Assessment of genetically modified maize NK603 × T25 × DAS-40278-9 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2019-164)

SCIENTIFIC OPINION ADOPTED: 29 October 2021 doi: 10.2903/j.efsa.2021.6942 Assessment of genetically modified maize NK603 3T25 3DAS-40278-9 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2019-164) EFSA Panel on Genetically Modified Organisms (GMO), Ewen Mullins, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Hanspeter Naegeli, Francisco Javier Moreno, Fabien Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann, Fabio Veronesi, Michele Ardizzone, Giacomo De Sanctis, Antonio Fernandez Dumont, Silvia Federici, Andrea Gennaro, Jose Angel Gomez Ruiz, Dafni Maria Kagkli, Anna Lanzoni, Franco Maria Neri, Nikoletta Papadopoulou, Konstantinos Paraskevopoulos and Tommaso Raffaello Abstract Maize NK603 9T25 9DAS-40278-9 (three-event stack maize) was produced by conventional crossing to combine three single events: NK603, T25 and DAS-40278-9. The GMO Panel previously assessed the three single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the two 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 in the three-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the three-event stack maize, as described in this application, is as safe as the non-GM comparator and the selected non-GM reference varieties. In the case of accidental release of viable grains of the three-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 one of the 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 three-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the three-event stack maize. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the three-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: GMO, herbicide tolerant, maize (Zea mays), NK603, T25, DAS-40278-9, import and processing Requestor: Competent Authority of The Netherlands Question number: EFSA-Q-2019-00808 Correspondence: GMO_secretariat_appl[email protected] EFSA Journal 2021;19(12):6942www.efsa.europa.eu/efsajournal Panel members: Ewen Mullins, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Hanspeter Naegeli, Francisco Javier Moreno, Fabien Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Declarations of interest: The declarations of interest of all scientific experts active in EFSA’s work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgments: The Panel wishes to thank the members of the Working Groups on Molecular Characterisation, Food and Feed Safety Assessment and Working Group On Comparative Analysis and Environmental Risk Assessment for the preparatory work on this scientific output and EFSA staff members Fernando  Alvarez, Giuseppe Condorelli, Aleksandra Lewandowska, Sylvie Mestdagh, Lorenz Oberkofler, Pietro Piffanelli, Sonia Hern andez Valero, Irina Vlas and Riccardo Vriz for the support provided to this scientific output. Suggested citation: EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins E, Bresson J-L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Naegeli H, Moreno FJ, Nogu e F, Rostoks N, S anchez Serrano JJ, Savoini G, Veromann E, Veronesi F, Ardizzone M, De Sanctis G, Fernandez Dumont A, Federici S, Gennaro A, Gomez Ruiz AJ, Kagkli DM, Lanzoni A, Neri FM, Papadopoulou N, Paraskevopoulos K and Raffaello T, 2021. Scientific Opinion on the Assessment of genetically modified maize NK603 9T25 9DAS-40278-9 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2019-164). EFSA Journal 2021;19(12):6942, 35 pp. https://doi.org/10.2903/j.efsa.2021.6942 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, a European agency funded by the European Union. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 2 EFSA Journal 2021;19(12):6942 Summary Following the submission of application EFSA-GMO-NL-2019-164 under Regulation (EC) No 1829/ 2003 from Pioneer Hi-Bred International, Inc. as represented by Pioneer Overseas Corporation (referred to hereafter as ‘the applicant’), the Panel on Genetically Modified Organisms of the European Food Safety Authority (EFSA) (referred to hereafter as ‘GMO Panel’) was asked to deliver a Scientific Opinion on the safety of genetically modified (GM) glufosinate, glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides tolerant maize (Zea mays L.) NK603 9T25 9DAS-40278-9 (referred to hereafter as ‘three-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-2019-164 is for import, processing and food and feed uses within the European Union (EU) of maize NK603 9T25 9DAS440278-9 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 two of the events present in the three-event stack maize. The safety of subcombinations occurring as segregating progeny in the harvested grains of maize NK603 9T25 9DAS-40278-9 is evaluated in the context of the assessment of the three-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 three-event stack, are risk assessed separately in the present scientific opinion. The three-event stack maize was produced by conventional crossing to combine three single maize events: NK603 expressing CP4 EPSPS and CP4 EPSPS L214P to confer tolerance to glyphosatecontaining herbicides; T25 expressing PAT to confer tolerance to glufosinate-ammonium containing herbicides; and DAS-40278-9 expressing AAD-1 to catalyse the degradation of the general class of herbicides known as aryloxyphenoxypropionates (AOPP) and to confer tolerance to 2,4-Dcontaining herbicides. The GMO Panel evaluated the three-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-2019-164, 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-2019-164, previous assessments of the three single events (NK603, T25 and DAS-40278-9), and three of the subcombinations provided a basis for the assessment of the three-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 three 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 three-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 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 NK603 9T15 9DAS-40278-9 have retained their integrity. Protein expression analysis showed that the levels of the newly expressed proteins are similar in the three-event stack maize and in the single events. The comparative analysis of agronomic and phenotypic characteristics and grain and forage composition identified no differences between maize NK603 9T25 9DAS-40278-9 and the non-GM comparator that required further assessment except for the change in plant lodging. This change was further assessed for 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 in the three-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that maize NK603 9T25 9DAS-40278-9, as Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 3 EFSA Journal 2021;19(12):6942 described in this application, is as safe as the non-GM comparator and the selected commercial nonGM 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 NK603 9T25 9DAS-40278-9 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 two 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 subcombination included in the scope of application EFSA-GMO-NL-2019-164, no experimental data were provided. The GMO Panel assessed the possibility of interactions between the events in this subcombination and concludes that this subcombination would not raise safety concerns. This subcombination is therefore expected to be as safe as the single events, the previously assessed subcombinations and the three-event stack maize. Given the absence of safety concerns for foods and feeds from maize NK603 9T25 9DAS-40278-9 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 three-event stack maize and its subcombinations. The literature searches did not identify any relevant publications on maize NK603 9T25 9DAS-40278-9. 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 NK603 9T25 9DAS-40278-9 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 maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 4 EFSA Journal 2021;19(12):6942 Table of contents Abstract................................................................................................................................................... 1 Summary................................................................................................................................................. 3 1. Introduction................................................................................................................................7 1.1. Background ................................................................................................................................7 1.2. Terms of Reference as provided by the requestor .......................................................................... 7 2. Data and methodologies .............................................................................................................. 8 2.1. Data........................................................................................................................................... 8 2.2. Methodologies............................................................................................................................. 8 3. Assessment................................................................................................................................. 