Scientific opinion on an application by Dow AgroSciences LLC (EFSA‐GMO‐NL‐2012‐106) for the placing on the market of genetically modified herbicide‐tolerant soybean DAS‐44406‐6 for food and feed uses, import and processing under Regulation (EC) No 1829/2003

Abstract Soybean DAS‐44406‐6 expresses 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase (2mEPSPS), conferring tolerance to glyphosate‐based herbicides, aryloxyalkanoate dioxygenase (AAD‐12), conferring tolerance to 2,4‐dichlorophenoxyacetic acid (2,4‐D) and other related phenoxy herbicides, and phosphinothricin acetyl transferase (PAT), conferring tolerance to glufosinate ammonium‐based herbicides. The molecular characterisation data and bioinformatics analyses did not identify issues requiring assessment for food/feed safety. The agronomic and phenotypic characteristics revealed no relevant differences between soybean DAS‐44406‐6 and its conventional counterpart, except for pod count, seed count and yield. The compositional analysis identified no differences requiring further assessment, except for an increase (up to 31%) in lectin activity in soybean DAS‐44406‐6. Such increase is unlikely to raise additional concerns for food/feed safety and nutrition of soybean DAS‐44406‐6 as compared to its conventional counterpart and non‐GM reference varieties. There were no concerns regarding the potential toxicity and allergenicity of the three newly expressed proteins, and no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS‐44406‐6. Soybean DAS‐44406‐6 is as nutritious as its conventional counterpart and the non‐GM soybean reference varieties tested. There are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS‐44406‐6 plants, unless exposed to the intended herbicides. The likelihood of environmental effects from the accidental release of viable seeds from soybean DAS‐44406‐6 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‐44406‐6. In conclusion, the GMO Panel considers that the information available for soybean DAS‐44406‐6 addresses the scientific comments raised by Member States and that soybean DAS‐44406‐6, as described in this application, is as safe as its conventional counterpart and 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.

Summary seeds into the environment. The scope of the PMEM plan provided by the applicant and the reporting intervals are in line with the intended uses of soybean DAS-44406-6 and the GMO Panel guidelines on the PMEM of GM plants.
In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL-2012-106, 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-44406-6 addresses the scientific comments raised by Member States and that soybean DAS-44406-6, as described in this application, is as safe and as nutritious as its conventional counterpart and 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.

Introduction
Soybean DAS-44406-6 was developed to confer tolerance to glyphosate-based herbicides, 2,4dichlorophenoxyacetic acid (2,4-D) and glufosinate ammonium-based herbicides. Tolerance to glyphosate-based herbicides is provided by the expression of 5-enolpyruvyl-shikimate-3-phosphate synthase (2mEPSPS) from maize, while tolerance to 2,4-D and other related phenoxy herbicides is achieved by the expression of aryloxyalkanoate dioxygenase (AAD-12) from Delftia acidovorans. Tolerance to glufosinate ammonium-based herbicides is provided by the expression of phosphinothricin acetyl transferase (PAT) from Streptomyces viridochromogenes. 1 The assessment of potential consumer health risks resulting from 2,4-D residues and its metabolites in soybean DAS-44406-6 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 396/2005.

Background
On 16 February 2012, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands an application (Reference EFSA-GMO-NL-2012-106) for authorisation of GM soybean DAS-44406-6 (Unique Identifier DAS-4440Ø6-6), 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-2012-106, and in accordance with Articles 5(2)(b) and 17 (2) In the frame of contract OC/EFSA/UNIT/GMO/2013/01, CFT/EFSA/AMU/2011/01 and OC/EFSA/ UNIT/GMO/2014/01, the contractors performed preparatory work and delivered reports on the information provided and the methods applied by the applicant in performing bioinformatic analyses, statistical analyses and toxicological studies, respectively.
In giving its scientific opinion on soybean DAS-44406-6 to the European Commission (EC), 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 1829/2003. 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).

1.2.
Terms of Reference as provided by the requestor The GMO Panel was requested to carry out a scientific assessment of soybean DAS-44406-6 for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829 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

Data
In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL-2012-106, additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications.

