 0.13L17 Hmg 4 0.74 - 0.10 0.10 -1.32L33 Adh1 (135 bp) 4 1.11 0.15 0.10 0.10 -0.44L51 Adh1 (70 bp) 4 1.37 0.56 0.11 0.44 0.02L18 Hmg 5 1.48 (2) 0.18 0.08 0.10 0.19L32 Adh1 (135 bp) 5 0.98 (1) 0.45 0.04 0.10 -0.71L42 Adh1 (70 bp) 5 1.53 - 0.03 0.10 0.26L03 Invertase 6 1.09 (1) 0.45 0.05 0.05 -0.47L46 ZSSIIb 6 1.72 0.30 0.01 0.10 0.52


57% σLOD = 0.04 σLOQ = 0.12 0%



Table A4.6a-c. z-scores for maize event MON 863 Level 2 test item, laboratory category: (a), (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported.

(a)

Laboratory Number

Reference Gene


µR = 0.53L44 Adh1 (70 bp) 1 7.04 1.80 L1: 0.026% L1: 0.259% 3.83L01 Adh1 (70 bp) 3 0.91 (1) 0.14 - 0.10 1.21L02 Hmg 3 0.40 (2) 0.20 - - -0.57L07 - 3 0.49 (2) 0.15 - - -0.13L10 Adh1 (136 bp) 3 1.11 0.26 0.03 0.10 1.65L16 Hmg 3 0.60 0.20 0.04 0.08 0.31L21 Adh1 3 0.36 (1) 0.12 <0.04 <0.09 -0.80L23 Adh1 (136 bp) 3 1.51 (1) 0.45 0.02 0.09 2.31L24 Adh1 (70 bp) 3 1.72 0.58 <0.05 <=0.1 2.60L30 Adh1 (70 bp) 3 1.80 - - - 2.70L35 Hmg 3 0.38 (1) (3) 30 - - -0.68L37 Adh1 (70 bp) 3 0.81 (2) 0.24 0.03 0.10 0.96L38 Hmg 3 1.29 0.49 0.02 0.10 1.97L43 Hmg 3 0.57 (2) 0.20 - - 0.20L47 Hmg 3 0.44 0.22 0.01 0.10 -0.36L48 Adh1 (70 bp) 3 1.52 (1)0.65 - - 2.33L49 Adh1 (134 bp) 3 0.56 0.14 0.05 0.10 0.16

L53 Adh1 (70 bp) 3 2.13 (1) 0.47 0.10 0.10 3.06L54 ZSSIIb 3 0.47 (1) 0.12 0.05 0.10 -0.22L55 Hmg 3 0.51 (1) 0.14 0.02 0.10 -0.04L56 Adh1 (70 bp) 3 0.78 (3) 34.31 - - 0.88L57 Hmg 3 0.44 0.23 0.03 0.30 -0.36L58 Hmg 3 0.70 (2) (3) 48 0.7/1.7 - 0.64L59 Hmg 3 0.48 0.22 0.15 0.30 -0.17L63 Adh1 (70 bp) 3 2.01 (1) 0.39 - - 2.93L64 - 3 1 (1) 0.30 0.01 0.10 1.42


64% σLOD = 0.04 σLOQ = 0.08 27%

(b)

Laboratory Number

Reference Gene


µR = 0.53L06 Hmg 2 0.45 (1) 0.16 0.01 0.10 -0.32L08 Hmg 2 0.61 0.17 - - 0.34L09 Hmg 2 0.27 (3) 28.84 0.02 0.07 -1.42L11 Adh1 (70 bp) 2 1.11 0.16 0.03 0.10 1.65L13 Hmg 2 0.58 0.26 0.03 0.10 0.24L15 Adh1 (70 bp) 2 0.51 0.10 - - -0.04L19 Hmg 2 0.60 0.10 0.10 0.10 0.31L20 Hmg 2 0.50 0.10 <0.1 0.10 -0.09L22 Hmg 2 0.55 0.22 0.01 0.13 0.12L26 Hmg 2 0.38 (3) 29.50 0.02 0.15 -0.68L27 Hmg 2 0.46 0.06 0.05 0.20 -0.27L28 Hmg 2 0.40 0.08 - 0.10 -0.57L31 Hmg 2 0.55 (2) 0.07 0.01 0.10 0.12L34 Hmg 2 0.48 0.11 - - -0.17L36 Hmg 2 0.57 0.08 0.02 0.18 0.20L40 Invertase 2 0.63 0.01 0.05 0.10 0.42L45 Adh1 2 > 0.00 - 0.10 0.10 *L52 Hmg 2 0.52 0.09 0.05 0.20 0.00L60 Hmg 2 0.60 0.15 - - 0.31L61 Adh1 (134 bp) 2 0.8 0.11 - - 0.93L62 Adh1 (70 bp) 2 1.65 0.4 0.10 0.10 2.51


20% σLOD = 0.03 σLOQ = 0.04 5%



(c)

Laboratory Number

Reference Gene


µR = 0.53L14 Hmg 4 0.58 (2) 28.80 0.05 0.15 0.24L17 Hmg 4 0.34 - 0.10 0.10 -0.92L33 Adh1 (135 bp) 4 0.35 0.04 0.10 0.10 -0.86L51 Adh1 (70 bp) 4 0.44 0.20 0.11 0.44 -0.36L18 Hmg 5 0.47 (2) 0.18 0.08 0.10 -0.22L32 Adh1 (135 bp) 5 0.45 (1) 0.21 0.04 0.10 -0.32L42 Adh1 (70 bp) 5 0.20 - 0.03 0.10 -2.08L03 Invertase 6 0.42 (1) 0.19 0.05 0.05 -0.46L46 ZSSIIb 6 0.63 0.06 0.10 0.10 0.42


57% σLOD = 0.04 σLOQ = 0.12 11%



Table A4.7 z-scores for maize event MON 863 Level 1 test item for results reported in cp/cp %, all laboratory categories. - = not reported, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner.

Laboratory Number

Group Value Uncertainty LOD cp/cp LOQ cp/cpz-score based

on µR = 0.68L63 3 9.00 (1) 0.67 0.10 0.10 5.64L50 3 0.84 0.06 0.02 0.10 0.49L12 3 0.80 0.19 - 0.10 0.39L41 3 0.75 0.21 - - 0.25L05 4 0.59 (1) 0.15 0.01 0.10 -0.27L39 1 0.57 0.06 - - -0.35L25 4 0.53 (1) 0.18 - - -0.51L29 2 0.38 (2) 0.18 0.12 0.36 -1.23


50% σLOD = 0.06 σLOQ = 0.12 12%


Laboratory Number


on µR = 0.26L63 3 2.92 (1) 0.18 0.10 0.10 5.26L50 3 0.36 0.05 0.02 0.10 0.71L12 3 0.28 0.05 - 0.10 0.17L41 3 0.26 0.11 - - 0.01L39 1 0.24 0.03 - - -0.17L05 4 0.23 (1) 0.06 0.01 0.10 -0.26L25 4 0.21 (1) 0.07 - - -0.46L29 2 0.15 (2) 0.06 0.12 0.36 -1.19


50% σLOD = 0.06 σLOQ = 0.12 12%



Table A4.9a-c. z-scores for maize event MON 810 Level 1 test item for results reported in m/m %, laboratory category: (a), (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported.

