Assessment of genetically modified maize 1507 × MIR162 × MON810 × NK603 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2015-127)

SCIENTIFIC OPINION ADOPTED: 25 November 2020 doi: 10.2903/j.efsa.2021.6348 Assessment of genetically modified maize 1507 3MIR162 3MON810 3NK603 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2015-127) EFSA Panel on Genetically Modified Organisms (GMO), Hanspeter Naegeli, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann, Fabio Veronesi, Fernando  Alvarez, Michele Ardizzone, Giacomo De Sanctis, Yann Devos, Antonio Fernandez, Andrea Gennaro, Jose  Angel G omez Ruiz, Anna Lanzoni, Franco Maria Neri, Nikoletta Papadopoulou, Konstantinos Paraskevopoulos and Tommaso Raffaello Abstract Maize 1507 9MIR162 9MON810 9NK603 (four-event stack maize) was produced by conventional crossing to combine four single events: 1507, MIR162, MON810 and NK603. The GMO Panel previously assessed the four single events and six of the subcombinations and did not identify safety concerns. No new data on the single events or the six subcombinations that could lead to modification of the original conclusions on their safety were identified. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the four-event stack maize, as described in this application, is as safe as its non-GM comparator and the non-GM reference varieties tested. In the case of accidental release of viable seeds of the four-event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in the four maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the four-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the four-event stack maize. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the four-event stack maize and its subcombinations are as safe as the non-GM comparator and the tested non-GM reference varieties with respect to potential effects on human and animal health and the environment. ©2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: GMO, Zea mays, herbicide-tolerant, insect-resistant, stack events Requestor: European Commission Question number: EFSA-Q-2015-00841 Correspondence: [email protected] EFSA Journal 2021;19(1):6348www.efsa.europa.eu/efsajournal Panel members: Hanspeter Naegeli, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Acknowledgments: The Panel wishes to thank the members of the Working Groups on Molecular Characterisation, Food and Feed Safety Assessment and Working Group on Comparative Analysis and Environmental Risk Assessment for the preparatory work on this scientific output and EFSA staff members Irene Mu~ noz Guajardo, Irina Vlas, Lorenz Oberkofler and Sylvie Mestdagh for the support provided to this scientific output. Suggested citation: EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Bresson J-L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogu e F, Rostoks N, S anchez Serrano JJ, Savoini G, Veromann E, Veronesi F,  Alvarez F, Ardizzone M, De Sanctis G, Devos Y, Fernandez A, Gennaro A, G omez Ruiz J  A, Lanzoni A, Neri FM, Papadopoulou N, Paraskevopoulos K and Raffaello T, 2021. Scientific Opinion on the assessment of genetically modified maize 1507 9MIR162 9MON810 9NK603 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2015-127). EFSA Journal 2021;19(1):6348, 40 pp. https://doi.org/10.2903/j.efsa.2021.6348 ISSN: 1831-4732 ©2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 2 EFSA Journal 2021;19(1):6348 Summary Following the submission of application EFSA-GMO-NL-2015-127 under Regulation (EC) No 1829/2003 from Pioneer (hereafter referred to as ‘the applicant’), the Panel on Genetically Modified Organisms of the European Food Safety Authority (hereafter referred to as the ‘GMO Panel’) was asked to deliver a scientific opinion on genetically modified (GM) maize 1507 9MIR162 9MON810 9NK603 (referred to hereafter to as ‘the four-event stack maize’) and its subcombinations independently of their origin, according to the Commission Regulation (EU) No 503/2013 (referred to hereafter as ‘subcombinations’). The scope of application EFSA-GMO-NL-2015-127 is for import, processing, and food and feed uses within the European Union (EU) of maize 1507 9MIR162 9MON810 9NK603 and all its subcombinations independently of their origin, and does not include cultivation in the EU. The term ‘subcombination’refers to any combination of up to four of the events present in the fourevent stack maize. The safety of subcombinations occurring as segregating progeny in the harvested grains of maize 1507 9MIR162 9MON810 9NK603 is evaluated in the context of the assessment of the four-event stack maize. The safety of subcombinations that have either been or could be produced by conventional crossing through targeted breeding approaches, and which can be bred, produced and marketed independently of the four-event stack, are risk assessed separately in the present scientific opinion. The four-event stack maize was produced by conventional crossing to combine four single maize events: 1507 (expressing the Cry1F and PAT proteins), MON810 (expressing the Cry1Ab protein), MIR162 (expressing the Vip3Aa20 and PMI proteins) and NK603 (expressing the CP4 EPSPS and CP4 EPSPS L214P proteins) to confer resistance to certain lepidopteran pests and tolerance to glyphosateand glufosinate ammonium-based herbicides. The GMO Panel evaluated the four-event stack maize and its subcombinations with reference to the scope and appropriate principles described in its guidelines for the risk assessment of GM plants and derived food and feed, the environmental risk assessment of GM plants and the post-market environmental monitoring (PMEM) of GM plants. The GMO Panel considered the information submitted in application EFSA-GMO-NL-2016-127, additional information provided by the applicant during the risk assessment, the scientific comments submitted by the Member States (MS) and the relevant scientific literature. The previous assessments of the single events 1507, MON810, MIR162 and NK603 and six of the subcombinations provided a basis for the assessment of the four-event stack maize and the remaining four subcombinations. No safety concerns were identified by the GMO Panel in the previous assessments. No safety issue concerning the four single maize events was identified by the updated bioinformatic analyses, nor reported by the applicant since the publication of the previous GMO Panel scientific opinions. Therefore, the GMO Panel considers that its previous conclusions on the safety of the single maize events remain valid. For the four-event stack maize, the risk assessment included the molecular characterisation of the inserted DNA and analysis of protein expression. An evaluation of the comparative analysis of agronomic, phenotypic and compositional characteristics was undertaken, and the safety of the newly expressed proteins and the whole food and feed were evaluated with respect to potential toxicity, allergenicity and nutritional characteristics. An evaluation of environmental impacts and the postmarket environmental monitoring (PMEM) plan was also undertaken. The molecular data establish that the events stacked in maize 1507 9MIR162 9 MON810 9NK603 have retained their integrity. Protein expression analyses showed that the levels of the newly expressed proteins are similar in the four-event stack maize and in the single events. No indications of interactions that may affect the integrity of the events and the levels of the newly expressed proteins in this four-event stack maize were identified. The comparative analysis of forage and grain composition and agronomic and phenotypic characteristics identified no differences between maize 1507 9MIR162 9MON810 9NK603 and the non-GM comparator that required further assessment for food/feed safety or environmental impact. The molecular characterisation, the comparative analysis and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that maize 1507 9MIR162 9MON810 9NK603, as described in this application, is as safe as and nutritionally equivalent to its non-GM comparator and the non-GM reference varieties tested. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 3 EFSA Journal 2021;19(1):6348 Considering the combined events and their potential interactions, the outcome of the comparative analysis, and the routes and levels of exposure, the GMO Panel concludes that maize 1507 9 MIR162 9MON810 9NK603 would not raise safety concerns in the case of accidental release of viable GM maize grains into the environment. Since no new safety concerns were identified for the six previously assessed subcombinations, and no new data leading to the modification of the original conclusions on safety were identified, the GMO Panel considers that its previous conclusions on these maize subcombinations remain valid. For the remaining four subcombinations included in the scope of application EFSA-GMO-NL-2015-127, no experimental data were provided. The GMO Panel assessed the possibility of interactions among the events in the four subcombinations and concludes that these subcombinations would not raise safety concerns. These subcombinations are therefore expected to be as safe as and nutritionally equivalent to the single events, the previously assessed subcombinations and the four-event stack maize. Based on the relevant publications identified through the literature searches, the GMO Panel does not identify any safety issue pertaining to the intended uses of maize 1507 9MIR162 9MON810 9NK603 and its subcombinations. In the context of annual PMEM reports, the applicant could further fine-tune future literature searches according to the GMO Panel recommendations given in this scientific opinion. Given the absence of safety concerns for foods and feeds from maize 1507 9MIR162 9 MON810 9NK603 and its subcombinations, the GMO Panel considers that post-market monitoring of these products is not necessary. The PMEM plan and reporting intervals are in line with the intended uses of the four-event stack maize and its subcombinations. The GMO Panel concludes that maize 1507 9MIR162 9MON810 9NK603 and its subcombinations, as described in this application, are as safe as the non-GM comparator and the tested non-GM reference varieties with respect to potential effects on human and animal health and the environment. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 4 EFSA Journal 2021;19(1):6348 Table of contents Abstract................................................................................................................................................... 1 Summary................................................................................................................................................. 3 1. Introduction................................................................................................................................7 1.1. Background ................................................................................................................................7 1.2. Terms of Reference as provided by the requestor .......................................................................... 7 2. Data and methodologies .............................................................................................................. 8 2.1. Data........................................................................................................................................... 8 2.2. Methodologies............................................................................................................................. 8 3. Assessment................................................................................................................................. 8 3.1. Introduction................................................................................................................................8 3.2. Updated information on single events ........................................................................................... 9 3.3. Systematic literature review ......................................................................................................... 