Methodologies
The GMO Panel carried out an evaluation of the scientific risk assessment of soybean DAS-44406-6 for food and feed uses, import and processing in accordance with Articles 6(6) and 18 (6)  The comments raised by Member States are addressed in Annex G of the EFSA overall opinion 3 and were taken into consideration during the evaluation of risk assessment. Soybean DAS-44406-6 was developed by A. tumefaciens (also known as Rhizobium radiobacter)mediated transformation of cotyledonary nodes derived from germinated soybean (G. max) cv. Maverick seeds with plasmid vector pDAB8264. Glufosinate ammonium tolerance conferred by the PAT protein was used as a marker during the selection of transformants.
The plasmid pDAB8264 used for the transformation contained three expression cassettes between the right and left borders of the T-DNA: 2mepsps, aad-12 and pat.
The 2mepsps expression cassette contains the following genetic elements: a promoter and a 5'-untranslated region (UTR) from the Arabidopsis thaliana histone H4A748 gene; TPotp C, a chloroplast transit (targeting) peptide derived from the maize and sunflower RuBisCO small subunits; the maize 2mepsps gene with two amino acid changes at positions 102 (threonine to isoleucine) and 106 (proline to serine), and the 3'-UTR of the A. thaliana histone H4A748 gene, including a transcription terminator. The RB7-MAR matrix attachment region from Nicotiana tabacum was positioned next to the 2mepsps expression cassette, to facilitate its expression.
The aad-12 expression cassette contains the following genetic elements: the constitutive A. thaliana polyubiquitin UBQ10 promoter, 5'-UTR and intron; a codon-optimized version of the aad-12 gene from Delftia acidovorans; and the 3'-UTR from the ORF23 of A. tumefaciens pTi15955 (AtuORF23), which includes a transcription terminator.
The pat expression cassette contains the following genetic elements: the promoter and 5'-UTR from Cassava vein mosaic virus (CsVMV); a codon-optimized version of the pat gene from S. viridochromogenes; and the 3'-UTR from the ORF1 of A. tumefaciens pTi15955 (AtuORF1), which includes a transcription terminator.
The vector backbone contained elements necessary for the maintenance of the plasmid in bacteria.
3.1.1.2. Transgene constructs in the genetically modified plant 1 Molecular characterisation of soybean DAS-44406-6 was performed by Southern analysis, polymerase chain reaction (PCR) and DNA sequence analysis, in order to determine insert 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 analyses indicated that soybean DAS-44406-6 contains a single insert, which consists of a single copy of the T-DNA in the same configuration as in the pDAB8264 vector. The insert and copy number were confirmed by multiple restriction enzyme/probe combinations covering the T-DNA region and flanking regions. PCR analyses confirmed the results obtained by the Southern analyses. The absence of vector backbone sequences was demonstrated by Southern analysis using backbonespecific overlapping probes.
The nucleotide sequence of the entire insert of soybean DAS-44406-6 together with 1,494 bp of the 5' and 1,885 bp of the 3' flanking regions were determined. The insert of 10,280 bp is identical to the T-DNA of pDAB8264, except for the deletion of the 5' terminal end and the truncation of the 3' terminal end of the T-DNA, and the insertion of 3 bp. A comparison of the flanking regions with the pre-insertion locus indicated that 4,383 bp of the parental genomic sequence, containing fragments of putative retrotransposable element, were deleted at the insertion locus. Approximately 42% of the soybean genome is occupied by retrotransposon-related sequences. Most of these sequences are remnants of ancient retrotransposons and are no longer active. In addition, most of the retrotransposon-related individual sequences do not fulfil a specific role in the genome. Therefore, this deletion does not raise a particular concern. The possible interruption of known endogenous soybean genes by the insertion in event DAS-44406-6 was evaluated by bioinformatic analyses of the pre-insertion locus and of the genomic sequences flanking the insert. The results of these analyses did not reveal the interruption of any known endogenous gene in the soybean DAS-44406-6. 5 The results of segregation (see below) and bioinformatic analyses established that the insert is located in the nuclear genome.
Updated bioinformatic analyses of the amino acid sequences of the newly expressed 2mEPSPS, AAD-12 and PAT proteins revealed no significant similarities to known toxins and allergens. 5 In addition, updated bioinformatic analyses of the newly created open reading frames (ORFs) within the insert or spanning the junctions between the insert and genomic DNA did not indicate significant similarities to toxins and allergens. 5 In order to assess the possibility for horizontal gene transfer by homologous recombination (HR), the applicant performed a sequence identity analysis of the regions of bacterial origin in soybean DAS-44406-6. Two elements were identified with sufficient length and identity to support HR (EFSA, 2015), namely the 525 bp fragment of A. tumefaciens Ti plasmid pTi15955 containing the 3'-UTR of ORF23 and the 703 bp fragment from the same plasmid containing the 3'-UTR of ORF1. Double HR with these sequences and the Ti plasmid of A. tumefaciens is a possible scenario to consider.
The likelihood and potential consequences of plant-to-bacteria gene transfer are described in Section 3.4.1.1.

