SCIENTIFIC OPINION. Abstract

Transcription

1 SCIENTIFIC OPINION ADOPTED: 26 January 2017 doi: /j.efsa Scientific Opinion on an application by Dow AgroSciences LLC (EFSA-GMO-NL ) for the placing on the market of genetically modified herbicide-tolerant soybean DAS for food and feed uses, import and processing under Regulation (EC) No 1829/2003 EFSA Panel on Genetically Modified Organisms (GMO), Hanspeter Naegeli, Andrew Nicholas Birch, Josep Casacuberta, Adinda De Schrijver, Mikołaj Antoni Gralak, Huw Jones, Barbara Manachini, Antoine Messean, Elsa Ebbesen Nielsen, Fabien Nogue, Christophe Robaglia, Nils Rostoks, Jeremy Sweet, Christoph Tebbe, Francesco Visioli, Jean-Michel Wal, Michele Ardizzone, Yann Devos, Ana Gomes, Yi Liu, Franco Maria Neri and Irina Olaru Abstract Soybean DAS was developed by Agrobacterium tumefaciens-mediated transformation to express the aryloxyalkanoate dioxygenase-12 (AAD-12) protein, conferring tolerance to 2,4- dichlorophenoxyacetic acid (2,4-D) and other related phenoxy herbicides, and the phosphinothricin acetyltransferase (PAT) protein, conferring tolerance to glufosinate ammonium-based herbicides. The molecular characterisation data and bioinformatics analyses did not identify issues requiring further assessment for food/feed safety. The agronomic and phenotypic characteristics tested revealed no relevant differences between soybean DAS and its conventional counterpart, except for days to 50% flowering. The compositional analysis identified no differences requiring further assessment, except for an increase (up to 36%) in lectin activity in soybean DAS Such increase is unlikely to raise additional concerns for food/feed safety and nutrition for soybean DAS as compared to its conventional counterpart and the non-gm reference varieties. There were no concerns regarding the potential toxicity and allergenicity of the two newly expressed proteins, and no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS Soybean DAS is as nutritious as its conventional counterpart and the non-gm reference varieties. There are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS plants, unless these are exposed to the intended herbicides. The likelihood of environmental effects resulting from the accidental release of viable seeds from soybean DAS into the environment is therefore very low. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DAS The GMO Panel concludes that the information available addresses the scientific comments of the Member States and that soybean DAS , as described in this application, is as safe as its conventional counterpart and the tested non- GM reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: GMO, soybean (Glycine max), DAS , herbicide tolerance, AAD-12, PAT, Regulation (EC) No 1829/2003 Requestor: Competent Authority of the Netherlands Question number: EFSA-Q Correspondence: EFSA Journal 2017;15(3):4719

2 Panel members: Andrew Nicholas Birch, Josep Casacuberta, Adinda De Schrijver, Mikołaj Antoni Gralak, Philippe Guerche, Huw Jones, Barbara Manachini, Antoine Messean, Hanspeter Naegeli, Elsa Ebbesen Nielsen, Fabien Nogue, Christophe Robaglia, Nils Rostoks, Jeremy Sweet, Christoph Tebbe, Francesco Visioli and Jean-Michel Wal. Competing interests: In line with EFSA s policy on declarations of interest, Panel member Philippe Guerche did not participate in the development and adoption of this scientific opinion. Suggested citation: EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Jones H, Manachini B, Messean A, Nielsen EE, Nogue F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Ardizzone M, Devos Y, Gomes A, Liu Y, Neri FM and Olaru I, Scientific Opinion on an application by Dow AgroSciences LLC (EFSA- GMO-NL ) for the placing on the market of genetically modified herbicide-tolerant soybean DAS for food and feed uses, import and processing under Regulation (EC) No 1829/2003. EFSA Journal 2017;15(3):4719, 31 pp. doi: /j.efsa ISSN: 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. 2 EFSA Journal 2017;15(3):4719

