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The User Manual - background information and didactical guide for the participants attending the training courses on ‘The analysis of Food Samples for the Presence of Genetically Modified Organisms’ organised by the Joint Reseach Centre - provides the theoretical and detaied practical information on the methodologies and protocols for GMO detection used during the training. Structured in 12 Sessions, it covers a wide variety of techniques for the detection, identification, characterisation, and quantification of GMO., JRC.F.7-Knowledge for Health and Consumer Safety

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format., JRC.F.5-Food and Feed Compliance

The recent ruling of the European Court of Justice has confirmed that organisms obtained by mutagenesis techniques are genetically modified organisms (GMOs). However, in contrast to organisms originating from conventional mutagenesis techniques, those obtained by new mutagenesis techniques are not exempted from the obligations of the GMO EU regulatory framework. This ruling raises questions about the detectability of the corresponding GM food and feed products. A case study is used in this document to explain and discuss possibilities and limitations for the detection and quantification of (known and unknown) genetic modifications in plant products derived from new mutagenesis techniques. Many of the mutations induced by new mutagenesis techniques cannot be unequivocally distinguished from natural mutations because such genome editing technologies are able to create very precise and limited genome changes that mimic the result of potential naturally occurring mutations. Moreover, mutations obtained by genome editing technologies could also not be differentiated from those introduced by conventional mutagenesis techniques which have been incorporated in traditional breeding programs and are often not thoroughly documented. Products of genome editing could only be detected and identified in imports of commodity products by enforcement laboratories when prior knowledge on the altered genome sequence, a validated detection method with appropriate selectivity and certified reference materials are available, similarly as required for the authorisation of current transgenic GMOs. However, when the modification involves only a SNP or few nucleotide changes, it would not be possible to identify whether the mutation originated spontaneously or was induced by conventional or new (genome editing) mutagenesis techniques. Moreover, it is unlikely that methods for the quantification of GMO products with small genome modifications in complex food or feed materials provide the level of selectivity needed for the enforcement of legislation, such as the one on labelling. In the absence of prior knowledge on the genome-edited changes, it is likely that non-authorised genetically modified food and feed products obtained by genome editing would enter the EU market undetected. The EU control system for GMOs and corresponding food and feed products may not function as efficiently for unauthorised genome-edited products compared to transgenic GMOs. In particular, the principle of zero tolerance for unauthorised GMO on the EU market is more difficult to maintain., JRC.F.5-Food and Feed Compliance

It is important to guarantee that results expressing the GM content are reliable, comparable and fulfil the requirements of existing EU legislation. The use of different measurement units to express a GM content, the appearance of new analytical methods that do not require a calibrant and the composite EU legislation on GMOs have triggered the need for a document to clarify how to obtain reliable and comparable results. For this guidance document, past and current EU legislations have been reviewed with a special emphasis on what is meant by 'GM percentage' in the different legal texts. The metrological traceability of measurement results and the currently available guidance are explained and summarised. The particular case of botanical impurities and the genetic constitution of GM seeds are described and illustrated to better understand the complexity hidden behind this type of analysis. An overview of the different analytical methods based on DNA measurements and used for the expression of quantitative GM content results is provided, including the use of new techniques based on digital PCR (dPCR). A measuring system that allows for comparing results by making them traceable to the same reference system has been elaborated in detail. Needs and tools are described and a solution has been proposed to convert results expressing GM content to the required measurement unit, whenever this is needed. By following these recommendations, results obtained in GM copy number per haploid genome equivalent (cp/HGE) by dPCR can be converted into mass fraction percentage and compared to the results obtained by quantitative PCR (qPCR) either with a calibrant certified for its GM mass fraction or with a calibrant certified for its GM purity. The general principle is to relate a measurement result to a GM quantity embedded in a specified certified reference material (CRM) either directly or via one single conversion factor (CF) per event. This conversion factor and its related uncertainty need to be determined precisely for each CRM batch, preferably on the pure GM CRM (100 %), using, for example, dPCR. The estimated uncertainty associated with this conversion factor must be integrated into the measurement uncertainty of the final results expressed in GM mass fraction. CF are currently not yet established for most CRMs. CF values have been recently reported in a few pioneer dPCR studies. However, such proof of concept studies remain incomplete. Therefore, to avoid a gap between new technologies and current EU regulation, the working group recommends to launch a dedicated study to determine CF values. Such a study should involve a limited number of competent laboratories with a proven experience in dPCR. The study could be coordinated by the EURL-GMFF., JRC.F.5-Food and Feed Compliance

