Assessment of genetically modified maize MON 87427 × MON 87460 × MON 89034 × 1507 × MON 87411 × 59122 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2017-139)

SCIENTIFIC OPINION ADOPTED: 25 November 2020 doi: 10.2903/j.efsa.2021.6351 Assessment of genetically modified maize MON 87427 3 MON 87460 3MON 89034 31507 3MON 87411 359122 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2017-139) 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, Antonio Fernandez, Andrea Gennaro, Jose  Angel G omez Ruiz, Dafni Maria Kagkli, Anna Lanzoni, Franco Maria Neri, Nikoletta Papadopoulou, Konstantinos Paraskevopoulos and Tommaso Raffaello Abstract Maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 (six-event stack maize) was produced by conventional crossing to combine six single events: MON 87427, MON 87460, MON 89034, 1507, MON 87411 and 59122. The GMO Panel previously assessed the six single maize events and 17 of the subcombinations and did not identify safety concerns. No new data on the single maize events or the 17 subcombinations were identified that could lead to modification of the original conclusions on their safety. 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 and dsRNA in the six-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the six-event stack maize, as described in this application, is as safe as its non-GM comparator and the selected non-GM reference varieties. In the case of accidental release of viable grains of the six-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 39 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 six–event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the six-event stack maize. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the six-event stack maize and its subcombinations are as safe as the non-GM comparator and the selected 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: dsRNA, GMO, herbicide tolerant, Zea mays, insect resistant, stack events Requestor: European Commission Question number: EFSA-Q-2017-00115 Correspondence: GMO_Secretariat_Application[email protected] EFSA Journal 2021;19(1):6351www.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. Acknowledgements: 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 Sonia Hern andez Valero, Sylvie Mestdagh, Irene Mu~ noz-Guajardo and Lorenz Oberkofler for the support provided to this scientific output. Suggested citation: EFSA Panel on Genetically Modified Organisms (GMO), Naegeli H, Bresson J-L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogu eF, Rostoks N, S anchez Serrano JJ, Savoini G, Veromann E, Veronesi F,  Alvarez F, Ardizzone M, De Sanctis G, Fernandez A, Gennaro A, G omez Ruiz J  A, Kagkli DM, Lanzoni A, Neri FM, Papadopoulou N, Paraskevopoulos K and Raffaello T, 2021. Scientific Opinion on the assessment of genetically modified maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2017-139). EFSA Journal 2021;19(1):6351, 45 pp. https://doi.org/10.2903/j.efsa.2021.6351 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 MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 2 EFSA Journal 2021;19(1):6351 Summary Following the submission of application EFSA-GMO-NL-2017-139 under Regulation (EC) No 1829/2003 from Monsanto Company (referred to hereafter as ‘the applicant’), the Panel on Genetically Modified Organisms of the European Food Safety Authority (referred to hereafter as ‘GMO Panel’) was asked to deliver a Scientific Opinion on the safety of genetically modified (GM) glufosinate and glyphosate tolerant, insect resistant and drought tolerant maize (Zea mays L.) MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 (referred to hereafter as ‘six-event stack maize’) and its subcombinations independently of their origin, according to Regulation (EU) No 503/2013 (referred to hereafter as ‘subcombinations’). The scope of application EFSA-GMO-NL-2017-139 is for import, processing, and food and feed uses within the European Union (EU) of maize MON 87427 9 MON 87460 9MON 89034 91507 9MON 87411 959122 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 five of the events present in the six-event stack maize. The safety of subcombinations occurring as segregating progeny in the harvested grains of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 is evaluated in the context of the assessment of the six-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 six-event stack, are risk assessed separately in the present scientific opinion. The six-event stack maize was produced by conventional crossing to combine six single maize events: MON 87427 expressing the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein to confer tolerance to glyphosate-containing herbicides; MON 87460 expressing the cold shock protein B (CspB) (to confer drought tolerance) and the neomycin phosphotransferase II protein (NPTII) (used as a selectable marker); MON 89034 expressing the Cry1A.105 and Cry2Ab2 proteins (for protection against certain lepidopteran pests); 1507 expressing the Cry1F protein (for protection against certain lepidopteran pests) and the PAT protein (for tolerance to glufosinate-ammoniumcontaining herbicides); MON 87411 expressing the Cry3Bb1 protein and the DvSnf7 dsRNA (for protection against certain coleopteran pests) and the CP4 EPSPS protein (for tolerance to glyphosatecontaining herbicides); and 59122 expressing the Cry34Ab1 and Cry35Ab1 proteins (for protection against certain coleopteran pests) and the PAT protein. The GMO Panel evaluated the six-event stack maize and its subcombinations with reference to the scope and appropriate principles described in its applicable guidelines for the risk assessment of GM plants and the post-market environmental monitoring. The GMO Panel considered the information submitted in application EFSA-GMO-NL-2017-139, additional information provided by the applicant during the risk assessment, the scientific comments submitted by the Member States and the relevant scientific literature. For application EFSA-GMO-NL-2017-139, previous assessments of the six single events (MON 87427, MON 87460, MON 89034, 1507, MON 87411 and 59122), and 17 of the subcombinations provided a basis for the assessment of the six-event stack maize and all its subcombinations. No safety concerns were identified by the GMO Panel in the previous assessments. No safety issue concerning the six 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 six-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 carried out, and the safety of the newly expressed proteins, of the dsRNAs and the whole food and feed were evaluated with respect to potential toxicity, allergenicity and nutritional characteristics. Environmental impacts and post-market environmental monitoring (PMEM) plan were also evaluated. The molecular characterisation data establish that the events stacked in maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 have retained their integrity. Protein expression analysis showed that the levels of the newly expressed proteins are similar in the six-event stack maize and in the single events, except for CP4 EPSPS and PAT protein levels that are expected to be different because of the combination of events MON 87427 and MON 87411 (both producing CP4 EPSPS) and events 1507 and 59122 (both producing PAT) in the in the six-event stack maize. In addition, the provided data indicate that there is no impact of the dsRNAs on the expression Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 3 EFSA Journal 2021;19(1):6351 levels of the newly expressed proteins. No indications were identified of interactions that may affect the integrity of the events and the levels of the newly expressed proteins in this six–event stack maize. The comparative analysis of agronomic and phenotypic characteristics and grain and forage composition identified no differences between maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 and the non-GM comparator that required further assessment except for the changes in root lodged plants, in levels of acid detergent fibre (ADF) in forage and in levels of protein, arginine, glycine, leucine, lysine and manganese in grain. These changes were further assessed for food/feed safety and environmental impact and raised no concern. 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 and dsRNA in the six-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122, as described in this application, is as safe as the non-GM comparator and the selected commercial non-GM maize reference varieties (referred to hereafter as non-GM reference varieties). 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 MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 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 17 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 39 subcombinations included in the scope of application EFSA-GMO-NL-2017-139, no experimental data were provided. The GMO Panel assessed the possibility of interactions between the events in the 39 subcombinations and concludes that these subcombinations would not raise safety concerns. These subcombinations are therefore expected to be as safe as the single events, the previously assessed subcombinations and the six-event stack maize. Given the absence of safety concerns for foods and feeds from maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 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 six-event stack maize and its subcombinations. The literature searches did not identify any relevant publications on maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122. In the context of annual PMEM reports, the applicant could further fine tune future literature searches according to the GMO Panel recommendations provided in this scientific opinion. The GMO Panel concludes that maize MON 87427 9MON 87460 9MON 89034 91507 9 MON 87411 959122 and its subcombinations, as described in this application, are as safe as the non-GM comparator and the selected non-GM reference varieties with respect to potential effects on human and animal health and the environment. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 4 EFSA Journal 2021;19(1):6351 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 ........................................................................................... 11 3.3. Systematic literature review ......................................................................................................... 