3 Summary Following the submission of an application (EFSA-GMO-NL ) under Regulation (EC) No 1829/2003 from Dow AgroSciences LLC, the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific opinion on the safety of the genetically modified (GM) herbicide-tolerant soybean (Glycine max L.) DAS (Unique Identifier DAS ). The scope of application EFSA-GMO-NL is for import, processing, and food and feed uses of soybean DAS within the European Union (EU), but excludes cultivation in the EU. The GMO Panel evaluated soybean DAS with reference to the scope and appropriate principles described in its guidelines for the risk assessment of GM plants. The evaluation addressed the following components of the risk assessment: the molecular characterisation of the inserted DNA and analysis of the expression of the corresponding proteins; the comparative analyses of compositional, agronomic and phenotypic characteristics; the safety of the newly expressed proteins and the whole food/feed with respect to potential toxicity, allergenicity and nutritional characteristics; the environmental risk assessment; and the post-market environmental monitoring plan. Soybean DAS was developed by Agrobacterium tumefaciens-mediated transformation. It expresses the aryloxyalkanoate dioxygenase-12 (AAD-12) protein, conferring tolerance to 2,4- dichlorophenoxyacetic acid (2,4-D) and other related phenoxy herbicides, and the phosphinothricin acetyltransferase (PAT) protein, conferring tolerance to glufosinate ammonium-based herbicides. The molecular characterisation data established that soybean DAS contains one insert consisting of two expression cassettes, aad-12 and pat. No other parts of the plasmid used for transformation were detected in soybean DAS Bioinformatic analyses and genetic stability studies were performed and the results did not identify issues requiring further assessment for food/feed safety. The levels of the newly expressed proteins present in soybean DAS were obtained and reported adequately. The agronomic and phenotypic characteristics of soybean DAS tested revealed no relevant differences between soybean DAS and its conventional counterpart, except for days to 50% flowering for the GM soybean not treated with the intended herbicides. The difference observed in days to 50% flowering was further assessed for its potential environmental impact. No differences in composition requiring further assessment for food/feed safety were found between soybean DAS and its conventional counterpart, except for a higher lectin activity (up to 36%) in two of the four treatments of soybean DAS The increase in lectin activity is unlikely to raise additional concerns for food/feed safety and nutrition for soybean DAS as compared to its conventional counterpart and the non-gm commercial varieties. The safety assessment identified no concerns regarding the potential toxicity and allergenicity of the newly expressed PAT and AAD-12 proteins in soybean DAS , and found no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS The GMO Panel concludes that soybean DAS is as safe and as nutritious as its conventional counterpart and the non-gm soybean reference varieties. The GMO Panel considers that post-market monitoring of food/feed derived from soybean DAS is not necessary, given the absence of safety concerns identified. Considering the scope of this application, the environmental risk assessment is concerned with the accidental release into the environment of viable soybean DAS seeds (i.e. during transport and/or processing), and with the exposure of bacteria to recombinant DNA in the gastrointestinal tract of animals fed GM material and those present in environments exposed to their faecal material (manure and faeces). In the case of accidental release into the environment of viable seeds of soybean DAS , there are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS plants, unless these plants are exposed to the intended herbicides. This will not result in different environmental impacts compared to conventional soybean. Considering the scope of the application EFSA-GMO-NL , interactions with the biotic and abiotic environment are not considered to be relevant issues. Bioinformatic analyses of the inserted DNA identified sufficient sequence identity with bacterial DNA which could theoretically facilitate the transfer of a plant codonoptimised pat gene onto a plasmid of a soil bacterium. Illegitimate transfer of a plant-optimised aad-12 gene was also considered. Based on the functional proteins encoded by these genes and their expected prevalence in environmental bacteria, the GMO Panel did not identify a concern in relation to horizontal gene transfer to bacteria. Therefore, considering the introduced traits, the outcome of the 3 EFSA Journal 2017;15(3):4719

4 comparative analysis, the routes of exposure and the limited exposure levels, the GMO Panel concludes that soybean DAS would not raise safety concerns in the event of accidental release of viable GM soybean seeds into the environment. The scope of the post-market environmental monitoring plan provided by the applicant and the reporting intervals are in line with the intended uses of soybean DAS and the GMO Panel guidelines on the post-market environmental monitoring of GM plants. In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL , additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications. In conclusion, the GMO Panel considers that the information available for soybean DAS addresses the scientific comments raised by the Member States and that soybean DAS , as described in this application, is as safe as its conventional counterpart and the tested non-gm soybean reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application. 4 EFSA Journal 2017;15(3):4719

5 Table of contents Abstract... 1 Summary Introduction Background Terms of Reference as provided by the requestor Data and methodologies Data Methodologies Assessment Molecular characterisation Evaluation of relevant scientific data Transformation process and vector constructs Transgene constructs in the GM plant Information on the expression of the insert Inheritance and stability of inserted DNA Conclusion on molecular characterisation Comparative analysis Evaluation of relevant scientific data Choice of comparator and production of material for the comparative assessment Agronomic and phenotypic analysis Compositional analysis Conclusions on the comparative analysis Food/feed safety assessment Evaluation of relevant scientific data Effects of processing Toxicology Animal studies with the food/feed derived from GM plants Allergenicity Nutritional assessment of GM food/feed Post-market monitoring of GM food/feed Conclusions on the food/feed safety assessment Environmental risk assessment and monitoring plan Evaluation of relevant scientific data Environmental risk assessment Post-market environmental monitoring Conclusions on the environmental risk assessment and monitoring plan Conclusions Documentation provided to EFSA References Abbreviations EFSA Journal 2017;15(3):4719