10 3.4. Risk assessment of the four-event stack maize 1507 9MON 810 9MIR162 9NK603..................... 10 3.4.1. Molecular characterisation............................................................................................................ 10 3.4.1.1. Genetic elements and their biological function ............................................................................... 11 3.4.1.2. Integrity of the events in four-event stack..................................................................................... 12 3.4.1.3. Information on the expression of the inserts ................................................................................. 12 3.4.1.4. Conclusion of the molecular characterisation ................................................................................. 12 3.4.2. Comparative analysis ................................................................................................................... 13 3.4.2.1. Overview of studies conducted for the comparative analysis ........................................................... 13 3.4.2.2. Experimental field trial design and statistical analysis ..................................................................... 13 3.4.2.3. Suitability of selected test materials .............................................................................................. 14 3.4.2.4. Representativeness of the receiving environments ......................................................................... 15 3.4.2.5. Agronomic and phenotypic analysis .............................................................................................. 15 3.4.2.6. Compositional analysis................................................................................................................. 16 3.4.2.7. Conclusion on comparative analysis .............................................................................................. 17 3.4.3. Food/Feed safety assessment....................................................................................................... 17 3.4.3.1. Effects of processing ................................................................................................................... 17 3.4.3.2. Influence of Temperature and pH on newly expressed proteins....................................................... 17 3.4.3.3. Toxicology .................................................................................................................................. 17 3.4.3.4. Allergenicity ................................................................................................................................19 3.4.3.5. Dietary exposure assessment to new constituents.......................................................................... 20 3.4.3.6. Nutritional assessment of endogenous constituents........................................................................ 22 3.4.3.7. Conclusion of the food and feed safety assessment ....................................................................... 22 3.4.4. Environmental risk assessment ..................................................................................................... 22 3.4.4.1. Persistence and invasiveness of the GM plant ................................................................................ 22 3.4.4.2. Potential for gene transfer ........................................................................................................... 23 3.4.4.3. Interactions of the GM plant with target organisms........................................................................ 24 3.4.4.4. Interactions of the GM plant with non-target organisms ................................................................. 24 3.4.4.5. Interactions with abiotic environment and biogeochemical cycles .................................................... 24 3.4.4.6. Conclusion of the environmental risk assessment........................................................................... 24 3.4.5. Conclusion on the four-event stack maize 1507 9MON810 9MIR162 9NK603 ............................. 24 3.5. Risk assessment of the subcombinations ....................................................................................... 25 3.5.1. Subcombinations previously assessed ........................................................................................... 25 3.5.2. Subcombinations not previously assessed...................................................................................... 25 3.5.2.1. Stability of the events.................................................................................................................. 25 3.5.2.2. Expression of the events.............................................................................................................. 25 3.5.2.3. Potential functional interactions among the events......................................................................... 26 3.5.3. Conclusion .................................................................................................................................. 26 3.6. Post-market monitoring................................................................................................................ 26 3.6.1. Post-market monitoring of GM food/feed....................................................................................... 26 3.6.2. Post-market environmental monitoring.......................................................................................... 26 3.6.3. Conclusion on post-market monitoring .......................................................................................... 27 4. Overall conclusions ...................................................................................................................... 27 5. Documentation as provided to EFSA ............................................................................................. 28 References............................................................................................................................................... 28 Abbreviations ........................................................................................................................................... 33 Appendix A –List of additional studies ....................................................................................................... 34 Appendix B –Protein expression data ........................................................................................................ 36 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 5 EFSA Journal 2021;19(1):6348 Appendix C –List of relevant publications................................................................................................... 38 Appendix D –Statistically significant findings in the 90-day toxicity study in rats on the whole food/feed from maize 1507.............................................................................................................................................. 39 Appendix E –Animal dietary exposure ....................................................................................................... 40 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 6 EFSA Journal 2021;19(1):6348 1. Introduction The scope of application EFSA-GMO-NL-2015-127 is for food and feed uses, import and processing in the European Union (EU) of the genetically modified (GM) herbicide-tolerant and insect-resistant maize 1507 9MIR162 9MON810 9NK603 and all its subcombinations independently of their origin and does not include cultivation in EU. 1.1. Background On 18 December 2015, the European Food Safety Authority (EFSA) received from the Competent Authority of The Netherlands application EFSA-GMO-NL-2015-127 for authorisation of maize 1507 9MIR162 9MON810 9NK603 (Unique Identifier DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYNIR162-4 9MON-ØØ6Ø3-6), submitted by Pioneer Hi-Bred International, Inc. (hereafter referred to as ‘the applicant’) according to Regulation (EC) No 1829/2003 1 . Following receipt of application EFSA-GMO-NL-2015-127, EFSA informed EU Member States and the European Commission and made the summary of the application available to the public on the EFSA website. 2 EFSA checked the application for compliance with the relevant requirements of Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013 3 and, when needed, asked the applicant to supplement the initial application. On 9 February 2016, EFSA declared the application valid and made the application available to the Member States and the European Commission. From validity date, EFSA and its scientific Panel on Genetically Modified Organisms (hereafter referred to as ‘the GMO Panel’) endeavoured to respect a time limit of 6 months to issue a scientific opinion on application EFSA-GMO-NL-2015-127. Such time limit was extended whenever EFSA and/or its GMO Panel requested supplementary information to the applicant. According to Regulation (EC) No 1829/2003, any supplementary information provided by the applicant during the risk assessment was made available to the EU Member States and European Commission (for further details, see the section ‘Documentation’, below). In accordance with Regulation (EC) No 1829/2003, EFSA consulted the nominated risk assessment bodies of EU Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC 4 . The EU Member States had three months to make their opinion known on application EFSA-GMO-NL-2015-127 as of date of validity. 1.2. Terms of Reference as provided by the requestor According to Articles 6 and 18 of Regulation (EC) No 1829/2003, EFSA and its GMO Panel were requested to carry out a scientific risk assessment of maize 1507 9MIR162 9MON810 9NK603 and all its subcombinations independently of their origin according to the of application EFSA-GMO-NL2015-127. According to Regulation (EC) No 1829/2003, this scientific opinion is to be seen as the report requested under Articles 6(6) and 18(6) of that Regulation including the opinions of the nominated risk assessment bodies of EU Member States. 5 In addition to the present scientific opinion, EFSA and its GMO Panel were also asked to report on the particulars listed under Articles 6(5) and 18(5) of Regulation (EC) No 1829/2003. The relevant information is made available in the EFSA Register of Questions, 6 including the information required under Annex II to the Cartagena Protocol; a labelling proposal; a post-market environmental monitoring (PMEM) plan as provided by the applicant; the methods, validated by the Community reference laboratory, for detection, including sampling, identification of the transformation events in the food-feed and/or foods-feeds produced from it and the appropriate reference materials. 1 Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 18.10.2003, p. 1–23. 2 Available online: http://registerofquestions.efsa.europa.eu/roqFrontend/questionDocumentsLoader?question=EFSA-Q-2015-00841 3 Commission Implementing Regulation (EU) No 503/2013 of 3 April 2013 on applications for authorization of genetically modified food and feed in accordance with Regulation (EC) No 1829/2003 of the European Parliament and of the Council and amending Commission Regulations (EC) No 641/2004 and (EC) No 1981/2006. OJ L157, 8.6.2013, p. 1–48. 4 Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modifed organisms and repealing Council Directive 90/220/EEC. OJ L 106, 12.3.2001, p. 1–38. 5 Opinions of the nominated risk assessment bodies of EU Member States can be found at the EFSA Register of Questions (http://registerofquestions.efsa.europa.eu/roqFrontend/login), querying the assigned Question Number. 