In the EU, method validation is an essential part of the process that regulates the introduction of new GMOs as food and/or feed into the market. When the inter-laboratory validation study is completed, the method is ready to be implemented in routine testing laboratories. When implementing the new method, the laboratory has to verify that the method can be used for its intended purpose (method verification). The scope of this document is to provide guidance on how to carry out the method verification of inter-laboratory validated methods for the qualitative and quantitative detection of GMOs. Considering that the Polymerase Chain Reaction (PCR) is the method of choice in the EU for the identification and quantification of GMOs, this document refers exclusively to real time PCR. However, if novel methods are subsequently developed that fulfil legal requirements, then this document will be amended accordingly., JRC.F.5-Food and Feed Compliance

Testing seed lots for the unintended presence of genetically modified (GM) seeds is carried out in European Union Member States (MS). The aim of the testing of seeds for genetically modified organisms (GMOs) is to test whether GMOs are present in non-GM seed lots. Splitting samples of seeds taken from lots into subsamples, testing for the presence of GM seeds in each subsample, and counting the number of positive subsamples is a suitable method for estimating the proportion of GM seeds impurities with a specified probability. The detection of lower proportions of GM seeds in lots requires the analysis of larger seed samples and larger amounts of DNA. This entails more effort and cost to detect lower quantities of GM seed. This document aims at studying the relation between the impurity of GM seed that could be detected and the cost of the analyses required to detect the unintended presence of GM seed in conventional seed lots. A statistical model is elaborated to describe the relation between the impurity level of GMO seeds in seed lots that will, with a high probability, be reliably detected by test plans (the limit of detection) and the cost of the test plans needed to achieve this and effort devoted to the plan. Lowest-cost test plans were estimated for crops with test plan limits of detection at 5%, 0.9%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005% GM seed. As the limit of detection for a plan is reduced, an increasing number of subsamples are required and the change in estimated cost becomes inversely proportional to the change in the estimated limit of detection. A halving from a low limit of detection to a lower limit of detection approximately doubles the estimated cost of the laboratory analysis. For high test plan limits of detection this has no effect on laboratory costs because there is a certain minimum effort required to test working samples of any size. The analysis showed that the rate at which cost increases is determined by the properties of the seed being tested: specifically, the size of the seed and the number of genome copies per mass of DNA., JRC.I.3-Molecular Biology and Genomics

Choline Chloride 60% (CC) is a feed additive that is imported in significant quantities from China. In a number of cases GM rice, harbouring the event Bt63, has been found in imported CC batches but not in derived pre-mixes. The Member States were asked to provide positive CC samples and derived pre-mixes to the EU-RL GMFF in order to allow the EU-RL GMFF establishing practical approaches to DNA extraction and the subsequent testing for presence of (a) rice and (b) the Bt63 rice event. This technical note has been derived based on data provided from National Control Laboratories and experience made by the EU-RL GMFF when re-testing nine feed additive samples and ten pre-mixes samples. The EU-RL GMFF found that the extraction of DNA from the feed additive Choline Chloride (CC) does not normally pose specific problems. It can be carried out following the available protocols or using available standard DNA extraction kits. The extraction of DNA from derived pre-mixes (PMCC) is, however, posing problems because of observed strong inhibition and the presence of DNA from additional sources than those present in the CC. The EU-RL GMFF has tried a number of protocols and extraction kits on PMCC samples. On this basis it is concluded that it is advisable to carefully purify the DNA, to verify the possible presence of inhibition effects and eventually to try to reduce any inhibition observed. Concerning testing the extracted DNA for rice and Bt63 rice event presence, this does not pose a problem in case of the CC while for PMCC rice was not anymore detectable or was detected in trace amounts. In any case testing for Bt63 presence was impossible. A possible explanation could be that much of the extracted DNA is from other plants than rice and hence the concentration of rice DNA is below the LOD of the rice-taxon specific method. In order to verify this, the EU-RL GMFF has analysed the available rice (taxon-) specific methods by means of bio-informatics and (partly) in the laboratory and concludes that they all should be suitable for specifically and reliably detecting rice from the Oryza sativa species. The re-testing by the EU-RL GMFF of CC and PMCC samples has confirmed the results of the initial tests., JRC.I.3-Molecular Biology and Genomics