Protein characterisation and equivalence
Soybean DAS-44406-6 expresses three new proteins AAD-12, 2mEPSPS and PAT. Given the technical restraints in producing large enough quantities for safety testing from plants, these proteins were recombinantly produced in Pseudomonas fluorescens or Escherichia coli. 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.

AAD-12 characterisation and equivalence 6
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 proteinspecific antibodies. In addition, glycosylation detection analysis demonstrated that none of the AAD-12 proteins were 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 the microbe-derived protein was deleted but that only a fraction of the plant-derived protein was similarly truncated. A fraction of the plant protein was acetylated in alanine 2. Such modifications are common in eukaryotic proteins (e.g. Polevoda and Sherman, 2000). The C-termini of the plant and the microbial-derived protein 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. Microbial-produced AAD-12 protein was also screened for its ability to utilise certain endogenous plant substrates and none of them were metabolised by  2mEPSPS characterisation and equivalence 6 SDS-PAGE and Western blot analysis showed that plant and microbe-derived 2mEPSPS proteins had the expected molecular weight of~47.5 kDa and were comparably immunoreactive to 2mEPSPS protein-specific antibodies. In addition, glycosylation detection analysis demonstrated that none of the 2mEPSPS proteins were glycosylated. Amino acid sequence analysis by mass spectrometry methods showed that both proteins matched their expected sequence. These data also showed that besides the N-terminal methionine which was truncated, both N-and C-termini were identical for the plant and microbe-derived proteins. Functional equivalence was demonstrated by a biochemical in vitro activity assay which showed that both proteins had comparable activity.

PAT characterisation and equivalence 8
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 2mEPSPS, AAD-12 and PAT proteins indicate that these proteins are equivalent. Therefore, the GMO Panel accepts the use of the 2mEPSPS, AAD-12 and PAT proteins expressed in bacteria for the safety studies.

Information on the expression of the insert 9
Protein levels of the 2mEPSPS, AAD-12 and PAT were analysed by enzyme-linked immunosorbent assay (ELISA) in material harvested from replicated field trials across 10 locations in the United States during the 2010 growing season. Samples analysed included leaves (V5 and V10-V12), root (R3), forage (R3) and seeds (R8-maturity) both treated and non-treated with glyphosate, 2,4-D, glufosinate or a combination of the three. The mean values, standard deviations and ranges of protein expression levels in seeds and forage (n = 40) of the 2mEPSPS, AAD-12 and PAT proteins are summarised in Table 1. 6 Dossier: Part II -Section A4.2 (Study ID 101707). 7 Additional information received on 18/02/2016 (Study ID: 101617). 8 Study ID: 102098 and additional information received on 13/5/2016. 9 Dossier: Part II -Section A 2.2.3.

Inheritance and stability of inserted DNA 10
Genetic stability of the soybean DAS-44406-6 insert was assessed by Southern analysis of genomic DNA from five 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 glufosinate tolerance trait of soybean DAS-44406-6. The results supported the presence of a single insertion, segregating in a Mendelian fashion.

Conclusion on the molecular characterisation
The molecular characterisation data establish that soybean DAS-44406-6 contains a single insert consisting of one copy of the 2mepsps, 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 stability of the inserted DNA and of the introduced herbicide tolerance traits was confirmed over several generations. The levels of the 2mEPSPS, AAD-12 and PAT protein were obtained and reported adequately. The protein characterisation data comparing the structural, biochemical and functional properties of plant and microbial derived 2mEPSPS, AAD-12 and PAT proteins indicate that these proteins are equivalent and the microbial derived proteins can be used in the safety studies.