6 http://registerofquestions.efsa.europa.eu/roqFrontend/questionDocumentsLoader?question=EFSA-Q-2015-00841 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 7 EFSA Journal 2021;19(1):6348 2. Data and methodologies 2.1. Data The GMO Panel based its scientific risk assessment of maize 1507 9MIR162 9MON810 9NK603 on the valid application EFSA-GMO-NL-2015-127, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU Member States and relevant peer-reviewed scientific publications. As part of this comprehensive information package, the GMO Panel received additional unpublished studies submitted by the applicant in order to comply with the specific provisions of Regulation (EU) No 503/2013. A list of these additional unpublished studies is provided in Appendix A. 2.2. Methodologies The GMO Panel conducted its assessment in line with the principles described in Regulation (EU) No 503/2013, its applicable guidelines (i.e. EFSA GMO Panel, 2010a, 2011a,b, 2015, 2017a) and explanatory notes and statements (i.e. EFSA GMO Panel, 2010b; EFSA, 2014, 2017a,b, 2019) for the risk assessment of GM plants. For the assessment of 90-day animal feeding studies, the GMO Panel took into account the criteria included in the EFSA guidance (EFSA Scientific Committee, 2011) and the explanatory statement for its applicability (EFSA, 2014). The GMO Panel also assessed the applicant’s literature searches, which include a scoping review, in accordance with the recommendations on literature searching outlined in EFSA (2010, 2017a). In the frame of the contracts OC/EFSA/GMO/2013/01, OC/EFSA/GMO/2014/01 and OC/EFSA/GMO/ 2018/02 contractors performed preparatory work and delivered reports on the methods applied by the applicant in performing bioinformatic, statistical and toxicological analyses, respectively. 3. Assessment 3.1. Introduction Application EFSA-GMO-NL-2015-127 covers the four-event stack maize 1507 9MON810 9MIR162 9NK603 and all its 10 subcombinations independently of their origin (Table 1). The term ‘subcombination’refers to any combination of up to three of the maize events 1507, MON810, MIR162 and NK603. The safety of subcombinations occurring as segregating progeny in harvested grains of maize 1507 9MON810 9MIR162 9NK603 is evaluated in the context of the assessment of the four-event stack maize in Section 3.5 of the present scientific opinion. ‘Subcombination’also covers combinations that have either been or could be produced by conventional crossing through targeted breeding approaches (EFSA GMO Panel, 2011a). These are Table 1: Eleven combinations of the events covered by the scope of the application EFSA-GMO-NL2015-127 Degree of stacking Events Four-event stack MON810 91507 9NK603 9MIR162 Three-event stack 1507 9NK603 9MIR162 MON810 9NK603 9MIR162 MON810 91507 9MIR162 MON810 91507 9NK603 Two-event stack NK603 9MIR162 1507 9MIR162 1507 9NK603 MON810 9MIR162 MON810 9NK603 MON810 91507 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 8 EFSA Journal 2021;19(1):6348 maize stacks that can be bred, produced and marketed independently of the four-event stack maize. These subcombinations are assessed in Section 3.5 of this scientific opinion. The four-event stack maize was produced by conventional crossing to combine four single maize events: 1507 (expressing the Cry1F and PAT proteins), MON810 (expressing the Cry1Ab protein), MIR162 (expressing the Vip3Aa20 and PMI proteins) and NK603 (expressing the CP4 EPSPS and CP4 EPSPS L214P proteins) to confer resistance to certain lepidopteran pests and tolerance to glyphosateand glufosinate ammonium-based herbicides. It should be noted that the assessment of herbicide residues in maize herbicide-tolerant crops relevant for this application has been investigated by the EFSA Pesticides Unit (EFSA, 2018a). All four single maize events, the two-event stacks MON810 91507, MON810 9NK603, 1507 9NK603, 1507 9MIR162 and NK603 9MIR162, and the three-event stack MON810 91507 9NK603 have been previously assessed by the GMO Panel (see Table 2) and no safety concerns were identified. 3.2. Updated information on single events 7 Since the publication of the scientific opinions on the single maize events by the GMO Panel (see Table 2), no safety issue concerning the four single events has been reported by the applicant. The applicant clarified that the maize 1507 sequence reported in this application is identical to the corrected maize 1507 sequence. The evaluation of the corrected sequencing data and the Table 2: Single maize events and subcombinations of maize 1507 9MON 810 9MIR162 9NK603 previously assessed by the GMO Panel Events Application or mandate Reference MON810 EFSA-GMO-RX-MON810 EFSA (2009a) 1507 EFSA-Q-2004-011 EFSA (2004a) EFSA-GMO-NL-2004-02 EFSA (2005a) EFSA-Q-2006-00330 EFSA (2005b) EFSA-GMO-RX-1507 EFSA (2009b) EFSA-GMO-RX-001 EFSA GMO Panel (2017b) NK603 Art4_NK603 EFSA (2004b) CE/ES/00/01 EFSA (2007) EFSA-GMO-NL-2005-22 EFSA (2009c) EFSA-GMO-RX-NK603 EFSA (2009c) MIR162 EFSA-GMO-DE-2010-82 EFSA GMO Panel (2012) MON810 91507 EFSA-GMO-NL-2011-92 EFSA GMO Panel (2017c) MON810 9NK603 EFSA-GMO-UK-2004-01 EFSA (2005c) C/GB/02/M3/3 EFSA (2005d) EFSA-GMO-NL-2011-92 EFSA GMO Panel (2017c) EFSA-GMO-RX-007 EFSA GMO Panel (2018a) 1507 9NK603 EFSA-GMO-UK-2004-05 EFSA (2006) EFSA-GMO-NL-2009-65 EFSA GMO Panel (2010c) M-2011-0066 EFSA GMO Panel (2011c) EFSA-GMO-NL-2011-92 EFSA GMO Panel (2017c) EFSA-GMO-NL-2013-112 EFSA GMO Panel (2019a) EFSA-GMO-RX-008 EFSA GMO Panel (2018b) 1507 9MIR162 EFSA-GMO-DE-2010-86 EFSA GMO Panel (2018c) EFSA-GMO-DE-2011-103 EFSA GMO Panel (2019b) NK603 9MIR162 EFSA-GMO-NL-2016-131 EFSA GMO Panel (2019c) EFSA-GMO-NL-2016-134 EFSA GMO Panel (2019d) MON810 91507 9NK603 EFSA-GMO-NL-2011-92 EFSA GMO Panel (2017c) 7 Dossier: Part II - Section 1.2.2.2.v and vi; additional information: 8/3/2017 and 16/4/2020; spontaneous information: 17/11/2017, 17/12/2019 and 18/3/2020. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 9 EFSA Journal 2021;19(1):6348 seed germination (see section 3.4.2.3). Whether the differences can lead to an environmental adverse effect is considered in Section 3.4.4. 3.4.2.6. Compositional analysis Maize forage and grains harvested from the field trials in the USA and Canada in 2012 were analysed for 84 different constituents (nine in forage and 75 in grains), including the key constituents recommended by the Organisation for Economic Co-operation and Development (OECD) (OECD, 2002). Thirteen grain constituents having more than 50% of the observations below the limit of quantification were excluded from the statistical analysis. 17 The test of difference and the test of equivalence could be applied to the remaining 71 constituents (nine in forage 18 and 62 in grains 19 ), with the following results (Table 6): •For maize 1507 9MON810 9MIR162 9NK603 (not treated), the test of difference identified statistically significant differences from the non-GM comparator for 40 constituents (two in forage and 38 in grains). The test of equivalence between maize 1507 9MON810 9 MIR162 9NK603 and the non-GM maize reference varieties indicated that all 40 constituents fell under equivalence category I or II. •For maize 1507 9MON810 9MIR162 9NK603 (treated), statistically significant differences were also identified for 40 constituents (3 in forage and 37 in grains). All 40 constituents fell under equivalence category I or II. Table 6: Outcome of the comparative compositional analysis in grains and forage for maize 1507 9MON810 9MIR162 9NK603. The table shows the number of endpoints in each category Test of difference (a) Not treated (c) Treated (c) Not different Significantly different Not different Significantly different Test of equivalence (b) Category I/II 31 40 (d) 31 40 (d) Category III/IV –––– Total endpoints 71 71 (a): Comparison between maize 1507 9MON810 9MIR162 9NK603 and the non-GM comparator. (b): Four different outcomes: category I (indicating full equivalence to the non-GM reference varieties); category II (equivalence is more likely than non-equivalence); category III (non-equivalence is more likely than equivalence); and category IV (indicating non-equivalence). (c): Not treated/treated with the intended herbicides glyphosate and glufosinate ammonium (Section 3.4.2.2). (d): Endpoints with significant differences between maize 1507 9MON810 9MIR162 9NK603 and the non-GM comparator falling under equivalence category I-II (treated and not treated). In grain, both treated and not treated: alanine, aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, serine, threonine, valine, phytic acid, palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linolenic acid (C18:3), eicosenoic acid (C20:1), copper, iron, phosphorus, potassium, ADF, ash, carbohydrates, crude fat, crude fibre, crude protein, moisture, NDF, ferulic acid, pcoumaric acid, pyridoxine, c-tocopherol; only treated: glycine, histidine, proline, tyrosine, b-carotene; only not treated: lysine, trypsin inhibitor, lignoceric acid (C24:0), magnesium, total tocopherols, thiamine. In forage, both treated and not treated: neutral detergent fibre (NDF); only treated: crude fat, crude fibre; only not treated: calcium. 17 These were: caprylic acid (C8:0), lauric acid (C12:0), myristic acid (C14:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), heptadecadienoic acid (C17:2), (9,15) isomer of linoleic acid (C18:2), nonadecanoic acid (C19:0), eicosadienoic acid (C20:2), heneicosanoic acid (C21:0), behenic acid (C22:0), tricosanoic acid (C23:0). 18 Crude protein, crude fat, crude fibre, acid detergent fibre (ADF), neutral detergent fibre (NDF), ash, carbohydrates, calcium, phosphorus. 19 Moisture, crude protein, crude fat, crude fibre, acid detergent fibre (ADF), neutral detergent fibre (NDF), ash, carbohydrates, alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, calcium, manganese, phosphorus, iron, magnesium, copper, potassium, sodium, zinc, caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), myristoleic acid (C14:1), pentadecanoic acid (C15:0), pentadecenoic acid (C15:1), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), heptadecadienoic acid (C17:2), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), (9,15) isomer of linoleic acid (C18:2), linolenic acid (C18:3), c-linolenic acid (C18:3), nonadecanoic acid (C19:0), arachidic acid (C20:0), eicosenoic acid (C20:1), eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3), arachidonic acid (C20:4), heneicosanoic acid (C21:0), behenic acid (C22:0), erucic acid (C22:1), tricosanoic acid (C23:0), lignoceric acid (C24:0), b-carotene, thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, folic acid, a-tocopherol, b-tocopherol, c-tocopherol, d-tocopherol, p-coumaric acid, inositol, ferulic acid, furfural, phytic acid, raffinose, trypsin inhibitor. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 16 EFSA Journal 2021;19(1):6348 The GMO Panel assessed all the significant differences between maize 1507 9MON810 9 MIR162 9NK603 and its non-GM comparator, taking into account the potential impact on plant metabolism and the natural variability observed for the set of non-GM reference varieties. No endpoints showing significant differences between the four-event stack maize and the non-GM comparator and falling under category III/IV were identified. 3.4.2.7. Conclusion on comparative analysis Taking into account the natural variability observed for the set of non-GM reference varieties, the GMO Panel concludes that: •The differences identified in the agronomic and phenotypic characteristics tested between the four-event stack maize and the non-GM comparator do not need further assessment for environmental safety, with the exception of the changes noted in regard to early stand count and yield, which are considered in Section 3.4.4. •The differences identified in forage and grain composition between the four-event stack maize and the non-GM comparator do not need further assessment in regard to food and feed safety. 3.4.3. Food/Feed safety assessment 3.4.3.1. Effects of processing Processed products Maize 1507 9MON810 9MIR162 9NK603 will undergo existing production processes used for conventional maize. No novel production process is envisaged. Based on the outcome of the comparative assessment, processing of the four-event stack maize 1507 9MON810 9MIR162 9 NK603 into food and feed products is not expected to result in products being different from those of conventional non-GM maize varieties. 