A joint application was submitted by Bayer CropScience AG and Monsanto Company to request the authorisation of genetically modified stack (GM stack) MS8xRF3xGT73 oilseed rape (tolerant to glufosinate ammonium and glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) NoN° 1829/2003 on GM Food and GM Feed. The unique identifier assigned to GM stack MS8xRF3xGT73 oilseed rape is ACS-BNØØ5-8xACS-BNØØ3-6xMON-ØØØ73-7. The GM stack MS8xRF3xGT73 oilseed rape has been obtained by conventional crossing between three genetically modified oilseed rape events: MS8, RF3 and GT73, without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events MS8, RF3 and GT73 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack MS8xRF3xGT73 oilseed rape. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack MS8xRF3xGT73 oilseed rape., JRC.I.3-Molecular Biology and Genomics

The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single line soybean events MON 87705 and MON 89788 and has published the corresponding reports (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean. The hereby reported in-house verification study led to the conclusion that the individual methods meet the ENGL requirements also when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean., JRC.I.3-Molecular Biology and Genomics

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), accredited under ISO/IEC 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 rice noodles and commercial soybeans spiked with ground powder of soybean event DP-356043-5 in two different concentrations (Level 1 and 2). Participants were required to perform species identification and test for the presence of any GM event in the two test items. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL GMFF calculated the robust means (R) for Level 1 and 2 test items accordingly. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.2 for the event, 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. Eighty-eight laboratories from 42 countries registered for this CT round, of which 85 laboratories from 41 countries returned at least qualitative test results. When performing species identification, almost all laboratories correctly identified soybean and rice in both test items, and a few laboratories also detected maize and/or oilseed rape. In total 71 laboratories reported the presence of GM material in the test items, but 14 laboratories failed in this task. All of the 71 laboratories, except eight, correctly identified soybean event DP-356043-5 in the test items. Results of the quantitative evaluation of the GM content were satisfactory for both measurement units, with only two NRLs appointed under Regulation (EC) No 1981/2006 (one measuring in m/m % and one in cp/cp %) obtaining unsatisfactory z-scores (|z| ≥ 2.0) for both test items. Despite the overall satisfactory outcome of this CT round, only 58 % of participants provided information on measurement uncertainty in a complete and consistent manner, and further improvement in this crucial area is needed., JRC.I.3-Molecular Biology and Genomics

In accordance with relevant EU legislation , Dow AgroSciences LLC provided to the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) a DNA extraction method for soybean grains and the relevant samples (ground soybean grains). In line with its mandate , the EU RL GMFF has conducted an in-house validation of this DNA extraction method. To this end it tested the DNA extraction method on the samples provided and evaluated its performance in terms of DNA yield, integrity and quality. The in-house validation study confirmed that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL , and that it satisfies the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004. The method is therefore fit for the purpose of producing soybean DNA of suitable quantity and quality for subsequent PCR-based analysis., JRC.I.3-Molecular Biology and Genomics

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., JRC.I.3-Molecular Biology and Genomics