3.2.
Comparative analysis 3.2.1. Evaluation of relevant scientific data 3.2.1.1. Choice of comparator and production of materials for the comparative assessment 11 Application EFSA-GMO-NL-2012-106 presents data on agronomic and phenotypic characteristics, as well as data on forage and seed composition of soybean DAS-44406-6 derived from field trials performed at 11 sites in the United States in 2010 and 2012 (Table 2). Field trials for the comparative assessment of soybean DAS-44406-6 were performed in the United States at 10 sites in 2010 and one site in 2012, all located in the major soybean-growing regions of the country. 12 All field trials were managed according to local requirements. At each field trial site, the materials were grown in a randomised complete block design with four replicates. The materials were: DAS-44406-6 with no additional herbicide treatment (DAS-44406-6/untreated); DAS-44406-6 additionally sprayed with 2,4-D (DAS-44406-6/2,4-D); DAS-44406-6 additionally sprayed with glyphosate (DAS-44406-6/glyphosate); DAS-44406-6 additionally sprayed with glufosinate (DAS-44406-6/glufosinate); DAS-44406-6 additionally sprayed with 2,4-D, glyphosate and glufosinate (DAS-44406-6/2,4-D + glyphosate + glufosinate); the comparator Maverick; and three non-GM commercial soybean reference varieties. 13,14 Soybean DAS-44406-6 was obtained using the non-GM soybean variety Maverick as recipient variety (see Section 3.1.1.1). As documented by the pedigree, the line of soybean DAS-44406-6 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 of soybean DAS-44406-6. The GMO Panel considers that this non-GM line is the appropriate conventional counterpart.

Statistical analysis of field trial data
The statistical analysis of the agronomic, phenotypic and compositional data from the 2010/2012 field trials followed the recommendations of the GMO Panel (EFSA GMO Panel, 2010a, 2011a. This included, for each of the five treatments of soybean DAS-44406-6, 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). 15

Agronomic and phenotypic analysis 16
Fourteen agronomic and phenotypic characteristics were analysed. Of those, nine 17 could be analysed with the combination of difference and equivalence testing (see Section 3.2.1.1). The outcome of the test of difference and test of equivalence is listed below: • For DAS-44406-6/untreated, one significantly different endpoint was found: yield (GM: 2,087 g; Maverick: 2,257 g), falling under equivalence category II.
• For DAS-44406-6/2,4-D, no differences were identified. • For DAS-44406-6/glyphosate, two significantly different endpoints: pod count (GM: 403.9; Maverick: 361.1) and seed count (GM: 881; Maverick: 801.7), both falling under equivalence category II. For the remaining five endpoints, 18 which did not fulfil the assumptions for parametric testing, the differences between the GM soybean and the conventional counterpart were tested using a Wilcoxon signed rank test. Statistically significant differences were identified for DAS-44406-6/2,4-D (lodging) and DAS-44406-6/2,4-D + glyphosate + glufosinate (seedling vigour); however, the average values for the GM soybean were within the range of the non-GM commercial reference varieties.
The observed differences in pod count, seed count and yield were considered relevant and are further assessed for their potential environmental impact in Section 3.4.1.1.

Agronomic and phenotypic characteristics tested under controlled conditions 16
Seed germination of soybean DAS-44406-6 was compared with that of its conventional counterpart under warm and cold conditions in two different studies. In each study, four replicates of 100 seeds for each line, in a complete randomised design, were tested for each of the temperature treatments. The warm treatment consisted of exposure to a constant temperature of 25°C for 5 or 7 days, while the cold treatment consisted of exposure to 10°C for 7 days, followed by additional exposure to 25°C for 7 days. The germination rate of soybean DAS-44606-6 seeds under warm and cold conditions did not differ significantly from that of its conventional counterpart.