3.4.3.2. Influence of Temperature and pH on newly expressed proteins Effects of temperature and pH on the newly expressed proteins in this four-event stack maize have been previously evaluated by the GMO Panel (Table 2). No additional studies were provided in the context of this application. 3.4.3.3. Toxicology Testing of newly expressed proteins Six proteins (Cry1F, Cry1Ab, Vip3Aa20, PAT, PMI and CP4 EPSPS, including the variant CP4 EPSPS L214P) are newly expressed in the four-event stack maize 1507 9MON810 9MIR162 9NK603 (Section 3.4.1). The GMO Panel has previously assessed these proteins in the context of the single events (Table 2), and no safety concerns were identified for humans and animals. The GMO Panel is not aware of any new information that would change this conclusion. The potential for a functional interaction among the proteins newly expressed in maize 1507 9MON810 9MIR162 9NK603 has been assessed with regard to human and animal health. The three enzymatic proteins (CP4 EPSPS, PAT and PMI) catalyse distinct biochemical reactions, acting on unrelated substrates and are not expected to interact. The CP4 EPSPS proteins act on the shikimic acid pathway for the biosynthesis of aromatic amino acids in plants, showing high substrate specificity. PAT acts on the herbicide glufosinate and PMI (used as selectable marker) is involved in carbohydrate metabolism in plant, allowing maize cells to use mannose as a sole carbon source. The insecticidal proteins Cry1F and Cry1Ab are delta-endotoxins acting through cellular receptors found in target insect species. It is reported that the gastrointestinal tract of mammals, including humans, lacks receptors with high specificaffinity to Cry proteins (Hammond et al., 2013; Koch et al., 2015). The Vip3Aa20 protein is a protein secreted by B. thuringiensis during its vegetative phase acting in target insects via a mechanism similar to that of Cry proteins (Chakroun et al., 2016; Bel et al., 2017). On the basis of the known biological function of the individual newly expressed proteins (Table 4), there is currently no expectation for possible interactions relevant to the food and feed safety of the four-event stack maize 1507 9MON810 9MIR162 9NK603. In vitro protein degradation studies on Cry1F, Cry1Ab, Vip3Aa20, PAT, PMI and CP4 EPSPS (including its variant CP4 EPSPS L214P) proteins have been previously evaluated by the GMO Panel (Table 2). No new information has been provided in the context of this application. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 17 EFSA Journal 2021;19(1):6348 The GMO Panel concludes that there are no safety concerns to human and animal health related to the newly expressed proteins Cry1F, Cry1Ab, Vip3Aa20, PAT, PMI, CP4 EPSPS and its variant CP4 EPSPS L214P in the four-event stack maize 1507 9MON810 9MIR162 9NK603. Testing of new constituent other than proteins No new constituents other than newly expressed proteins have been identified in in grain and forage from the four-event stack maize 1507 9MON810 9MIR162 9NK603. Therefore, no further food/feed safety assessment of components other than the newly expressed proteins is required. Information on altered levels of food and feed constituent The four-event stack maize did not show any compositional differences to the non-GM comparator that would require further assessment (Section 3.4.2.6). Testing of the whole genetically modified food and feed Based on the outcome of the molecular characterisation, comparative analysis and toxicological assessment, no indication of findings relevant to food/feed safety related to the stability and expression of the inserts or to interaction between the transformation events, and no modifications of toxicological concern in the composition of maize 1507 9MON810 9MIR162 9NK603 have been identified (see Sections 3.4.1,3.4.2 and 3.4.3.3). Therefore, animal studies on food/feed derived from the four-stack are not necessary (EFSA GMO Panel, 2011a). In accordance to Regulation (EU) No 503/2013, the applicant provided a 90-day oral repeated-dose toxicity study in rats on whole food and feed from each of the maize single event composing the fourevent stack maize. 90-Day studies on maize MIR162 and NK603 The GMO Panel had previously concluded that these studies are in line with Regulation (EU) No 503/2013 and do not show adverse effects related to diets incorporating the single-event maize MIR162 and NK603 (EFSA GMO Panel, 2019c). 90-Day studies on maize MON810 A 90-day study on maize MON810 had been previously assessed by the GMO Panel in the context of the single-event renewal application dossier (EFSA, 2009a). Upon EFSA’s request to fulfil the requirements of Regulation (EU) No 503/2013, the applicant provided details of the histopathological findings of a number of organs and tissues, not originally examined according to OECD test guideline 408 (OECD, 1998). 20 Sporadic histopathological findings were seen in the additional tissues and organs examined both in male and female rats, compatible with the spontaneous background pathology of rats of this strain and age. A spontaneous mammary adenocarcinoma reported in a single female (F2019) fed diet containing grains (33%) from maize MON810 was concluded to be an incidental occurrence. The GMO Panel concludes that this study is in line with the legal requirements and confirms that there are no indications of adverse effects related to the 90-day administration to rats of diets including grains from maize MON810. The incorporation rate of maize selected in the study with maize MON810 is up to 33%, in line with commercially available rodent diets. It has been recently reported that a diet incorporating 50% maize may be tolerated without inducing nutritional imbalances in rats after 90-day administration (Steinberg et al., 2019), but the GMO Panel considers that further scientific confirmation is needed before this 50% maize incorporation rate is applicable in future studies. 90-Day study on maize 1507 21 The GMO Panel had previously assessed a 90-day study on maize 1507 in the context of the singleevent application (EFSA, 2005a). Kernels used in that study were obtained from 1507 maize plants that had not been treated with the intended herbicide (glufosinate-based herbicide). Upon EFSA’s request to fulfil the requirements of Regulation (EU) No 503/2013, the applicant provided a new 90day study on 1507 maize. Pair-housed Crl:CD(SD) rats (16/sex per group; 2 rats/cage) were allocated to six groups using a randomised complete block design with 5 replications/sex. Groups were fed diets containing 50% by weight grains either from maize 1507 plants treated with the intended herbicide 20 Additional information 31/10/2018. 21 Additional information 24/2/2020 and 13/7/2020. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 18 EFSA Journal 2021;19(1):6348 (test material, high dose), from the conventional counterpart (control material), or one of three nontransgenic commercial reference maize hybrids. 22 An additional group was fed diets containing 33% by weight grains from maize 1507 treated with the intended herbicide (test material, low dose) and 17% by weight maize grain from the conventional counterpart. The study was adapted from OECD test guideline 408 (OECD, 2018), aligned with EFSA Scientific Committee guidance (EFSA Scientific Committee, 2011) and complied with the principles of good laboratory practice (GLP) with some deviations not impacting the study results and interpretation (i.e. test item stability, homogeneity and concentration), which are detailed below. Event-specific PCR analysis confirmed the presence of the event 1507 in both the GM maize grains and diets and excluded the presence of the event in the respective controls. ELISA analyses also confirmed the presence of event DAS-1507 (i.e. Cry1F concentration) in the GM maize grains and GM diets. Both GM and control maize grains and diets were analysed for nutrients, antinutrients and potential contaminants (e.g. selected heavy metals, mycotoxins and pesticides). Balanced diets were formulated based on the specifications for PMI Certified Rodent LabDiet ® 5002. The stability of the test and control materials was not verified; however, in accordance to product expiration declared by the diet manufacturer, the constituents of the diets are considered stable for the duration of the treatment. The GMO Panel considered this justification acceptable. Diet preparation procedures and regular evaluations of the mixing methods guaranteed the homogeneity and the proper concentration of the test or control substances in them. The applicant provided information on concentration of Cry1F protein in the formulated test diets, further supporting the homogeneity of the formulations. Feed and water were provided ad libitum. Inlife procedures and observations and terminal procedures were conducted in accordance to OECD test guideline 408 (OECD, 2018). In the statistical analysis, rats consuming the lowand high-dose test diets were compared with those consuming the control diet. For continuous parameters, a linear mixed model was applied to data from individual animals for the two sexes combined (fixed effects: diet, sex and sex-by-diet interaction; random effects: block-within-sex and cage). Test-control comparisons were done both across sexes and separately for males and females; in case a significant sex-by-diet interaction was identified, only the sex-specific results were considered for the assessment. The model was modified as needed for the analysis of sex-specific endpoints and cage-level data (food consumption and food efficiency). There were no test diet-related incidents of mortality or clinical signs. All animals survived to scheduled euthanasia except one male from the reference group XL5840 that was found dead on test day 69 without any preceding clinical signs. Although a cause of death could not be conclusively determined, the incidental death of an untreated animal did not impact the interpretation of the study. No test diet-related adverse findings were identified in any of the investigated parameters. A small number of statistically significant findings were noted but these were not considered adverse effects of treatment for one or more of the following reasons: –were present at the low dose but not in the high-dose groups; –were within the normal variation for the parameter in rats of this age; –were of small magnitude; –were identified at only a small number of time intervals with no impact on the overall value; –exhibited no consistent pattern with related parameters or end-points. Detailed description of statistically significant findings identified in rats given diets containing maize DAS-1507 is reported in Appendix D. No gross pathology findings related to the administration of the test diets were observed at necropsy, and the microscopic examinations of a wide range of organs and tissues did not identify relevant differences in the incidence and severity of the histopathological findings related to the administration of the test diet compared to the control group. The GMO Panel concludes that this study is in line with the requirements of Regulation (EU) No 503/ 2013 and that no test diet-related adverse effects were observed in rats after feeding diets including maize 1507 up to 50% for 90 days. 3.4.3.4. Allergenicity For allergenicity assessment, a weight-of-evidence approach was followed, taking into account all of the information obtained on the newly expressed proteins, as no single piece of information or 22 P0760, P0589 and XL5840. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 19 EFSA Journal 2021;19(1):6348 experimental method yields sufficient evidence to predict allergenicity and adjuvanticity (Codex Alimentarius, 2009; EFSA GMO Panel, 2011a; Commission Regulation (EU) No 503/2013). Assessment of allergenicity of the newly expressed proteins For allergenicity, the GMO Panel has previously evaluated the safety of the Cry1F, Cry1Ab, Vip3Aa20, PAT, PMI and CP4 EPSPS (including the variant CP4 EPSPS L214P) proteins individually, and no evidence of allergenicity was identified in the context of the applications assessed (Table 2). No new information on allergenicity of the newly expressed proteins in this four-event stack maize that might change the previous conclusions of the EFSA GMO Panel has become available. Based on current knowledge, and as there is no evidence of allergenicity of the newly expressed proteins, there are no expected concerns of allergenicity as a consequence of their potential interaction in this fourevent stack maize. Furthermore, the GMO Panel has previously evaluated the safety of the newly expressed proteins, and no concerns on adjuvanticity in the context of the applications assessed were identified (Table 2). More recently, this aspect has been discussed in detail by EFSA (EFSA, 2018b; Parenti et al., 2019). To date there is no evidence for adjuvanticity in the GMOs assessed by the Panel. This four-event stack maize has comparable levels of the individual Bt proteins as those in the respective single maize events (see Section 3.4.1). The GMO Panel did not find indications that the Bt proteins at the levels expressed in this four-event stack maize might be adjuvants able to enhance an allergic reaction. Assessment of allergenicity of GM plant products The GMO Panel regularly reviews the available publications on food allergy to maize. However, maize is not considered a common allergenic food 23 (OECD, 2002). Therefore, the GMO Panel does not request experimental data to analyse the allergen repertoire of GM maize. In the context of this application and considering the data from the molecular characterisation, the compositional analysis and the assessment of the newly expressed proteins (Sections 3.4.1,3.4.2 and 3.4.3), the GMO Panel identified no indications of a potentially increased allergenicity of food and feed derived from this four-event stack maize with respect to that derived from its non-GM comparator. 3.4.3.5. Dietary exposure assessment to new constituents In line with Regulation (EU) No 503/2013 the applicant provided dietary exposure estimates to Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins newly expressed in 1507 9MON810 9 MIR162 9NK603 maize. Dietary exposure was estimated based on protein expression levels reported in this application for the four-event stack maize treated with the intended herbicides, the current available consumption data and feed practices, the foods and feeds currently available in the market and the described processing conditions. For the purpose of estimating dietary exposure, the levels of newly expressed proteins in 1507 9MON810 9MIR162 9NK603 maize grains, forage and pollen were derived from replicated field trials (four replicates from four locations) in the 2012 US growing season (see section 3.4.2). When for a particular newly expressed protein all the data were below the limit of quantification (LOQ), this LOQ was used as expression value to estimate dietary exposure. Table 7describes the protein expression levels used to estimate both human and animal dietary exposure. 23 Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 20 EFSA Journal 2021;19(1):6348 Human dietary exposure 24 As per request of the GMO Panel, chronic and acute dietary exposure to the newly expressed proteins in 1507 9MON810 9MIR162 9NK603 maize grains were provided. The applicant followed the methodology described by EFSA to estimate dietary exposure in high consumers using summary statistics (EFSA, 2015). Human dietary exposure was estimated across different European countries on different population groups: young population (infants, toddlers, ‘other children’), adolescents, adult population (adults, elderly and very elderly) and special populations (pregnant and lactating women). Since no specific consumption data were available on commodities containing, consisting of or obtained from 1507 9MON810 9MIR162 9NK603 maize grains, a conservative scenario with 100% replacement of conventional maize by the GM maize was considered. Consumption figures for all relevant commodities (e.g. corn flakes, sweet corn, popcorn, etc.) were retrieved from the EFSA Comprehensive European Food Consumption Database (EFSA consumption database). 25 Corn oil was excluded from the assessment since no proteins are expected to be present in the oil. Mean protein expression values on fresh weight basis are considered as the most adequate to estimate human dietary exposure (EFSA, 2019). However, dietary exposure was provided using expression values on dry weight basis; this results in more conservative exposure estimations which is considered acceptable. Since Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins were not analysed in processed foods, the concentration of the newly expressed proteins in these commodities was estimated using the ratio between the total protein content in processed foods and in maize grains. 26 This is a conservative approach as neither recipes nor the effect of processing is considered on the final concentration of newly expressed proteins, except for corn oil which is eventually excluded from the exposure estimations. The highest acute dietary exposure was estimated in the age class ‘Toddlers’with exposure estimates of 48.1 lg/kg body weight (bw) per day, 1.0 lg/kg bw per day, 2.7 lg/kg bw per day, 737 lg/kg bw per day, 24.1 lg/kg bw per day and 180 lg/kg bw per day for Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS, respectively. The main average contributor to the exposure in the dietary survey with the highest estimates was corn chips. The highest chronic dietary exposure was estimated in the age class ‘Infants’with exposure estimates of 10.8 lg/kg bw per day, 0.2 lg/kg bw per day, 0.6 lg/kg bw per day, 165 lg/kg bw per day, 5.4 lg/kg bw per day, and 40.5 lg/kg bw per day for Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS, respectively. The main average contributor to the exposure in the dietary survey with the highest estimates was corn flour. Table 7: Mean values (n =16, lg/g dry weight and lg/g fresh weight) for newly expressed proteins in grains, forage and pollen from 1507 9MON810 9MIR162 9NK603 maize treated with the intended herbicides (a) Protein Tissue/developmental stage Grains/R6 (lg/g dry weight) Pollen/R1 (lg/g fresh weight) (b) Forage/R4 (lg/g dry weight) Cry1F 3.2 29.1 14 PAT <LOQ (c) <LOQ (c) 1.1 Cry1Ab 0.18 <LOQ (c) 7.1 Vip3Aa20 49 132 250 PMI 1.6 5.2 5.3 CP4 EPSPS 12 489 140 LOQ: limit of quantification. (a): Intended herbicide: glufosinate and glyphosate. (b): Concentrations values in pollen were adjusted to 6% moisture content before using them to estimate dietary exposure to the different newly expressed protein via the consumption of pollen supplements. (c): All samples were below the limit of quantification: for PAT protein in grain (LOQ =0.069 lg/g dry weight), for PAT protein in pollen (LOQ =0.26 lg/g fresh weight), for Cry1Ab protein in pollen (LOQ =0.16 lg/g fresh weight). 24 Additional information: 7/5/2018. 25 https://www.efsa.europa.eu/en/applications/gmo/tools. Data accessed April 2018. 26 The protein content of maize grain and maize foods was obtained from the EuroFIR Food Composition Databases. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 21 EFSA Journal 2021;19(1):6348 An ad hoc dietary exposure scenario was carried out for consumers of pollen supplements under the assumption that these supplements might be made of pollen from 1507 9MON810 9MIR162 9 NK603 maize. Consumption data on pollen supplements are available for few consumers across nine different European countries 27 ; the low number of consumers available adds uncertainty to the exposure estimations and prevents to estimate exposure for high consumers of pollen supplements. In average consumers of pollen supplements, the highest acute dietary exposure would range from 0.12 lg/kg bw per day for Cry1Ab to 362 lg/kg bw per day for CP4 EPSPS, in the elderly population. Similarly, the highest chronic dietary exposure in average consumers would range from 0.08 lg/kg bw per day for Cry1Ab to 242 lg/kg bw per day for CP4 EPSPS, also in the elderly population. Animal dietary exposure Dietary exposure to Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins in maize 1507 9MON810 9MIR162 9NK603 was estimated across different animal species as below described, assuming the consumption of maize products commonly entering the feed supply chain (i.e. maize grains and forage). A conservative scenario with 100% replacement of conventional maize products by the four-event stack maize products was considered. Mean levels (dry weight) of the newly expressed proteins in grains and forage from the four-event stack maize treated with the intended herbicide used for animal dietary exposure are listed in Table 7. The applicant estimated dietary exposure to Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins in livestock (i.e. poultry, swine, cattle and sheep), based on estimates for body weights, daily feed intakes and inclusion rates (percentage) of maize grains and forage in diets/rations (OECD, 2009). Estimated dietary exposure in livestock animals was calculated based on the consumption of maize grain and forage alone or in combination, as reported in Appendix E. 3.4.3.6. Nutritional assessment of endogenous constituents The intended traits of 1507 9MON810 9MIR162 9NK603 maize are herbicideand insect resistance, with no intention to alter nutritional parameters. Comparison of the composition of the four-event stack maize, with the non-GM comparator and non-GM reference varieties did not identify differences that would require further safety assessment. From these data, the GMO Panel concludes that 1507 9MON810 9MIR162 9NK603 maize is nutritionally equivalent to the non-GM comparator and the non-GM reference varieties used. 3.4.3.7. Conclusion of the food and feed safety assessment The newly expressed proteins Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins in 1507 9MON810 9MIR162 9NK603 maize do not raise safety concerns for human and animal health. Interactions among the newly expressed proteins Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins raising food and feed safety concerns (in terms of toxicology, allergenicity and adjuvanticity) are not expected. There is no evidence that the genetic modification might change the overall allergenicity of the four-event stack maize. Based on the outcome of the animal and human nutritional assessments, the consumption of 1507 9MON810 9MIR162 9NK603 maize does not represent any nutritional concern, in the context of the scope of this application. 3.4.4. Environmental risk assessment Considering the scope of application EFSA-GMO-NL-2015-127, which excludes cultivation, the environmental risk assessment (ERA) of maize 1507 9MON810 9MIR162 9NK603 mainly takes into account: 1) the exposure of microorganisms to recombinant DNA in the gastrointestinal tract of animals fed GM material and of microorganisms present in environments exposed to faecal material of these animals (manure and faeces); and 2) the accidental release into the environment of viable fourevents stack maize grains during transportation and/or processing (EFSA GMO Panel, 2010a). 3.4.4.1. Persistence and invasiveness of the GM plant Maize is highly domesticated, not winter hardy in colder regions of Europe, and generally unable to survive in the environment without appropriate management. Occasional feral GM maize plants may occur outside cultivation areas in the EU (e.g. Pascher, 2016), but survival is limited mainly by a combination of low competitiveness, absence of a dormancy phase and susceptibility to plant 27 https://www.efsa.europa.eu/en/food-consumption/comprehensive-database. Data accessed April 2020. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 22 EFSA Journal 2021;19(1):6348 pathogens, herbivores and cold climate conditions (OECD, 2003). Field observations indicate that maize grains may survive and overwinter in some EU regions, resulting in volunteers in subsequent crops (e.g. Gruber et al., 2008; Palaudelm as et al., 2009; Pascher, 2016). However, maize volunteers have been shown to grow weakly and flower asynchronously with the maize crop (Palaudelm as et al., 2009). Thus, the establishment and survival of feral and volunteer maize in the EU is currently limited and transient. It is unlikely that the intended traits of event 1507 9MON810 9MIR162 9NK603 will provide a selective advantage to maize plants, except when they are exposed to glyphosateand/or glufosinatecontaining herbicides or infested by insect pests that are susceptible to the Cry1F, Cry1Ab and/or Vip3Aa20 proteins. The GMO Panel considers that the fitness advantage provided by the intended traits, and that the observed agronomic and phenotypic differences observed on early stand count and yield (see Section 3.4.2.5) will not allow the GM plant to overcome other biological and abiotic factors (described above) limiting plant’s persistence and invasiveness. Therefore, the presence of the intended traits and other observed differences will not affect the persistence and invasiveness of the GM plant. In conclusion, the GMO Panel considers it unlikely that maize 1507 9MON810 9MIR162 9NK603 will differ from conventional maize hybrid varieties in its ability to survive until subsequent seasons, or to establish occasional feral plants under European environmental conditions in case of accidental release into the environment of viable maize 1507 9MON810 9MIR162 9NK603 grains. 3.4.4.2. Potential for gene transfer A prerequisite for any gene transfer is the availability of pathways for the transfer of genetic material, either through HGT of DNA, or through vertical gene flow via cross-pollination from feral plants originating from spilled grains. Plant-to-microorganism gene transfer The probability and potential adverse effects of HGT of the recombinant DNA have been assessed in previous GMO Panel Scientific Opinions for the single events (see Table 2). This assessment included consideration of homology-based recombination processes, as well as non-homologous end joining and microhomology-mediated end joining. Possible fitness advantages that the bacteria in the receiving environments would gain from acquiring recombinant DNA were considered. No concern as a result of an unlikely, but theoretically possible, HGT of the recombinant genes to bacteria in the gut of domesticated animals and humans fed GM material or other receiving environments was identified. The applicant submitted an updated bioinformatic analysis for each of the single events to assess the possibility for HGT by homologous recombination. The updated bioinformatics analyses of events 1507, MIR162 and NK603 do not reveal any new DNA sequence that could provide sufficient length and identity which could facilitate HGT by double homologous recombination, confirming the conclusions of previous Scientific Opinions (EFSA GMO Panel, 2019b,d,e). The updated bioinformatic analysis of MON810 confirmed the absence of sequences in the recombinant DNA which would provide sufficient sequence identity to facilitate HGT to bacteria. Synergistic effects of the recombinant genes, for instance due to combinations of recombinogenic sequences, which would cause an increase in the likelihood for HGT or a selective advantage were not identified. Therefore, the GMO Panel concludes that the unlikely, but theoretically possible, horizontal transfer of recombinant genes from this number-event stack maize to bacteria does not raise any environmental safety concern. Plant-to-plant gene transfer The potential for occasional feral maize 1507 9MON810 9MIR162 9NK603 plants originating from grain import spills to transfer recombinant DNA to sexually compatible plants and the environmental consequences of this transfer were considered. For plant-to-plant gene transfer to occur, imported GM maize grains need to germinate and develop into plants in areas containing sympatric wild relatives and/or cultivated maize with synchronous flowering and environmental conditions favouring cross-pollination. Maize is an annual predominantly cross-pollinating crop. Cross-fertilisation occurs mainly by wind (OECD, 2003). Vertical gene transfer from maize is limited to Zea species. Wild relatives of maize outside cultivation are not known/reported in Europe (Eastham and Sweet, 2002; EFSA, 2016; OECD, Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 23 EFSA Journal 2021;19(1):6348 2003; Trtikova et al., 2017). Therefore, potential vertical gene transfer is restricted to maize and weedy Zea species, such as teosintes, and/or maize-teosinte hybrids, occurring in cultivated areas (EFSA, 2016; Le Corre et al., 2020; Trtikova et al., 2017). The potential of spilled maize grains to establish, grow and produce pollen is extremely low and transient (see Section 3.4.4.1). Therefore, the likelihood/frequency of cross-pollination between occasional feral GM maize plants resulting from grain spillage, and weedy or cultivated Zea plants is considered extremely low (EFSA, 2016). Even if cross-pollination would occur, the GMO Panel is of the opinion that environmental effects as a consequence of the spread of genes from occasional feral GM maize plants in Europe will not differ from that of conventional maize varieties for the reasons given in Section 3.4.4.1 even if exposed to the intended herbicides. 3.4.4.3. Interactions of the GM plant with target organisms Taking the scope of the application EFSA-GMO-NL-2015-127 into account (no cultivation), potential interactions of occasional feral four-event stack maize plants arising from grain import spills with the target organisms are not considered a relevant issue by the GMO Panel. 3.4.4.4. Interactions of the GM plant with non-target organisms Given that environmental exposure of non-target organisms to spilled GM grains or occasional feral GM maize plants arising from spilled four-event stack maize grains is limited and because ingested proteins are degraded before entering the environment through faecal material of animals fed GM maize, potential interactions of the four-event stack maize with non-target organisms are not considered by the GMO Panel to raise any environmental safety concern. Interactions that may occur among the Cry and Vip proteins (as mentioned in Section 3.4.1.4) would not alter this conclusion. 3.4.4.5. Interactions with abiotic environment and biogeochemical cycles Given that environmental exposure to spilled grains or occasional feral four-event stack maize plants arising from grain import spills is limited, and because ingested proteins are degraded before entering the environment through faecal material of animals fed GM maize, potential interactions with the abiotic environment and biogeochemical cycles are not considered by the GMO Panel to raise any environmental safety concern. 3.4.4.6. Conclusion of the environmental risk assessment The GMO Panel concludes that it is unlikely that the four-event stack maize would differ from conventional maize varieties in its ability to persist under EU environmental conditions. Considering the scope of the application EFSA-GMO-NL-2015-127, interactions of occasional feral four-event stack maize plants with the biotic and abiotic environment are not considered to be relevant issues. The analysis of HGT from the four-event stack maize to bacteria does not indicate a safety concern. Therefore, considering the combined traits and their interactions, the outcome of the agronomic and phenotypic analysis, and the routes and levels of exposure, the GMO Panel concludes that the fourevent stack maize would not raise safety concerns in the event of accidental release of viable GM maize grains into the environment. 3.4.5. Conclusion on the four-event stack maize 1507 3MON810 3MIR162 3 NK603 No new data on the four single maize events 1507, MON810, MIR162 and NK603 that would lead to a modification of the original conclusions on their safety were identified. The combination of maize events 1507, MON810, MIR162 and NK603 in the four-event stack maize did not give rise to issues concerning the molecular, agronomic, phenotypic or compositional characteristics of the four-event stack maize that would be of concern for food and feed safety and nutrition. The newly expressed proteins in the four-event stack maize do not raise safety concerns for human and animal health and the environment in light of the scope of this application. No indications of interactions among the events based on the biological functions of the newly expressed proteins that would raise a safety issue were identified in maize 1507 9MON810 9MIR162 9NK603. Comparison of the levels of the newly expressed proteins between the four-event stack maize and those of the single maize events did not reveal an interaction at protein expression level. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 24 EFSA Journal 2021;19(1):6348 Considering the combined traits and their interactions, the outcome of the agronomic and phenotypic analysis, and routes and levels of exposure, the GMO Panel concludes that the four-event maize would not raise safety concerns in the event of accidental release of viable GM maize grains into the environment. No scientific information that could change the conclusions on this four-event stack maize was retrieved through systematic literature searches covering the 10 years before submission of the application and the period since the time of validity of the application. The GMO Panel concludes that maize 1507 9MON810 9MIR162 9NK603, as described in this application, is nutritionally equivalent to and as safe as the comparator and the non-GM reference varieties tested. 3.5. Risk assessment of the subcombinations 24 Subcombinations previously assessed in the frame of other applications are discussed in Section 3.5.1. The subcombinations that have not been previously assessed are discussed in Section 3.5.2. 3.5.1. Subcombinations previously assessed The GMO Panel has previously assessed six subcombinations and no safety concerns were identified: the two-event maize stacks MON810 91507, MON810 9NK603, 1507 9NK603, 1507 9MIR162 and NK603 9MIR162; the three-event stack maize MON810 91507 9NK603 and all its subcombinations (see Table 2). Literature searches covering the 10 years before submission of the application and the period since the time of validity of the application revealed no new scientific information relevant to the risk assessment of these maize stacks. 28 Consequently, the GMO Panel considers that its previous conclusions on these subcombinations remain valid. 3.5.2. Subcombinations not previously assessed Four of the 10 subcombinations included in the scope of this application have not been previously assessed by the GMO Panel (Table 8). In this case, following the strategy defined by the GMO Panel, 29 the risk assessment takes as its starting point the assessment of the single maize events, and uses the data generated for the four-event stack as well as all the additional data available on subcombinations previously assessed by the GMO Panel (Table 2) and the additional studies provided by the applicant (Appendix A). 3.5.2.1. Stability of the events The genetic stability of the inserted DNA over multiple generations in the four single maize events was demonstrated previously (see Table 2). Integrity of the events was demonstrated in the four-event stack maize 1507 9MON810 9MIR162 9NK603 (Section 3.4.1.2) and the previously assessed maize subcombinations (Table 2). The GMO Panel finds no reasons to expect the loss of integrity of the events in the maize subcombinations not previously assessed (see Table 8). 