An application was submitted by Monsanto Company to request the authorisation of genetically modified stack (GM stack) MON 87708 × MON 89788 soybean (tolerant to dicamba and to glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to GM stack MON 87708 × MON 89788 soybean is MON-877Ø8-9 x MON-89788-1. The GM stack MON 87708 × MON 89788 soybean has been obtained by conventional crossing between two genetically modified soybean events: MON 87708 and MON 89788, without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events MON 87708 and MON 89788 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack MON 87708 × MON 89788 soybean. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack MON 87708 × MON 89788 soybean., JRC.I.3-Molecular Biology and Genomics

In support to the Commission Implementing Decision 2013/287/EU , amending Decision 2011/884/EU , the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) prepared a revision of the previously published guidance document. This document provides further guidance on the correct use of the methods indicated in the Decision, including measures aimed at improving the specificity of the detection approach. This revised guidance, as its previous version, is exclusively meant for the implementation of Decision 2013/287/EU and should not be used for other screening activities. Laboratories should apply it only in conjunction with good standard practices for testing for the presence of GMOs (e.g. use of appropriate controls)., JRC.I.3-Molecular Biology and Genomics

The JRC as European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), established by Regulation (EC) No 1829/2003, in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the MON 88017 transformation event in maize DNA (unique identifier MON-88Ø17-3). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and with Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto provided the detection method and the samples (maize seeds containing the transformation event and conventional maize seeds). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004, JRC.I.3-Molecular Biology and Genomics

The JRC as European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), established by Regulation (EC) No 1829/2003, in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the MON 88017 transformation event in maize DNA (unique identifier MON-88Ø17-3). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and with Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto provided the detection method and the samples (maize seeds containing the transformation event and conventional maize seeds). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004, JRC.I.3-Molecular Biology and Genomics

In response to a request of DG SANCO to provide National Reference Laboratories (NRLs) as soon as possible with a method to test soft white wheat consignments for the presence of unauthorised GM glyphosate-resistant wheat harbouring the event MON71800, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) developed, in collaboration with the European Network of GMO Laboratories (ENGL), a testing strategy intended to be immediately implementable by EU NRLs. The testing strategy is based on a combination of three validated screening methods that allow excluding (detectable) presence of Monsanto’s GM glyphosate-resistant wheat (MON71800) in wheat grain or food/feed products and confirming its presence whenever other GMOs can be excluded. The present report describes the results of the tests carried out by the EU-RL GMFF to verify the testing strategy proposed; the tests were conducted using the positive control sample represented by a crude DNA lysate of MON71800 provided by Monsanto and genomic DNA samples of genetically modified organisms harbouring the CTP2-CP4epsps element for which a validated event-specific method is available. The sensitivity of the three methods was assessed by verifying the relative limit of detection (LODrel) on MON71800 wheat DNA. The LODrel is approximately 0.03% for the P-35S and for T-nos methods and 0.06% for the CTP2-CP4epsps method in 300 nanograms of wheat genomic DNA. Further experimental evidence confirmed that the three methods react against genomic DNA extracted from GM events containing the CTP2-CP4epsps element for which a validated event-specific method is available. The experimental verification hereby reported confirmed the validity of the EU-RL GMFF guidance on testing for GM glyphosate-resistant wheat (MON71800) in wheat grain or in food/feed products containing wheat flour originating or consigned from the US, provided that DNA of acceptable quality can be obtained., JRC.I.3-Molecular Biology and Genomics

In line with its mandate the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), in collaboration with the European Network of GMO Laboratories (ENGL), has validated an event-specific polymerase chain reaction (PCR) method for detecting and quantifying oilseed rape event MON88302 (unique identifier MON-883Ø2-9). The validation study was conducted according to the EU-RL GMFF validation procedure [http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm] and the internationally accepted guidelines (1, 2). In accordance with current EU legislation , Monsanto has provided the detection method and the positive and negative control samples (genomic DNA extracted from seeds harbouring the MON-883Ø2-9 event as positive control DNA, genomic DNA extracted from seeds of conventional oilseed rape as negative control DNA). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at different GM percentage [DNA/DNA]), organised an international collaborative study and analysed the results. The study confirms that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL and according to Annex I-2.C.2 to Regulation (EC) No 641/2004 and it fulfils the analytical requirements of Regulation (EU) No 619/2011 ., JRC.I.3-Molecular Biology and Genomics