12 3.4. Risk assessment of the six-event stack maize MON 87427 9MON 87460 9MON 89034 91507 9 MON 87411 959122 .................................................................................................................. 13 3.4.1. Molecular characterisation............................................................................................................ 13 3.4.1.1. Genetic elements and their biological function ............................................................................... 13 3.4.1.2. Integrity of the events in the six-event stack maize MON 87427 9MON 87460 9MON 89034 9 1507 9MON 87411 959122...................................................................................................... 15 3.4.1.3. Information on the expression of the inserts ................................................................................. 16 3.4.1.4. Conclusions of the molecular characterisation................................................................................ 16 3.4.2. Comparative analysis ................................................................................................................... 16 3.4.2.1. Overview of studies conducted for the comparative analysis ........................................................... 16 3.4.2.2. Experimental field trial design and statistical analysis ..................................................................... 16 3.4.2.3. Suitability of selected test materials .............................................................................................. 17 3.4.2.4. Representativeness of the receiving environments ......................................................................... 17 3.4.2.5. Agronomic and phenotypic analysis .............................................................................................. 18 3.4.2.6. Compositional analysis................................................................................................................. 18 3.4.2.7. Conclusions on the comparative analysis ....................................................................................... 20 3.4.3. Food/Feed safety assessment....................................................................................................... 20 3.4.3.1. Effects of processing ................................................................................................................... 20 3.4.3.2. Influence of temperature and pH on newly expressed proteins ....................................................... 20 3.4.3.3. Toxicology .................................................................................................................................. 21 3.4.3.4. Allergenicity ................................................................................................................................23 3.4.3.5. Dietary exposure assessment to new constituents.......................................................................... 24 3.4.3.6. Nutritional assessment of endogenous constituents........................................................................ 26 3.4.3.7. Conclusion on the food/feed safety assessment ............................................................................. 28 3.4.4. Environmental risk assessment ..................................................................................................... 28 3.4.4.1. Persistence and invasiveness of the GM plant ................................................................................ 28 3.4.4.2. Potential for gene transfer ........................................................................................................... 28 3.4.4.3. Interactions of the GM plant with target organisms........................................................................ 29 3.4.4.4. Interactions of the GM plant with non-target organisms ................................................................. 29 3.4.4.5. Interactions with abiotic environment and biogeochemical cycles .................................................... 29 3.4.4.6. Conclusion of the environmental risk assessment........................................................................... 30 3.4.5. Conclusion on the six-event stack maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122........................................................................................................................... 30 3.5. Risk assessment of the subcombinations ....................................................................................... 30 3.5.1. Subcombinations previously assessed ........................................................................................... 30 3.5.2. Subcombinations not previously assessed...................................................................................... 32 3.5.2.1. Stability of the events.................................................................................................................. 32 3.5.2.2. Expression of the events.............................................................................................................. 32 3.5.2.3. Potential functional interactions between the events ...................................................................... 32 3.5.3. Conclusion .................................................................................................................................. 32 3.6. Post-market monitoring................................................................................................................ 32 3.6.1. Post-market monitoring of GM food/feed....................................................................................... 32 3.6.2. Post-market environmental monitoring.......................................................................................... 33 3.6.3. Conclusion on post-market monitoring .......................................................................................... 33 4. Overall conclusions ...................................................................................................................... 33 5. Documentation as provided to EFSA ............................................................................................. 34 References............................................................................................................................................... 34 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 5 EFSA Journal 2021;19(1):6351 Abbreviations ........................................................................................................................................... 39 Appendix A –Additional studies................................................................................................................. 41 Appendix B –Protein expression data ........................................................................................................ 43 Appendix C –Statistically significant findings in the 90-day toxicity study in rats on the whole food/feed from maize 1507.............................................................................................................................................. 44 Appendix D –Animal dietary exposure ....................................................................................................... 45 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 6 EFSA Journal 2021;19(1):6351 1. Introduction The scope of application EFSA-GMO-NL-2017-139 is for food and feed uses, import and processing within the European Union (EU) of the genetically modified (GM) herbicide-tolerant, insect-resistant and drought-tolerant maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 9 59122 and all its subcombinations independently of their origin and does not include cultivation in EU. 1.1. Background On 21 February 2017, the European Food Safety Authority (EFSA) received from the Competent Authority of The Netherlands application EFSA-GMO-NL-2017-139 for authorisation of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 (hereafter referred to as ‘the six-event stack maize’) (Unique Identifier MON-87427-7 9MON-8746Ø-4 9MON-89Ø34-3 9 DAS-Ø15Ø7-1 9MON-87411-9 9DAS-59122-7), submitted by Monsanto Europe S.A. (hereafter referred to as ‘the applicant’) according to Regulation (EC) No 1829/2003 1 . Following receipt of application EFSA-GMO-NL-2017-139, EFSA informed the Member States (MS) 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 31 May 2017, EFSA declared the application valid and made the application available to MS and the EC. From the 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-2017-139. 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 MS 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-2017-139 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 MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 and all its subcombinations independently of their origin according to the of application EFSA-GMO-NL-2017-139. 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, 2 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 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 at the EFSA Register of Questions: http://registerofquestions.efsa.europa.eu/roqFrontend/questionDocume ntsLoader?question=EFSA-Q-2017-00115 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 modified 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. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 7 EFSA Journal 2021;19(1):6351 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. 2. Data and methodologies 2.1. Data The GMO Panel based its scientific assessment of maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 on the valid application EFSA-GMO-NL-2017-139, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU MS 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 (EFSA GMO Panel, 2010a, 2011a,b, 2015a), explanatory notes and statements (EFSA GMO Panel, 2010b; EFSA, 2014, 2017a,b, 2019) for the risk assessment of GM plants. During its risk assessment the GMO Panel considered all additional unpublished studies as listed in Appendix Afor potential effects on human and animal health and the environment. 