6 1. Introduction Soybean DAS was developed to confer tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate ammonium-based herbicides. Tolerance to 2,4-D and other related phenoxy herbicides is provided by the expression of the aryloxyalkanoate dioxygenase-12 (AAD-12) protein from Delftia acidovorans. Tolerance to glufosinate ammonium-based herbicides is provided by the expression of the phosphinothricin acetyltransferase (PAT) protein from Streptomyces viridochromogenes. 1 The assessment of potential consumer health risks resulting from 2,4-D residues and its metabolites in soybean DAS is outside the remit of the GMO Panel and needs to be performed upon request of an applicant in the framework of Regulation (EC) No 396/ Background On 25 January 2011, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands an application (Reference EFSA-GMO-NL ) for authorisation of genetically modified (GM) soybean DAS (Unique Identifier DAS ), submitted by Dow AgroSciences LLC within the framework of Regulation (EC) No 1829/2003 on GM food and feed. 2 After receiving the application EFSA-GMO-NL , and in accordance with Articles 5(2)(b) and 17(2)(b) of the Regulation (EC) No 1829/2003, EFSA informed the Member States and the European Commission, and made the summary of the application publicly available on the EFSA website. 3 EFSA initiated a formal review of the application to check compliance with the requirements laid down in Articles 5(3) and 17(3) of the Regulation (EC) No 1829/2003. On 23 March 2011, 5 May 2011, 27 June 2011 and 19 August 2011 EFSA received additional information requested under completeness check (on 4 March 2011, 12 April 2011, 24 May 2011 and 20 July 2011, respectively). On 8 September 2011, EFSA declared the application as valid in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003. EFSA made the valid application available to the Member States and the European Commission, and consulted nominated risk assessment bodies of Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC 4 following the requirements of Articles 6(4) and 18(4) of Regulation (EC) No 1829/2003, to request their scientific opinion. Member States had 3 months after the date of receipt of the valid application (until 8 December 2012) to make their opinion known. The GMO Panel requested additional information from the applicant on 5 December 2011, 30 January 2012, 20 April 2012, 7 September 2012, 1 July 2014, 28 November 2014, 16 February 2015, 19 February 2015, 6 March 2015, 1 April 2015, 24 June 2015, 15 September 2015, 2 October 2015, 23 March 2016, 26 April 2016 (EURL-JRC), 26 May 2016 and 29 September The applicant provided the requested information on 13 April 2012, 15 May 2012, 18 October 2013, 2 September 2014, 16 December 2014, 19 February 2015, 12 March 2015, 16 March 2015, 11 June 2015, 22 July 2015, 25 September 2015, 26 April 2016 (EURL-JRC), 29 April 2016, 13 May 2016, 13 June 2016 and 26 October The applicant also spontaneously provided additional information on 7 August 2012, 27 August 2012, 22 December 2015, 31 March 2016 (EURL sequence info) and 13 May In the frame of contract OC/EFSA/UNIT/GMO/2013/01 CT01, the contractor performed preparatory work and delivered reports on the methods applied by the applicant in performing bioinformatic analyses. On 7 September 2016, the European Union Reference Laboratory (EURL-JRC) submitted to EFSA the report on the verification of sequencing data on event DAS received from the applicant. In giving its scientific opinion on soybean DAS to the European Commission, Member States and the applicant, and in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, EFSA has endeavoured to respect a time limit of 6 months from the acknowledgement of the valid application. As additional information was requested by the GMO Panel, the time limit of 6 months was extended accordingly, in line with Articles 6(1), 6(2), 18(1) and 18(2) of Regulation (EC) No 1829/ Dossier: Part I Section D1. 2 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, , p Available online: 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, , p EFSA Journal 2017;15(3):4719

7 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 and thus will be part of the EFSA overall opinion in accordance with Articles 6(5) and 18(5) Terms of Reference as provided by the requestor The GMO Panel was requested to carry out a scientific assessment of soybean DAS for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003. Where applicable, any conditions or restrictions which should be imposed on the placing on the market and/or specific conditions or restrictions for use and handling, including post-market monitoring requirements based on the outcome of the risk assessment and, in the case of GMOs or food/feed containing or consisting of GMOs, conditions for the protection of particular ecosystems/environment and/or geographical areas should be indicated in accordance with Articles 6(5)(e) and 18(5)(e) of Regulation (EC) No 1829/2003. The GMO Panel was not requested to give an opinion on information required under Annex II to the Cartagena Protocol. Furthermore, the GMO Panel did not consider proposals for labelling and methods of detection (including sampling and the identification of the specific transformation event in the food/feed and/or food/feed produced from it), which are matters related to risk management. 2. Data and methodologies 2.1. Data In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL , additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications Methodologies The GMO Panel carried out a scientific risk assessment of soybean DAS for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/ The GMO Panel took into account the appropriate principles described in its guidelines for the risk assessment of GM plants and derived food and feed (EFSA, 2006a; EFSA GMO Panel, 2011a), the environmental risk assessment (ERA) of GM plants (EFSA GMO Panel, 2010a) and the post-market environmental monitoring (PMEM) of GM plants (EFSA GMO Panel, 2011b). The comments raised by Member States are addressed in Annex G of EFSA s overall opinion 3 and were taken into consideration during the scientific risk assessment. 3. Assessment 3.1. Molecular characterisation Evaluation of relevant scientific data Transformation process and vector constructs Soybean DAS was developed by Agrobacterium tumefaciens (also known as Rhizobium radiobacter)-mediated transformation of cotyledonary nodes of soybean (Glycine max (L.) Merr.) line Maverick with the A. tumefaciens strain EHA101 containing the binary plasmid pdab4468. The plasmid pdab4468 contained two expression cassettes, aad-12 and pat, between the right and left borders of the T-DNA. 5 The aad-12 expression cassette contains the following genetic elements: the constitutive Arabidopsis thaliana polyubiquitin UBQ10 promoter, 5 -untranslated region and intron; a codonoptimised version of the aad-12 gene from D. acidovorans; and the 3 -untranslated region from the open reading frame (ORF) 23 of A. tumefaciens pti15955 (AtuORF23), which includes a transcription terminator. The RB7-MAR matrix attachment region from Nicotiana tabacum was positioned next to the aad-12 expression cassette to increase expression of the aad-12 gene. 5 Dossier: Part I Sections C2 and C EFSA Journal 2017;15(3):4719