8 3.1. Introduction................................................................................................................................8 3.2. Updated information on single events ........................................................................................... 9 3.3. Systematic literature review ......................................................................................................... 9 3.4. Risk assessment of the three-event stack maize NK603 9T25 9DAS-40278-9 ............................... 9 3.4.1. Molecular characterisation............................................................................................................ 9 3.4.1.1. Genetic elements and their biological function ............................................................................... 10 3.4.1.2. Integrity of the events in the three-event stack maize NK603 9T25 9DAS-40278-9....................... 11 3.4.1.3. Information on the expression of the inserts ................................................................................. 11 3.4.1.4. Conclusions of the molecular characterisation................................................................................ 11 3.4.2. Comparative analysis ................................................................................................................... 11 3.4.2.1. Overview of studies conducted for the comparative analysis ........................................................... 11 3.4.2.2. Experimental field trial design and statistical analysis ..................................................................... 12 3.4.2.3. Suitability of selected test materials .............................................................................................. 12 3.4.2.4. Representativeness of the receiving environments ......................................................................... 13 3.4.2.5. Agronomic and phenotypic analysis .............................................................................................. 13 3.4.2.6. Compositional analysis................................................................................................................. 14 3.4.2.7. Conclusions on the comparative analysis ....................................................................................... 15 3.4.3. Food/Feed safety assessment....................................................................................................... 15 3.4.3.1. Effects of processing ................................................................................................................... 15 3.4.3.2. Stability of newly expressed proteins ............................................................................................ 15 3.4.3.3. Toxicology .................................................................................................................................. 15 3.4.3.4. Allergenicity ................................................................................................................................18 3.4.3.5. Dietary exposure assessment to new constituents.......................................................................... 19 3.4.3.6. Nutritional assessment of endogenous constituents........................................................................ 21 3.4.3.7. Conclusion on the food/feed safety assessment ............................................................................. 21 3.4.4. Environmental risk assessment ..................................................................................................... 21 3.4.4.1. Persistence and invasiveness of the GM plant ................................................................................ 21 3.4.4.2. Potential for gene transfer ........................................................................................................... 22 3.4.4.3. Interactions of the GM plant with target organisms........................................................................ 22 3.4.4.4. Interactions of the GM plant with non-target organisms ................................................................. 22 3.4.4.5. Interactions with the abiotic environment and biogeochemical cycles .............................................. 23 3.4.4.6. Conclusion of the environmental risk assessment........................................................................... 23 3.5. Risk assessment of the subcombinations ....................................................................................... 23 3.5.1. Subcombinations previously assessed ........................................................................................... 23 3.5.2. Subcombinations not previously assessed...................................................................................... 23 3.5.2.1. Stability of the events.................................................................................................................. 23 3.5.2.2. Expression of the events.............................................................................................................. 23 3.5.2.3. Potential functional interactions between the events ...................................................................... 24 3.5.3. Conclusions................................................................................................................................. 24 3.6. Post-market monitoring................................................................................................................ 24 3.6.1. Post-market monitoring of GM food/feed....................................................................................... 24 3.6.2. Post-market environmental monitoring.......................................................................................... 24 3.6.3. Conclusion on post-market monitoring .......................................................................................... 25 4. Overall conclusions ...................................................................................................................... 25 5. Documentation as provided to EFSA ............................................................................................. 25 References............................................................................................................................................... 26 Abbreviations ........................................................................................................................................... 29 Appendix A –List of additional studies ....................................................................................................... 31 Appendix B –Protein expression data ........................................................................................................ 32 Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 5 EFSA Journal 2021;19(12):6942 Appendix C –Statistical analysis and statistically significant findings in the 90-day toxicity studies in rats on the whole food/feed ................................................................................................................................. 33 Appendix D –Animal dietary exposure....................................................................................................... 35 Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 6 EFSA Journal 2021;19(12):6942 1. Introduction The scope of application EFSA-GMO-NL-2019-164 is for food and feed uses, import and processing in the European Union (EU) of the genetically modified (GM) herbicide-tolerant maize NK603 9T25 9DAS-40278-9 and all its subcombinations independently of their origin. 1.1. Background On 13 December 2019, the European Food Safety Authority (EFSA) received from the Competent Authority of The Netherlands application EFSA-GMO-NL-2019-164 for authorisation of maize NK603 9T25 9DAS-40278-9 (hereafter referred to as ‘the three-event stack maize’) (Unique Identifier MON-ØØ6Ø3-6 9ACS-ZMØØ3-2 9DAS-4Ø278-9), submitted by Pioneer Hi-Bred International, Inc. as represented by Pioneer Overseas Corporation (hereafter referred to as ‘the applicant’) according to Regulation (EC) No 1829/2003 1 . Following receipt of application EFSA-GMO-NL-2019-164, EFSA informed EU Member States and the European Commission, and made the application available to them. Simultaneously, EFSA published the summary of the application. 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 26 March 2020, EFSA declared the application valid. 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-2019-164. Such time limit was extended whenever EFSA and/or its GMO Panel requested supplementary information to the applicant. According to Regulation (EC) No 1829/2003, any supplementary information provided by the applicant during the risk assessment was made available to the EU Member States and European Commission (for further details, see the Section ‘Documentation’, below). In accordance with Regulation (EC) No 1829/2003, EFSA consulted the nominated risk assessment bodies of EU Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC 4 . The EU Member States had 3 months to make their opinion known on application EFSA-GMO-NL-2019-164 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 NK603 9T25 9DAS-40278-9 and all its subcombinations independently of their origin according to the scope of application EFSA-GMO-NL2019-164. 