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/2014/01 and OC/EFSA/GMO/2018/02 contractors performed preparatory work and delivered report on the methods applied by the applicant in performing statistical and toxicological analyses, respectively. 3. Assessment 3.1. Introduction Application EFSA-GMO-NL-2017-139 covers the six-event stack maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 and all its 56 subcombinations independently of their origin (Table 1). Table 1: Stacked maize events covered by the scope of application EFSA-GMO-NL-2017-139 Degree of stacking Event Unique identifiers Six-event stack MON 87427 9MON 87460 9MON 89034 9 1507 9MON 87411 959122 MON-87427-7 9MON-8746Ø-4 9MON-89Ø34-3 9DAS-Ø15Ø7-1 9MON-87411-9 9DAS-59122-7 Five-event stack 59122 9MON 89034 9MON 87460 9 MON 87427 9MON 87411 DAS-59122-7 9MON-89Ø34-3 9MON-8746Ø-4 9 MON-87427-7 9MON-87411-9 1507 9MON 89034 9MON 87460 9 MON 87427 9MON 87411 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-8746Ø-4 9MON-87427-7 9MON-87411-9 1507 959122 9MON 87460 9MON 87427 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-8746Ø-4 9 MON-87427-7 9MON-87411-9 1507 959122 9MON 89034 9MON 87427 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-87427-7 9MON-87411-9 1507 959122 9MON 89034 9MON 87460 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-8746Ø-4 9MON-87411-9 1507 959122 9MON 89034 9MON 87460 9MON 87427 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-8746Ø-4 9MON-87427-7 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 8 EFSA Journal 2021;19(1):6351 Degree of stacking Event Unique identifiers Four-event stack MON 89034 9MON 87460 9MON 87427 9 MON 87411 MON-89Ø34-3 9MON-8746Ø-4 9MON-87427-7 9MON-87411-9 59122 9MON 87460 9MON 87427 9 MON 87411 DAS-59122-7 9MON-8746Ø-4 9MON-87427-7 9 MON-87411-9 59122 9MON 89034 9MON 87427 9 MON 87411 DAS-59122-7 9MON-89Ø34-3 9MON-87427-7 9 MON-87411-9 59122 9MON 89034 9MON 87460 9 MON 87411 DAS-59122-7 9MON-89Ø34-3 9MON-8746Ø-4 9 MON-87411-9 59122 9MON 89034 9MON 87460 9 MON 87427 DAS-59122-7 9MON-89Ø34-3 9MON-8746Ø-4 9 MON-87427-7 1507 9MON 87460 9MON 87427 9 MON 87411 DAS-Ø15Ø7-1 9MON-8746Ø-4 9MON-87427-7 9 MON-87411-9 1507 9MON 89034 9MON 87427 9 MON 87411 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-87427-7 9 MON-87411-9 1507 9MON 89034 9MON 87460 9 MON 87411 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-8746Ø-4 9MON-87411-9 1507 9MON 89034 9MON 87460 9 MON 87427 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-8746Ø-4 9MON-87427-7 1507 959122 9MON 87427 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-87427-7 9 MON-87411-9 1507 959122 9MON 87460 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-8746Ø-4 9 MON-87411-9 1507 959122 9MON 87460 9MON 87427 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-8746Ø-4 9 MON-87427-7 1507 959122 9MON 89034 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-87411-9 1507 959122 9MON 89034 9MON 87427 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-87427-7 1507 959122 9MON 89034 9MON 87460 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 9 MON-8746Ø-4 Three-event stack MON 87460 9MON 87427 9MON 87411 MON-8746Ø-4 9MON-87427-7 9MON-87411-9 MON 89034 9MON 87427 9MON 87411 MON-89Ø34-3 9MON-87427-7 9MON-87411-9 MON 89034 9MON 87460 9MON 87411 MON-89Ø34-3 9MON-8746Ø-4 9MON-87411-9 MON 89034 9MON 87460 9MON 87427 MON-89Ø34-3 9MON-8746Ø-4 9MON-87427-7 59122 9MON 87427 9MON 87411 DAS-59122-7 9MON-87427-7 9MON-87411-9 59122 9MON 87460 9MON 87411 DAS-59122-7 9MON-8746Ø-4 9MON-87411-9 59122 9MON 87460 9MON 87427 DAS-59122-7 9MON-8746Ø-4 9MON-87427-7 59122 9MON 89034 9MON 87411 DAS-59122-7 9MON-89Ø34-3 9MON-87411-9 59122 9MON 89034 9MON 87427 DAS-59122-7 9MON-89Ø34-3 9MON-87427-7 59122 9MON 89034 9MON 87460 DAS-59122-7 9MON-89Ø34-3 9MON-8746Ø-4 1507 9MON 87427 9MON 87411 DAS-Ø15Ø7-1 9MON-87427-7 9MON-87411-9 1507 9MON 87460 9MON 87411 DAS-Ø15Ø7-1 9MON-8746Ø-4 9MON-87411-9 1507 9MON 87460 9MON 87427 DAS-Ø15Ø7-1 9MON-8746Ø-4 9MON-87427-7 1507 9MON 89034 9MON 87411 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-87411-9 1507 9MON 89034 9MON 87427 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-87427-7 1507 9MON 89034 9MON 87460 DAS-Ø15Ø7-1 9MON-89Ø34-3 9MON-8746Ø-4 1507 959122 9MON 87411 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-87411-9 1507 959122 9MON 87427 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-87427-7 1507 959122 9MON 87460 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-8746Ø-4 1507 959122 9MON 89034 DAS-Ø15Ø7-1 9DAS-59122-7 9MON-89Ø34-3 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 9 EFSA Journal 2021;19(1):6351 3.4.1.3. Information on the expression of the inserts CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 protein levels were analysed by enzyme-linked immunosorbent assay (ELISA), in material harvested in afield trial across five locations in the USA in 2014. Samples analysed included leaf (V3-V4), root (V3V4), forage (R5) and grain (R6) both those treated and not treated with glyphosate and/or glufosinate-ammonium. In order to assess changes in protein expression levels which may result from potential interactions between the events, protein levels were determined for the six-event maize stack and the corresponding single events in different parts of the plant. The levels of all the proteins newly expressed in the six-event stack maize and the corresponding singles were comparable in all tissues, except for CP4 EPSPS and PAT protein levels that are expected to be different because of the combination of events MON 87427 and MON 87411 both producing CP4 EPSPS and events 1507 and 59122 both producing PAT protein in the six-event stack maize (Appendix B). In addition, the potential impact of the DvSnf7 dsRNA on the levels of the newly expressed proteins was assessed by comparing the protein expression levels in the six-event stack and the respective singles. The data indicate that there is no impact of the DvSnf7 dsRNA on the expression level. Therefore, there is no indication of an interaction that may affect the levels of the newly expressed proteins in this stack. 3.4.1.4. Conclusions of the molecular characterisation The molecular data establish that the events stacked in maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 have retained their integrity. Protein expression analyses showed that the levels of the newly expressed proteins are similar in the six-event stack maize and in the single events. CP4 EPSPS and PAT shows the expected higher level in the stack resulting from the combination of events MON 87427 and MON 87411, and 1507 and 59122, respectively. Therefore, there is no indication of an interaction that may affect the integrity of the events and the levels of the newly expressed proteins in this stack. Based on the known biological function (Table 3) of the newly expressed proteins, the only foreseen interactions at the biological level are between the Cry proteins in susceptible insects, which will be dealt with in Sections 3.4.4. 3.4.2. Comparative analysis 10 3.4.2.1. Overview of studies conducted for the comparative analysis Application EFSA-GMO-NL-2017-139 presents data on agronomic and phenotypic characteristics, as well as on forage and grain composition of maize MON 87427 9MON 87460 9MON 89034 9 1507 9MON 87411 959122 (Table 5). 3.4.2.2. Experimental field trial design and statistical analysis At each field trial site, the following materials were grown in a randomised complete block design with four replicates: the six-event stack maize exposed to the intended herbicides glyphosate and glufosinate-ammonium (treated), the six-event stack maize not exposed to the intended herbicides Table 5: Overview of the comparative analysis studies to characterise the six-event stack maize in application EFSA-GMO-NL-2017-139 Study focus Study details Comparator Non-GM reference varieties Agronomic and phenotypic analysis Field study, US, 2014, eight sites (a) MPA640B (b) 18 (c) Compositional analysis GM: genetically modified. (a): The field trials were located in Boone, IA; Jefferson, IA; Vermilion, IL; Warren, IL; Shelby, IL; Pawnee, KS; Miami, OH; and Berks, PA. (b): MPA640B refers to LH244 9LH287. (c): Non-GM reference varieties used in the 2014 field trials were LG2540, LG2548, Phillips 717, Channel 211-97, Midland Phillips 7B15P, Seed Consultants 1112, Stine 9724, Dekalb DKC62-06, Mycogen 2H721, NC+5220, NH6280, Stewart S602, Stewart S588, Channel 213-88, Dekalb DKC63-43, Mycogen 2J790, Gateway 6158, NH6769. 10 Dossier: Part II –Section 1.3 and additional information: 3/9/2018 and 29/8/2019. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 16 EFSA Journal 2021;19(1):6351 (not treated), the comparator MPA640B and four non-GM maize reference varieties (hereafter referred to as ‘non-GM reference varieties’). The agronomic, phenotypic and compositional data were analysed as specified by the EFSA GMO Panel (2010b, 2011a). This includes, for each of the two treatments of the six-event stack maize, the application of a difference test (between the GM maize and the non-GM comparator) and an equivalence test (between the GM maize and the set of non-GM commercial reference varieties). The results of the equivalence test are categorised into four possible outcomes (I–IV, ranging from equivalence to non-equivalence). 