8 The pat expression cassette consisted of the following elements: the promoter and 5 -untranslated region from the Cassava vein mosaic virus (CsVMV); a codon-optimised version of the pat gene from the bacterium S. viridochromogenes; and the 3 -untranslated region from the ORF1 of A. tumefaciens pti15955 (AtuORF1), which includes a terminator and a polyadenylation site. The vector backbone contained elements necessary for the maintenance and selection of the plasmid in bacteria Transgene constructs in the GM plant 6 Molecular characterisation of soybean DAS was performed by Southern analysis, polymerase chain reaction (PCR) and DNA sequence analysis, in order to determine copy number, size and organisation of the inserted sequences and to confirm the absence of plasmid backbone sequences. The approach used was acceptable both in terms of coverage and sensitivity. Southern analysis indicated that soybean DAS contains a single insert, which consists of a single copy of the T-DNA in the same configuration as in the pdab4468 vector. The insert and copy number were confirmed using multiple combinations of restriction endonucleases and 19 probes that covered all elements of the plasmid. No elements from the vector backbone were detected. 7 The nucleotide sequence of the entire insert of soybean DAS , together with 2,730 bp of the 5 and 1,082 bp of 3 flanking regions, was determined. The EURL-JRC checked the compliance of the sequencing data provided for event DAS with the requirements of its guidance. 8 The insert of 6,400 bp is identical to the T-DNA of pdab4468, except for the insertion of 9 bp at the 3 end of the insert. A comparison of the sequences of the flanking regions with that of the pre-insertion locus indicated that 55 bp were deleted from soybean genomic DNA. No evidence was found for the interruption of any known endogenous gene in the soybean genome. The results of segregation (Section ) and bioinformatic analyses established that the insert is located in the nuclear genome. 9 Updated bioinformatic analyses of the amino acid sequences of the newly expressed AAD-12 and PAT proteins revealed no significant similarities to toxins and allergens. In addition, updated bioinformatics analyses of the newly created ORFs present within the insert or spanning the junctions between the insert and genomic DNA did not indicate significant similarities to toxins and allergens Information on the expression of the insert 11 Protein levels of AAD-12 and PAT were analysed by enzyme-linked immunosorbent assay (ELISA) in material harvested from field trials performed at eight locations in the USA during the 2009 growing season (also used for comparative assessment, Section 3.2.1). Samples analysed included leaf (V5 and V10 12), root (R3), forage (R3) and seed (R8-maturity) from soybean DAS treated and not treated with 2,4-D, glufosinate ammonium-based herbicides or a combination of the two. The mean values, standard deviations and ranges of protein expression levels of AAD-12 and PAT in seed and forage are summarised in Table 1. 6 Dossier: Part I Section D2. 7 Dossier: Part I Section D2; additional information: 13/4/ Guideline for the submission of DNA sequences derived from genetically modified plants and associated annotations within the framework of Directive 2001/18/EC and Regulation (EC) No 1829/2003 ( 9 Dossier: Part I Section D2; additional information: 13/4/2012, 16/12/2014 and 22/12/ Dossier: Part I Section D2; additional information: 22/12/ Dossier: Part I Section D EFSA Journal 2017;15(3):4719

9 Table 1: Protein expression data (lg/g dry weight) for AAD-12 and PAT in soybean DAS seed and forage Seed Forage AAD-12 PAT AAD-12 PAT Untreated Glufosinate treated 2,4-D treated Glufosinate and 2,4-D treated (a) 4.17 (b) ( ) (c) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (< ) ( ) ( ) ( ) 2,4-D: 2,4-dichlorophenoxyacetic acid; AAD-12: aryloxyalkanoate dioxygenase-12; PAT: phosphinothricin acetyltransferase. Number of seed and forage samples is 28 both for untreated (unsprayed) and for herbicide treated plants. (a): Mean. (b): Standard deviation. (c): Range Inheritance and stability of inserted DNA 12 Genetic stability of the soybean DAS insert was assessed by Southern analysis of genomic DNA from three consecutive generations. The restriction enzyme/probe combinations used were sufficient to conclude that all the plants tested retained the single copy of the insert and flanking regions, which were stably inherited in subsequent generations. Phenotypic stability was observed by segregation analysis of the 2,4-D tolerance trait of soybean DAS The results supported the presence of a single insertion, segregating in a Mendelian fashion Conclusions on molecular characterisation The molecular characterisation data established that soybean DAS contains a single insert consisting of one copy of the aad-12 and pat expression cassettes. Bioinformatic analyses of the sequences encoding the newly expressed proteins and other ORFs present within the insert or spanning the junctions between the insert and genomic DNA did not indicate significant similarities to toxins and allergens. The levels of the AAD-12 and PAT proteins were obtained and reported adequately. The stability of the inserted DNA and of the introduced herbicide tolerance traits was confirmed over several generations Comparative analysis Evaluation of relevant scientific data Choice of comparator and production of material for the comparative assessment 13 Application EFSA-GMO-NL presents data on agronomic and phenotypic characteristics, as well as forage and seed composition, of soybean DAS derived from field trials performed at eight sites in the USA in 2009 (Table 2). Table 2: Overview of comparative assessment studies with soybean DAS provided in application EFSA-GMO-NL Study focus Study details Comparators Agronomic and phenotypic characteristics; composition Agronomic and phenotypic characteristics Field trials, 2009, USA (eight locations) Maverick Seed germination test Maverick None Commercial reference varieties Six non-gm varieties 12 Dossier: Part I Section D5. 13 Dossier: Part I Section D EFSA Journal 2017;15(3):4719