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 Open. EFSA, 6 including the information required under Annex II to the Cartagena Protocol, a labelling proposal, a post-market environmental monitoring (PMEM) plan as provided by the applicant; the methods, validated by the European Union Reference Laboratory, for detection, including sampling, identification of the transformation events in the food-feed and/or foods-feeds produced from it and the appropriate reference materials. 1 Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 18.10.2003, p. 1–23. 2 Available online: https://open.efsa.europa.eu/questions/EFSA-Q-2019-0080 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 https://open.efsa.europa.eu/ questions/EFSA-Q-2019-00808 6 https://open.efsa.europa.eu/questions/EFSA-Q-2019-00808 Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 7 EFSA Journal 2021;19(12):6942 2. Data and methodologies 2.1. Data The GMO Panel based its scientific assessment of the three-event stack maize on the valid application EFSA-GMO-NL-2019-164, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU Member States and relevant peer-reviewed scientific publications. As part of this comprehensive information package, the GMO Panel received an additional unpublished study submitted by the applicant in order to comply with the specific provisions of Regulation (EU) No 503/2013. This additional unpublished study is provided in Appendix A. 2.2. Methodologies The GMO Panel conducted its assessment in line with the principles described in Regulation (EU) No 1829/2003, the applicable guidelines (i.e. EFSA GMO Panel, 2010a,2011a,b,2015a; EFSA Scientific Committee, 2011) and explanatory notes and statements (i.e. EFSA GMO Panel, 2010b; EFSA, 2010, 2014, 2017a,b, 2019a,b) for the risk assessment of GM plants. For this application, in the context of the contracts OC/EFSA/GMO/2018/04, OC/EFSA/GMO/2018/02 and EOI/EFSA/SCIENCE/2020/01 –CT02GMO the contractors performed preparatory work for the evaluation of the applicant`s literature search methods applied for the statistical analysis and statistical analysis of the 90-day toxicity study on T25 maize. 3. Assessment 3.1. Introduction Application EFSA-GMO-NL-2019-164 covers the three-event stack maize NK603 9T25 9DAS40278-9 and all its three subcombinations independently of their origin (Table 1). The safety of subcombinations occurring as segregating progeny in harvested grains of maize NK603 9T25 9DAS-40278-9 is evaluated in the context of the assessment of the three-event stack maize in Section 3.5 of the present scientific opinion. ‘Subcombination’also covers combinations that have either been or could be produced by conventional crossing through targeted breeding approaches (EFSA GMO Panel, 2011a). These are maize stacks that can be bred, produced and marketed independently of the three-event stack maize. These subcombinations are assessed in Section 3.5 of this scientific opinion. The three-event stack maize was produced by conventional crossing to combine three single maize events: NK603 expressing CP4 EPSPS and CP4 EPSPS L214P to confer tolerance to glyphosate-containing herbicides; T25 expressing PAT to confer tolerance to glufosinate-ammonium-containing herbicides; and DAS-40278-9 expressing AAD-1 to catalyse the degradation of the general class of herbicides known as aryloxyphenoxypropionates (AOPP) and to confer tolerance to 2,4-Dcontaining herbicides. All three single events were assessed previously (see Table 2) and no concerns for human and animal health or environmental safety were identified. Table 1: Stacked maize events covered by the scope of application EFSA-GMO-NL-2019-164 Degree of stacking Event Unique identifiers 3-event stack NK603 9T25 9DAS-40278-9 MON-ØØ6Ø3-6 9ACS-ZMØØ3-2 9DAS-4Ø278-9 2-event stack NK603 9T25 MON-ØØ6Ø3-6 9ACS-ZMØØ3-2 NK603 9DAS-40278-9 MON-ØØ6Ø3-6 9DAS-4Ø278-9 T25 9DAS-40278-9 DAS-4Ø278-9 9ACS-ZMØØ3-2 The term ‘subcombination’refers to any combination of up to two of the maize events NK603, T25 and DAS-40278-9. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 8 EFSA Journal 2021;19(12):6942 3.2. Updated information on single events Since publication of the scientific opinions on the single maize events by the GMO Panel (see Table 2), no safety issue concerning the three single events has been reported by the applicant. According to the Implementing Regulation EC 503/2013, sequencing information of the three events provided in the stack was assessed and confirms the originally submitted sequences of the single events (Section 3.4.1.2). Updated bioinformatic analysis of the junction regions for maize events NK603, T25 and DAS40278-9 confirmed that no known endogenous genes were disrupted by any of the inserts. Updated bioinformatic analyses of the amino acid sequence of the newly expressed CP4 EPSPS, CP4 EPSPS L214P, PAT and AAD-1 proteins confirmed previous results indicating no significant similarities to known toxins and allergens. Updated bioinformatic analyses of the newly created open reading frames (ORFs) within the inserts or spanning the junctions between the insert and the flanking regions for events NK603, T25 and DAS-40278-9 confirmed previous analyses (Table 2). These analyses indicate that the production of a new peptide showing significant similarities to toxins or allergens for any of the events in maize NK603 9T25 9DAS-40278-9 is highly unlikely. In order to assess the possibility for horizontal gene transfer (HGT) by homologous recombination (HR), the applicant performed a sequence identity analysis with microbial DNA for events NK603, T25 and DAS-40278-9. The likelihood and potential consequences of plant-to-bacteria gene transfer are described in Section 3.4.4.2. Based on the above information, the GMO Panel considers that its previous conclusions on the safety of the single maize events remain valid. 3.3. Systematic literature review The GMO Panel assessed the applicant’s literature searches on maize NK603 9T25 9DAS-402789, which include a scoping review, according to the guidelines given in EFSA (2010, 2019b). A systematic review as referred to in Regulation (EU) No 503/2013 has not been provided in support to the risk assessment of application EFSA-GMO-NL-2019-164. Based on the outcome of the scoping review, the GMO Panel agrees that there is limited value of undertaking a systematic review for maize NK603 9T25 9DAS-40278-9 at present. The GMO Panel considered the overall quality of the performed literature searches is acceptable. The literature searches identified no relevant publications on maize NK603 9T25 9DAS-40278-9. 3.4. Risk assessment of the three-event stack maize NK603 3T25 3DAS-40278-9 3.4.1. Molecular characterisation 7 In line with the requirements laid down by Regulation (EU) 503/2013, the possible impact of the combination of the events on the integrity of the events, the expression levels of the newly expressed proteins or the biological functions conferred by the individual inserts are considered below. Table 2: Single maize events and subcombinations of maize NK603 9T25 9DAS-40278-9 previously assessed by the GMO Panel Event Application or mandate EFSA Scientific Opinion NK603 EFSA-GMO-NL-2005-22 EFSA GMO Panel (2009) Art4_NK603 EFSA (2004) CE/ES/00/01 EFSA GMO Panel (2007) EFSA-GMO-RX-NK603 EFSA GMO Panel (2009) T25 EFSA-GMO-NL-2007-46 EFSA GMO Panel (2013) EFSA-GMO-RX-T25 EFSA GMO Panel (2013) DAS-40278-9 EFSA-GMO-NL-2010-89 EFSA GMO Panel (2016) NK603 3T25 EFSA-GMO-NL-2010-80 EFSA GMO Panel (2015b) NK603 3DAS-40278-9 EFSA-GMONL-2013-112 EFSA GMO Panel (2019a) 7 Dossier: Part II –Section 1.2 and additional information 7/8/2020, 30/11/2020, and 1/10/2021. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 9 EFSA Journal 2021;19(12):6942 shikimic acid pathway for the biosynthesis of aromatic amino acids in plants, showing high substrate specificity. The PAT enzyme acts on the glufosinate-ammonium-based herbicides and the AAD-1 enzyme degrades 2,4-dichlorophenoxyacetic acid (2,4-D) and AOPP class of herbicides. On the basis of the known biological function of the individual newly expressed proteins (Table 4), there is currently no expectation for their possible interactions relevant to the food and feed safety of the three-event stack maize NK603 9T25 9DAS-40278-9. The GMO Panel concludes that there are no safety concerns to human and animal health related to the newly expressed proteins CP4 EPSPS, including the variant CP4 EPSPS L214P, PAT, and AAD-1 in the three-event stack maize NK603 9T25 9DAS-40278-9. Testing of new constituent other than proteins No new constituents other than newly expressed proteins have been identified in seed and forage from maize NK603 9T25 9DAS-40278-9. Therefore, no further food/feed safety assessment of components other than the newly expressed proteins is required. Information on altered levels of food and feed constituent No altered levels of food/feed constituents have been identified in seed and forage from maize NK603 9T25 9DAS-40278-9. Therefore, no further food/feed safety assessment of components other than the newly expressed proteins is required. Testing of the whole genetically modified food and feed Based on the outcome of the molecular characterisation, comparative analysis and toxicological assessment, no indication of findings relevant to food/feed safety related to the stability and expression of the inserts or to interaction between the transformation events, and no modifications of toxicological concern in the composition of maize NK603 9T25 9DAS-40278-9 have been identified (see Sections 3.4.1,3.4.2 and 3.4.3.3). Therefore, animal studies on food/feed derived from the three stacks are not necessary (EFSA GMO Panel, 2011a). In accordance to Regulation (EU) No 503/2013, the applicant provided a 90-day oral repeated-dose toxicity study in rats on whole food and feed from each of the maize single event composing the three-event stack maize. 