11 3.4.2.3. Suitability of selected test materials Selection of the test materials To produce the GM stack maize, the single events MON 87427, MON 87460, MON 89034, 1507 and 59122 were transferred in the genetic background of the non-GM inbred lines LH287. Event MON 87411 was obtained transforming the non-GM inbred line LH244 and was maintained in this genetic background. In subsequent subsections, maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 refers to hybrid (F 1 ) obtained crossing GM inbred line LH244 (carrying MON 87411) with GM inbred line LH287 (carrying MON 87427 9MON 87460 9MON 89034 91507 959122). The comparator selected in the field trials is the hybrid maize MPA640B that was obtained by crossing the non-GM inbred lines LH244 and LH287. As documented by the pedigree, the EFSA GMO Panel considers the selected comparator suitable for the comparative analysis. Maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122 and the non–GM comparator, both with a comparative relative maturity (CRM) of 110, are appropriate for growing in a range of environments across North America. Several non-GM reference varieties (see Table 5) with a CRM ranging from 108 to 115 were selected by the applicant and, at each selected site, four reference varieties were tested. On the basis of the provided information on relative maturity classes, the GMO Panel considers the selected non-GM reference varieties appropriate for the comparative assessment. Seed production and quality Seeds of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 and the comparator used in the 2014 field trials were produced, harvested and stored under similar conditions. The seed lots were verified for their purity via event specific quantitative polymerase chain reaction analysis. The mean germination rates of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 9 59122 and of the comparator were 100% and 99%, respectively. The GMO Panel considers that the starting seed used as test material in the agronomic, phenotypic and compositional studies was of suitable quality. Conclusion on suitability The GMO Panel is of the opinion that the six-event stack maize, the non-GM comparator and the non-GM reference varieties were properly selected and are of sufficient quality. Therefore, the test materials are considered acceptable for the comparative analysis. 3.4.2.4. Representativeness of the receiving environments Selection of field trial sites The selected field trial sites were located in commercial maize-growing regions of the US. The soil characteristics of the selected fields were diverse, 12 corresponding to optimal and near-optimal conditions for maize cultivation (Sys et al., 1993). The GMO Panel considers that the selected sites reflect commercial maize-growing regions in which the test materials are likely to be grown. Meteorological conditions Maximum and minimum mean temperatures and sum of precipitations were provided on a monthly basis. No exceptional weather conditions were reported at any of the selected sites; therefore, the GMO Panel considers that the meteorological dataset falls within the range of climatic conditions normally occurring at these sites. 11 In detail, the four outcomes are: 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). 12 Soil types of the field trials were silty clay loam, loam and silty loam; soil organic matter ranged from 0.9% to 3.8%. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 17 EFSA Journal 2021;19(1):6351 Management practices The field trials included plots containing six-event stack maize, plots with the comparator and plots with non-GM reference varieties, managed according to local agricultural practices. In addition, the field trials included plots containing the six-event stack maize managed following the same agricultural practices, plus exposed to the glyphosate-containing and glufosinate-ammonium-containing herbicides. Glyphosateand glufosinate-ammonium-containing herbicides were applied separately at the V2-V4 and V5-V7 growth stage, respectively. The GMO Panel considers that the management practices including sowing, harvesting and application of plant protection products were appropriate. Conclusion on representativeness The GMO Panel concludes that the geographical locations, soil characteristics, meteorological conditions and management practices of the field trials are typical for receiving environments where the tested materials could be grown. 3.4.2.5. Agronomic and phenotypic analysis Thirteen 13 agronomic and phenotypic endpoints plus information on abiotic stressors, disease incidence and arthropod damage were collected from the field trials (Table 5). Of those, the endpoint dropped ears was not analysed as described in Section 3.4.2.2 because more than 90% of the values were 0. The outcome of the analysis for the remaining 12 endpoints was as follows: •For the six-event stack maize (not treated with the intended herbicides), statistically significant differences with the non-GM comparator were identified for early stand count, days to 50% pollen shed, days to 50% silking, root lodged plants, final stand count and test weight. All these endpoints fell under equivalence category I except for root lodged plants, for which the test of equivalence was not applied (because the variation between the non-GM commercial varieties was estimated to be 0). 14 •For the six-event stack maize (treated with the intended herbicides), statistically significant differences with the non-GM comparator were identified for early stand count, days to 50% pollen shed, days to 50% silking, ear height, root lodged plants, final stand count and test weight. All these endpoints fell under equivalence category I or II except for root lodged plants, for which the test of equivalence was not applied. 14 For the endpoint root lodged plants, significant differences were found between the six-event stack maize (for both treatments) and the non-GM comparator and equivalence could not be determined. Because the mean values for root lodged plants were very small for all the materials, 14 the GMO Panel considered that this result does not indicate issues in the materials used for the comparative analysis. Whether these differences could have an adverse environmental impact is discussed in Section 3.4.4.1. 3.4.2.6. Compositional analysis Forage and grain harvested from the field trials in the US in 2014 (Table 5) were analysed for 78 different constituents (9 in forage and 69 in grain), including the key constituents recommended by the OECD (2002). A total 15 grain constituents were excluded from the statistical analysis since more than 50% of the observations were below the limit of quantification. 15 The statistical analysis was applied to the remaining 63 constituents (9 in forage 16 and 54 in grain 17 ); a summary of the outcome of the test of difference and the test of equivalence is presented in Table 6. 13 Early stand count, days to 50% pollen shed, days to 50% silking, stay green rating, ear height, plant height, dropped ears, stalk lodged plants, root lodged plants, final stand count, grain moisture, test weight and yield. 14 Estimated means for root lodged plants (number of plants/two rows): 1.61 (untreated GM maize), 0.67 (treated GM maize), 2.89 (non-GM comparator) and 0.25 (non-GM reference varieties). 15 Sodium, furfural, 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), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), c-linolenic acid (C18:3), eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3) and arachidonic acid (C20:4). 16 Ash, carbohydrates, moisture, protein, total fat, acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium and phosphorus. 17 Proximates and fibre content (ash, carbohydrates, moisture, protein, total fat, ADF, NDF and total dietary fibre (TDF)), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium and zinc), vitamins (ß-carotene, thiamine, riboflavin, niacin, pyridoxine, folic acid and a-tocopherol), amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), fatty acids (palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), eicosenoic acid (C21:0) and behenic acid (C22:0)) and other compounds (ferulic acid, p-coumaric acid, phytic acid and raffinose). Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 18 EFSA Journal 2021;19(1):6351 •For the six-event stack maize not treated with the intended herbicides, statistically significant differences in the comparison with MPA640B were identified for 30 endpoints (3 in forage and 27 in grains). All these endpoints fell under equivalence category I or II except for acid detergent fibre (ADF) in forage, for which the test of equivalence was not applied (because the variation between the non-GM commercial varieties was estimated to be 0), and arginine levels in grain which fell under equivalence category III (Table 6). •For the six-event stack maize treated with the intended herbicides, statistically significant differences in the comparison with MPA640B were identified for 39 endpoints (3 in forage and 36 in grains). All these endpoints fell under equivalence category I or II except for the levels of protein, arginine, glycine, leucine, lysine and manganese in grain, which fell under category III or IV (Table 6). The GMO Panel assessed all significant differences between the six-event stack maize 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. Quantitative results for the endpoints showing significant differences between the six-event stack maize and its non-GM comparator and not falling under equivalence category I/II are given in Table 7. Table 6: Summary of the outcome of the comparative analysis in grain and forage from maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 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 28 (d) 21 33 (d) Category III/IV 1 (e) 1 (f) 1 (e) 6 (f) Not categorised 1 (g) 1 (h) 2 (g) – Total endpoints 63 63 (a): Comparison between the six-event stack maize 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). Not categorised means that the test of equivalence was not applied because of the lack of variation among the non-GM reference varieties. (c): Treated/not treated with glyphosate and glufosinate-ammonium-containing herbicides. (d): Endpoints with significant differences between the six-event stack maize and its non-GM comparator and falling in equivalence category I-II. For forage, not treated only: carbohydrates and protein. Treated only: moisture, calcium and phosphorus. Both treated and not treated: none. For grains, not treated only: protein, glycine, leucine and manganese. Treated only: ash, moisture, NDF, cystine, tryptophan, potassium, zinc and thiamine. Both treated and not treated: carbohydrates, total fat, TDF, alanine, glutamic acid, isoleucine, methionine, serine, threonine, valine, linolenic acid (C18:3), arachidic acid (C20:0), eicosenoic acid (C20:1), calcium, copper, iron, magnesium, pyridoxine, ß-carotene, a-tocopherol, ferulic acid and raffinose. (e): Proline levels in grain fell under equivalence category III (for both treated and not treated GM maize), but no significant differences were identified between the GM maize and the non-GM comparator. (f): Endpoints with significant differences between the six-event stack maize and its non-GM comparator and falling in equivalence category III/IV. In forage, none. In grain, untreated only: none. Treated only: protein, glycine, leucine, lysine and manganese. Both treated and untreated: arginine. Quantitative results for these endpoints are reported in Table 7. (g): Endpoints that were not categorised for equivalence and for which no significant differences were identified between the six-event stack maize and the non-GM comparator: ADF in forage (treated only) and NDF in forage (both treated and not treated). (h): Level of ADF in forage (not treated only) was not categorised for equivalence and a significant difference was identified between the six-event stack maize and the non-GM comparator. Quantitative results are reported in Table 7. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 19 EFSA Journal 2021;19(1):6351 3.4.2.7. Conclusions on the comparative analysis Taking into account the natural variability observed for the set of non-GM reference varieties, the GMO Panel concludes that: •None of the differences identified in the agronomic and phenotypic characteristics between the six-event stack maize and the non-GM comparator needs further assessment, except for the changes in root lodged plants. These differences are further assessed for their potential environmental impact in Section 3.4.4. •None of the differences identified in forage and grain composition between the six-event stack maize and the non-GM comparator needs further food/feed safety assessment except for the changes in levels of ADF in forage and protein, arginine, glycine, leucine, lysine and manganese in grain. These differences are further discussed in Section 3.4.3. 3.4.3. Food/Feed safety assessment 3.4.3.1. Effects of processing Maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 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 six-event stack maize 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 The effects of temperature and pH on proteins CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 newly expressed in this six-event stack maize have been previously evaluated by the GMO Panel (Table 1). No new information has been provided in the context of this application. Table 7: Quantitative results (estimated means and equivalence limits) for compositional endpoints in maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 that are further assessed based on the results of the statistical analysis Endpoint Maize MON 87427 3MON 87460 3 MON 89034 31507 3 MON 87411 359122 Non-GM comparator Non-GM reference varieties Not treated (a) Treated (a) Mean Equivalence limits Forage ADF (% dw) 25.78* 25.05 24.18 24.00 – Grain Protein (% dw) 10.78* 11.08* 10.21 9.88 8.82–10.94 Arginine (% AA) 4.57* 4.45* 4.70 4.90 4.58–5.22 Glycine (% AA) 3.56* 3.46 * 3.65 3.76 3.47–4.06 Leucine (% AA) 13.39* 13.57* 13.10 12.90 12.33–13.48 Lysine (% AA) 2.59 2.47* 2.64 2.83 2.58–3.08 Manganese (mg/kg dw) 6.87* 7.38* 6.54 5.98 4.69–7.28 For maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122, significantly different values are marked with an asterisk, while the outcomes of the test of equivalence are differentiated by greyscale backgrounds. A white background is used for equivalence category I and II and for ADF in forage (which was not categorised for equivalence); light and dark grey backgrounds correspond to equivalence category III and IV, respectively. dw: dry weight; %AA: percentage total amino acid. (a): Treated/not treated with glyphosateand glufosinate-ammonium-containing herbicides. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 20 EFSA Journal 2021;19(1):6351 3.4.3.3. Toxicology Testing of newly expressed proteins 18 Ten proteins (CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1) are newly expressed in the six–event stack maize (Section 3.4.1). The GMO Panel has previously assessed these proteins in the context of the single maize events and no safety concerns were identified for humans and animals. The GMO Panel is not aware of any other new information that would change its previous conclusions on the safety of these proteins. The potential for a functional interaction between the proteins newly expressed in the six-event stack maize was assessed by the GMO Panel with regard to human and animal health. The CP4 EPSPS and PAT proteins are enzymes that catalyse distinct biochemical reactions and act on unrelated substrates in the plant with high substrate specificity. The CspB protein is an RNA chaperone associated with enhanced abiotic stress tolerance in bacteria and plants, through its interaction with RNA secondary structures, limiting their misfolding and allowing cells to maintain cellular functions under various stress conditions (Phadtare et al., 2002a,b; Castiglioni et al., 2008). The NPTII protein inactivates by phosphorylation a range of aminoglycoside antibiotics (Fraley et al., 1983). The insecticidal proteins Cry1A.105, Cry2Ab2, Cry1F, Cry3Bb1, Cry34Ab1 and Cry35Ab1 are deltaendotoxins acting through cellular receptors found in target insect species. It is reported that the gastrointestinal tract of mammals, including humans, lacks receptors with high affinity to Cry proteins (Hammond et al., 2013; Koch et al., 2015). On the basis of the known biological function of the individual newly expressed proteins (Table 3), there is currently no expectation for their possible interactions relevant to the food and feed safety of this six-event stack maize. In vitro protein degradation studies on CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 proteins have been previously evaluated by the GMO Panel (Table 1) and no indications of safety concerns were identified. The GMO Panel concludes that there are no safety concerns to human and animal health related to the proteins CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 newly expressed in maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122. Testing of new constituent other than proteins 19 No new constituents other than the newly expressed proteins have been identified in maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122, with the exception of the intended expression of DvSnf7 dsRNA and derived siRNAs, designed to control coleopteran pests via RNAi. The GMO Panel has previously assessed these compounds in the context of the single maize event MON 87411 (EFSA, 2018) and concluded that no safety concerns are associated with the presence of these compounds. The GMO Panel is not aware of any other new information that would change its previous conclusions on the safety of these compounds. On the basis of the known biological function of these constituents (Table 3), there is currently no expectation for their possible interactions with other new compounds (newly expressed proteins) or other constituents relevant to the food and feed safety of this six–event stack maize. Information on altered levels of food and feed constituent 20 Acid detergent fibre in forage (not treated with the intended herbicides), and protein, glycine, leucine, lysine and manganese in grain (treated with the intended herbicides) and arginine in grain (under both herbicide regimens treatments) were significantly different in the six-stack maize when compared with its non-GM comparator and showed a lack of equivalence with the non-GM reference varieties (Section 3.4.2.6). No toxicological concern is identified regarding these compounds. Further information on safety is provided in Section 3.4.3.6. 18 Dossier: PartII –Section 1.4.1. 19 Dossier: PartII –Section 1.4.2. 20 Dossier: PartII –Section 1.4.3. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 21 EFSA Journal 2021;19(1):6351 Testing of the whole genetically modified food and feed 21 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 interactions between the transformation events, and no modifications of toxicological concern in the composition of the six-stack maize have been identified (see Sections 3.4.1.4,3.4.2.7 and 3.4.3) Therefore, animal studies on food/feed derived from the six-event stack maize are not necessary (EFSA GMO Panel, 2011a). In accordance to Regulation (EU) No 503/2013, the applicant provided 90-day oral repeated-dose toxicity studies in rats on whole food and feed from each of the maize single-events composing the six-event stack maize. 90-Day studies on maize MON 87427, MON 87460, MON 89034, and MON 87411 The GMO Panel had previously concluded that these studies are in line with Reg (EU) No 503/2013 and do not show adverse effects related to diets incorporating the single-event maize crops (MON 87427, MON 87460 and MON 89034 in EFSA GMO Panel (2019a); MON 87411 in EFSA GMO Panel (2018a,b,c). 90-Day study on maize 59122 A 90-day study on maize 59122 had been previously assessed by the GMO Panel in the context of the single-event application dossier (EFSA, 2007). Upon EFSA’s request to fulfil the requirements of Regulation (EU) No 503/2013, the applicant confirmed that the test material was treated with the intended herbicide (glufosinate-ammonium-containing herbicide). Therefore, the GMO Panel concluded that this study is in line with the legal requirements and confirmed that do not show adverse effects related to a diet incorporating the single-event maize 59122. The incorporation rate of maize in this study is around 35%, 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 The GMO Panel had previously assessed a 90-day study on maize 1507 in the context of the single-event application (EFSA, 2005a). Kernels used in that study were obtained from 1507 maize plants that had not been treated with the intended herbicide (glufosinate-ammonium-containing herbicide). Upon EFSA’s request to fulfil the requirements of Regulation (EU) No 503/2013, the applicant provided a new 90-day 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 (test material, high dose), from the conventional counterpart (nonGM comparator, control material), or one of three non-transgenic 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 (2018), aligned with EFSA Scientific Committee guidance (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 polymerase chain reaction (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 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 date 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 21 Dossier: PartII –Section 1.4.4; additional information 10/12/2018; 17/1/2019; 14/3/2019; 27/2/2020 and 16/7/2020; spontaneous information 20/8/2019. 22 Non-transgenic commercial reference maize hybrids: P0760, P0589 and XL5840. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 22 EFSA Journal 2021;19(1):6351 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. In-life procedures and observations and terminal procedures were conducted in accordance to 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 endpoints. Detailed description of statistically significant findings identified in rats given diets containing maize 1507 is reported in Appendix C. 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 the allergenicity assessment, a weight-of-evidence approach was followed, taking into account all the information obtained on the newly expressed proteins, as no single piece of information or experimental method yields sufficient evidence to predict allergenicity and adjuvanticity (Codex Alimentarius, 2009; EFSA GMO Panel, 2011a; Commission Regulation (EU) No 503/2013). Furthermore, an assessment of specific newly expressed proteins in relation to their potential to cause celiac disease was also performed (EFSA GMO Panel, 2017e). Assessment of allergenicity of the newly expressed proteins 23 For allergenicity, the GMO Panel has previously evaluated the safety of CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 proteins individually, and no evidence of allergenicity was identified in the context of the applications assessed (Table 1). No new information on allergenicity of the proteins newly expressed in this six-event stack maize that might change the previous conclusions of the GMO Panel has become available. Based on the 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 interaction in this six–event stack maize. The GMO Panel has previously evaluated the safety of the newly expressed proteins, and no evidence of adjuvanticity were identified in the context of the applications assessed (Table 2). More recently, this aspect has been discussed in detail by EFSA (EFSA, 2018; Parenti et al., 2019). To date, there is no evidence for adjuvanticity in the GMOs assessed by the Panel. This six–event stack maize 23 Dossier: Part II –Sections 1.5.1 and 1.5.3. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 23 EFSA Journal 2021;19(1):6351 has similar 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 six-event stack maize might be adjuvants able to enhance an allergic reaction. The applicant provided spontaneous information on the safety of the CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2 and Cry3Bb1 proteins regarding their potential to cause a celiac disease response. 24,25 For such assessment, the applicant followed the principles described in the EFSA GMO Panel guidance document (EFSA GMO Panel, 2017a–e). The assessment of these proteins identified no perfect or relevant partial matches with known celiac disease peptide sequences. These partial matches have been previously assessed by the EFSA GMO Panel (2019a,b,f, 2020), and no indications of safety concerns were identified. Assessment of allergenicity of the whole GM plant 26 The GMO Panel regularly reviews the available publications on food allergy to maize. However, maize is not considered a common allergenic food 27 (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 (see Sections 3.4.1,3.4.2 and 3.4.3), the GMO Panel identifies no indications of a potentially increased allergenicity of food and feed derived from this six-event stack maize with respect to that derived from the 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 CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 proteins newly expressed in MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 maize. Dietary exposure was estimated based on protein expression levels reported in this application for the six-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. Table 8describes the protein expression levels derived from replicated field trials in United States during 2014 (five locations, four replicates) and used to estimate both human and animal dietary exposure to CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 proteins. Table 8: Mean values (n =20, lg/g dry weight and lg/g fresh weight) for newly expressed proteins in grains and forage from MON 87427 3MON 87460 3MON 89034 3 1507 3MON 87411 359122 maize treated with a combination of the intended herbicides (a) Protein Tissue/developmental stage Grains/R6 (b) (lg/g dry weight and lg/g fresh weight) Forage/R5 (lg/g dry weight) CP4 EPSPS (c) 12/10 200 CspB 0.11/0.099 0.13 NPTII 0.0058/0.0053 (d) 0.24 Cry1A.105 (e) 15/14 43 Cry2Ab2 2.4/2.2 40 Cry1F 2.6/2.3 7.3 24 It is pointed out that the requirements laid down in the EFSA guidance on allergenicity (EFSA GMO Panel, 2017a–e) are not applicable to this dossier, as described in Section 1.5 ‘Transition period’of the above guidance document. 25 Additional information: 18/6/2019 and 29/5/2020. 26 Dossier: Part II –Section 1.5.2. 27 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 MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 24 EFSA Journal 2021;19(1):6351 Human and animal dietary exposure assessment to DvSnf7 dsRNA and its derived siRNAs was not conducted because these molecules are generally rapidly denaturated, depurinated and degraded shortly after ingestion, and therefore they are considered generally not to exert any biological effects once ingested by humans and animals (EFSA GMO Panel, 2018a). Human dietary exposure 28 Human dietary exposure was estimated across different European countries on different population groups 29 : young population (infants, toddlers, ‘other children’), adolescents, adult population (adults, elderly and very elderly) and special populations (pregnant and lactating women). Mean protein expression values on fresh weight basis (Table 8) are considered as the most adequate to estimate human dietary exposure (both acute and chronic) when working with raw primary commodities that are commonly consumed as processed blended commodities (EFSA, 2019). Since no specific consumption data were available on commodities containing, consisting of or obtained from the six-event stack 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). 30 Corn oil was excluded from the assessment since no proteins are expected to be present in the oil. For the acute dietary exposure estimations, the applicant directly assigned to processed commodities the mean value reported for the concentration of the newly expressed proteins in maize grains (Table 8). Overall, this is a conservative approach as neither recipes nor the effect of processing on the final concentration of newly expressed proteins are considered, except for corn oil which is eventually excluded from the exposure estimations. Summary statistics from the EFSA consumption database were used. 31 Acute dietary exposure in high consumers within each dietary survey and age class (toddlers, ‘other children’, adolescents, adults, elderly) was estimated by summing the exposure derived from the 95th percentile consumption for the dominant food commodity 32 among consumers only and those exposures derived from the mean consumption of the remaining food categories in the total population (EFSA, 2015). The highest acute dietary exposure was estimated in the age class ‘Toddlers’with exposure estimates that ranged between 0.04 lg/kg body weight (bw) per day and 260 lg/kg bw per day for NPTII and Cry34Ab1 proteins, respectively. The most relevant food commodities in terms of contribution to the dietary exposure were sweet corn. The GMO Panel estimated chronic dietary exposure to CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2, Cry1F, PAT, Cry3Bb1, Cry34Ab1 and Cry35Ab1 proteins. Individual consumption data of the Protein Tissue/developmental stage Grains/R6 (b) (lg/g dry weight and lg/g fresh weight) Forage/R5 (lg/g dry weight) PAT (c) 0.025 (f) 0.7 Cry3Bb1 5.1/4.6 46 Cry34Ab1 36/32 87 Cry35Ab1 1.1/1.0 21 (a): Intended herbicides: glyphosate and glufosinate–ammonium. (b): Fresh weight values for CP4 EPSPS, CspB, NPTII, Cry1A.105, Cry2Ab2 and Cry3Bb proteins used to estimate human dietary exposure were calculated by multiplying the dry weight values by a dry weight correction factor (DWCF). Similarly, a DWCF was also used to convert the fresh weight values for Cry1F, PAT, Cry34Ab1, and Cry35Ab1 to dry weight values. (c): CP4 EPSPS levels are the result of CP4 EPSPS protein expressed in MON 87427 and MON 87411. Likewise, PAT levels are the result of PAT protein expressed in maize 1507 and 59122. (d): N =15 since five samples were reported as below the LOQ (LOQ =0.005 lg/g fw). (e): Expression levels for Cry1A.105 are not corrected for the cross-reactivity between Cry1F and Cry1A.105 observed in the ELISA for Cry1A.105. As a consequence, the Cry1A.105 expression levels are overestimated by ~7%. (f): PAT protein levels were below the limit of detection in all grain samples (LOD =0.025 lg/kg). The reported LOD was used for both human and animal dietary exposure estimations. 28 Dossier: Part II –Section 2.4 and Additional information 26/8/2019. 29 For infants, very elderly population and vulnerable groups only chronic dietary exposure estimations are provided. 30 http://www.efsa.europa.eu/en/data/food-consumption-data 31 Summary statistics from the EFSA Comprehensive European Food Consumption Database accessed in September 2016. 32 Dominant food commodity refers to the food that will lead to the highest exposure among all consumed foods. Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 25 EFSA Journal 2021;19(1):6351 3.5.2.1. Stability of the events The genetic stability of the inserted DNA over multiple generations in the six single maize events was demonstrated previously (see Table 2). Integrity of the events was demonstrated in the six-event stack maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 (Section 3.4.1.2) and the previously assessed maize subcombinations (see 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 9). 