(a)

Laboratory Number


µR = 0.91L44 1 1.00 0.14 L1: 0.01% L1: 0.105% 0.27L01 3 1.00 (1) 0.20 - 0.10 0.27L02 3 0.90 (2) 0.30 - - 0.04L07 3 0.65 (2) 0.23 - - -0.66L10 3 1.11 0.20 0.03 0.10 0.50L21 3 0.64 (1) 0.24 <0.04 <0.09 -0.70L23 3 2.05 (1) 0.62 0.02 0.09 1.83L24 3 1.24 0.65 <=0.1 <=0.1 0.74L30 3 0.48 - - - -1.32L35 3 0.78 (1) (3) 30.00 - - -0.27L37 3 0.60 (2) 0.09 0.07 0.10 -0.84L38 3 1.47 0.56 0.02 0.10 1.11L43 3 0.93 (2) 0.10 - - 0.12L47 3 0.92 0.62 0.09 0.09 0.09L48 3 0.92 (1) 0.44 - - 0.09L49 3 1.19 0.35 0.05 0.10 0.65L53 3 0.82 (1) 0.18 0.10 0.10 -0.16L54 3 1.04 (1) 0.20 0.05 0.10 0.36L55 3 1.15 (1) 0.16 0.02 0.10 0.58L56 3 0.62 (3) 53.95 - - -0.77L57 3 0.61 0.23 0.02 0.20 -0.80L58 3 1.10 (2) (3) 100.00 1.0/2.1 - 0.48L59 3 1.25 0.48 0.15 0.30 0.76L63 3 0.55 (1) 0.08 - - -1.03L64 3 0.93 (1) 0.28 0.01 0.05 0.12


67% σLOD = 0.04 σLOQ = 0.06 0%

(b)

Laboratory Number


µR = 0.91L06 2 0.88 (1) 0.28 0.01 0.10 0.00L08 2 1.06 0.03 - - 0.41L09 2 2.50 (3) 21.41 0.05 0.16 2.26L11 2 1.02 0.11 0.03 0.10 0.32L13 2 0.72 0.28 0.03 0.10 -0.44L15 2 0.73 0.14 - - -0.41L19 2 1.40 0.30 0.10 0.10 1.00L20 2 1.40 0.20 <0.1 0.10 1.00L22 2 0.66 0.30 0.01 0.03 -0.63L26 2 0.86 (3) 16.70 0.02 0.09 -0.05L27 2 0.65 0.04 0.09 0.18 -0.66L28 2 1.10 0.12 - 0.05 0.48L31 2 0.56 (2) 0.12 0.01 0.10 -0.99L34 2 0.67 0.25 0.10 0.30 -0.60L36 2 1.29 0.18 0.02 0.10 0.83L40 2 1.04 0.19 0.02 0.04 0.36L45 2 > 0.00 - 0.10 0.10 *L52 2 0.80 0.11 0.05 0.10 -0.21L61 2 2.26 0.95 - - 2.04L62 2 0.74 0.12 0.10 0.10 -0.38


21% σLOD = 0.04 σLOQ = 0.06 11%



(c)

Laboratory Number


µR = 0.91L14 4 0.57 (2) (3) 25.60 0.05 0.10 -0.95L17 4 0.83 - 0.10 0.10 -0.13L33 4 0.81 0.13 0.10 0.10 -0.18L51 4 0.43 0.31 0.06 0.24 -1.56L18 5 1.01 (2) 0.21 0.07 0.10 0.29L32 5 1.30 (1) 0.60 0.04 0.10 0.84L42 5 0.65 - 0.03 0.10 -0.66L03 6 0.29 (1) 0.21 0.05 0.05 -2.42L46 6 0.81 0.07 0.01 0.10 -0.18


57% σLOD = 0.03 σLOQ = 0.05 11%



Table A4.10a-c. z-scores for maize event MON 810 Level 2 test item for results reported in m/m %, laboratory category: (a) , (b), and (c). - = not reported, * = no z-score attributed, (1) Uncertainty (U) and/or coverage factor k was reported in an inconsistent manner, (2) U was reported in an incomplete manner, (3) U seems to be a relative value. Descriptive statistics for LOD and LOQ were estimated using only data for which a value was clearly reported.

(a)

Laboratory Number


µR = 0.36L44 1 0.31 0.04 - - -0.17L01 3 0.44 (1) 0.07 - 0.10 0.59L02 3 0.30 (2) 0.10 - - -0.24L07 3 0.31 (2) 0.10 - - -0.17L10 3 0.45 0.11 0.03 0.10 0.64L21 3 0.27 (1) 0.10 <0.04 <0.09 -0.47L23 3 0.76 (1) 0.23 0.02 0.09 1.78L24 3 0.41 0.26 <=0.1 <=0.1 0.44L30 3 0.18 - - - -1.35L35 3 0.26 (1) (3) 30.00 - - -0.55L37 3 0.18 (2) 0.05 0.07 0.10 -1.35L38 3 1.15 0.44 0.02 0.10 2.68L43 3 0.48 (2) 0.15 - - 0.78L47 3 0.39 0.27 0.09 0.09 0.33L48 3 0.33 (1) 0.16 - - -0.03L49 3 0.42 0.17 0.05 0.10 0.49L53 3 0.30 (1) 0.07 0.10 0.10 -0.24L54 3 0.35 (1) 0.07 0.05 0.10 0.10L55 3 0.40 (1) 0.16 0.02 0.10 0.39L56 3 0.22 (3) 60.82 - - -0.91L57 3 0.20 0.09 0.02 0.20 -1.12L58 3 0.60 (2) (3) 100.00 1.0/2.1 - 1.27L59 3 0.49 0.28 0.15 0.30 0.83L63 3 0.19 (1) 0.06 - - -1.23L64 3 0.43 (1) 0.13 0.01 0.05 0.54


67% σLOD = 0.04 σLOQ = 0.06 4%

(b)