90-Day studies on maize NK603 The GMO Panel had previously concluded that this study is in line with Regulation (EU) No 503/2013 and do not show adverse effects related to diets incorporating the single-event maize NK603 (EFSA GMO Panel, 2019b). 90-Day studies on maize T25 A 90-day study on maize T25 has been previously assessed by the GMO Panel in the context of the single-event renewal application dossier (EFSA GMO Panel, 2013), and was not consider adequate because of the low number of experimental units per treatment (two cages per sex with five animals per cage), reducing the power of the statistical analysis; moreover, the study was conducted with grains which were harvested from T25 maize plants not treated with the intended herbicide; upon EFSA’s request to fulfil the requirements of Regulation (EU) No 503/2013, the applicant provided a new 90-day toxicity study on T25 maize. 20 In this study, pair-housed Crl: WI (Han) rats (16/sex per group; 2 rats/cage) were allocated to three groups using a randomised complete block design with eight replications/sex. Groups were fed diets containing 50% of incorporation rate of grains from maize T25 treated with the intended herbicide (glufosinate-ammonium-containing herbicide) (test material), the non-GM comparator (NIL, control material) or the non-GM commercial reference maize variety (5385, reference material). The study was adapted from OECD test guideline 408 (OECD, 2018), aligned with EFSA Scientific Committee guidance (EFSA Scientific Committee, 2011) and complied with the principles of good laboratory practice (GLP) with some deviations not impacting the study results and interpretation (i.e. test item stability, homogeneity and concentration), which are detailed below. Event-specific PCR analysis confirmed the presence of the event T25 in both the GM ground maize grains (meal) and diets and excluded the presence of the event in the respective controls. ELISA 20 Additional information: 12/3/2021. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 16 EFSA Journal 2021;19(12):6942 analyses also confirmed the presence of event T25 (i.e. PAT 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 Kliba-modified maintenance diet. The stability of the test and control materials was not verified; however, in accordance to product expiration declared by the diet manufacturer, the constituents of the diets are considered stable for the duration of the treatment. The GMO Panel considered this justification acceptable. Diet preparation procedures and regular evaluations of the mixing methods guaranteed the homogeneity and the proper concentration of the test or control substances in them. The applicant provided information on concentration of proximates and fibre 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 test guideline 408 (OECD, 2018). An appropriate range of statistical tests was performed on the results of the study. Detailed description of the methodology and of statistically significant findings identified in rats given a diet containing T25 maize is reported in Appendix C. There were no test diet-related incidents of mortality or clinical signs. 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 within the normal variation 21 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. No gross pathology findings related to the administration of the test diet were observed at necropsy, and the microscopic examinations of a wide range of organs and tissues did not identify relevant differences in the incidence or severity of the histopathological findings related to the administration of the test diet compared to the control group. A notable, but not statistically significant, increase in basophilic renal tubules was noted in rats receiving T25 diet, but these animals had lower levels of lymphoid infiltration and tubular degeneration/regeneration and no indications of altered blood or urinary changes associated with impaired renal function. Overall, it is concluded that T25 did not have an adverse effect on the rat kidney. The GMO Panel concludes that this study is in line with the requirements of Regulation (EU) No 503/2013 and that no treatment-related adverse effects were observed in rats after feeding diets including 50% grains from T25 maize for 90 days. 90-Day study on maize DAS-40278-9 In accordance with Regulation (EU) No 503/2013, the applicant provided a 90-day feeding study in rats receiving diets derived from maize DAS-40278-9. In this study, pair-housed Crl: CD (SD) rats (12/sex per group; 2 rats/cage) were allocated to six groups with six replications/sex per group. 22 Groups were fed diets incorporating DAS-40278-9 maize grains sprayed with 2,4-Dand quizalofopcontaining herbicides at 33% and 11% of inclusion level (the latter supplemented with 22% of the non-GM comparator maize), the non-GM comparator (inclusion level 33%) or one of three nontransgenic commercial reference maize varieties (inclusion level 33%) (Master’s choice commercial meal MC-535RF, Blue River commercial meal 71M36RF, Pioneer commercial meal 34M78RF). 21 Although animals used in a toxicology study are of the same strain, from the same supplier and are closely matched for age and body weight at the start of the study, they exhibit a degree of variability in the parameters investigated during the study. This variability is evident even within control groups. To help reach a conclusion on whether a statistically significant finding in a test group is ’adverse’account is taken of whether the result in the test group is outside the normal range for untreated animals of the same strain and age. To do this, a number of sources of information are considered, including the standardized effect size, the standard deviations and range of values within test and control groups in the study and, if applicable, data from other studies performed in the same test facility within a small timeframe and under almost identical conditions (Historic Control Data). 22 The treatment groups were arranged in blocks, with all diets equally represented in each block; there was no information available, however, on the randomisation of treatment groups within blocks. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 17 EFSA Journal 2021;19(12):6942 The study was adapted from OECD test guideline 408 (OECD, 1998), aligned with EFSA Scientific Committee guidance (EFSA Scientific Committee, 2011) and complied with the principles of good laboratory practice (GLP) with some deviations not impacting the study results and interpretation (i.e. test item stability, homogeneity and concentration), which are detailed below. Event-specific PCR analysis confirmed the presence of the event DAS-40278-9 in both the GM maize grains and diets and excluded the presence of the event in the non-GM comparator. ELISA analyses also confirmed the presence of event DAS-40278-9 (i.e. AAD-1 concentration) in the GM maize grains and GM diets. Both GM and control maize grains and diets were analysed for nutrients, antinutrients and potential contaminants (e.g. selected heavy metals, mycotoxins and pesticides). Balanced diets were formulated based on the specifications for PMI Certified Rodent LabDiet ® 5002. The stability of the test and control materials was not verified; however, in accordance to product expiration declared by the diet manufacturer, the constituents of the diets are considered stable for the duration of the treatment. The GMO Panel considered this justification acceptable. Diet preparation procedures and regular evaluations of the mixing methods guaranteed the homogeneity and the proper concentration of the test or control substances in them. Feed and water were provided ad libitum. In-life procedures and observations and terminal procedures were conducted in accordance to OECD test guideline 408 (OECD, 1998). An appropriate range of statistical tests was performed on the results of the study. Detailed description of the methodology and of statistically significant findings identified in rats given a diet containing DAS-40278-9 maize is reported in Appendix C. There were no test diet-related incidents of mortality or clinical signs. 