10 The field trials were conducted in major soybean growing areas of the USA, 14 representing regions of diverse agronomic practices and environmental conditions. At each site, the following materials were grown in a randomised complete block design with four replicates: soybean DAS (DAS / untreated), the non-gm comparator Maverick and three non-gm soybean reference varieties, all treated with required maintenance pesticides (including conventional herbicides); and soybean DAS treated with 2,4-D (DAS /2,4-D), with glufosinate ammonium (DAS /glufosinate) and with both 2,4-D and glufosinate ammonium (DAS /2,4-D + glufosinate). In total (across sites), six non-gm soybean reference varieties 15 were included in the field trials for agronomic/phenotypic characteristics and composition (Table 2). Soybean DAS was obtained using the non-gm soybean variety Maverick as recipient variety (Section ). As documented by the pedigree, the line of soybean DAS used in the field trials was not crossed with other soybean lines. Maverick was used as comparator in the field trials (Table 2), and has the same genetic background as the line of soybean DAS used. The GMO Panel considers that this non-gm line is the appropriate conventional counterpart. Statistical analysis of field trials data The statistical analysis of the agronomic, phenotypic and compositional data from the 2009 field trials followed the recommendations of the GMO Panel (EFSA GMO Panel, 2010b, 2011a). This included, for each of the four treatments of soybean DAS , the application of a difference test (between the GM soybean and its conventional counterpart) and an equivalence test (between the GM soybean and the set of non-gm soybean reference varieties). The results of the equivalence test are categorised into four possible outcomes (I IV, ranging from equivalence to non-equivalence) Agronomic and phenotypic analysis 17 Agronomic and phenotypic characteristics tested under field conditions 18 The agronomic and phenotypic parameters evaluated in the 2009 field trials were: (early) stand count, early population (as % planted seeds), days to 50% flowering, days to maturity, plant height, number of pods, number of seeds, final population (plant count), yield, seedling vigour, plant vigour (crop injury from herbicide application, scored at V4, R1 and R2) and lodging. Additionally, visually observable responses to naturally occurring diseases (disease incidence) and arthropod damage were recorded, in order to provide indications of altered stress responses of soybean DAS as compared with its conventional counterpart. Of the 16 endpoints evaluated, nine 19 could be analysed with the combination of difference and equivalence testing described in Section , with the following results: For soybean DAS /untreated, the test of difference identified statistically significant differences from the conventional counterpart for three endpoints ( number of seeds, stand count and days to 50% flowering ). The test of equivalence between soybean DAS / untreated and the non-gm soybean reference varieties indicated that number of seeds and stand count fell under equivalence category I, while days to 50% flowering 20 fell under equivalence category III (non-equivalence is more likely than equivalence); For DAS /2,4-D, a statistically significant difference was identified for the endpoint number of seeds, which fell under equivalence category I; For DAS /glufosinate, statistically significant differences were identified for five endpoints ( days to maturity, number of seeds, stand count, early population and final population ), which all fell under equivalence category I; For DAS /2,4-D + glufosinate, no statistically significant differences were identified. 14 One site each in Arkansas, Iowa, Indiana and Nebraska, and two sites each in Illinois and Missouri. 15 Pioneer 93M62, LG Seeds C3884N, Arise 9E394, Phillips 363, Hisoy 38C60 and Hoffman H 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). 17 Dossier: Part I Section D7.4; additional information: 18/10/2013 and 2/9/ Dossier: Part I Section D7.4; additional information: 7/8/2012, 27/8/2012, 18/10/2013 and 2/9/ The endpoints were: early population, days to 50% flowering, days to maturity, plant height, number of pods, number of seeds, final population, stand count and yield. 20 Soybean DAS /untreated: heat units; conventional counterpart: heat units EFSA Journal 2017;15(3):4719