Compositional analysis
Soybean forage and seeds harvested from the field trials in the United States in 2010 and 2012 were analysed for 87 different constituents (nine from forage 19 and 78 from seeds 20 ), including the key constituents recommended by the OECD (OECD, 2001). Considering the data on substrate specificity for AAD-12 (Section 3.1.1.3), the GMO Panel concluded that the spectrum of constituents chosen by the applicant is adequate. Seventeen seed constituents having more than 50% of the observations below the limit of quantification were excluded from the statistical analysis. 21 Of the remaining 70 constituents, the test of equivalence could not be applied to one forage endpoint neutral detergent fibre (NDF) and two seed endpoints (levels of isoleucine and lysine) because the variation associated with the commercial reference genotypes was too small. Among those three endpoints, only lysine level for DAS-44406-6/2,4-D was significantly different from that of the conventional counterpart (Table 3).
www.efsa.europa.eu/efsajournal under equivalence category I or II and five endpoints under equivalence category III or IV (Table 3).
• For DAS-44406-6/2,4-D, 25 significantly different endpoints (24 in seeds and one in forage) were identified. 23 Of these, 21 endpoints fell under equivalence category I or II and four endpoints under equivalence category III or IV (Table 3).
• For DAS-44406-6/glyphosate, 22 significantly different endpoints (21 from seeds and one from forage) were identified. 24 Of these, 20 endpoints fell under equivalence category I or II and two endpoints under equivalence category III or IV (Table 3).
• For DAS-44406-6/glufosinate, 22 significantly different endpoints (21 from seeds and one from forage) were identified. 25 Of these, 19 seed endpoints fell under equivalence category I or II and three endpoints under equivalence category III or IV (Table 3).
• For DAS-44406-6/2,4-D+glyphosate+glufosinate, 23 significantly different endpoints (22 from seeds and one from forage) were identified. 26 Of these, 21 endpoints fell under equivalence category I or II and two endpoints under equivalence category III or IV (Table 3). No further assessment was deemed necessary for the differences in the five amino acids, total fat and calcium levels (Table 3), owing to the known biochemical roles of the compounds involved and to the small absolute magnitude of the reported changes.
Lectin activity 27 in soybean DAS-44406-6 was significantly different (15-31% higher) than that of the conventional counterpart for all five treatments and fell under equivalence category III for four of the five treatments (Table 3). 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 3.3.1.

Conclusion on comparative analysis
The increase in lectin activity (up to 31%) observed in soybean DAS-44406-6 with respect to its conventional counterpart is further discussed in Section 3.3.1. The GMO Panel concludes that none of the other differences identified in forage and seed composition between soybean DAS-44406-6 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 agronomic and phenotypic characteristics of soybean DAS-44406-6 tested, no relevant differences were observed between soybean DAS-44406-6 and its conventional counterpart, except for higher pod count, higher seed count and lower yield for soybean DAS-44406-6. These differences are further assessed for their potential environmental impact in Section 3.4.1.1.

Processed products
Soybean DAS-44406-6 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 31%) in DAS-44406-6 seeds compared to that of the conventional counterpart. Food/feed processing (e.g. soaking, heating, fermentation) is known to reduce the content and/or activity of soybean endogenous anti-nutrients, 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-44406-6 were strongly reduced compared to those from unprocessed seeds (Table 3)