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 within the normal variation 21 for the parameter in rats of this age; •were of small magnitude; •were identified at only a small number of time intervals with no impact on the overall value; •exhibited no consistent pattern with related parameters or end-points. •no consistency with increasing dietary incorporation level. No gross pathology findings related to the administration of the test diet were observed at necropsy, and the microscopic examinations of a wide range of organs and tissues did not identify relevant differences in the incidence or 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 treatment-related adverse effects were observed in rats after feeding diets including 11% or 33% grains from DAS 40278-9 maize for 90 days. Although evidence is increasing that 50% maize incorporation rate is generally applicable as the high dose (EFSA 2014; Steinberg et al., 2019; EFSA GMO Panel, 2021b), the GMO Panel considers that further scientific evidence is needed that test diets incorporating 50% would not induce nutritional imbalance. 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 the newly expressed proteins in relation to their potential to cause coeliac disease was also performed (EFSA GMO Panel, 2017). Assessment of allergenicity of the newly expressed proteins 23 For allergenicity, the GMO Panel has previously evaluated the safety of CP4 EPSPS, PAT and AAD-1 proteins individually, and no evidence of allergenicity was identified in the context of the applications assessed (Table 2). No new information on allergenicity of the proteins newly expressed in this threeevent stack maize that might change the previous conclusions of the GMO Panel has become available. 23 Technical dossier and additional information 18 December 2020. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 18 EFSA Journal 2021;19(12):6942 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 three-event stack maize. The GMO Panel has previously evaluated the safety of the newly expressed proteins and has not found evidence of adjuvanticity (ability to enhance an allergic response) in the context of the applications assessed (Table 2). There was no indication that the proteins at the levels expressed in this three-event stack maize act as adjuvants. Furthermore, the applicant provided information on the safety of CP4 EPSPS, PAT and AAD-1 proteins regarding their potential hazard to cause a coeliac disease response. For such assessment, the applicant followed the principles described in the EFSA GMO Panel guidance document (EFSA GMO Panel, 2017). The assessment of the CP4 EPSPS and AAD-1 proteins identified no perfect or relevant partial matches with known coeliac disease peptide sequences. The assessment of the PAT protein revealed partial matches containing the Q/E-X1-P-X2 motif and required further investigations. Based on additional considerations on position and nature of amino acids flanking the ELPA motif, such as the presence of two consecutive prolines and the charge and size of adjacent amino acids (EFSA GMO Panel, 2017), the two relevant peptides containing the motif do not raise concern as they fail to mimic gluten sequences. Therefore, no indications of safety concern were identified by the GMO Panel. Assessment of allergenicity of the whole GM plant The GMO Panel regularly reviews the available publications on food allergy to maize. However, maize is not considered a common allergenic food 24 (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 three-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, PAT and AAD-1 proteins newly expressed in NK603 9T25 9DAS-40278-9 maize. Dietary exposure was estimated based on protein expression levels reported in this application for the threeevent stack maize treated with the intended herbicides, the current available consumption data and feed practices, the foods and feeds currently available in the market and the described processing conditions. For the purpose of estimating dietary exposure, the levels of newly expressed proteins in NK603 9T25 9DAS-40278-9 maize grains, forage and pollen were derived from replicated field trials (four replicates from six locations) in 2018 in the United States and Canada (see Section 3.4.1.3). Table 7 describes the protein expression levels used to estimate both human and animal dietary exposure. Table 7: Mean values (n =24, lg/g dry weight and lg/g fresh weight) for newly expressed proteins in grains, forage and pollen from NK603 9T25 9DAS-40278-9 maize treated with the intended herbicides (a) Protein Tissue/developmental stage Grains/R6 (lg/g dry weight/lg/g fresh weight) Pollen/R1 (lg/g fresh weight) (b) Forage/R4 (lg/g dry weight) CP4 EPSPS 14/11 385.4 110 PAT 0.27/0.21 0.12 (c) 18 AAD-1 4.3/3.4 88.4 9 (a): Intended herbicides: glufosinate, glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and aryloxyphenoxypropionate (AOPP) herbicides. 24 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 608/200. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 19 EFSA Journal 2021;19(12):6942 Human dietary exposure Chronic and acute dietary exposure to CP4 EPSPS, PAT and AAD-1 proteins newly expressed in NK603 9T25 9DAS-40278-9 maize was provided. The applicant followed the methodology described in the EFSA Statement ‘Human dietary exposure assessment to newly expressed protein in GM foods’ (EFSA, 2019a) to estimate human dietary exposure in average and high consumers making use of summary statistics of consumption. Human dietary exposure was estimated across different European countries on different population groups: young population (infants, toddlers, ‘other children’), adolescents, adult population (adults, elderly and very elderly) and special populations (pregnant and lactating women). Since no specific consumption data were available on commodities containing, consisting of or obtained from NK603 9T25 9DAS-40278-9 maize grains, a conservative scenario with 100% replacement of conventional maize by the GM maize was considered. Consumption figures for all relevant commodities (e.g. corn flakes, sweet corn, popcorn, etc.) were retrieved from the EFSA Comprehensive European Food Consumption Database (EFSA consumption database). 25 Corn oil was excluded from the assessment since no proteins are expected to be present in the oil. Mean protein expression values on fresh weight basis are considered as the most adequate to estimate human dietary exposure (both acute and chronic) when working with raw primary commodities that are commonly consumed as processed blended commodities (EFSA, 2019a). Different recipes and factors were considered to estimate the amount of maize in the consumed commodities before assigning newly expressed protein levels to the relevant commodities. 26 No losses in the newly expressed proteins during processing were considered, except for certain commodities excluded from the exposure estimations (maize oil, corn starch, corn syrup). The highest acute dietary exposure (high consumers) was estimated in the age class ‘Other children’with exposure estimates of 167 µg/kg body weight (bw) per day, 3.2 µg/kg bw per day and 51.7 µg/kg bw per day for CP4 EPSPS, PAT and AAD-1 proteins, respectively. The main contributor to the exposure in the dietary survey with the highest estimates was corn grains. The highest chronic dietary exposure (high consumers) was estimated in the age class ‘Infants’ with exposure estimates of 62.1 µg/kg bw per day, 1.2 µg/kg bw per day and 19.2 µg/kg bw per day for CP4 EPSPS, PAT and AAD-1 proteins, respectively. The main contributor to the exposure in the dietary survey with the highest estimates was sweet corn. An ad hoc dietary exposure scenario was carried out for consumers of pollen supplements under the assumption that these supplements might be made of pollen from NK603 9T25 9DAS-40278-9 maize. Consumption data on pollen supplements are available for few consumers across eight different European countries. 27 The low number of consumers available adds uncertainty to the exposure estimations which should be carefully interpreted, and it prevents from estimating exposure for high consumers of pollen supplements. In average consumers of pollen supplements, the highest acute dietary exposure would range from 0.09 µg/kg bw per day for PAT to 285.7 µg/kg bw per day for CP4 EPSPS, in the elderly population. Similarly, the highest chronic dietary exposure in average consumers would range from 0.06 µg/kg bw per day for PAT to 190.5 µg/kg bw per day for CP4 EPSPS, also in the elderly population. Animal dietary exposure Dietary exposure to CP4 EPSPS, PAT and AAD-1 proteins in maize NK603 9T25 9DAS-40278-9 was estimated across different animal species as below described, assuming the consumption of maize products commonly entering the feed supply chain (i.e. maize grains and forage). A conservative scenario with 100% replacement of conventional maize products by the three-event stack maize products was considered. (b): Values refer to a moisture content of 6% derived from the reported concentrations in dry weight (study reports PHI-2018017 and PHI-R066-Y19). These values were used to estimate dietary exposure to the different newly expressed protein via the consumption of pollen supplements. (c): A total of 22 out of 24 samples were reported below the limit of quantification (LOQ, 0.22 lg/g). Half the LOQ value was assigned to the left-censored data to calculate the mean. 25 https://www.efsa.europa.eu/en/applications/gmo/tools. Data accessed: September 2019. 