The JRC as Community Reference Laboratory (CRL) for the GM Food and Feed (see Regulation EC 1829/2003), in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the H7-1 transformation event in sugarbeet DNA (unique identifier KM-ØØØH71-4). The collaborative trial was conducted according to internationally accepted guidelines. Monsanto provided the method-specific samples (seeds of sugarbeet line H7-1, 100% event H7-1 and seeds of non-GM conventional sugarbeet line, 0% event H7-1) whereas the JRC, upon extraction of the respective DNA, prepared the validation samples (calibration samples and blind samples at unknown GM percentage). The trial involved thirteen laboratories from ten European countries. The results of the collaborative trial fully met ENGL's performance requirements and the scientific understanding about satisfactory method performance. Therefore, the JRC as Community Reference Laboratory considers the method validated as fit for the purpose of regulatory compliance. The results of the collaborative study are publicly available under http://gmo-crl.jrc.it/. The method will also be submitted to CEN, the European Standardisation body, to be considered as international standard., JRC.I.3-Molecular Biology and Genomics

An application was submitted by Syngenta Crop Protection AG to request the authorisation of genetically modified Bt11 x MIR162 x MIR604 x GA21 maize (resistant to lepidopteran and coleopteran pests, able to utilise mannose as the only primary carbon source and tolerant to glufosinate ammonium and glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to Bt11 x MIR162 x MIR604 x GA21 maize is SYN-BTØ11-1x YN-IR162-4xSYN-IR6Ø4-5xMON-ØØØ21-9. The genetically modified maize line Bt11 x MIR162 x MIR604 x GA21 maize has been obtained by conventional crossing of four genetically modified maize events: Bt11, MIR162, MIR604, and GA21 without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events Bt11, MIR162, MIR604 and GA21 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from Bt11 x MIR162 x MIR604 x GA21. The hereby reported the in-house verification study lead to the conclusion that the individual methods meet the ENGL criteria also when applied to DNA extracted from the GM maize stack Bt11 x MIR162 x MIR604 x GA21, JRC.I.3-Molecular Biology and Genomics

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF (Regulation (EC) No 1829/2003(1)) that is also mandated as EU-RL by Regulation (EC) 882/2006(2) organised a comparative testing round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004(2). Participation was open and free of charge for NRLs nominated under Regulation (EC) No 1981/2006(3), for all members of the European Network of GMO Laboratories (ENGL), and for official control laboratories from the EU and third countries. The EU-RL is accredited under ISO 17043 (‘General requirements for proficiency testing’(4)) and this comparative testing round met this ISO Standard(4). The test items were produced in-house from dried leaves of MON 88017 (MON-88Ø17-3) and seeds of soybean event 40-3-2 (MON-Ø4Ø32-6) provided by Monsanto, by spiking a compound feedstuff provided by a Belgian NRL. Participants were required to screen two test items (feedstuff Levels 1 and 2) for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL calculated the robust means (R) of Level 1 and 2 test items accordingly. In addition, "target" values () were assigned by the EU-RL on the basis of its homogeneity study(8) for m/m % data. These values were included in the uncertainty budget. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.15 (log10 value) for soybean event 40-3-2 and at 0.20 for maize event MON 88017 based on experience from previous CT rounds. This target standard deviation was used to derive z-scores for the participants’ results. Ninety laboratories from 43 countries registered for this CT round of which 82 from 35 countries returned at least qualitative test results. The results of the qualitative evaluation of the GM content indicated that most of the laboratories correctly detected soybean event 40-3-2 and maize event MON 88017 thus resulting in a very good performance overall. The results of the quantitative evaluation of GM content were found to be satisfactory overall for both events, with 94% of the laboratories submitting results in mass/mass % with a z-score, estimated on the basis of the robust mean, lying within the range of -2 to +2. This percentage decreased to 79% for results expressed in copy/copy %. When asked to repeat experimental work, most of the underperforming laboratories obtained satisfactory results. Only ~57% of participants provided information on measurement uncertainty in a complete and consistent manner, it is apparent therefore that despite the overall satisfactory outcome of this CT round, there is still improvement needed in this crucial area., JRC.I.3-Molecular Biology and Genomics