3.5.2.2. Expression of the events The GMO Panel assessed whether any combination of the four events by conventional crossing could result in significant changes in expression levels of the newly expressed proteins, as this could indicate an unexpected interaction among the events. Based on current knowledge of the molecular elements introduced, there is no reason to expect interactions that would affect the levels of the newly Table 8: Maize stacks not previously assessed and covered by the scope of application EFSA-GMONL-2015-127 Degree of stacking Events Three-event stack 1507 9NK603 9MIR162 MON 810 9NK603 9MIR162 MON 810 91507 9MIR162 Two-event stack MON 810 9MIR162 28 Dossier: Part II –Section 7; additional information: 17/11/2017, 8/1/2018 and 13/7/2020. 29 115th GMO Panel meeting (Annex 1 of the minutes: http://www.efsa.europa.eu/sites/default/files/event/170517-m.pdf). Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 25 EFSA Journal 2021;19(1):6348 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Bresson JL, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogu e F, Rostoks N, S anchez Serrano JJ, Savoini G, Veromann E, Veronesi F,  Alvarez F, Ardizzone M, De Sanctis G, Fern andez Dumont A, Gennaro A, G omez Ruiz JA, Lanzoni A, Papadopoulou N and Paraskevopoulos K, 2019d. Scientific Opinion on the assessment of genetically modified maize MON 87427 9MON 87460 9MON 89034 9MIR162 9NK603 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL2016-134). EFSA Journal 2019;17(8):5774, 36 pp. https://doi.org/10.2903/j.efsa.2019.5774 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Bresson J-L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogu e F, Rostoks N, Serrano S anchez JJ, Savoini G, Veromann E, Veronesi F,  Alvarez F, Ardizzone M, De Sanctis G, Dumont AF, Gennaro A, G omez Ruiz J  A, Lanzoni A, Neri FM, Papadopoulou N and Paraskevopoulos K, 2019e. Scientific Opinion on the assessment of genetically modified maize MON 87427 9MON 89034 9MIR162 9MON 87411 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2017-144). EFSA Journal 2019;17(11):5848, 33 pp. https://doi.org/10.2903/j.efsa.2019.5848 EFSA Scientific Committee, 2011. EFSA guidance on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed. EFSA Journal 2011;9(12):2438, 21 pp. https://doi.org/10.2903/j.efsa.2011.2438 Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA and Koziel MG, 1996. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proceedings of the National Academy of Sciences, 93, 5389–5394. Fang J, Xu X, Wang P, Zhao JZ, Shelton AM, Cheng J, Feng MG and Shen Z, 2007. Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Applied and Environmental Microbiology, 73, 956–961. Gruber S, Colbach N, Barbottin A and Pekrun C, 2008. Post-harvest gene escape and approaches for minimizing it. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, 3, 1–7. Hammond B, Kough J, Herouet-Guicheney C, Jez JM and behalf of the ILSI International Food Biotechnology Committee Task Force on the Use of Mammalian Toxicology Studies in the Safety Assessment of GM Foods, 2013. Toxicological evaluation of proteins introduced into food crops. Critical Reviews in Toxicology, 43(Suppl. 2), 25–42. Herrmann KM, 1995. The Shikimate Pathway: early steps in the biosynthesis of aromatic compounds. Plant Cell, 7, 907–919. Koch MS, Ward JM, Levine SL, Baum JA, Vicini JL and Hammond BG, 2015. The food and environmental safety of Bt crops. Frontiers in Plant Science, 6, 283. Le Corre V, Siol M, Vigouroux Y, Tenaillon MI and D ely C, 2020. Adaptive introgression from maize has facilitated the establishment of teosinte as a noxious weed in Europe. Proceedings of the National Academy of Sciences of the United States of America, 117, 25618–25627. Lecoq E, Holt K, Janssens J, Legris G, Pleysier A, Tinland B and Wandelt C, 2007. General surveillance: roles and responsibilities the industry view. Journal f€ ur Verbraucherschutz und Lebensmittelsicherheit-Journal of Consumer Protection and Food Safety, 2(S1), 25–28. Markovitz A, Sydiskis RJ and Lieberman MM, 1967. Genetic and biochemical studies on mannose-negative mutants that are deficient in phosphomannose isomerase in Escherichia coli K-12. Journal of Bacteriology, 94, 1492–1496. Negrotto D, Jolley M, Beer S, Wenck AR and Hansen G, 2000. The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports, 19, 798–803. OECD (Organisation for Economic Co-operation and Development), 1998. OECD Guideline for the testing of chemicals - Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD Publishing, Paris. OECD (Organisation for Economic Co-operation and Development), 2002. Consensus Document on compositional considerations for new varieties of maize (Zea mays): key food and feed nutrients, anti-nutrients and secondary plant metabolites. Series on the Safety of Novel Food and Feeds (ENV/JM/MONO(2002)25), 6, 1–42. OECD (Organisation for Economic Co-operation and Development), 2003. Consensus Document on the biology of Zea mays subsp. mays (Maize). Series on Harmonisation of Regulatory Oversight in Biotechnology (ENV/JM/ MONO(2003)11), 27, 1–49. OECD (Organisation for Economic Co-operation and Development), 2009. Guidance document on overview of residue chemistry studies (as revised in 2009). Series on Testing and Assessment number 64 and Series on Pesticides number 32, OECD Environment, Health and Safety Publications, Paris, ENV/JM/MONO, 31, 93 pp. OECD (Organisation for Economic Co-operation and Development), 2018. OECD Guideline for the testing of chemicals - Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD Publishing, Paris. Palaudelm as M, Pe~ nas G, Mel e E, Serra J, Salvia J, Pla M, Nadal A and Messeguer J, 2009. Effect of volunteers on maize gene flow. Transgenic Research, 18, 583–594. Parenti MD, Santoro A, Del Rio A and Franceschi C, 2019. Literature review in support of adjuvanticity/ immunogenicity assessment of proteins. EFSA supporting publication 2019;EN-1551, https://doi.org/10.2903/ sp.efsa.2019.en-1551 Pascher K, 2016. Spread of volunteer and feral maize plants in Central Europe: recent data from Austria. Environmental Sciences Europe, 28, 30. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 32 EFSA Journal 2021;19(1):6348 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH, 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, 62, 775–806. Steinberg P, van der Voet H, Goedhart PW, Kleter G, Kok EJ, Pla M, Nadal A, Zeljenkov aD,Al  a cov a R, Babincov aJ, Rollerov a E, Jad’ud’ov a S, Kebis A, Szabova E, Tulinsk aJ,L  ı  skov aA,Tak  acsov a M, Lehotsk a Miku sov aM, Krivo s ıkov aZ,Sp € ok A, Racovita M, de Vriend H, Alison R, Alison C, Baumg€ artner W, Beck-er K, Lempp C, Schmicke M, Schrenk D, P€ oting A, Schiemann J and Wilhelm R, 2019. Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats. Archives of Toxicology, 93, 1095. https://doi.org/10.1007/s00204-019-02400-1 Sys C, Van Ranst E, Debaveye J and Beernaert F, 1993. Land Evaluation. Part III: crop requirements. Agricultural Publication No. 7. Brussels, General Administration for Development Cooperation. 199 pp. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M and Botterman J, 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. The EMBO journal, 6, 2519–2523. Trtikova M, Lohn A, Binimelis R, Chapela I, Oehen B, Zemp N, Widmer A and Hilbeck A, 2017. Teosinte in Europe – searching for the origin of a novel weed. Scientific Reports, 71, 1560. Windels P, Alcalde E, Lecoq E, Legris G, Pleysier A, Tinland B and Wandelt C, 2008. General surveillance for import and processing: the EuropaBio approach. Journal of Consumer Protection and Food Safety, 3(S2), 14–16. Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E and P€uhler A, 1988. Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes T€ u494 and its expression in Nicotiana tabacum. Gene, 70, 25–37. Abbreviations ADF acid detergent fibre bw body weight CaMV cauliflower mosaic virus CRM comparative relative maturity CTP chloroplast transit peptide ELISA enzyme-linked immunosorbent assay EPSPS 5-enolpyruvylshikimate-3-phosphat synthase ERA environmental risk assessment GM genetically modified GMO genetically modified organism GMO Panel EFSA Panel on Genetically Modified Organisms HGT horizontal gene transfer HR homologous recombination hsp heat shock protein LOQ limit of quantification NDF neutral detergent fibre Nos nopaline synthase OECD Organisation for Economic Co-operation and Development ORF open reading frame PAT phosphinothricin-acetyl-transferase PCR polymerase chain reaction PMEM post-market environmental monitoring PMI phosphomannose isomerase ract rice actin TSH thyroid hormones US United States UTR untranslated region Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 33 EFSA Journal 2021;19(1):6348 Appendix A –List of additional studies List of additional studies performed by or on behalf of the applicant with regard to the evaluation of the safety of maize 1507 9MON810 9MIR162 9NK603 for humans, animal or the environment. Study identification Title PHI-2004-094 Evaluation of Cold Tolerance of 1507 9NK603 Maize Seedlings PHI-2005-026 Sample Generation of Hybrid Maize Lines Containing cry34Ab1, cry35Ab1, cry1F, pat, and cp4 epsps Genes: Greenhouse Location PHI-2005-042 Quantitative ELISA Characterization of Hybrid Maize Lines Containing cry34Ab1, cry35Ab1, cry1F, pat, and/or cp4 epsps Genes (Greenhouse) PHI-2008-077 Agronomic Characteristics and Nutrient Composition of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9MON-ØØ6Ø3-6: U.S. Test Sites PHI-2008-103 Molecular Characterization of DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9MON-ØØ6Ø3-6 Maize Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction PHI-2010-061 Agronomic Characteristics, Expressed Trait Protein Concentration, and Nutrient Composition of Maize Lines Containing Event DAS-Ø15Ø7-1, MON-ØØ81Ø-6, and the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6: US and Canada Test Sites PHI-2011-013 Expressed Trait Protein Concentration of a Maize Line Containing Events DAS-Ø15Ø7-1, MON-ØØ81Ø-6, SYN-IR162-4, MON-ØØ6Ø3-6 and the Combined Trait Product DAS-Ø15Ø719MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6: US Test Sites PHI-2011-014 Agronomic Characteristics and Nutrient Composition of a Maize Line Containing Events DASØ15Ø7-1, MON-ØØ81Ø-6, SYN-IR162-4, MON-ØØ6Ø3-6, and the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6: US Test Sites PHI-2011-094 Expressed Trait Protein Concentration of a Maize Line Containing Events DAS-Ø15Ø7-1, SYN-IR162-4, and the Combined Trait Product DAS-Ø15Ø7-1 9SYN-IR162-4: US Test Sites PHI-2011-118 Expressed Trait Protein Concentration of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6: Chile Test Sites PHI-2011-119 Agronomic Characteristics and Nutrient Composition of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6: Chile Test Sites PHI-2012-023/011 Expressed Trait Protein Concentration of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4: U.S. and Canada Test Sites PHI-2012-164/701 Southern Blot Analysis of Maize Hybrid XY696 Lines Containing Events DAS-Ø15Ø7-1, MONØØ81Ø-6, and MON-ØØ6Ø3-6 PHI-2012-185 Expressed Trait Protein Concentration in Leaf, Tassel, Silk, Pollen, Husk, and Grain Tissue of Three Hybrid Maize Lines XYG35H, XYG35YH, and XYG35YHR (Greenhouse) PHI-2012-186 Expressed Trait Protein Concentration in Leaf, Tassel, Silk, Pollen, Husk, and Grain Tissue of Two Hybrid Maize Lines XYA91H and XYA91YH (Greenhouse) PHI-2012-194 Agronomic Characteristics, Expressed Trait Protein Concentration, and Nutrient Composition of Maize Lines Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9 SYN-IR162-4 and DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6: Brazil Test Sites PHI-2012-287 Six Week Poultry Feeding Study with Grains from Maize Hybrid Containing the Combined Trait Product TC1507 9NK603 PHI-2013-019 Expressed Trait Protein Concentration of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4: United States Test Sites PHI-2013-020 Agronomic Characteristics and Nutrient Composition of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4: United States Test Sites PHI-2013-121 Genetic Stability and Equivalency of DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 Maize Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction PHI-2013-167 Rodent Diet Formulation