3.5.2.2. Expression of the events The GMO Panel assessed whether any combination of the six events by conventional crossing could result in significant changes in expression levels of the newly expressed proteins, as this could indicate an unexpected interaction between 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 expressed proteins in the 39 subcombinations compared with those in the single maize events. This assumption was confirmed by comparing the levels of the newly expressed proteins of each single maize event with those of the six-event stack maize. The levels were similar in the six-event stack maize and in the single events except for CP4 EPSPS and PAT, which showed, in general, the expected higher level in the stack resulting from the combination of the single events MON 87427 and MON 87411 for the CP4 EPSPS and the combination of 1507 and 59122 for the PAT (Section 3.4.1.3 and Appendix B). In addition, the potential impact of the DvSnf7 dsRNA on the levels of the newly expressed proteins was assessed by comparing the protein expression levels in the six-event stack and the respective singles. The data indicate that there is no impact of the DvSnf7 dsRNA on the expression levels of the newly expressed proteins. This supports the conclusion that interactions affecting the expression levels of the newly expressed proteins are not expected in the 39 subcombinations not previously assessed and included in the scope of application EFSA-GMO-NL-2017-139. 3.5.2.3. Potential functional interactions between the events The GMO Panel assessed the potential for interactions between maize events in the 39 subcombinations not previously assessed (Table 9), taking into consideration intended traits and unintended effects. Based on the known biological functions of the individual newly expressed proteins and dsRNA (Table 4), there is currently no expectation for possible interactions relevant for the food and feed or environmental safety between these proteins in those subcombinations. The GMO Panel took into account all the intended and potential unintended effects considered in the assessment of the six single events, the previously assessed subcombinations (Table 2) and the six-event stack maize. It is concluded that none of these events would raise safety concerns when combined in any of these maize subcombinations. The GMO Panel considers that no further data are needed to complete the assessment of subcombinations from the six-event stack maize. 3.5.3. Conclusion Since no new safety concerns were identified for the previously assessed subcombinations, the GMO Panel considers that its previous conclusions on these maize subcombinations remain valid. For the remaining 39 subcombinations included in the scope of application EFSA-GMO-NL-2017-139, for which no experimental data have been provided, the GMO Panel assessed the possibility of interactions between the events and concluded that these combinations would not raise safety concerns. These subcombinations are therefore expected to be as safe as the single maize events, the previously assessed subcombinations and the six-event stack maize. 3.6. Post-market monitoring 3.6.1. Post-market monitoring of GM food/feed The GMO Panel concluded that the six-event stack maize, as described in this application, does not raise any nutritional concern and is as safe as the non-GM comparator and the selected non-GM reference varieties (Section 3.4.3.7). Seventeen of the subcombinations have been previously assessed and no safety concerns were identified. The 39 subcombinations not previously assessed and included in the scope of this application are expected to be as safe as the single maize events, the previously Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 32 EFSA Journal 2021;19(1):6351 assessed maize subcombinations and the six–event stack maize (Section 3.5.3). Therefore, the GMO Panel considers that post-market monitoring of food and feed from the six-event stack maize and its subcombinations, as described in this application, is not necessary. 3.6.2. Post-market environmental monitoring The objectives of a post-market environmental monitoring (PMEM) plan, according to Annex VII of Directive 2001/18/EC, are: 1) to confirm that any assumption regarding the occurrence and impact of potential adverse effects of the GMO, or its use, in the ERA are correct; and 2) to identify the occurrence of adverse effects of the GMO, or its use, on human health or the environment that were not anticipated in the ERA. Monitoring is related to risk management, and thus, a final adoption of the PMEM plan falls outside the mandate of EFSA. However, the GMO Panel gives its opinion on the scientific rationale of the PMEM plan provided by the applicant (EFSA GMO Panel, 2011b). As the ERA does not identify potential adverse environmental effects from the six-event stack maize, no case-specific monitoring is required. The PMEM plan proposed by the applicant for the six-event stack maize and its subcombinations includes: (1) the description of a monitoring approach involving operators (federations involved in import and processing), reporting to the applicant, via a centralised system, any observed adverse effect(s) of GMOs on human health and the environment; (2) a coordinating system established by EuropaBio for the collection of information recorded by the various operators; and (3) the review of relevant scientific publications retrieved from literature searches (Lecoq et al., 2007; Windels et al., 2008). The applicant proposes to submit a PMEM report on an annual basis and a final report at the end of the authorisation period. The GMO Panel considers that the scope of the PMEM plan provided by the applicant is consistent with the intended uses of the six-event stack maize. The GMO Panel agrees with the reporting intervals proposed by the applicant in its PMEM plan. The PMEM plan and reporting intervals are in line with the intended uses of the six-event stack maize and its subcombinations. In the context of PMEM, the applicant should improve the literature searches according to the GMO Panel recommendations given in Section 3.3. 3.6.3. Conclusion on post-market monitoring No PMM of food and feed is necessary. The scope of the PMEM plan provided by the applicant and the reporting intervals are in line with the intended uses of maize MON 87427 9MON 87460 9 MON 89034 91507 9MON 87411 959122. 4. Overall conclusions The GMO Panel was asked to carry out a scientific assessment of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 and subcombinations for import, processing and food and feed uses in accordance with Regulation (EC) No 1829/2003. No new information was identified on the six single maize events (MON 87427, MON 87460, MON 89034, 1507, MON 87411 and 59122) that would lead to a modification of the original conclusions on their safety. The molecular characterisation, the 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 and the dsRNA in the six-event stack maize does not give rise to food/feed safety and nutritional concerns. The GMO Panel concludes that the six-event stack maize, as described in this application, does not raise any nutritional concern and is as safe as its non-GM comparator and the selected non-GM reference varieties. The GMO Panel concludes that there is a very low likelihood of environmental effects resulting from the accidental release of viable grains from the six-event stack maize into the environment. Since no new data were identified on the 17 previously assessed subcombinations that would lead to a modification of the original conclusions on their safety, the GMO Panel considers that its previous conclusions on these maize stacks remain valid. For the remaining 39 subcombinations included in the scope of application EFSA-GMO-NL-2017-139, no information has been provided. The GMO Panel assessed the possible interactions between the events in the 39 subcombinations and concludes that these combinations of events MON 87427, MON 87460, MON 89034, 1507, MON 87411 and Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 33 EFSA Journal 2021;19(1):6351 59122 would not raise safety concerns. These subcombinations are therefore expected to be as safe as the maize single events, the previously assessed subcombinations and the six-event stack maize. The literature searches did not identify any relevant publications on maize MON 87427, MON 87460, MON 89034, 1507, MON 87411 and 59122. In the context of annual PMEM reports, the applicant could further fine-tune future literature searches according to the GMO Panel recommendations. In addition, the GMO Panel considered the additional unpublished studies listed in Appendix A. This new information does not raise any concern for human and animal health and the environment regarding the six-event stack maize and its subcombinations. Given the absence of safety and nutritional concerns for foods and feeds from the six-event stack maize and all its subcombinations, the GMO Panel considers that PMM of these products is not necessary. The PMEM plan and reporting intervals are in line with the intended uses of the six-event stack maize and its subcombinations. In conclusion, the GMO Panel considers that maize MON 87427 9MON 87460 9MON 89034 9 1507 9MON 87411 959122 and its subcombinations, as described in this application, are as safe as the non-GM comparator and the selected non-GM reference varieties with respect to potential effects on human and animal health and the environment. 5. Documentation as provided to EFSA 1) Letter from the Competent Authority of Netherlands received on 21 February 2017 concerning a request for authorisation of the placing on the market of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 (EFSA-GMO-NL-2017139) submitted in accordance with Regulation (EC) No 1829/2003 by Monsanto Company. 2) Application EFSA-GMO-NL-2017-139 validated by EFSA, 31 May 2017. 3) Request for supplementary information to the applicant, 1 June 2018. 4) Request for supplementary information to the applicant, 6 July 2018. 5) Request for supplementary information to the applicant, 17 July 2018. 6) Receipt of supplementary information from the applicant, 3 September 2018. 7) Receipt of supplementary information from the applicant, 17 September 2018. 8) Request for supplementary information to the applicant, 16 October 2018. 9) Request for supplementary information to the applicant, 13 November 2018. 10) Receipt of supplementary information from the applicant, 10 December 2018. 11) Receipt of supplementary information from the applicant, 19 January 2019. 12) Receipt of supplementary information from the applicant, 14 March 2019. 13) Request for supplementary information to the applicant, 18 March 2019. 14) Request for supplementary information to the applicant, 16 April 2019. 15) Request for supplementary information to the applicant, 21 May 2019. 