Laboratory Number


µR = 0.36L06 2 0.36 (1) 0.13 0.01 0.10 0.16L08 2 0.46 0.02 - - 0.69L09 2 1.00 (3) 14.13 0.05 0.16 2.38L11 2 0.36 0.05 0.03 0.10 0.16L13 2 0.29 0.14 0.03 0.10 -0.31L15 2 0.24 0.03 - - -0.72L19 2 0.60 0.20 0.10 0.10 1.27L20 2 0.60 0.15 <0.1 0.10 1.27L22 2 0.25 0.06 0.01 0.03 -0.63L26 2 0.30 (3) 32.00 0.02 0.09 -0.24L27 2 0.22 0.02 0.09 0.18 -0.91L28 2 0.40 0.06 - 0.05 0.39L31 2 0.22 (2) 0.05 0.01 0.10 -0.91L34 2 0.26 0.05 0.10 0.30 -0.55L36 2 0.49 0.08 0.02 0.10 0.83L40 2 0.55 0.11 0.02 0.04 1.08L45 2 0.00 - 0.10 0.10 *L52 2 0.35 0.10 0.05 0.10 0.10L61 2 0.88 0.37 - - 2.10L62 2 0.28 0.03 0.10 0.10 -0.39


21% σLOD = 0.04 σLOQ = 0.06 11%



(c)

Laboratory Number


µR = 0.36L14 4 0.19 (2) (3) 44.10 0.05 0.10 -1.23L17 4 0.27 - 0.10 0.10 -0.47

L33 4 0.13 0.03 0.10 0.10 -2.05L51 4 0.16 0.12 0.06 0.24 -1.60L18 5 0.46 (2) 0.21 0.07 0.10 0.69L32 5 0.52 (1) 0.24 0.04 0.10 0.96L42 5 0.14 - 0.03 0.10 -1.89L03 6 0.12 (1) 0.10 0.05 0.05 -2.23L46 6 0.37 0.02 0.01 0.10 0.22


57% σLOD = 0.03 σLOQ = 0.05 22%




Laboratory Number


on µR = 0.35L29 2 0.83 (2) 0.49 0.11 0.32 1.91L25 4 0.42 (1) 0.14 0.01 0.07 0.43L39 1 0.39 0.05 - - 0.27L63 3 0.36 (1) 0.03 0.10 0.10 0.10L50 3 0.35 0.04 0.02 0.10 0.03L41 3 0.33 0.25 - - -0.09L12 3 0.31 0.05 - 0.10 -0.23L60 2 0.30 0.13 - - -0.30L16 3 0.30 - 0.01 0.02 -0.30L05 4 0.28 (1) 0.1 0.01 0.10 -0.45


44% σLOD = 0.05 σLOQ = 0.09 0%


Laboratory Number


on µR = 0.13L29 2 0.51 (2) 0.2 0.11 0.32 3.00L39 1 0.15 0.02 - - 0.34L25 4 0.15 (1) 0.07 0.01 0.07 0.34L12 3 0.14 0.04 - 0.10 0.19L50 3 0.14 0.02 0.02 0.10 0.19L41 3 0.12 0.10 - - -0.15L05 4 0.12 (1) 0.05 0.01 0.10 -0.15L63 3 0.11 (1) 0.02 0.10 0.10 -0.33L60 2 0.10 0.05 - - -0.54L16 3 0.10 - 0.01 0.02 -0.54


44% σLOD = 0.05 σLOQ = 0.09 10%



Figure A4.1. z-scores for maize event 98140 Level 1 test item on the basis of a robust mean of 0.33 m/m % (◊◊◊◊).



Figure A4.2. z-scores for maize event 98140 Level 2 test item on the basis of a robust mean of 1.06 m/m % (◊◊◊◊).



Figure A4.3. z-scores for maize event 98140 Level 1 test item on the basis of a robust mean of 0.18 cp/cp % (◊◊◊◊).



Figure A4.4. z-scores for maize event 98140 Level 2 test item on the basis of a robust mean of 0.69 cp/cp % (◊◊◊◊).



Figure A4.5. z-scores for maize event MON 863 Level 1 test item on the basis of a robust mean of 1.36 m/m % (◊◊◊◊).



Figure A4.7. z-scores for maize event MON 863 Level 1 test item on the basis of a robust mean of 0.68 cp/cp % (◊◊◊◊).



Annex 5: Questionnaire data

1. Number of replicate DNA extractions from test materials:

No. of laboratories

a) 1 1

b) 2 41

c) 3 6

d) 4 10

e) Other 5

Other of which:

5 2

6 1

12 1

16 1

2. DNA extraction method? No. of laboratories

a) ISO/CEN published method 23

b) EURL validated method 8

c) National reference method 1

d) International literature 2

e) In-house developed and optimised 9

f) Commercial kit 17

g) Other 3

Other of which Answers referred to used kits, see Q4

2.3. Was the DNA extraction method used within the scope of your ISO/IEC 17025 accreditation?

No. of laboratories

a) Yes 51

b) No 12

3. Sample intake (in g) for the DNA extraction: No. of laboratories

a) < 0.1 0

b) 0.1 - 0.2 36

c) > 0.2 25

d) Other 2

Other of which:

0.5 1

1 1



4. DNA extraction method/kit used: No. of laboratories

a) CTAB 30

b) CTAB-derived 9

c) Biotecon 2

d) GeneScan GENESpin 3

e) Guanidine HCl with proteinase K 2

f) Macherey-Nagel Nucleospin 10

g) Promega Wizard 2

h) Qiagen DNeasy plant mini kit 2

i) TEPNEL kit 0

j) In-house developed and optimised 1

k) Other 3

Other of which:

SDS 1

MN NucleoMag 96 Plant, modified with RNAse, Amylase, Proteinase K

1

EN ISO 21571 CTAB method combined with Maxwell Preparation Kit (Fa. Promega)

1

5. How was the clean-up of the DNA performed? No. of laboratories

a) No DNA clean-up 35

b) Ethanol precipitation 11

c) Amersham MicroSpin S300 0

d) Promega Wizard DNA clean-up resin 5

e) Qiagen QIAQuick 4

f) Qiagen Genomic-Tip 20/G 0

g) Silica 3

h) Other 5

Other of which:

Maxwell 16 Nucleic Acid Extraction System from Promega 1

CTAB- and Isopropanol precipitation 1

Invisorb DNA Cleanup 1

JET QUICK SPIN COLUMN TECHNIQUE 1

Eurofins GeneScan Cleaning Columns 1



6. How have you quantified the DNA? No. of laboratories

a) Gel 1

b) UV spectrophotometer 41

c) Fluorometer 10

d) Other 5

e) Not applicable (i.e. DNA was not quantified) 6

Other of which:

Estimation is based on qPCR results 1

Nanodrop 3

QPCR 1

7. Dilution buffer? No. of laboratories

a) TE (10 mM Tris-HC1, 1 mM EDTA) 8

b) TE 0.1X (10 mM Tris-HC1, 0.1 mM EDTA) 9

c) TE low (1 mM Tris, 0.01 mM EDTA) 3

d) Water 35

e) Other 8

Other of which

TE 0.2X 1

TEx0,2(tris-HCl 2mM,pH 7,5 EDTA 0,2mM) 1

Elution Buffer (A828D) from Promega 1

No dilution applied 1

TE (10 mM TrisHCl; 0,2 mM EDTA) 1

0.2x TE (2 mM Tris-HCl, 0.2 mM EDTA, pH 8) 1

0.5X TE 1

Tris elution buffer without EDTA according to Maxwell Preparation Kit (Fa. Promega)

1

8. Screening method used for GM detection No. of laboratories

a) Combinatory SYBR® Green qPCR Screening (COSYPS) 0

b) In-house developed and optimised 1

c) International literature 5

d) ISO/CEN published method 11

e) National reference method 7

f) Pre-spotted plate 4

g) Qualitative PCR 8

h) Real-time PCR 41

i) SYBR® Green qPCR screening 4

j) Other 4



Other of which:

The lab does not apply a screening method when the events are mentioned by the client. SybrGreen real-time PCR identification is applied

1

No screening, 14 event specific q-PCR methods 1

Zeitler R., Pietsch K., Waiblinger H-U (2002) J. AOAC Int. 20; Brodmann et al, 2002 J. AOAC Int. 2002, 85(3), 646-653; P35S-Bar: JRC 2006; Konstr. P-ract1-CTP2-CP4-EPSPS: RKI 2003; cryIAb/cryIAc: BVL Leitfaden

1

8.3 PCR analytical screening method used within the

scope of your ISO/IEC 17025 accreditation? No. of laboratories

a) Yes 46

b) No 17

9. Principle of PCR product detection used for screening?

No. of laboratories

a) Gel 6

b) SYBR® Green 4

c) Taqman probe 51

d) Other 2

Other of which:

Gel or TaqMan Probe 1


10. Screening method used for GM detection: No. of laboratories

a) Multiplex PCR 11

b) Singleplex PCR 52

11. Elements / targets used for screening (P =

promoter, T = terminator): No. of laboratories

a) CP4 EPSPS 20

b) P35S 33

c) T-nos 33

d) Other 54

Other of which:

Event-specific methods 12

Event-specific elements 3

pat, bar 5

bar; p35S-pat 5

P34S-FMV, bar, pat, nptII 1

35S-pat, bar, Ly38 1

Targets present on pre-spotted plate 1



Pubi 1

not appl. 1

screening not performed 1

PAT 1

Screening table; Bundesamt für Verbraucherschutz und Lebensmittelsicherheit

1

35S/NOS-duplex + pat + bar + CTP2-EPSPS (§64 methods Germany); pat-bar-CTP2-EPSPS-triplex (inhouse)

1

event-specific: Querci et al., (2009) 1

Multiplex PCR reactions containing three or more events 1

35s_pat; bar; 35S/NOS is Multiplex 1

p35S-pat, p35S-nptII, bar 1

event-specific targets from methods listed in point 8.1, with DNA from reference materials as controls

1

P35S-PAT, PAT-BAR 1

PAT, bar, 35S-PAT 1

construct (Bt176) and event-specific real-time PCR (other events) 1

Cry1Ab, pat, bar 1

pFMV 1

Singleplex PCR: bar, 35S-pat; for species- and event-specific screening methods EU-RL validated methods were used

1

specific sequences 1

P-FMV 1

pat; bar; P35S-pat; P35S-Bar; P-ract1-CTP2-CP4-EPSPS; cryIAb/cryIAc

1

taxon-specific: adh, cruciferine, lectin 1

event methods were applied directly for these specific samples 1


Real Time PCR Based Ready to use multitarget analytical system for the detection of EU Authorised and unauthorised GM events (EURL JRC Ispra)

1

Endogenous and specific transgene primers/probe 1

All potential GM lines (as requested) were tested with EU-RL validated methods

1

12. Real-time PCR quantification method(s) No. of laboratories

a) EU-RL validated method(s) 56



d) ISO/CEN published method(s) 7

e) National reference method(s) 2

f) Other 9

Other of which:

GMOQuant Kit 1

Eurofins GMO Quant event(s) MON810, MON863 kits, verified by the laboratory

1

MON863: EU-RL method with HMG as endogenous gene 1



GeneScan : GMOQuant Event MON810 Corn; GMOQuant Event MON863 Corn

1

Brodmann et al. 2002: Journal of AOAC international Vol. 85(3):646-653

1

methods modified from EU-RL validated methods: CRL, 2006, CRL assessment on the validation of an event specific method for the relative quantitation of maize line MON810 DNA using real-time PCR as carried out by Federal Institute for Risk Assessment (BfR), CRL-VL-25/04VR; CRL, 2005, Event-specific method for the quantitation of maize line MON863 using real-time PCR

1

12.3. Real-time PCR quantification method used within

the scope of your ISO/IEC 17025 accreditation? No. of laboratories

a) Yes 43

b) No 16

Not reported 4

13. End-point PCR quantification method(s) No. of laboratories






f) Other 47

Other of which:

Not used 14

NA 4

Not relevant 1

rt-PCR 1

13.3. End-point PCR quantification method used within

the scope of your ISO/IEC 17025 accreditation? No. of laboratories

a) Yes 10

b) No 21

Not reported 32

14. Digital PCR quantification method(s) No. of laboratories








f) Other 53

Other of which:

Not used 15

NA 4

Not relevant 1

14.3. Digital PCR quantification method used within the

scope of your ISO/IEC 17025 accreditation? No. of laboratories

a) Yes 3

b) No 22

Not reported 38

15. Real-time PCR quantification method(s): No. of laboratories

a) Multiplex PCR 2

b) Singleplex PCR 61

16. Real-time PCR instrument: No. of laboratories

a) ABI 7000 1

b) ABI 7300 3

c) ABI 7500 24

d) ABI 7700 3

e) ABI 7900 HT 14

f) ABI StepOne & StepOne Plus real-time PCR system 0

g) BioRad icycler 1

h) Corbett Rotor-Gene 6000 1

i) Roche LightCycler 480 4

j) Roche Light Cycler 2.0 2

k) Stratagene Mx 3000/Mx 3005 7

l) Stratagene Mx4000 0

m) Other 10

Other of which:

BioRad CFX96 3

Qiagen Rotor-Gene 1

ABI 7500 FAST 1

Roche LightCycler 1.x 1

Viia 7 1

Corbett Rotor-Gene 3000, BioRad iQ5 1

Qiagen Rotor-Gene Q 1

ABI ViiA7 1



17. Real-time PCR Master Mix: No. of laboratories

a) ABI TaqMan® Universal PCR master mix 34

b) ABI TaqMan® Universal PCR master mix, no AmpErase® UNG

8

c) ABI TaqMan® Fast Universal PCR master mix 1

d) ABI TaqMan® Gold with Buffer A 5

e) Eurogentec: qPCR MasterMix 3

f) Eurogentec MESA GREEN qPCR MasterMix Plus for SYBR® Assay

0

g) Eurogentec qPCR MasterMix for SYBR® Green 0

h) Sigma JumpstartTM Taq ReadyMixTM 2

i) Qiagen: QuantiTect SYBR® Green PCR Kit 0

j) Qiagen: QuantiTect Probe PCR Kit 5

k) Roche: FastStart TaqMan® Probe Master (Rox) 1

l) Roche: FastStart Universal Probe Master (Rox) 2

m) Diagenode: Universal Mastermix 2

n) Fermentas: MaximaTM Probe/ROX qPCR Master Mix 2

o) Fermentas: MaximaTM SYBR Green/ROX qPCR Master Mix 1

p) Ampliqon: RealQ PCR 2 x Master Mix 0

q) Takara: SYBR® Premix Ex TaqTM 0

r) Takara: Premix Ex TaqTM 1

s) Other 15

Other of which:

Eurofins GeneScan MasterMix 1

in-house prepared master mix 1

Eurofins reaction mix 1

Qiagen: QuantiTect Mulitplex PCR Kit 1

Novazym: AmpliQ Real-time PCR Opti-probe Kit 1

iQ Supermix, BioRad 1

ABI TaqMan PCR Core Reagent Kit 1

GMOQuant Event MON810 Corn; GMOQuant Event MON863 Corn

1

Quanta Biosciences Perfecta qPCR Master Mix, UNG 1

LightCycler 480 Probes Master; Amplification reaction mixture for DAS-59122-7 specific system following instructions in CRL, Event-specific method for the quantitation of maize 59122 using real-time PCR, CRLVL03/05VP - corrected version 1

1

BioConnect 5X HOTFirePol Probe qPCR Mix Plus (No ROX) 1

Roche: LightCycler 480 Probes Master 1

ABI TaqMan Gene Expresion Master mix 1

Promega GoTaq Probe qPCR MasterMix 1

For MON810 no UMM 1



17.2. Number of reagents (i.e. DNA, primers, probe, water, …) involved?

No. of laboratories

a) 5 30

b) 6 29

c) 7 4

d) 8 3

e) Other 7

Other of which:

9 2

10 1

12 2

14 1

15 1

Q 18.1 Sample intake (in ng) per real-time PCR No. of laboratories

a) 0-50 9

b) 50-100 15

c) 100-200 32

d) > 200 6

e) DNA amount not quantified 6

Q18.2. Sample intake (in ng) per real-time PCR No. of laboratories

a) 0-50 2

b) 50-100 3

c) 100-200 9

d) > 200 2



a) 0-50 1

b) 50-100 2

c) 100-200 2

d) > 200 2



a) 0-50 0

b) 50-100 1

c) 100-200 2

d) > 200 1





a) 0-50 0

b) 50-100 0

c) 100-200 2

d) > 200 1


19. Number of PCR replicates per test item (biscuit flour level 1 and level 2):

No. of laboratories

a) 1 0

b) 2 7

c) 3 22

d) 4 18

e) 5 0

f) 6 7

g) Other 9

Other of which:

8 2

9 1

10 3

12 1

16 1

20. Real time PCR method(s) for quantification: No. of laboratories

a) Taqman probe 63

b) SYBR® Green 0

c) Other 0

21. Real-time PCR quantification method(s) used? No. of laboratories

a) DNA copy number standard curve using a dilution series 16

b) Mass/mass standard curve using a dilution series 46

c) Delta Ct method 3

d) Absolute quantification (end-point digital PCR) 0

e) Other 0



22. Real-time PCR quantification method(s): slope(s) endogenous gene

No. of laboratories per GM event

3272 Bt11 Bt176 59122 GA21 MIR604 MON 810 MON 863 NK603 1507 MON 88017 MON 89034 98140 MIR162

-4.1 ≤ slope < -3.6 0 0 0 0 0 0 4 2 0 0 0 0 1 0

-3.6 ≤ slope ≤ -3.1 1 1 1 3 2 2 52 52 2 2 1 1 49 1

-3.1 < slope < -2.6 0 0 0 0 0 0 2 3 0 0 0 0 0 0

Other 0 0 0 0 0 0 2 2 0 0 0 0 1 0

Not Applicable 25 25 25 23 24 24 2 3 24 24 25 25 5 24

23. Real-time PCR quantification method(s): slope(s) GM trait gene



-4.1 ≤ slope < -3.6 0 0 0 0 0 0 5 9 0 0 0 0 3 0

-3.6 ≤ slope ≤ -3.1 2 1 1 4 3 2 55 51 1 2 1 1 55 2

-3.1 < slope < -2.6 0 0 0 0 0 1 1 1 0 0 0 0 2 0

Other 0 0 0 0 0 0 2 2 0 0 0 0 1 0

Not Applicable 24 25 25 23 23 23 3 1 24 24 25 25 0 23

24. Real-time PCR quantification method(s): R2 coefficient(s) endogenous gene



0.97 ≤ R2 < 0.98 0 0 0 0 0 0 0 0 0 0 0 0 1 0

0.98 ≤ R2 < 0.99 0 0 0 0 1 0 11 12 0 0 0 0 7 0

0.99 ≤ R2 ≤ 1.00 1 1 1 3 1 2 45 43 1 2 1 1 41 1

Other 0 0 0 0 0 0 4 5 1 0 0 0 2 0

Not applicable 22 22 22 20 21 21 2 2 21 21 22 22 4 21



25. Real-time PCR quantification method(s): R2 coefficient(s) GM-trait gene



0.97 ≤ R2 < 0.98 0 0 0 0 0 0 0 2 0 0 0 0 0 0

0.98 ≤ R2 < 0.99 0 0 1 1 0 0 15 11 0 0 0 0 10 1

0.99 ≤ R2 ≤ 1.00 2 1 0 1 2 2 44 45 1 2 1 1 41 1

Other 0 0 0 1 1 1 4 4 1 0 0 0 1 0

Not applicable 21 22 22 20 20 20 0 0 21 21 22 22 2 20

26. Real-time PCR quantification method(s): endogenous target DNA sequence(s)


3272 Bt11 Bt176 59122 GA21 MIR604 MON 810

MON 863

NK603 1507 MON

88017 MON

89034 98140 MIR162

adh1 0 0 0 1 1 1 8 26 1 0 0 0 6 0

hmg 1 1 1 2 1 1 48 31 1 2 1 1 43 1

Invertase 0 0 0 0 0 0 1 2 0 0 0 0 1 0

Zein 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ZSSIIb 0 0 0 0 0 0 1 1 0 0 0 0 0 0