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these activities. This report describes the outcome of the third comparative testing round ILC-EURL-GMFF-CT-01/11. Participants had to determine the GM content in two test items denoted soybean powder levels 1 and 2, containing different GM percentages of soybean event 40-3-2 flour. This comparative testing round was organised in collaboration with the Food Safety and Quality Unit of the Institute for Reference Materials and Measurements (Geel, BE). The soybean event 40-3-2 test items were produced in-house. The Food Safety and Quality Unit managed the on-line registration and submission of results. A total of 155 laboratories were invited to participate in ILC-EURL-GMFF-CT-01/11. Eight National Reference Laboratories declined participation, of which one was no longer a National Reference Laboratory. One hundred and two laboratories from 43 countries returned results, of which 62 were National Reference Laboratories, 11 were members of the European Network of GMO Laboratories only, eight were only Official control laboratories and 21 were laboratories from third countries. Seven laboratories including one National Reference Laboratory, one European Network of GMO Laboratory and five laboratories from a third country did not submit results. Participants could report the results of the exercise either in mass/mass % or in copy/copy %. In this third comparative testing round greater than 86 % of participants gained a satisfactory z-score in the range of -2 to +2 for both soybean powder levels 1 and 2 regardless of the calibration method and the measurement unit., JRC.I.3-Molecular Biology and Genomics

Suntory Holdings Ltd has submitted an application for marketing (C/NL/09/02) of a genetically modified carnation line 26407 (Unique identifier: IFD-26407-2). In this context, the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) was asked to carry out a singlelaboratory validation of the performance of a polymerase chain reaction (PCR)-based method for detecting and identifying the carnation GM line 26407, developed by the applicant. This report describes the results of this validation, carried out by the EU-RL GMFF with control samples provided by the applicant. The method is a duplex end-point PCR, where a carnation (taxon) target and a transgenic sequence are detected simultaneously. The limit of detection (LOD) of the method has been established to be at least 10 copies for the taxon-specific target and between 50 and 10 copies for the GM target, based on haploid genome copy number. The event-specificity of the method was assessed by the applicant as being sufficient. The EU-RL could verify that the taxon-specific primers correctly detect the endogenous gene target in genomic DNA of a conventional carnation line (negative control) and in the genomic DNA of the GM carnation line, while the GM target is detected by the GM specific primers only in genomic DNA of 26407 GM line (positive control)., JRC.I.3-Molecular Biology and Genomics

In line with its mandate the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), in collaboration with the European Network of GMO Laboratories (ENGL), has validated an event-specific real-time polymerase chain reaction (qPCR) method for detecting and quantifying the soybean event MON87708 (unique identifier MON-877Ø8-9). The validation study was conducted according to the EU-RL GMFF validation procedure [http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm] and the relevant internationally accepted guidelines. In accordance with current EU legislation, Monsanto Company has provided the detection method and the positive and negative control samples (genomic DNA from soybean seeds harbouring the MON87708 event as positive control DNA, genomic DNA from conventional soybean seeds as negative control DNA). The EU-RL GMFF verified the method performance data provided by the developer, prepared the validation samples (calibration samples and blind samples at different GM percentage [DNA/DNA]), organised an international collaborative study and analysed the results. The study confirms that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL and laid down in Annex I-2.C.2 to Regulation (EC) No 641/2004 and it fulfils the analytical requirements of Regulation (EU) No 619/2011. This report is published at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm., JRC.I.3-Molecular Biology and Genomics