26 Example: 100 grams of maize bread are made with approximately 74 g of maize flour, and a reverse yield factor of 1.22 from the conversion of maize grains into flour is used. This results in 9.9 µg of CP4 EPSPS per gram of maize bread as compared to the 11 µg/g reported as mean concentration in the maize grains. 27 https://www.efsa.europa.eu/en/food-consumption/comprehensive-database. Data accessed: March 2021. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 20 EFSA Journal 2021;19(12):6942 Mean levels (dry weight) of the newly expressed proteins in grains and forage from the three-event stack maize treated with the intended herbicide used for animal dietary exposure are listed in Table 7. The applicant estimated dietary exposure to CP4 EPSPS, PAT and AAD-1 proteins in livestock (i.e. poultry, swine, cattle and sheep), based on estimates for body weights, daily feed intakes and inclusion rates (percentage) of maize grains and forage in diets/rations (OECD, 2013). Estimated dietary exposure in livestock animals was calculated based on the consumption of maize grain and forage alone or in combination, as reported in Appendix D. 3.4.3.6. Nutritional assessment of endogenous constituents The intended traits of maize NK603 9T25 9DAS-40278-9 are herbicide tolerance, with no intention to alter nutritional parameters. Comparison of the seed and forage composition of maize NK603 9T25 9DAS-40278-9 with the conventional counterpart and the commercial non-GM maize reference varieties did not identify differences that would require a nutritional assessment as regards food/feed (see Section 3.4.2.7). From these data, the GMO Panel concludes that maize NK603 9T25 9DAS-40278-9 is nutritionally equivalent to the non-GM comparator and the non-GM reference varieties used. 3.4.3.7. Conclusion on the food/feed safety assessment The CP4 EPSPS, PAT and AAD-1 proteins newly expressed in maize NK603 9T25 9DAS-40278-9 do not raise safety concerns for human and animal health. No interactions between the newly expressed proteins relevant for food and feed safety were identified, and no overall toxicological concerns on the three-event stack maize were identified. Similarly, the GMO Panel did not identify indications of safety concerns regarding allergenicity or adjuvanticity related to the presence of the newly expressed proteins in the three-stack maize NK603 9T25 9DAS-40278-9, or regarding the overall allergenicity of this three-event stack maize. Based on the outcome of the comparative assessment and the nutritional assessment, the GMO Panel concludes that the consumption of maize NK603 9T25 9DAS-40278-9 does not represent any nutritional concern, in the context of the scope of this application. 3.4.4. Environmental risk assessment Considering the scope of application EFSA-GMO-NL-2019-164, which excludes cultivation, the environmental risk assessment (ERA) of maize NK603 9T25 9DAS-40278-9 mainly takes into account: (1) the exposure of microorganisms to recombinant DNA in the gastrointestinal tract of animals fed GM material and of microorganisms present in environments exposed to faecal material of these animals (manure and faeces); and (2) the accidental release into the environment of viable three-event stack maize grains during transportation and/or processing (EFSA GMO Panel, 2010a). 3.4.4.1. Persistence and invasiveness of the GM plant Maize is highly domesticated, not winter hardy in colder regions of Europe, and generally unable to survive in the environment without appropriate management. Survival is limited mainly by a combination of low competitiveness, absence of a dormancy phase and susceptibility to plant pathogens, herbivores and cold climate conditions (OECD, 2002), even though occasional feral GM maize plants may occur outside cultivation areas in the EU (e.g. Pascher, 2016). Field observations indicate that maize grains may survive and overwinter in some EU regions, resulting in volunteers in subsequent crops (e.g. Gruber et al., 2008; Palaudelm as et al., 2009; Pascher, 2016). However, maize volunteers have been shown to grow weakly and flower asynchronously with the maize crop (Palaudelm as et al., 2009). Thus, the establishment and survival of feral and volunteer maize in the EU are currently limited and transient. It is unlikely that the intended traits of the three-event stack maize and the observed decrease in lodged plants (see Section 3.4.2.5) will provide a selective advantage to maize plants, except when they are exposed to glyphosateand/or glufosinate ammoniumand/or 2,4-Dand/or AOPP-containing herbicides. However, if this was to occur, this fitness advantage will not allow the three-event stack maize to overcome other biological and abiotic factors (described above) limiting plant’s persistence and invasiveness. Therefore, the presence of the intended traits and the observed differences in lodging will not affect the persistence and invasiveness of the GM plant. In conclusion, the GMO Panel considers that the three-event stack maize will be equivalent to conventional maize hybrid varieties in their ability to survive until subsequent seasons, or to establish Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 21 EFSA Journal 2021;19(12):6942 occasional feral plants under European environmental conditions in case of accidental release into the environment of viable three-event stack maize grains. 3.4.4.2. Potential for gene transfer A prerequisite for any gene transfer is the availability of pathways for the transfer of genetic material, either through horizontal gene transfer (HGT) of DNA or through vertical gene flow via crosspollination from feral plants originating from spilled grains. Plant to microorganism gene transfer The probability and potential adverse effects of HGT of the recombinant DNA have been assessed in previous GMO Panel Scientific Opinions for the single events (see already assessed APs in Table 2). This assessment included consideration of homology-based recombination processes, as well as nonhomologous end joining and microhomology-mediated end joining. Possible fitness advantages that the bacteria in the receiving environments would gain from acquiring recombinant DNA were considered. No concern as a result of an unlikely, but theoretically possible, HGT of the recombinant genes to bacteria in the gut of domesticated animals and humans fed GM material or other receiving environments was identified. The applicant submitted an updated bioinformatic analysis for each of the single events in order to assess the possibility for HGT by homologous recombination. The updated bioinformatic analyses for NK603, T25 and DAS-40278-9 confirm the assessments provided in the context of previous Scientific Opinions (EFSA GMO Panel, 2015b, 2019a, 2021b). Synergistic effects of the recombinant genes, for instance due to combinations of recombinogenic sequences, which would cause an increase in the likelihood for horizontal gene transfer or a selective advantage are not identified. Therefore, the GMO Panel concludes that the unlikely, but theoretically possible, horizontal transfer of recombinant genes from this three-event stack maize to bacteria does not raise any environmental safety concern. Plant-to-plant gene transfer The GMO Panel assessed the potential for occasional feral three-event stack maize plants originating from grain import spills to transfer recombinant DNA to sexually compatible plants; the environmental consequences of this transfer were also considered. For plant-to-plant gene transfer to occur, imported GM maize grains need to germinate and develop into plants in areas containing sympatric wild relatives and/or cultivated maize with synchronous flowering and environmental conditions favouring cross-pollination. Maize is an annual predominantly cross-pollinating crop. Cross-fertilisation occurs mainly by wind (OECD, 2003). Vertical gene transfer from maize is limited to Zea species. Wild relatives of maize outside cultivation are not known/reported in Europe (Eastham and Sweet, 2002; OECD, 2003; EFSA, 2016; Trtikova et al., 2017). Therefore, potential vertical gene transfer is restricted to maize and weedy Zea species, such as teosintes and/or maize-teosinte hybrids, occurring in cultivated areas (EFSA, 2016; Trtikova et al., 2017; Le Corre et al., 2020). The potential of spilled maize grains to establish, grow and produce pollen is extremely low and transient (see Section 3.4.4.1). Therefore, likelihood/frequency of cross-pollination between occasional feral GM maize plants resulting from grain spillage, and weedy or cultivated Zea plants is considered extremely low (EFSA, 2016). Even if cross-pollination would occur, the GMO Panel is of the opinion that environmental effects as a consequence of the spread of genes from occasional feral GM maize plants in Europe will not differ from that of conventional maize varieties for the reasons given in Section 3.4.4.1, even if exposed to the intended herbicides. 3.4.4.3. Interactions of the GM plant with target organisms Taking the scope of application EFSA-GMO-NL-2019-164 (no cultivation) and thus the absence of target organisms into account, potential interactions of occasional feral three-event stack maize plants arising from grain import spills with target organisms are not considered a relevant issue. 