11 The remaining seven endpoints 21 did not fulfil the assumptions for parametric testing and were analysed with the Wilcoxon Signed Rank (WSR) test. Significant differences with the conventional counterpart were identified for DAS /glufosinate ( plant vigour at stage V4) and DAS / 2,4-D + glufosinate ( disease incidence and plant lodging ); however, the average values for the GM soybean were within the range of the non-gm commercial reference varieties. In conclusion, none of the agronomic and phenotypic differences between soybean DAS and its conventional counterpart observed at field level were considered relevant, except for days to 50% flowering for soybean DAS /untreated. The difference identified in days to 50% flowering is therefore further assessed for its potential environmental impact in Section Agronomic and phenotypic characteristics tested under controlled conditions 22 Seed germination of soybean DAS was compared with that of its conventional counterpart under warm and cold conditions. Four replicates of 100 seeds for each line, in a randomised complete block design, were tested for each of the temperature treatments. The warm treatment consisted of exposure to a constant temperature of 25 C for 5 days, while the cold treatment consisted of exposure to 10 C for 7 days, followed by additional exposure to 25 C for 5 days. The germination rate of soybean DAS seeds under warm and cold conditions did not differ significantly from that of its conventional counterpart Compositional analysis 23 Soybean forage and seeds harvested from the field trials in the USA in 2009 were analysed for 87 different constituents (nine in forage 24 and 78 in seeds 25 ), including the key constituents recommended by the OECD (OECD, 2001). Considering the data on substrate specificity for AAD-12 (Section ), the GMO Panel concluded that the spectrum of constituents chosen by the applicant was adequate. Seventeen seed constituents having more than 50% of the observations below the limit of quantification were excluded from the statistical analysis. 26 Of the remaining 70 constituents, the test of equivalence could not be applied to a forage endpoint (NDF) and to five seed endpoints 27 because the estimated variation among the non-gm reference varieties was too small. Among those six endpoints, only iron level for DAS /glufosinate was significantly different from that of the conventional counterpart (Table 3). The test of difference and the test of equivalence could be applied to the remaining 64 endpoints, with the following results: For soybean DAS /untreated, the test of difference identified statistically significant differences from the conventional counterpart for 22 constituents (2 in forage and 20 in seeds). The test of equivalence between soybean DAS /untreated and the non-gm soybean reference varieties indicated that the level of 18 of the 22 constituents 28 fell under equivalence category I or II, while the level of four seed constituents fell under equivalence category III or IV (Table 3). 21 The endpoints were: disease incidence, insect damage, plant lodging, seedling vigour and plant vigour. 22 Additional information: 2/9/ Dossier: Part I Section D7.1; additional information: 13/4/2012, 7/8/2012, 27/8/2012, 18/10/2013, 2/9/2014 and 16/3/ Proximates (crude protein, crude fat, ash, and moisture), carbohydrates by calculation, fibre fractions (acid detergent fibre (ADF) and neutral detergent fibre (NDF)), calcium, and phosphorus. 25 Protein, fat, ash, moisture, carbohydrates, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre, calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc, alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), myristoleic acid (14:1), pentadecanoic acid (15:0), pentadecenoic acid (15:1), palmitic acid (16:0), palmitoleic acid (16:1), heptadecanoic acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), c-linolenic acid (18:3), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatrienoic acid (20:3), arachidonic acid (20:4), behenic acid (22:0), b-carotene, thiamine HCl, riboflavin, niacin, pantothenic acid, pyridoxine HCl, folic acid, ascorbic acid, a-tocopherol, b-tocopherol, c-tocopherol, d-tocopherol, total tocopherol, total daidzein equivalent, total genistein equivalent, total glycitein equivalent, lectin (activity), phytic acid, raffinose, stachyose and trypsin inhibitor. 26 These were: sodium, caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), myristoleic acid (14:1), pentadecanoic acid (15:0), pentadecenoic acid (15:1), palmitoleic acid (16:1), heptadecanoic acid (17:0), heptadecenoic acid (17:1), c-linolenic acid (18:3), eicosadienoic acid (20:2), eicosatrienoic acid (20:3), arachidonic acid (20:4), b-carotene and b-tocopherol. 27 Aspartic acid, proline, serine, NDF and iron. 28 In forage: ash and moisture. In seeds: isoleucine, leucine, methionine, phenylalanine, raffinose, stachyose, oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0), ash, moisture, total fat, riboflavin, pantothenic acid and folic acid EFSA Journal 2017;15(3):4719