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these activities. This preliminary report describes the outcome of the fourth comparative testing round ILC-EURL-GMFF-CT-02/11. Participants were required to screen two test items denoted maize powder levels 1 and 2, for the presence of maize events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603 and TC1507. Any events detected were then to be quantified. This comparative testing round was organised in collaboration with the Food Safety and Quality Unit of the Institute for Reference Materials and Measurements (Geel, BE). The maize test items were produced in-house. The Food Safety and Quality Unit managed the on-line registration and submission of results. A total of 159 laboratories were invited to participate in ILC-EURL-GMFF-CT-02/11. Participants could report the results of the exercise either in mass/mass % or in copy/copy %. In this fourth comparative testing round greater than 86 % of participants gained a satisfactory z-score in the range of -2 to +2 for the results expressed in mass/mass % for both maize powder levels 1 and 2 regardless of the GM event. However, a lower percentage (43–86 %) of z-scores within the working range of -2 to +2 was calculated for those participants that expressed the results in copy/copy %., JRC.I.3-Molecular Biology and Genomics

An application was submitted by Pioneer Overseas Corporation to request the authorization of the genetically modified maize stack 1507 x 59122 x MON 810 x NK603, resistant against certain lepidopteran pests, protected against corn rootworm larvae, and glufosinate-ammonium and glyphosate tolerant, and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to 1507 x 59122 x MON 810 x NK603 maize is DAS-Ø15Ø7-1xDAS-59122-7xMON-ØØ81Ø-6xMON-ØØ6Ø3-6. The genetically modified maize line 1507 x 59122 x MON 810 x NK603 has been obtained by conventional crossing of four genetically modified single maize events: 1507, 59122, MON 810 and NK603 without any new genetic modification. The EU-RL GMFF has previously validated, and declared fit for purpose, the detection methods for the single events 1507, 59122, MON 810 and NK603 (see: http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from 1507 x 59122 x MON 810 x NK603. The herewith reported in-house verification study lead to the conclusion that the individual methods meet the ENGL performance criteria also when applied to DNA extracted from the GM maize stack 1507 x 59122 x MON 810 x NK603., JRC.I.3-Molecular Biology and Genomics

In the European Union nations, and other countries including Japan, Australia and Malaysia, it is a legal requirement that food products containing genetically modified organism (GMO) materials are labelled as such in order that customers may make informed purchasing decisions. For manufacturers and consumers to be confident about these assertions, systems must be in place along the entire food chain which support the co-existence of GM and non GM materials whilst maintaining a strict segregation between the two. This book is an output of a European Union-funded project entitled "Co-Extra: GM and non-GM food and feed supply chains: their Co-Existence and Traceability". The objective of this four year project is to provide practical tools and methods for implementing co-existence that will: enable the co-existence of genetically modified (GM) and non-GM crops enable the segregation and tracing of genetically modified organism (GMO) materials and derived products along the food and feed chains anticipate the future expansion of the use of GMOs The project is designed to foster a robustly science-based debate amongst all of the stakeholders involved in the food and feed chains, and the tools will be assessed not only from a technical point of view but with regard to the economic and legal aspects. It also surveys the GMO-related legal regimes and practices that exist in and beyond the EU. This book examines the practical tools and methods available to implement the co-existence and traceability of GM and non-GM food materials along the entire food and feed chains, as demanded by consumers and by legislation in force in the EU and elsewhere. GM and Non-GM Supply Foods is a source of valuable information for food manufacturers, food research institutions and regulatory bodies internationally.