3.4.4.4. Interactions of the GM plant with non-target organisms Given that environmental exposure of non-target organisms to spilled GM grains or occasional feral GM maize plants arising from spilled GM grains is limited and because most proteins are degraded before entering the environment through faecal material of animals fed GM maize, potential interactions with non-target organisms are not considered a relevant issue by the GMO Panel. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 22 EFSA Journal 2021;19(12):6942 3.4.4.5. Interactions with the abiotic environment and biogeochemical cycles Given that environmental exposure to spilled grains or occasional feral maize NK603 9T25 9DAS40278-9 plants arising from grain import spills is limited and because most proteins are degraded before entering the environment through faecal material of animals fed GM maize, potential interactions with the abiotic environment and biogeochemical cycles are not considered a relevant issue by the GMO Panel. 3.4.4.6. Conclusion of the environmental risk assessment The GMO Panel concludes that it is unlikely that the maize NK603 9T25 9DAS-40278-9 would differ from conventional maize varieties in its ability to persist under EU environmental conditions. Considering the scope of the application EFSA-GMO-NL-2019-164, interactions of occasional feral maize NK603 9T25 9DAS-40278-9 plants with the biotic and abiotic environment are not considered to be relevant issues. The analysis of horizontal gene transfer from maize NK603 9T25 9DAS-40278-9 to bacteria does not indicate a safety concern. Therefore, considering the combined traits and their interactions, the outcome of the agronomic and phenotypic analysis, the routes and levels of exposure, the GMO Panel concludes that maize NK603 9T25 9DAS-40278-9 would not raise safety concerns in the event of accidental release of viable GM maize grains into the environment. 3.5. Risk assessment of the subcombinations Subcombinations previously assessed in the frame of other applications are discussed in Section 3.5.1. The subcombination that has not been previously assessed is discussed in Section 3.5.2. 3.5.1. Subcombinations previously assessed The GMO Panel has previously assessed two subcombinations (see Table 1) and no safety concerns were identified. Literature searches covering the 10 years before submission of the application and the period since the time of validity of the application (January 2009–April 2021) revealed no new scientific information relevant to the risk assessment of these maize stacks. 28 Consequently, the GMO Panel considers that its previous conclusions on these subcombinations remain valid. 3.5.2. Subcombinations not previously assessed One of the three subcombinations included in the scope of this application has not been previously assessed by the GMO Panel, and no experimental data were provided for this maize stack (see Table 8). In this case, following the strategy defined by the GMO Panel, 29 the risk assessment takes as its starting point the assessment of the single maize events, and uses the data generated for the three-event stack as well as all the additional data available on subcombinations previously assessed by the GMO Panel (Table 2) and the additional studies provided by the applicant (Appendix A). 3.5.2.1. Stability of the events The genetic stability of the inserted DNA over multiple generations in the three single maize events was demonstrated previously (see Table 2). Integrity of the events was demonstrated in the threeevent stack maize NK603 9T25 9DAS-40278-9 (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 subcombination not previously assessed (see Table 8). 3.5.2.2. Expression of the events The GMO Panel assessed whether any combination of the three-events by conventional crossing could result in significant changes in expression levels of the newly expressed proteins, as this could Table 8: Maize stacks not previously assessed and covered by the scope of application EFSA-GMONL-2019-164 Degree of stacking Event 2-event stack DAS-40278-9 9T25 28 Dossier: Part II –Section 7; additional information: 7/8/2020. 29 115th GMO Panel meeting (Annex 1 of the minutes: https://www.efsa.europa.eu/sites/default/files/event/170517-m.pdf). Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 23 EFSA Journal 2021;19(12):6942 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 this subcombination 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 three-event stack maize. The levels were comparable in the three-event stack maize and in the single events (Section 3.4.1.3 and Appendix B). This supports the conclusion that interactions affecting the expression levels of the newly expressed proteins are not expected in the subcombination not previously assessed and included in the scope of application EFSA-GMO-NL2019-164. 3.5.2.3. Potential functional interactions between the events The GMO Panel assessed the potential for interactions between maize events in the subcombination not previously assessed (Table 8), taking into consideration intended traits and unintended effects. Based on the known biological functions of the individual newly expressed proteins (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 three single events, the previously assessed subcombinations (Table 2) and the three-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 three-event stack maize. 3.5.3. Conclusions 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 subcombination included in the scope of application EFSA-GMO-NL-2019-164, 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 three-event stack maize. 3.6. Post-market monitoring 3.6.1. Post-market monitoring of GM food/feed The GMO Panel concluded that the three-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 non-GM reference varieties tested (Section 3.4.3). Two of the subcombinations have been previously assessed and no safety concerns were identified. The subcombination not previously assessed and included in the scope of this application is expected to be as safe as the single maize events, the previously assessed maize subcombinations and the three-event stack maize (Section 3.5.2). Therefore, the GMO Panel considers that post-market monitoring of food and feed from the three-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 three-event stack maize, no case-specific monitoring is required. The PMEM plan proposed by the applicant for the three-event stack maize and its subcombinations includes: (1) the description of a monitoring approach involving operators (federations involved in Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 24 EFSA Journal 2021;19(12):6942 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 Croplife Europe 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 three-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 three-event stack maize and its subcombinations. 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 NK603 9T25 9DAS-40278-9. 4. Overall conclusions The GMO Panel was asked to carry out a scientific assessment of maize NK603 9T25 9DAS40278-9 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 three single maize events (NK603, T25 and DAS-40278-9) 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 in the threeevent stack maize does not give rise to food/feed safety and nutritional concerns. The GMO Panel concludes that the three-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 three-event stack maize into the environment. Since no new data were identified on the two 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 subcombination included in the scope of application EFSA-GMO-NL-2019-164, no information has been provided. The GMO Panel assessed the possible interactions between the events in this subcombination and concludes that these combinations of events NK603, T25 and DAS-40278-9 would not raise safety concerns. This subcombination is therefore expected to be as safe as the maize single events, the previously assessed subcombinations and the three-event stack maize. The literature searches did not identify any relevant publications on maize NK603, T25 and DAS40278-9. 