. 28
Newly expressed proteins a) Effect of temperature on newly expressed proteins The thermal stability of the bacterial 2mEPSPS protein was evaluated by heating protein solutions for 30 min at 25, 37, 55, 75, and 95°C in a buffer solution, respectively. At temperatures at or above 55°C, the enzymatic activity was reduced by ≥ 73% and the protein lost ≥ 90% of its immunoreactivity. The molecular mass (approximately 47 kDa) was unchanged. 29 These data are in line with those observed in another study where the temperature dependence of bacterial 2mEPSPS activity was examined at nine different temperatures, ranging from 0 to 69°C, showing a considerable enzyme activity from 40 to 58°C, significantly decreased at temperatures over 58°C. 30 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 (approximately 32 kDa) was unchanged. The temperature dependence of bacterial 27 The biological activity of soybean lectins was quantified using a haemagglutination assay with animal red blood cells (RBCs) (Liener, 1955). The activity is measured in haemagglutination units (HU): one HU corresponds to the level of test solution (serially diluted) that gives agglutination of 50% of the RBCs. The results of the haemagglutination assay are known to be significantly influenced by the different types/batches of red blood cells (RBC) used: therefore, the results in terms of absolute values (HU) cannot be considered reliable enough for the risk assessment. However, the applicant showed that the method is reliable in quantifying relative differences between test materials; additional information: 7/3/2016, 13/6/2016 and 1/9/2016. 28 Dossier: Part II -Section A6.2. 29 Dossier: Part II -Section A4.2 (Study ID 110461). 30 Additional information received on 13/5/2013 (Study ID 130470). AAD-12 protein activity was examined after 6 min at different temperatures, ranging from 1 to 60°C. The temperature dependence of bacterial AAD-12 protein activity was examined after 6 minutes at different temperatures, ranging from 1-60°C, using 2,4-D as a substrate. Considerable activity remained up to 40°C but decreased significantly at the temperatures of 50 and 60°C. 31 The thermal stability of the bacterial PAT protein was evaluated by heating protein solutions for 30 min at 25, 37, 55, 75, and 95°C in a buffer solution. The molecular mass of the PAT protein (approximately 20 kDa) was unchanged at temperatures ≤ 55°C. At temperatures at or above 55°C, > 99% of the enzymatic activity was lost with no residual activity detected above 75°C. At temperatures at or above 37°C, the soluble PAT protein lost ≥ 91% of its immunoreactivity. 32 b) Effect of pH on newly expressed proteins The effect of pH on the in vitro activity of the bacterial 2mEPSPS was assessed using a mixed buffer system with pH varying from 3 to 9.5. Activity was observed over a narrow window between pH 5 and 6 with an optimum at pH 5.5. 33 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 pH 7. 34 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 the highest activity at pH 8; the molecular mass (approximately 20 kDa) was unchanged at acidic, neutral and basic pH values. 35

Toxicology
The three proteins (2mEPSPS, AAD-12 and PAT) newly expressed in soybean DAS-444Ø6-6 have been extensively characterised and showed the expected molecular weight, immunoreactivity versus specific antibodies, amino acid sequence and functional activity (Section 3.1.1.3). Newly expressed 2mEPSPS and PAT proteins have been previously assessed by the GMO Panel (e.g. EFSA, 2007EFSA, , 2009bEFSA GMO Panel, 2011c and no safety concerns for humans and animals were identified. Updated bioinformatics analysis did not reveal similarities of the 2mEPSPS and PAT proteins to known toxins (Section 3.1.1.2). The GMO Panel is not aware of any new information that would change these conclusions. The GMO Panel concludes that the 2mEPSPS and PAT proteins do not raise safety concerns.
The newly expressed AAD-12 protein is assessed below.
The applicant provided new studies on 2mEPSPS, AAD-12 and PAT proteins from bacterial recombinant systems, which were considered equivalent to the plant-produced proteins (Section 3.1.1.3).