16) Receipt of supplementary information from the applicant, 19 June 2019. 17) Supplementary information submitted spontaneously by the applicant, 20 August 2019. 18) Receipt of supplementary information from the applicant, 27 August 2019. 19) Supplementary information submitted spontaneously by the applicant, 29 August 2019. 20) Receipt of supplementary information from the applicant, 30 August 2019. 21) Request for supplementary information to the applicant, 15 October 2019. 22) Receipt of supplementary information from the applicant, 17 December 2019. 23) Receipt of supplementary information from the applicant, 27 February 2020. 24) Supplementary information submitted spontaneously by the applicant, 18 March 2020. 25) Request for supplementary information to the applicant, 4 May 2020. 26) Supplementary information submitted spontaneously by the applicant, 29 May 2020. 27) Supplementary information submitted spontaneously by the applicant, 16 July 2020. 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Scientific opinion on an application for renewal of authorisation for continued marketing of maize 1507 and derived food and feed submitted under Articles 11 and 23 of Regulation (EC) No 1829/2003 by Pioneer Overseas Corporation and Dow AgroSciences LLC. EFSA Journal 2017;15(1):4659, 11 pp. https://doi.org/10.2903/j.efsa. 2017.4659 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F,Wal J-M, Alvarez F, Ardizzone M and Paraskevopoulos K, 2017b. Scientific opinion on an application for renewal of authorisation for continued marketing of maize 59122 and derived food and feed submitted under articles 11 and 23 of Regulation (EC) No 1829/2003 by Pioneer Overseas Corporation and Dow AgroSciences LLC. EFSA Journal 2017;15(6):4861, 10 pp. https://doi.org/10.2903/j.efsa.2017.4861 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 36 EFSA Journal 2021;19(1):6351 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Alvarez F, Lanzoni A and Paraskevopoulos K, 2017c. Scientific Opinion on application EFSA-GMO-BE-2013-118 for authorisation of genetically modified maize MON 87427 9MON 89034 91507 9MON 88017 959122 and subcombinations independently of their origin, for food and feed uses, import and processing submitted under Regulation (EC) No 1829/2003 by Monsanto Company. EFSA Journal 2017;15(8):4921, 32 pp. https://doi.org/10.2903/j.efsa.2017.4921 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Gennaro A, Neri FM and Paraskevopoulos K, 2017d. Scientific Opinion on application EFSA-GMO-BE-2013-117 for authorisation of genetically modified maize MON 87427 9MON 89034 9NK603 and subcombinations independently of their origin, for food and feed uses, import and processing submitted under Regulation (EC) No 1829/2003 by Monsanto Company. EFSA Journal 2017;15 (8):4922, 26 pp. https://doi.org/10.2903/j.efsa.2017.4922 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Messean A, Nielsen EE, Nogue F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Eigenmann P, Epstein M, Hoffmann-Sommergruber K, Koning F, Lovik M, Mills C, Moreno FJ, van Loveren H, Selb R and Fernandez Dumont A, 2017e. Guidance on allergenicity assessment of genetically modified plants. EFSA Journal 2017;15(5):4862, 49 pp. https://doi.org/10.2903/j.efsa.2017.4862 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak AM, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Ardizzone M, De Sanctis G, Fernandez Dumont A, Gennaro A, G omez Ruiz JA, Lanzoni A, Neri FM, Papadopoulou N, Paraskevopoulos K and Ramon M, 2018a. Scientific Opinion on the assessment of genetically modified maize MON 87411 for food and feed uses, import and processing, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2015-124). EFSA Journal 2018;16(6):5310, 29 pp. https://doi.org/10.2903/j.efsa.2018.5310 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Ardizzone M, Federici S, Fernandez Dumont A, Gennaro A, Gomez Ruiz J A, Lanzoni A, Neri FM, Papadopoulou N and Paraskevopoulos K, 2018b. Scientific Opinion on the assessment of genetically modified maize Bt11 9MIR162 91507 9GA21 and three subcombinations independently of their origin, for food and feed uses under Regulation (EC) No 1829/2003 (application EFSA-GMO-DE-2010-86). EFSA Journal 2018;16(7):5309, 35 pp. https://doi.org/10.2903/j.efsa.2018.5309 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Mess ean A, Nielsen EE, Nogu e F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F,Wal J-M, Alvarez F, Ardizzone M, De Sanctis G, Fernandez Dumont A, Ruiz Gomez JA, Lanzoni A, Papadopoulou N and Paraskevopoulos K, 2018c. Scientific Opinion on the assessment of genetically modified maize 1507 9NK603 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA-GMORX-008). EFSA Journal 2018;16(7):5347, 11 pp. https://doi.org/10.2903/j.efsa.2018.5347 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 and Raffaello T, 2019a. Assessment of genetically modified maize MON 89034 for renewal authorisation under Regulation (EC) No 1829/2003 (application EFSAGMO-RX-015). EFSA Journal 2019;17(11):5845, 10 pp. https://doi.org/10.2903/j.efsa.2019.5845 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, 2019b. 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 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, Neri FM, Papadopoulou N and Paraskevopoulos K, 2019c. 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-NL2017-144). EFSA Journal 2019;17(11):5848, 33 pp. https://doi.org/10.2903/j.efsa.2019.5848 Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 37 EFSA Journal 2021;19(1):6351 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, Fernandez Dumont A, Gennaro A, G omez Ruiz JA, Lanzoni A, Neri FM, Papadopoulou N and Ramon M, 2019d. Scientific Opinion on the assessment of genetically modified maize MON 89034 91507 9NK603 9DAS-40278-9 and subcombinations independently of their origin for food and feed uses, import and processing, under Regulation (EC) No 1829-2003 (application EFSAGMONL-2013-112). EFSA Journal 2019;17(1):5522, 30 pp. https://doi.org/10.2903/j.efsa.2019.5522 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, Ardizzone M, Fernandez Dumont A, Gennaro A, G omez Ruiz JA, Lanzoni A, Neri FM, Papadopoulou N and Ramon M, 2019e. Scientific Opinion on the assessment of genetically modified maize MON 89034 91507 9MON 88017 959122 9DAS-40278-9 and subcombinations independently of their origin for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2013-113). EFSA Journal 2019;17(1):5521, 30 pp. https://doi.org/10.2903/j.efsa.2019.5521 EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Bresson JL, Dalmay JL, 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, Dumont AF, Gennaro A, G omez Ruiz J  A, Lanzoni A, Neri FM, Papadopoulou N, Paraskevopoulos K and Raffaello T, 2019f. Scientific Opinion on the assessment of genetically modified soybean MON 87751 9MON 87701 9MON 87708 9MON 89788 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2016-128). 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Journal Animal Science Biotechnology, 5, 34. https://doi.org/10.1186/2049-1891-5-34 Abbreviations AA amino acids ADF acid detergent fibre Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 39 EFSA Journal 2021;19(1):6351 BUN blood urea nitrogen bw body weight CaMV cauliflower mosaic virus CspB cold shock protein B CRM comparative relative maturity CTP chloroplast transit peptide dsRNA double strand RNA dw dry weight ELISA enzyme-linked immunosorbent assay EPSPS 5-enolpyruvylshikimate-3-phosphat synthase ERA environmental risk assessment FMV Figwort Mosaic Virus fw fresh weight GM genetically modified GMO genetically modified organism GMO Panel EFSA Panel on Genetically Modified Organisms HGT horizontal gene transfer hsp heat shock proteins LOQ limit of quantification MS Member States NDF neutral detergent fibre NOAEL no-observed-adverse-effect level nos nopaline synthase NPTII neomycin phosphotransferase II 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 RNA ribonucleic acid RNAi RNA interference siRNA small interfering RNA TDF total dietary fibre TSH thyroid hormones UL tolerable upper intake level UTR untranslated region WCR western corn rootworm Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 40 EFSA Journal 2021;19(1):6351 Appendix A –Additional studies List of additional studies performed by or on behalf of the applicant with regard to the evaluation of the safety of maize MON 87427 9MON 87460 9MON 89034 91507 9MON 87411 959122 for humans, animal or the environment Study identification Title Hoi and Asiimwe (2015) Phenotypic Evaluation and Environmental Interactions of Maize MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS-59122-7 in 2014 U.S. Field Trials. Hoi (2015) Phenotypic Evaluation of Maize MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS-59122-7 with Herbicide Treatments in 2014 U.S. Field Trials. Joshi et al. (2016) Comparison of Lipid Transfer Protein (LTP) Expression Levels from MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS–59122–7 with Conventional Control Maize. Klusmeyer et al. (2016a) Compositional Analyses of Maize Forage and Grain from MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS-59122-7 Grown in the United States in 2014. Klusmeyer et al. (2016b) Compositional Analyses of Maize Forage and Grain from MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS–59122–7 Grown in the United States in 2014: Individual Site Analysis. Mueller and Uffman (2016) An evaluation of the potential for interaction between MON 87460 and the insecticidal traits in the combined maize product MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS–59122–7 with the European Corn Borer (Ostrinia nubilalis). Skottke and Malven (2015a) Amended Report for MSL0026334: Southern Blot Analyses to Confirm the Presence of TC1507 and DAS–59122–7 in the Combined Trait Maize Product MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS–59122–7. Skottke and Malven (2015b) Southern Blot Analyses to Confirm the Presence of MON 87427, MON 87460, MON 89034 and MON 87411 in the Combined Trait Maize Product MON 87427 9MON 87460 9MON 89034 9TC1507 9MON 87411 9DAS–59122–7. MSL0027263 An Acute Oral Gavage Toxicity Study of E. coli-produced Cry3Bb1 protein in CD–1 Mice Assessment of GM maize MON 87427 3MON 87460 3MON 89034 31507 3MON 87411 359122 www.efsa.europa.eu/efsajournal 41 EFSA Journal 2021;19(1):6351