Other 0 0 0 0 0 0 2 0 0 0 0 0 0 0

Other of which

Zm Zm1

179np SSIIb-3 SSIIb-3

SSIIb-3



27. Real-time PCR quantification method(s): GM trait target DNA sequence(s)



P35S 0 1 1 1 0 0 1 1 1 1 1 2 0 0

CP4 EPSPS 1 1 1 1 1 1 1 1 2 1 2 1 1 1

T-nos 1 1 0 0 1 1 0 1 1 0 1 1 0 1

3272 event-specific 4 0 0 0 0 0 0 0 0 0 0 0 0 0

Bt11 event-specific 0 2 0 0 0 0 0 0 0 0 0 0 0 0

Bt176 event-specific 0 0 2 0 0 0 0 0 0 0 0 0 0 0

59122 event-specific 0 0 0 4 0 0 0 0 0 0 0 0 0 0

GA21 event-specific 0 0 0 0 5 0 0 0 0 0 0 0 0 0

MIR604 event-specific 0 0 0 0 0 5 0 0 0 0 0 0 0 0

MON 810 event-specific 0 0 0 0 0 0 56 2 0 0 0 0 1 0


NK603 event-specific 0 0 0 0 0 0 0 1 3 0 0 1 0 0

1507 event-specific 0 0 0 0 0 0 0 0 0 3 0 0 0 0



98140 event-specific 0 0 0 0 0 0 0 0 0 0 0 0 44 0

MIR162 event-specific 0 0 0 0 0 0 0 0 0 0 0 0 0 4

Other 1 1 1 1 1 1 1 1 1 1 1 1 5 1

Other of which DUPLEX 35S NOS 82



28. Which reference material(s) was (were) used for calibration?

No. of laboratories

a) ERM-BF410 series 3

b) ERM-BF410k series 2

c) ERM-BF425 series 1

d) AOCS 0406-A 1

e) AOCS 0406-D 2

f) AOCS 0906-A 1

g) AOCS 0906-B 0

h) AOCS 0906-E 1

i) Non-modified maize leaf tissue DNA AOCS 0306-C2 0

j) Non-modified soybean leaf tissue DNA AOCS 0707-A3 0

k) Single-target plasmid(s) 5

l) Multiple target plasmid(s) 0

n) Eurofins GeneScan 1

o) Other 55

Other of which:

ERM-BF427, ERM-BF416, ERM-BF413 21

ERM-BF413gk, ERM-BF416d, ERM-BF427d 11

ERM-BF413, ERM-BF416 5

ERM BF413f, ERM BF427d, ERM BF416d 3

ERM-BF413k, ERM-BF416, ERM-BF427 2

ERM BF416d, BF427d, AD413 2

ERM-BF418d; ERM-BF420c; ERM-BF424d; ERM-BF427d;ERM-BF414f;AOCS1208-A; ERM-BF423d; ERM-BF413gk; ERM-BF416d; AOCS0304-B

1

CRM FROM IRMM 1

98140: ERM-BF427 series; MON 863: ERM-BF416 series; MON 810: dual-target plasmids from IRMM

1

GA21 : ERM-BF414; MON 810 : ERM-BF413; MON 863: ERM-BF416; MIR604 : ERM-BF423; 98140 : ERM-BF427

1

ERM-BF 413 f; ERM-BF 416 d 1

ERM-BF416d, ERM-AD413 1

98140: ERM-BF427d; MON 810: IRMM-413-5; MON 863: ERM-BF416d

1

ERM-BF-427d; ERM-BF-413; ERM-BF-416 1

IRMM ERM BF 413 (ek), 416 (c) and 427 (c) 1

ERM-BF417 and ERM-BF427 1

ERM-BF427B; ERM-BF413; ERM-BF416 1

* Most laboratories used several reference materials



29. Which reference material(s) was (were) used for quality control?

No. of laboratories

a) ERM-BF410 series 3

b) ERM-BF410k series 2

c) ERM-BF425 series 1

d) AOCS 0406-A 2

e) AOCS 0406-D 5

f) AOCS 0906-A 2

g) AOCS 0906-B 0

h) AOCS 0906-E 1

i) Non-modified maize leaf tissue DNA AOCS 0306-C2 0

j) Non-modified soybean leaf tissue DNA AOCS 0707-A3 0

k) Single-target plasmid(s) 5

l) Multiple target plasmid(s) 1

n) Eurofins GeneScan 1

o) Other 54

Other of which:

ERM-BF413, ERM-BF416, ERM-BF427 17

ERM-BF413, ERM-BF416 4

ERM-BF413k, ERM-BF416, ERM-BF427 3

ERM-BF413ck; ERM-BF427b; ERM-BF416b 2

ERM-AD413f; ERM-BF416d; ERM-BF415f; ERM-BF427d; ERM-BF424d; ERM-BF414f; AOCS 1208-A; AOCS 0406-D; AOCS 0906-E; ERM-BF412f; ERM-BF411f; ERM-BF423d; ERM-BF418d

1

ERM-BF413series, ERM-AD427, ERM-BF416series, ERM-BF411,ERM-BF412, ERM-BF414, ERM-BF415; ERM-BF418; ERM-BF423; ERM-BF420;ERM-BF424;AOCS1208-A

1

ERM-BF413ck, ERM-BF416b, ERM-BF427b, ERM-BF418d, ERM-BF420c, ERM-BF424d, ERM-BF412f, ERM-BF411f, ERM-BF414f,ERM-BF424d,ERM-BF423d, AOCS 0406-D,AOCS 0906-E, ERM-BF415f, ERM-BF410bf, ERM-BF412bf, ERM-BF415bf, seeds of Ms1xRf1 from LUA-SA, seeds of Liberator pHoe6/Ac from BezReg DT

1

ERM-BF413 series; ERM-BF416 seríes; ERM-BF427b,d; ERM-BF420b,c; ERM-BF412b,e; ERM-BF411b,e; ERM-BF424b,d; ERM-BF414b,f; ERM-BF423b,d; ERM-BF415b,f; ERM-BF418b,d; pENGL-00-16/05-01; pENGL-00-06/06-01; AOCS1208-A

1

ERM- BF427b and samples from previous proficiency testings. 1

98140 : ERM-BF427 c, MON 810 : ERM-BF413 d, MON 863 : ERM-BF416 c 1

ERM-BF413, BF416, BF427, AOCS 1208-A, AOCS 0610A 1

ERM BF 424c, 418c, 416c, 411d, 412d, 414d, 423c, 415d,413d 1

Standard mix - CRM (inhouse) 1

CRM FROM IRMM 1



ERMBF411 series, ERMBF412 series, ERMBF415 series, ERMBF418 series 1 0 %, 0.1 %, 1 % MON 810; 0 %, 0.1 %, 1 %MON 863; 0 %, 0.1 %, 2 % 98140 1