12 For DAS /2,4-D, statistically significant differences were identified for 23 constituents (2 in forage and 21 in seeds). The level of 19 of the 23 constituents 29 fell under equivalence category I or II, while the level of four seed constituents fell under equivalence category III or IV (Table 3). For DAS /glufosinate, statistically significant differences were identified for 26 constituents (2 in forage and 24 in seeds). The level of 19 of the 26 constituents 30 fell under equivalence category I or II, while the level of seven seed constituents fell under equivalence category III or IV (Table 3). For DAS /2,4-D + glufosinate, statistically significant differences were identified for 20 constituents (1 in forage and 19 in seeds). The level of 16 of the 20 constituents 31 fell under equivalence category I or II, while the level of four seed constituents fell under equivalence category III or IV (Table 3). Table 3: Compositional endpoints that are further discussed based on the results of the statistical analysis: means (for the conventional counterpart and the GM soybean) and equivalence limits (from the non-gm reference varieties) estimated from the 2009 field trials Endpoint Conventional counterpart Soybean DAS Untreated (a) 2,4-D (b) Glufosinate (c) 2,4-D + glufosinate (d) Equivalence limits from non-gm reference varieties Moisture (e) (%FW) * 12.82* 13.13* 12.74* (12.79, 14.31) Arginine (%AA) * 7.476* 7.466* 7.456* (7.528, 7.840) Glutamic acid (%AA) * 16.92* 16.83* (17.02, 17.80) Histidine (%AA) * (2.507, 2.699) Leucine (%AA) * 7.754* 7.779* 7.762* (7.615, 7.767) Stearic acid (18:0) (%FA) * 4.353* 4.29* (3.547, 4.186) Folic acid (mg/kg DM) * 2.53* 2.465* 2.589* (2.477, 3.375) Lectin activity (HU/mg protein) * * (21.89, 52.77) Raffinose (%DM) * * (0.544, 0.768) Calcium (mg/g DM) * 3.053* 3.222* (2.201, 3.144) Iron (mg/kg DM) * ,4-D: 2,4-dichlorophenoxyacetic acid; FW: fresh weight; DM: dry matter; %AA: percentage of total amino acids; %FA: percentage of total fatty acids; HU: haemagglutination unit; : the equivalence test was not applied because the estimated variation among the non-gm reference varieties was too small. For the GM soybean, significantly different entries are marked with an asterisk, while the outcomes of the test of equivalence are differentiated by greyscale backgrounds: white (equivalence categories I and II and iron, for which the test was not applied), light grey (equivalence category III) and dark grey (equivalence category IV). (a): Sprayed only with conventional herbicides. (b): Sprayed with 2,4-D. (c): Sprayed with glufosinate ammonium-based herbicides. (d): Sprayed with 2,4-D and glufosinate ammonium-based herbicides. (e): Mean values shown for moisture were re-calculated by EFSA for higher numerical precision. Regarding the differences in moisture, stearic acid and calcium and in four amino acids (Table 3), no further assessment was deemed necessary owing to the known biochemical roles of the compounds involved and to the small absolute magnitude of the reported changes. The increase in iron content in DAS /glufosinate is not of concern (or benefit), considering the low absorption of iron (2% to < 5%) from phytate-rich legumes like soybean (Hurrell, 2003). Folic acid decreased in all GM soybean treatments; however, considering that soybean is not a relevant source of folic acid in 29 In forage: ash and protein. In seeds: isoleucine, leucine, tryptophan, tyrosine, total glycitein equivalent, oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0), eicosenoic (20:1), moisture, protein, total fat, calcium, zinc, pyridoxine HCl and folic acid. 30 In forage: ash and protein. In seeds: alanine, isoleucine, tryptophan, tyrosine, stachyose, total glycitein equivalent, oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0), ash, moisture, protein, total fat, phosphorus, riboflavin and pantothenic acid. 31 In forage: ash, protein and phosphorus. In seeds: alanine, isoleucine, leucine, total glycitein equivalent, oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0), protein, total fat, riboflavin, pantothenic acid and folic acid EFSA Journal 2017;15(3):4719

13 the human diet, and that animal feed is usually supplemented with folic acid, no concern was identified for food and feed safety and nutrition. As raffinose is considered an antinutrient, the decrease in raffinose observed in two out of four treatments of soybean DAS did not pose any food/feed safety concern. Lectin activity 32 in soybean DAS was significantly different from that in the conventional counterpart for two of the four treatments (36% higher in DAS /2,4-D and 16% higher in DAS /2,4-D + glufosinate) and fell under equivalence category III or IV. Because of the known antinutritional properties of soybean lectins, the increase in lectin activity is further assessed for potential impact on food and feed safety in Section Conclusions on the comparative analysis The increase in lectin activity (up to 36%) observed in soybean DAS with respect to its conventional counterpart is further discussed in Section The GMO Panel concludes that none of the other differences identified in forage and seed composition between soybean DAS and the conventional counterpart, and none of those identified in the agronomic and phenotypic characteristics, needs further assessment regarding food and feed safety. Based on the tested agronomic and phenotypic characteristics of soybean DAS , no relevant differences were observed between soybean DAS and its conventional counterpart, except for days to 50% flowering for soybean DAS not treated with the intended herbicides. The difference in days to 50% flowering is further assessed for its potential environmental impact in Section Food/feed safety assessment Evaluation of relevant scientific data Effects of processing Processed products Soybean DAS will undergo existing production processes used for conventional soybean. No novel production process is envisaged. Compositional analysis identified an increase in lectin activity (up to 36%) in DAS seeds compared to the conventional counterpart. Food/feed processing (e.g. soaking, heating, fermentation) is known to reduce the content and/or activity of soybean endogenous antinutrients, including lectins (Liener, 1994; Duranti and Gius, 1997; OECD, 2012). The applicant provided data on toasted meal, showing that levels of lectin activity in toasted meal from DAS were strongly reduced compared to those in unprocessed DAS seeds (Table 3). 33 Newly expressed proteins a) Effect of temperature on newly expressed proteins 34 The thermal stability of the bacterial AAD-12 protein was evaluated by heating protein solutions for 30 min at 50, 70 and 95 C in a phosphate-based buffer solution. At all heating conditions (50 95 C) the enzymatic activity was eliminated and the protein lost more than 99% of its immunoreactivity. The molecular mass (~ 32 kda) was unchanged. The temperature dependence of bacterial AAD-12 protein activity was examined after 6 min at different temperatures (1 60 C), using 2,4-D as a substrate, revealing considerable activity up to 40 C, and significantly decreased activity at 50 and 60 C. The thermal stability of the bacterial PAT protein was evaluated by heating protein solutions for 30 min at different temperatures (25 95 C) in a buffer solution. The molecular mass of the PAT protein (~ 20 kda) was unchanged at temperatures 55 C. At temperatures 55 C, > 99% of the enzymatic activity was lost with no residual activity detected above 75 C. At temperatures 37 C, the soluble PAT protein lost 91% of its immunoreactivity. 32 The biological activity of soybean lectins was quantified using a haemagglutination assay with rabbit red blood cells (RBCs) (Liener, 1955). The activity was measured in haemagglutination units (HU): one HU corresponds to the level of test solution (serially diluted) that gives agglutination of 50% of the RBCs. 33 Dossier: Part I Section D7.10.2; additional information: 29/4/ Dossier: Part I Section D7.8.1; additional information: 18/10/ EFSA Journal 2017;15(3):4719