Following the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS) announcement that test results confirmed the finding of unauthorised GM glyphosate-resistant wheat "volunteer" plants harbouring the event MON71800 on a farm in Oregon, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) was requested to provide as soon as possible a method to test wheat consignments for the presence of this genetically modified organism (GMO) to the National Reference Laboratories (NRLs) for GMOs of the EU Member States. In response, the EU-RL put together a testing strategy, based on readily available screening tests which was published here (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm). Upon request, Monsanto provided in May 2013 the EU-RL with the procedure “Roundup Ready® Wheat MON71800 Event Specific Endpoint TaqMan® PCR with acc Internal Control for Seed Pools of 1:15” that had previously been made available to, and was used by USDA. The EU-RL GMFF tested this protocol on positive control samples consisting of MON71800 crude lysate, also provided by Monsanto. Our results can be summarised as follow: The method is apparently event-specific. Our specificity-tests did not show cross-reactivity on genomic DNA from a wide selection of similar GMO. The sensitivity of the method was found to be in agreement with previous findings of USDA, i.e. the relative limit of detection lies at 0.5% in a background of 301 ng of total wheat genomic DNA. The absolute limit of detection (LODabs) was determined between 5 and 10 copies of MON71800 target. The latter was not indicated by the USDA. For seed/grains the application of a sub-sampling strategy could allow detection below 0.5% but would require significant additional efforts, including the analysis of numerous sub-samples. Our tests also indicated that the duplex PCR system at the tested stage of optimisation is characterised by poor efficiency at increasing background DNA concentration in reaction. Based on the scientific evidence described in the present report, the EU-RL suggest that its testing strategy (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm), making use of validated element and construct-specific methods and found to be more sensitive, is used to test for presence of MON71800 GM-wheat. The verified event specific method of Monsanto could be used to confirm positive findings at GM-target concentration equal or above 0.5% or it could be used for detection of GM-event MON71800 below 0.5% but it would require a costly sub-sampling strategy, which, in addition, is only possible in seeds/grains., JRC.I.3-Molecular Biology and Genomics

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these activities. This report describes the outcome of the fourth comparative testing round ILC-EURL-GMFF-CT-02/11. Participants were required to screen two test items denoted maize powder levels 1 and 2, for the presence of maize events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603 and TC1507. Any events detected were then to be quantified. This comparative testing round was organised in collaboration with the Food Safety and Quality Unit of the Institute for Reference Materials and Measurements (Geel, BE). The maize test items were produced in-house. The Food Safety and Quality Unit managed the on-line registration and submission of results. A total of 159 laboratories were invited to participate in ILC-EURL-GMFF-CT-02/11. Ninety-three laboratories from 40 countries returned results, of which 62 were National Reference Laboratories, seven were members of the European Network of GMO Laboratories only, eight were only Official control laboratories and 16 were laboratories from third countries. Nine laboratories including one National Reference Laboratory, one European Network of GMO Laboratory and seven laboratories from a third country did not submit results. In this fourth comparative testing round greater than 86 % of participants gained a satisfactory z-score in the range of -2 to +2 for the results expressed in mass/mass % for both maize powder levels 1 and 2 regardless of the GM event. However, a lower percentage (43 – 86 %) of z-scores within the working range of -2 to +2 was calculated for those participants that expressed the results in copy/copy %., JRC.I.3-Molecular Biology and Genomics

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these activities. This report describes the outcome of the fifth comparative testing round ILC-EURL-GMFF-CT-01/12. Participants had to determine the content of oilseed rape event GT73 and maize event 59122 in two test items denoted genomic DNA levels 1 and 2, containing different GM percentages of both GM events. This comparative testing round was organised in collaboration with the Food Safety and Quality Unit of the Institute for Reference Materials and Measurements (Geel, BE). The test items were produced in-house. The Food Safety and Quality Unit managed the on-line registration and submission of results. A total of 160 laboratories were invited to participate in ILC-EURL-GMFF-CT-01/12. Eighty laboratories from 36 countries returned results, of which 59 were National Reference Laboratories, six were only members of the European Network of GMO Laboratories, three were only Official control laboratories and 12 were laboratories from third countries. Five laboratories including one National Reference Laboratory and four laboratories from third countries did not submit results. In this fifth comparative testing round 92 % to 98 % of participants gained a satisfactory z-score in the range of -2 to +2 for the results expressed in mass/mass % depending on the GM content and the GM event. However, a lower percentage (38 – 93 %) of z-scores within the working range of -2 to +2 was calculated for those participants that expressed the results in copy/copy %., JRC.I.3-Molecular Biology and Genomics