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 study listed in Appendix A. This new information does not raise any concern for human and animal health and the environment regarding the three-event stack maize and its subcombinations. Given the absence of safety and nutritional concerns for foods and feeds from the three-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 three-event stack maize and its subcombinations. In conclusion, the GMO Panel considers that maize NK603 9T25 9DAS-40278-9 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 •Letter from the Competent Authority of the Netherlands received on 13 December 2019 concerning a request for authorization of the placing on the market of maize Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 25 EFSA Journal 2021;19(12):6942 Appendix B –Protein expression data Table B.1: Mean, standard deviation and range of protein levels (lg/g dry weight) from maize NK603 9T25 9DAS-40278-9 (not treated) and NK603, T25 and DAS-40278-9 (not treated), from field trials performed across six locations in USA and Canada in 2018 (n =24) (a) Protein Event(s) Leaf (V2–V4) Leaf (V9) Leaf (R1) Pollen (R1) Root (R1) Forage (R4) Grain (R6) CP4 EPSPS (f) NK603 3T25 3 DAS-40278-9 310 (b) 87 (c) (160–490) (d) 200 34 (120–260) 220 29 (150–280) 280 43 (150–340) 81 19 (39–120) 98 32 (54–180) 8.6 2.5 (3.4–13) NK603 330 85 (190–470) 200 38 (130–290) 220 41 (150–290) 280 48 (120–350) 60 12 (33–75) 96 22 (61–150) 8.0 2.1 (4.2–13) PAT NK603 3T25 3 DAS-40278-9 50 15 (23–91) 50 11 (30–70) 55 17 (15–91) <LOQ (e) 13 4.4 (4.1–23) 18 7.6 (8.5–33) 0.24 0.13 (<0.054–0.51) T25 53 11 (33–77) 50 9.8 (34–77) 59 13 (38–98) 0.13 0.03 (e) (<LOQ–0.26) 16 3.9 (9–25) 19 6.8 (10–33) 0.22 0.09 (0.073–0.48) AAD-1 NK603 3T25 3 DAS-40278-9 13 5.2 (4.6–22) 6.6 2.5 (2.8–11) 10 2.5 (4.9–13) 99 19 (57–120) 5.4 2.2 (0.87–10) 8.6 1.6 (5.3–13) 3.9 1.1 (2.0–6.4) DAS-40278-9 12 5.6 (4.5–22) 6.1 1.8 (3.0–9.0) 8.9 3.1 (4.2–16) 100 18 (71–130) 5.4 1.9 (1.6–9.8) 9.0 1.3 (7.1–12) 3.7 1.3 (1.7–6.4) (a): Number of samples is n =24. (b): Mean. (c): Standard deviation. (d): Range. (e): Some, but not all, sample results were below the LLOQ. A value equal to half the LLOQ value was assigned to those samples to calculate the mean and standard deviation. LOQ =0.22; LOQ: limit of quantification. (f): EPSPS levels in the maize NK603 9T25 9DAS-40278-9 are a sum of two protein variants CP4 EPSPS and CP4 EPSPS L214P, both expressed in maize NK603. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 32 EFSA Journal 2021;19(12):6942 Appendix C –Statistical analysis and statistically significant findings in the 90-day toxicity studies in rats on the whole food/feed C.1. Statistical analysis of the 90-day study on T25 in rats The following endpoints were statistically analysed: body weights, body weight changes, water and food consumption, clinical pathology values (as applicable), absolute and relative organ weights, functional observational battery (FOB) data, locomotor activity, ophthalmological examinations and histopathological data. For all continuous endpoints, mean, standard deviation in terms of the standardised effect sizes (SES) of each dose group for each sex, variable, and period or time interval were reported. The main statistical analysis compared rats consuming the test diet (T25) with those consuming the control diet (NIL). The statistical analysis was performed using linear mixed models with treatment, sex and their interaction as fixed effects whereas the random effects included only the block. If the interaction of the treatment per sex was significant, the comparison of the test vs. the control group was conducted separately for the two genders. In case of pathology and clinical pathology data where a statistical difference between test (T25) and test the control group (NIL) was identified, an additional comparison between the test (T25) and the reference (5385) group was performed. If this latest analysis also shows a significant difference, a direct comparison of test data (T25) to the historical control data was provided. Historical control data were provided for endpoints belonging to clinical pathology and pathology. Missing data were considered by the Panel and found not impacting the results. Table C.1: Statistically significant findings in 90-day study on T25 in rats Statistically significant parameter/endpoint Finding GMO Panel interpretation RBC count and haematocrit & haemoglobin levels Statistically significant decreases in males (<5%) Low magnitude, within normal variation. Not an adverse effect of treatment with maize T25. Basophil count Statistically significant decrease in females Within normal variation. Not an adverse effect of treatment with maize T25. Monocyte (% of WBC) Statistically significant increase in both sexes combined (<20%) Low magnitude, absolute count not changed significantly. Within normal variation. Not an adverse effect of treatment. Blood urea Statistically significant decrease in females (25%) Decrease is not adverse in isolation. Within normal variation. Not an adverse effect of treatment. Creatinine Statistically significant decrease in both sexes combined (20%) Decrease is not adverse in isolation. Within normal variation Not an adverse effect of treatment. Total bilirubin Statistically significant increase in both sexes combined (15%) Low magnitude, within normal variation. No consistent pattern with other markers of liver toxicity. Not an adverse effect of treatment. Albumin Statistically significant increase in females (1%) Low magnitude, within normal variation. Not an adverse effect of treatment. Sodium Statistically significant increase in females (1%) Low magnitude, within normal variation. Not an adverse effect of treatment. Urinary pH Statistically significant increase in both sexes combined (20%) Value is within the normal physiological range. Not an adverse effect of treatment. Kidney weight (absolute and relative to body weight) Statistically significant increase in males and females (10–20%) Low magnitude, within normal variation. No pattern of adverse effects on other markers of kidney function. Not an adverse effect of treatment. Pituitary weight (relative to body weight) Statistically significant decrease in both sexes combined (20%) Low magnitude, within normal variation. Not an adverse effect of treatment. No associated pathological changes. Thyroid weight (relative to body weight) Statistically significant decrease in both sexes combined (15%) Low magnitude, within normal variation. Not an adverse effect of treatment. No associated pathological changes. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 33 EFSA Journal 2021;19(12):6942 C.2. Statistical analysis of the 90-day study on DAS 40278-9 in rats The following endpoints were statistically analysed: body weight, body weight gain, feed consumption and feed efficiency, forelimb and hindlimb strength, motor activity data, sensory evaluation data, rectal temperature, haematology, coagulation, urinalysis and clinical chemistry values, absolute and relative organ weights. For all continuous endpoints, mean and standard deviation were provided for each dose group for each sex, variable and period or time interval. 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 for 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). The data for the three reference groups (not included in the linear mixed model analysis) were used to calculate ranges of variability for the parameters. For each comparison, point estimates and 95% confidence intervals of the SES were reported to aid the assessment. Missing data were considered by the Panel and found not to affect the results. Statistically significant parameter/endpoint Finding GMO Panel interpretation Uterus weight Statistically significant increase (40%) Within normal variation. No associated pathological changes. Not an adverse effect of treatment. Table C.2: Statistically significant findings in 90-day study on DAS-40278-9 in rats Statistically significant parameter/endpoint Finding GMO Panel interpretation Potassium Statistically significant increase in males and females at the top dose (<10%) Within normal variation compared with reference diet results. Not an adverse effect of treatment with maize DAS 40278-9. Brain weight (absolute) Statistically significant decrease (5%) in low dose males Low magnitude. No significant change in top dose males. Within normal variation. Not an adverse effect of treatment. Spleen weight (relative to body weight) Statistically significant increase in low dose groups in both sexes (15%) Low magnitude, within normal variation. No significant change at top dose. No associated haematology or pathological findings. Not an adverse effect of treatment. Thymus weight (relative to body weight) Statistically significant increase in low dose groups in both sexes (<20%) Low magnitude. No significant change at top dose. No associated haematology or pathological findings Not an adverse effect of treatment. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 34 EFSA Journal 2021;19(12):6942 Appendix D –Animal dietary exposure Table D.1: Dietary exposure to CP4 EPSPS, PAT and AAD-1 proteins (mg/kg bw per day) in livestock, based on the consumption of maize grain and forage Dietary exposure (mg/kg bw per day) CP4 EPSPS PAT AAD-1 Grain (G) Forage (F) G+FGrain (G) Forage (F) G+FGrain (G) Forage (F) G+F Broiler 0.69 NA NA 0.013 NA NA 0.21 NA NA Layer 0.67 75.3 75.9 0.013 12.3 12.3 0.21 6.2 6.4 Turkey 0.50 NA NA 0.0096 NA NA 0.15 NA NA Breeding pigs 0.23 50.8 51 0.0044 8.3 8.3 0.069 4.2 4.2 Finishing pigs 0.29 NA NA 0.0057 NA NA 0.090 NA NA Beef cattle (a) 0.27 2.11 2.38 0.0052 0.35 0.35 0.083 0.17 0.26 Dairy cattle 0.16 2.54 2.70 0.0031 0.42 0.42 0.050 0.21 0.26 Ram/ewe 0.14 NA NA 0.0027 NA NA 0.043 NA NA Lamb 0.18 1.40 1.58 0.0034 0.23 0.23 0.055 0.11 0.17 (a): The inclusion rate for beef cattle would be 160% of the diet, resulting the DDE to each protein an overestimation. NA indicates that a forage inclusion rate was not provided in the reference, and therefore, no exposure calculations were done. Assessment of maize NK603 3T25 3DAS-40278-9 www.efsa.europa.eu/efsajournal 35 EFSA Journal 2021;19(12):6942