Study Using Maize Grains Containing the Combined Trait Product TC1507 9NK603, TC1507 9MON810 9NK603 and Non-transgenic Maize Grains Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 34 EFSA Journal 2021;19(1):6348 Study identification Title PHI-2014-162 Thirteen week rat feeding study with maize grain containing the combined trait products TC1507 9NK603 and TC1507 9MON810 9NK603 (guidelines Ministry of Science and Technology Government of India, 2008) PHI-2013-175 Agronomic Characteristics, Expressed Trait Protein Concentration, and Nutrient Composition of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9SYN-IR162-4 9 MON-ØØ6Ø3-6: Brazil Test Sites PHI-2014-064 Evaluation of Germination and Viability of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4: Controlled Environment Test Site PHI-2014-098 Genetic Stability and Equivalency of DAS–Ø15Ø7–19MON–ØØ81Ø-6 Maize Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction PHI-2014-107 Evaluation of Germination and Viability of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6: Controlled Environment Test Site PHI-2014-109 Genetic Stability and Equivalency of DAS-Ø15Ø7-1 9SYN-IR162-4 Maize Using Southern Blot Analysis and Event-specific Polymerase Chain Reaction PHI-2014-110 Genetic Stability and Equivalency of DAS-Ø15Ø7-1 9SYN-IR162-4 9MON-ØØ6Ø3-6 Maize Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction PHI-2014-131 Analysis of Single (HX1) and Triple Stack Maize (HX1 9MON810 9NK603) Samples of Spring 2014 from Various Trial Locations of Pakistan for Quantification of Cry1F, Cry1Ab and CP4 EPSPS Proteins by ELISA PHI-2015-007/001 Evaluation of Agronomic Characteristics and Yield for a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4 9MON-ØØ6Ø3-6 PHI-2014-046 Yield Characteristics of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9SYN-IR162-4: U.S. Test Sites PHI-2012-023_020 Agronomic Characteristics, Expressed Trait Protein Concentration, and Nutrient Composition of a Maize Line Containing the Combined Trait Product DAS-Ø15Ø7-1 9MON-ØØ81Ø-6 9 SYN-IR162-4 9MON-ØØ6Ø3-6: U.S. and Canada Test Sites (EU Study Format) PHI 2012-012 Nutritional Equivalency Study of the Combined Trait Product DAS-01507-1 9MON-00810-6 9SYN-1R162-4 9MON-00603-6 Poultry Feeding Study Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 35 EFSA Journal 2021;19(1):6348 Appendix B –Protein expression data Mean, standard deviation and range of protein levels (lg/g dry weight) from maize 1507 9MON810 9MIR162 9NK603, 1507, MON810, MIR162 and NK603, not treated with intended herbicides, from field trials performed across four locations in USA in 2012 (n =16 30 ). Protein Event(s) Leaf (V6) Root (V9) Leaf (V9) Whole plant (V9) Root (R1) Leaf (R1) Pollen (R1) Stalk (R1) Whole plant (R1) Root (R4) Leaf (R4) Forage (R4) Root (R6) Leaf (R6) Whole plant (R6) Grain (R6) Cry1F 1507 3 MON810 3 MIR162 3 NK603 32 (a)  9.9 (b) (22–55) (c) 12 2.8 (6.9–16) 18 6.1 (14–33) 27 5.2 (20–34) 10 3.4 (6.6–16) 18 3.3 (13–25) 31 3.9 (28–42) 12 1.5 (9.4–15) 18 3.4 (13–26) 8.5 2.3 (4.2–12) 46 12 (25–60) 13 1.3 (11–16) 8.7 3.5 (0.48–14) 8.2 9.9 (<0.14–31) 13 4.0 (6.2–20) 2.9 0.80 (1.9–4.2) 1507 21 3.8 (14–26) 8.9 2.0 (4.8–12) 16 4.3 (9.6–24) 18 2.4 (14–22) 7.2 1.7 (4.5–11) 17 4.9 (10–26) 26 2.1 (22–30) 9.2 0.77 (8.0–11) 15 1.5 (13–18) 5.8 2.4 (1.7–11) 23 16 (1.0–41) 11 1.7 (8.6–15) 6.3 1.7 (2.8–9.0) 11 12 (0.17–37) 9.2 3.3 (5.2–17) 2.4 0.61 (1.5–3.6) PAT 1507 3 MON810 3 MIR162 3 NK603 6.5 2.2 (3.4–12) 0.42 0.17 (0.16–0.69) 3.7  0.93 (2.0–5.2) 4.7 1.4 (2.6–7.6) 0.37  0.14 (0.19– 0.60) 4.2 1.1 (2.3–7.2) <LOQ (d) 0.042 0.029 (<0.046– 0.13) 3.2 1.2 (1.7–5.0) 0.36 0.19 (<0.069– 0.60) 2.6 1.1 (0.72–4.3) 0.92 0.29 (0.5–1.5) 0.28 0.18 (<0.069– 0.60) 0.15  0.18 (<0.14– 0.60) 0.22 0.16 (<0.046– 0.56) <LOQ 1507 6.9 1.8 (4.4–10) 0.40 0.22 (0.11–0.84) 5.7 1.5 (2.8–7.2) 5.6 1.7 (3.0–8.0) 0.33  0.13 (0.13– 0.54) 6.0 2.2 (2.8–11) <0.28 <0.28 0.058 0.088 (<0.046– 0.38) 3.7 0.71 (2.6–5.0) 0.31 0.21 (0.093– 0.72) 3.4 1.5 (0.96–5.8) 1.1 0.36 (0.58–1.7) 0.29 0.17 (<0.069– 0.57) 0.50  0.73 (<0.14– 2.0) 0.32 0.29 (<0.046– 0.84) <LOQ Cry1Ab 1507 3 MON810 3 MIR162 3 NK603 35 9.3 (25–52) 9.3 1.8 (6.6–13) 17 3.1 (14–23) 20 3.5 (16–26) 8.3 2.9 (5.4–14) 15 2.4 (12–20) <LOQ 5.5 1.0 (4.2–7.2) 12 2.4 (8.8–16) 7.4 2.7 (2.4–12) 18 6.1 (7.2–24) 6.2 1.0 (4.2–8.6) 8.0 4.2 (0.36–14) 5.0 5.3 (0.72–17) 5.8 2.7 (1.5–10) 0.21 0.062 (0.12–0.30) MON810 37 8.1 (28–56) 8.8 2.1 (3.0–11) 21 4.2 (12–30) 22 4.6 (17–30) 8.2 2.8 (4.8–14) 19 4.8 (11–26) <LOQ 5.7 0.98 (4.6–7.6) 14 2.4 (10–18) 6.2 3.7 (0.63–11) 15 9.7 (1.7–28) 7.1 1.7 (4.8–9.6) 10 5.5 (0.99–19) 7.1 8.3 (0.59–23) 6.4 3.1 (1.7–12) 0.20 0.074 (0.13–0.42) Vip3Aa20 1507 3 MON810 3 MIR162 3 NK603 160 72 (78–310) 48 14 (20–75) 99 29 (66–170) 170 27 (140–220) 34 13 (13–54) 130 35 (66–180) 150 15 (120–170) 60 14 (40–92) 140 24 (110–190) 32 13 (13–51) 400 140 (190–660) 230 38 (170–320) 55 32 (0.87–99) 110 150 (0.59–500) 130 57 (48–260) 100 80 (14–280) MIR162 120 37 (66–170) 38 9.4 (23–51) 96 29 (60–170) 160 31 (110–200) 29 9.0 (15–45) 160 57 (54–220) 130 13 (100–140) 73 11 (58–98) 150 28 (100–200) 26 13 (7.8–51) 360 110 (140–530) 190 74 (130–440) 52 28 (1.7–87) 160 200 (<LOQ– 600) 180 110 (22–400) 110 41 (45–180) 30 Except the following tissues, for which n =15: Root (V9) of MON810, MIR162 and NK603, for all proteins analysed for each event; Root (R1) of 1507 9MON810 9MIR162 9NK603, for all analysed proteins. www.efsa.europa.eu/efsajournal 36 EFSA Journal 2021;19(1):6348 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 Protein Event(s) Leaf (V6) Root (V9) Leaf (V9) Whole plant (V9) Root (R1) Leaf (R1) Pollen (R1) Stalk (R1) Whole plant (R1) Root (R4) Leaf (R4) Forage (R4) Root (R6) Leaf (R6) Whole plant (R6) Grain (R6) PMI 1507 3 MON810 3 MIR162 3 NK603 8.5 3.4 (4.4–16) 2.9 0.87 (1.3–4.5) 5.0 1.4 (3.7–8.4) 5.9 1.0 (4.6–7.6) 2.0 0.72 (1.1–3.3) 5.1  0.76 (3.6–6.6) 4.6 0.40 (4.0–5.3) 3.0 0.78 (2.2–4.4) 4.8 0.66 (3.8–5.6) 1.4 0.58 (0.51–2.3) 10 2.7 (5.0–14) 4.9 0.68 (3.6–6.2) 2.7 1.6 (<LOQ– 5.1) 1.7 2.5 (<LOQ– 9.0) 3.7 1.7 (0.54–7.4) 1.6 0.63 (0.81–2.7) MIR162 7.2 2.0 (4.3–9.6) 2.7 0.69 (1.6–3.9) 5.1 1.2 (3.7–7.8) 5.7 1.1 (3.8–7.2) 1.7 0.68 (0.93– 3.3) 6.4 1.2 (4.0–7.8) 4.6 0.57 (3.8–5.9) 3.3 0.71 (2.2–4.6) 4.8 0.73 (3.8–6.2) 0.93 0.64 (<LOQ– 2.0) 11 3.4 (5.0–16) 5.0 1.3 (4.0–9.2) 2.6 1.4 (<LOQ– 4.2) 3.0 4.1 (<LOQ– 13) 3.6 2.4 (0.60–9.0) 1.5 0.45 (0.84–2.2) CP4 EPSPS 1507 3 MON810 3 MIR162 3 NK603 230 65 (140–400) 110 44 (36–160) 160 36 (110– 230) 260 60 (160–440) 93 31 (57–140) 170 19 (140– 210) 460 78 (250–580) 110 35 (76–180) 180 29 (130–220) 75 12 (51–100) 340 100 (160–500) 120 24 (86–180) 79 41 (0.51–150) 78 94 (<LOQ– 250) 110 54 (19–220) 11 3.7 (6.0–17) NK603 230 46 (170–330) 79 13 (51–96) 190 22 (140– 220) 260 35 (200–340) 87 32 (36–140) 270 69 (180– 480) 400 84 (200–520) 130 45 (80–220) 250 46 (170–320) 56 19 (36–110) 370 140 (140–660) 110 28 (78–170) 97 43 (19–160) 100 110 (<LOQ– 320) 120 57 (38–220) 8.5 2.3 (5.4–13) (a): Mean. (b): Standard deviation. (c): Range. (d): LOQ –limit of quantification. www.efsa.europa.eu/efsajournal 37 EFSA Journal 2021;19(1):6348 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 Appendix C –List of relevant publications List of relevant publications identified by the applicant through systematic literature searches (January 2005–May 2020) References Bowers E, Munkvold G and Hellmich RL, 2013. Vip3Aa and Cry1Ab proteins in maize reduce Fusarium ear rot and fumonisins by deterring kernel injury from multiple Lepidopteran pests. World Mycotoxin Journal, 6, 127–135. Castan M, Ben-Ali SE, Hochegger R, Ruppitsch W, Haslberger AG and Brandes C, 2017 Analysis of the genetic stability of event NK603 in stacked corn varieties using highresolution melting (HRM) analysis and Sanger sequencing. European Food Research and Technology, 243, 353–365. EFSA (European Food Safety Authority), 2011. Statement complementing the EFSA GMO Panel scientific opinion on maize MON 89034 91507 9NK603 (application EFSA-GMO-NL-2009-65), to cover all sub-combinations independently of their origin. EFSA Journal 2011;9(9):2377, 8 pp. https://doi.org/10.2903/j.efsa.2011.2377 Graser G, Walters FS, Burns A, Sauve A and Raybould A, 2017. A general approach to test for interaction among mixtures of insecticidal proteins which target different orders of insect pests. Journal of Insect Science, 17, 1–12. Park KW, Lee B, Kim C-G and Kim D-Y, 2010. Monitoring the occurrence of genetically modified maize at a grain receiving port and along transportation routes in the Republic of Korea. Food Control, 21, 456–461. Ridley W, Harrigan GG, Breeze ML, Nemeth MA, Sidhu RS and Glenn KC, 2011. Evaluation of compositional equivalence for multitrait biotechnology crops. Journal of Agricultural and Food Chemistry, 59, 5865–5876. Waminal NE, Ryu KH, Choi SH and Kim HH, 2013. Randomly detected genetically modified (GM) maize (Zea mays L.) near a transport route revealed a fragile 45S rDNA phenotype. PLoS ONE, 8, e74060. Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 38 EFSA Journal 2021;19(1):6348 Appendix D –Statistically significant findings in the 90–day toxicity study in rats on the whole food/feed from maize 1507 Statistically significant parameter/endpoint Finding GMO Panel interpretation Mean body weight gain Increased Not of toxicological relevance –sporadic, certain time points only –no effect on overall body weight or body weight gain Forelimb grip strength Reduced in high-dose animals Not of toxicological relevance –small magnitude (< 20% males, < 10% females) –within normal variability for this parameter Motor activity Reduced in low-dose females increased in low-dose males Not of toxicological relevance –within the normal variability for this parameter –no consistent pattern –no significant change in high dose animals Red blood cell and white blood cell parameters Changes in both dose groups Not of toxicological relevance –of low magnitude –no consistent pattern of findings within a group or between dose group Thyroid hormones (TSH, T4) Increases in high-dose females Not of toxicological relevance –of low magnitude (12%) –no pathological changes in thyroid glands Urine volume and specific gravity Increased urine volume and decreased specific gravity in females Not of toxicological relevance –values are within the physiological range –no associated changes in blood urea nitrogen (BUN) or kidney pathology Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 39 EFSA Journal 2021;19(1):6348 Appendix E –Animal dietary exposure Animal dietary exposure to Cry1F, PAT, Cry1Ab, Vip3Aa20, PMI and CP4 EPSPS proteins (mg/kg bw per day) in livestock, based on the consumption of maize grain and forage. Dietary exposure (mg/kg bw per day) Cry1F PAT Grain (G) Forage (F) G +F Grain (G) Forage (F) G +F Broiler 0.16 NA NA 0.0034 NA NA Layer 0.15 NA NA 0.0033 NA NA Turkey 0.11 NA NA 0.0025 NA NA Breeding pigs 0.05 NA NA 0.0011 NA NA Finishing pigs 0.07 NA NA 0.0014 NA NA Beef cattle 0.06 0.27 0.33 0.0013 0.021 0.022 Dairy cattle 0.04 0.32 0.36 0.0008 0.025 0.026 Ram/ewe 0.03 NA NA 0.0007 NA NA Lamb 0.04 0.18 0.22 0.0009 0.014 0.015 Dietary exposure (mg/kg bw per day) Cry1Ab Vip3Aa20 Grain (G) Forage (F) G +F Grain (G) Forage (F) G +F Broiler 0.009 NA NA 2.42 NA NA Layer 0.009 NA NA 2.35 NA NA Turkey 0.006 NA NA 1.75 NA NA Breeding pigs 0.003 NA NA 0.79 NA NA Finishing pigs 0.004 NA NA 1.03 NA NA Beef cattle 0.004 0.14 0.14 0.94 4.80 5.74 Dairy cattle 0.002 0.16 0.17 0.57 5.77 6.33 Ram/ewe 0.002 NA NA 0.49 NA NA Lamb 0.002 0.09 0.09 0.62 3.19 3.81 Dietary exposure (mg/kg bw per day) PMI CP4 EPSPS Grain (G) Forage (F) G +F Grain (G) Forage (F) G +F Broiler 0.079 NA NA 0.59 NA NA Layer 0.077 NA NA 0.57 NA NA Turkey 0.057 NA NA 0.43 NA NA Breeding pigs 0.026 NA NA 0.19 NA NA Finishing pigs 0.034 NA NA 0.25 NA NA Beef cattle 0.031 0.10 0.13 0.23 2.7 2.9 Dairy cattle 0.018 0.12 0.14 0.14 3.2 3.4 Ram/ewe 0.016 NA NA 0.12 NA NA Lamb 0.020 0.07 0.09 0.15 1.8 1.9 Assessment of GM maize 1507 3MIR162 3MON810 3NK603 www.efsa.europa.eu/efsajournal 40 EFSA Journal 2021;19(1):6348