14 b) Effect of ph on newly expressed proteins 35 The effect of ph on the in vitro activity of the bacterial AAD-12 was assessed using 2,4-D as a substrate and a mixed buffer system with ph varying from 5.5 to 9.5. Considerable activity after 6 min was observed over a narrow window, between ph 6 and 7.5 with an optimum at 7. The effect of ph on the in vitro activity of the bacterial PAT was assessed using acetyl-coa and glufosinate as substrates and a mixed buffer system with ph at 3, 8 and 11. The enzyme activity was significantly reduced after 10 min, at ph 3 and 11, showing highest activity at ph 8; the molecular mass (~ 20 kda) was unchanged at acidic, neutral, and basic ph s Toxicology Soybean DAS expresses two new proteins, AAD-12 and PAT (see Section 3.1.1). Proteins used for safety assessment Given the technical restraints in producing large enough quantities of the proteins from plants for safety testing, these proteins were recombinantly produced in Pseudomonas fluorescens. Prior to safety studies, a set of biochemical methods was employed to demonstrate the equivalence between soybean- and microbe-derived proteins. Purified proteins from these two sources were characterised and compared in terms of their physicochemical, structural and functional properties. a) AAD-12 characterisation and equivalence 36 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis showed that plant- and microbe-derived AAD-12 proteins had the expected molecular weight of ~ 32 kda and were comparably immunoreactive to AAD-12 protein specific antibodies. In addition, glycosylation detection analysis demonstrated that the AAD-12 proteins were not glycosylated. Amino acid sequence analysis by mass spectrometry methods showed that both proteins matched their expected sequence. These data also showed that the N-terminal methionine of both proteins was truncated, while alanine 2 of the plant protein was also acetylated. Additional variants of the plant protein were identified that were truncated up to threonine 9. Such modifications are common in eukaryotic proteins (e.g. Polevoda and Sherman, 2000). The C-termini of the plant- and microbial-derived proteins were identical and fully matched the theoretical AAD-12 sequence. Functional equivalence was demonstrated by a biochemical in vitro activity assay which showed that both proteins had comparable activity for the intended herbicide. Plant- and microbial-produced AAD-12 proteins were also screened for their ability to utilise certain endogenous plant substrates and none of them were metabolised by AAD-12. b) PAT characterisation and equivalence 37 The equivalence between the plant- and microbe-derived PAT proteins was demonstrated by SDS- PAGE and western blot analysis. The results from these analyses showed that both proteins migrated to the expected molecular weight of ~ 20.5 kda. In addition, western blot analysis showed that both proteins were comparably immunoreactive to PAT specific antibodies. Functional equivalence was demonstrated by a biochemical in vitro activity assay which showed that both proteins had comparable activity for the intended herbicide. The protein characterisation data comparing the structural, biochemical and functional properties of plant- and microbial-derived AAD-12 and PAT proteins indicate that these proteins are equivalent. Therefore, the GMO Panel accepts the use of the AAD-12 and PAT proteins expressed in bacteria for the safety studies. Toxicological assessment of newly expressed proteins The PAT protein has been previously assessed by the GMO Panel (e.g. EFSA 2007; EFSA GMO Panel, 2011c, 2013a,b), and no safety concerns for humans and animals were identified. Updated bioinformatics analyses did not reveal similarities of the PAT protein to known toxins. 38 The GMO Panel is not aware of any new information that would change these conclusions. The GMO Panel concludes that the PAT protein does not raise safety concerns. 35 Additional information: 18/10/ Dossier: Part I Section D7.8.1; additional information: 12/3/2012, 11/6/2015 and 13/5/ Dossier: Part I Section D7.8.1; additional information: 13/5/ Dossier: Part I Section D7.8.1; additional information: 18/10/2013, 2/9/2014 and 22/12/ EFSA Journal 2017;15(3):4719