ERM-BF416b, ERM-BF416c, ERM-BF415d, AOCS0306, ERM-BF413ek, ERM-BF410dk 1

ERM BF -413d, 416c, 427c, 420b, 412d, 411d, 424c, 414d, 423c, 415d, 418c 1

ERM-BF 427d ERM-BF 413d ERM-BF 416c 1

ERM-BF416c ERM-BF413d ERM-BF427b 1

ERM-BF416d, ERM-AD413 1

ERM-BF413 series, ERM-BF416 series, ERM-BF424 series 1

ERM-BF413ek , ERM-BF416c , ERM-BF427b 1

ERM-BF417 and ERM-BF427 1

ERM-BF427B; ERM-BF413; ERM-BF416 1

for MON 810 ERM-AD413EK;for 98140 ERM-BF427d and ERM-427a;for MON 863 BF416c 1

ERM-BF-427d; ERM-BF-413; ERM-BF-416 1

98140: ERM-BF427b; MON810: ERM-BF413b, IRMM-413-3; MON863: ERM-BF416b, ERM-BF416c 1

ERM-BF413gk; ERM-BF416d; ERM-BF427d 1

MON 810: ERM-BF413ek, ERM-BF413ck; MON 863: ERM-BF416c, ERM-BF416b; 98140: ERM-BF427c, ERM-BF427b 1

ERM-BF416c, ERM-BF413ck, ERM-BF413ek, ERM-BF427b 1

ERM-BF427b, ERM-BF416b, ERM413b 1

* Most laboratories used several reference materials

30a. Practical LOD and LOQ (in %) of

the GM content determination in

mass/mass or DNA copy number ratio?

No. of laboratories - event 1507

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.01 1

0.02 1 1

0.025 1

0.03 3

0.04 1

0.05 2 1

0.08 2

0.1 3 6 1

<0.1 1

0.13 1

0.15 1

0.3 2

0.31 1

Not reported 48 52 59 60



30b. Practical LOD and LOQ (in %) of

the GM content determination in mass/mass or DNA copy number

ratio?


LOD (m/m) LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 2

0.03 4

0.04 1

0.05 3

0.09 1

0.1 1 6 1 1

<0.1 1

0.11 1

0.15 1

0.2 1

0.3 1

0.42 1

Not determined 0 1 0 0


30c. Practical LOD and LOQ (in %) of




LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 1

0.03 3

0.05 4 1

0.08 1 1

0.09 1

0.1 2 6 1

<0.1 1

0.12 1

0.15 1

0.25 1

0.27 1

0.3 1




30d. Practical LOD and LOQ (in %) of




LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.001 1

0.008 1

0.01 6 1

L1: 0,012 %; L2: 1

0.02 6 1

L1:0,024 %; L2: 1

0.03 3

0.04 1 1

<=0.04 1

<0.04 1

0.05 4 2

0.07 1 2

0.075 1

0.08 1

<=0.08 1

0.09 1

0.1 4 19 1 2

<0.1 1

0.14 1

0.15 1

0.18 1

0.2 1

0.24 1

0.28 1

0.3 1

0.5 1 1

0.9/1.6 1





30e. Practical LOD and LOQ (in %)

of the GM content determination in mass/mass or DNA copy number

ratio?

No. of laboratories - event Bt11

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 1

0.03 4

0.05 4 1

0.09 1

0.1 2 6 1

<0.1 1

0.13 1

0.15 1

0.18 1 1

0.3 2


30f. Practical LOD and LOQ (in %) of the GM content determination in

mass/mass or DNA copy number

ratio?

No. of laboratories - event Bt176

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 1

0.03 4

0.05 4 1 1

0.1 2 6 1

<0.1 1

0.11 1

0.15 1

0.27 1

0.3 2




30g. Practical LOD and LOQ (in %)


ratio?

No. of laboratories - event GA21

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.01 1

0.02 1 1

0.025 1

0.03 3

0.05 4 1

0.08 1

0.1 2 7 1

<0.1 1

0.11 1

0.15 1

0.2 1 1

0.3 2


30h. Practical LOD and LOQ (in %)


ratio?

No. of laboratories - event MIR162

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 1

0.03 3

0.05 3

0.1 1 7 1

<0.1 1

0.14 1

0.15 1

0.2 1

0.25 1

0.3 1

0.37 1





30i. Practical LOD and LOQ (in %)


ratio?

No. of laboratories - event MIR604

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.09 1

0.02 1 1

0.025 1

0.03 4

0.05 3 1

0.07 1

0.08 1

0.09 1 1

0.1 3 6 1

<0.1 1

0.15 1

0.2 1

0.21 1

0.3 2


30j. Practical LOD and LOQ (in %) of the GM content determination in


No. of laboratories - event MON 810

LOD (m/m)

LOQ (m/m)

LOD (cp/cp)

LOQ (cp/cp)

0.009 1

0.01 5 2

L1: 0.01 %; L2: 1

0.02 7 1 1

0.025 1

0.03 3 1

0.04 1 1

<0.04 1

0.05 6 3

0.06 1

0.07 2 1

0.09 2 3

< 0.09 1

0.1 7 25 1 4

<0.1 1

<=0.1 1 1

L1: 0.105 %; L2: 1

0.11 1

0.15 1



0.16 1

0.18 1

0.2 1

0.24 1

0.3 2

0.31 1

1.0/2.1 1


30k. Practical LOD and LOQ (in %) of the GM content determination in mass/mass or DNA copy number ratio?


LOD (m/m)

LOQ (m/m)

LOD (cp/cp)

LOQ (cp/cp)

0.01 6 1

0.02 6 1

0.025 1

L1: 0.026 %; L2: 1

0.03 5

0.04 1

<0.04 1

0.05 7 1

<0.05 1

for L1 0.05;for L2 0.04 1

0.07 1

0.08 1

0.09 1

<0.09 1

0.1 6 26 1 4

<0.1 1

<=0.1 1

for L1 0.1; for L2 0.08 1

0.11 1

0.12 1

0.13 1

0.15 1 2

0.18 1

0.2 2

L1: 0,259 %; L2: 1

0.3 2

0.36 1

0.44 1

0.7/1.7 1





30l. Practical LOD and LOQ (in %) of the GM content determination in

mass/mass or DNA copy number

ratio?


LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.01 1

0.02 1 1

0.025 1

0.03 4

0.05 4 1

0.1 1 6 1 1

<0.1 1

0.11 1

0.15 1

0.25 1

0.3 1



30m. Practical LOD and LOQ (in %) of the GM content determination in



LOD (m/m)

LOQ (m/m)

LOD (cp/cp)

LOQ (cp/cp)

0.01 1

0.02 1 1

0.025 1

0.03 2

0.05 5 1

0.08 1

0.1 1 5 1

<0.1 1

0.11 1

0.15 1

0.2 1

0.29 1

0.3 1

0.4 1




30n. Practical LOD and LOQ (in %)


ratio?

No. of laboratories - event NK603

LOD

(m/m)

LOQ

(m/m)

LOD

(cp/cp)

LOQ

(cp/cp)

0.02 1 1

0.025 2

0.03 4

0.05 3 1

0.08 1

0.09 1

0.1 2 6 1

<0.1 1

0.15 1

0.21 1

0.3 2


31.1. Does the uncertainty correspond to a repeatability standard deviation?

No. of laboratories

a) Yes 38

b) No 14

c) Not applicable 7

Not reported 4

31.2. Does the uncertainty correspond to a within-laboratory reproducibility standard deviation?

No. of laboratories

a) Yes 24

b) No 25

c) Not applicable 9

Not reported 5

31.3. Does the uncertainty include a contribution from the

DNA extraction step? No. of laboratories

a) Yes 27

b) No 21

c) Not applicable 10

Not reported 5

31.5. Did you report an expanded uncertainty including a

coverage factor? No. of laboratories

a) Yes 50

b) No 5

c) Not applicable 4

Not reported 4



31.6. If applicable, please specify the coverage factor used (k = 1 for a 66.67 % confidence level, k= 2 for a 95 %

confidence level, k = 3 for a 99 % confidence level):

No. of laboratories

a) k = 1 0

b) k = 2 45

c) k = 3 0

d) Other 7

Other of which:

2.31; 2.57 for the 98140 quantification 1

no 1

Coverage factor dependent upon degrees of freedom and vary between 2.37 to 2.57

1

The coverage factor depend on the number of measurements (n=7 factor 2.45, n=8 factor 2.37, n=15 factor 2.15, n=16 factor 2.13) for P=95 % and f=n-1

1

2.57 1

3.18 1

32. Biscuit flour level 1: species identification

No. of laboratories per species

Soybean Maize Oilseed Rape Others

Present 28 62 2 0

Absent 26 0 52 1

NA 9 1 9 62

33. Biscuit flour level 2: species identification

No. of laboratories per species

Soybean Maize Oilseed Rape Others

Present 24 62 2 0

Absent 30 0 52 1

NA 9 1 9 62



Annex 6: Invitation letter



Annex 7: Accompanying letter to shipment of samples



Annex 8: Confirmation of shipment

Dear participant, Your test parcels related to the seventh comparative testing round ILC-EURL-GMFF-CT-01/13 has left our premises today by TNT courier. For your convenience, please find herewith the corresponding tracking number you could refer to in order to track the relevant materials on the Web:

13751 6064

The parcel with test items that you will receive should contain:

• Two plastic containers each containing approximately 5 g of test item; • An “acknowledgement of reception” form, that should be returned to the EU-RL GMFF by fax

(+39 0332 786159) or as scanned pdf by E-mail; • An accompanying letter entitled ‘Participation in ILC-EURL-GMFF-CT-01/13’.

The accompanying letter contains your personal password for on-line submission of your results to the reporting website https://web.jrc.ec.europa.eu/ilcReportingWeb. The “acknowledgement of reception” form indicates your lab code (Lxx) in the upper right side of the page; please keep it for future uses in this CT round. The deadline for submission of your results is 25 October 2013 .

Please contact the functional mailbox [email protected] for all issues related to this comparative testing round

Thank you for your collaboration in this comparative testing round.

Lorella Vidmar

On behalf of

The Comparative Testing staff

European Commission

DG Joint Research Centre

Institute for Health and Consumer Protection

Unit I.3 Molecular Biology and Genomics

TP 201 Via E. Fermi 2749

I-21027- Ispra (VA) Italy

Functional mailbox: [email protected]



Annex 9: Acknowledgement of receipt

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European Commission

EUR 26823 EN – Joint Research Centre – Institute for Health and Consumer Protection

Title: Comparative Testing Report on the Detection and Quantification of GM events in biscuit powder – Version b

Author(s): Niccolo Bassani, Adam Niedzwiecki, Angelo Collotta, Matteo Maretti, Marco Mazzara, Joachim Kreysa

Luxembourg: Publications Office of the European Union

2014 – 94 pp. – 21.0 x 29.7 cm

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

ISBN 978-92-79-40073-5 (PDF)

doi: 10.2788/14356

Abstract

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), accredited under ISO 17043,

organised a comparative testing (CT) round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No

882/2004, with voluntary participation of other official control laboratories.

The test items consisted of biscuit powder made from conventional maize and wheat grain spiked with powder of 98140 maize

and MON 863 X MON 810 maize in different concentrations (Level 1 and 2). Participants were required to perform species

identification and test for the presence of maize events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603,

1507, MON 88017, MON 89034, 98140 and MIR162 in the two test items. Any event detected then had to be quantified.

Results could be reported in mass/mass % or copy/copy % and the EU-RL calculated the robust means (R) of Level 1 and 2 test

items accordingly. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.20 for all

events, based on the experience of previous CT rounds. The robust means and target standard deviation were used to derive z-

scores for the participants’ results.

Sixty-four laboratories from 28 countries registered for this CT round, of which all but one returned at least qualitative test

results.

The EU-RL GMFF issued a final report on this CT round in April 2014. After the final report was prepared, it was discovered that

the distribution of the quantitative data obtained from laboratories for event MON 863 deviated from normality because some

laboratories used the adh1 reference gene (with target amplicon of 70 bp) for quantification of MON 863 maize, which is known

to carry a nucleotide polymorphism that may affect amplification. Therefore, the robust means for event MON 863 were re-

calculated based on the data from laboratories that had used a reference gene different from adh1-70 bp. The original final

report has been re-called; the current version contains the re-calculated z-scores for maize event MON 863.

When performing species identification, almost all laboratories correctly identified maize species in each test item. In addition,

approximately half of laboratories also identified soybean and a few oilseed rape.

The majority of laboratories correctly detected the three GM events included in the Level 1 and Level 2 test samples. Results of

the quantitative evaluation of the GM content were compromised by the use of the adh1 gene for MON 863 quantification by a

number of laboratories. Forty five laboratories (71 %) obtained satisfactory z-scores (|z| ≤ 2.0) for the three GM events in both

test items. Among the remaining 18 laboratories, half had unsatisfactory z-scores for MON 863.

Only 46 % of participants provided information on measurement uncertainty in a complete and consistent manner and further

improvement in this crucial area is needed.

ISBN 978-92-79-40073-5

LB

-NA

-26

82

3-E

N-N

comparative testing report on the detection and...

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