a) In vitro degradation studies 36
The resistance to degradation by pepsin of the bacterial 2mEPSPS, AAD-12 and PAT proteins was investigated in solutions at pH~1.2 in three independent studies. The integrity of the test proteins in samples of the incubation mixture taken at various time points was analysed by SDS-PAGE gel electrophoresis followed by protein staining or by Western blotting. The 2mEPSPS and PAT proteins were degraded by pepsin within 1 min. The AAD-12 protein was degraded by pepsin within 30 s. b) Acute oral toxicity testing 37 A bacterial 2mEPSPS protein was administered at the dose of 5,000 mg/kg bw to male and female Crl:CD1(ICR) mice. No adverse effects related to the 2mEPSPS protein were observed.
The bacterial AAD-12 protein was administered by oral gavage at a dose of 2,000 mg/kg bw to male and female Crl:CD1(ICR) mice. No adverse effects related to the AAD-12 protein were observed.
A bacterial PAT protein was administered by oral gavage at a dose of 5,000 mg/kg bw to male and female Crl:CD1 mice. No adverse effects related to the PAT protein were observed. 38 The GMO Panel is of the opinion that acute toxicity testing of the newly expressed proteins is of little additional value for the risk assessment of the repeated consumption of food and feed from GM plants by humans and animals. c) 28-day repeated dose toxicity study The applicant provided a 28-day oral repeated dose toxicity study in mice 37 to investigate the potential toxicity of the AAD-12 protein. However, the GMO Panel did not consider the overall study design adequate to identify the potential hazard of the AAD-12 protein, because of the low doses of AAD-12 protein tested (highest target dose level approximately 47 mg/kg bw per day, corresponding to an actual dose of approximately 17 mg/kg bw per day) and the limited number of animals used in treatment groups (five per sex per group) which is not considered sufficient to obtain an adequate statistical power (EFSA GMO Panel, 2011a).
The GMO Panel requested another 28-day oral repeated dose toxicity study in rodents, to support the safety assessment of the AAD-12 protein, with a sufficient number of animals to obtain an adequate statistical power, selecting the doses according to OECD TG 407 39 in order to induce adverse effects at the highest dose, or following a limit test approach if toxicity is not expected. In the second 28-day repeated dose toxicity study using mice, 40 the number of animals per treatment group was in line with the guidance document of the EFSA GMO Panel (2011a), but the highest target dose selected (142 mg/kg bw per day) was too low for an adequate hazard identification, as was also the case in the first 28-day study submitted by the applicant. Therefore the GMO Panel did not consider this study in the risk assessment. The study was withdrawn by the applicant. 41 The applicant provided a new 28-day oral repeated dose toxicity study in mice, 42 which was conducted in accordance with OECD TG 407 and in compliance with the principles of Good Laboratory Practice (GLP). Groups of singly caged Crl:CD1(ICR) mice (11 per sex per group, approximately 8 weeks old at study start) were administered by gavage the AAD-12 protein (in 0.5% METHOCEL TM ) at a targeted nominal dose of 1,100 mg/kg bw per day (AAD-12 protein group), the vehicle alone (vehicle control group), or bovine serum albumin (BSA) at a targeted nominal dose of 1,100 mg/kg bw per day (BSA control group).
The AAD-12 protein and BSA protein dosing formulations were prepared daily. Samples of the AAD-12 protein and BSA protein dosing formulations along with vehicle were taken from the first mix, from a mix near the middle, and from a mix towards the end of the study for dose confirmation and homogeneity analyses.
Feed and water were provided ad libitum. During the treatment period, the animals were checked daily for mortality and general clinical signs. Detailed clinical observations were conducted on all animals pre-treatment and then weekly. Ophthalmoscopy was carried out before the start and at the end of the treatment period. Body weights were recorded on test days 1, 2, 3, 4, 8, 15, 22 and 29 (terminal body weight) and body weight gains were calculated relative to test day 1. Feed consumption was determined on test days 1-2, 2-3, 3-4, 4-8, 8-15 and 22-29. At the end of the treatment period, blood samples were taken and haematological, coagulation and clinical chemistry analyses were performed. All animals were sacrificed and underwent a detailed necropsy examination with selected organs weighed. Organs and tissues from all animals were subjected to a comprehensive histological examination.
The GMO Panel noted that the AAD-12 protein formulations were prepared daily from the powdered test material stored at approximately 4°C until use. Stability tests on the powdered test material (i.e. the lyophilized AAD-12 protein) were not performed as part of this study. According to the study report, 42 the lyophilized AAD-12 protein was determined to be stable for 81 months under refrigerated storage conditions, as part of previous studies 42 ; however, the GMO Panel noted that stability of the AAD-12 protein was not documented in these previous studies. Therefore, the concentration of AAD-12 protein of 33.1% in the powdered test material following storage has not been confirmed. However, as dose confirmation analyses were performed on the dosing formulations both at the start and towards the end of the study, the GMO Panel considered that this is not a major limitation compromising the 28-day study.
The GMO Panel noted that haematology and clinical chemistry analyses were conducted on six mice/gender per group and coagulation (prothrombin time) was conducted on the remaining five animals in each group. The reasoning provided by the applicant was practical limitations in obtaining sufficient quantities of blood from mice for haematology and clinical chemistry, and coagulation examinations in the same animal. However, it is well known that when mice are used as the test animal, additional animals may be needed in each dose group to conduct all required determinations. The GMO Panel also noted that the animals were not fasted prior to necropsy and blood collection, as recommended in OECD TG 407.
The AAD-12 protein group was statistically compared to the BSA control group; the latter was also compared to the vehicle control group in order to assess potential effects of the higher protein intake. For all the continuous parameters a two-way analysis of variance (ANOVA; factors: sex and dose) for the two sexes combined, in order to account for sex-dose interactions, and a one-way ANOVA (factor: dose) separately for each sex were performed. For all parameters, in case a statistically significant dose effect was found with the one-or two-way ANOVA, each individual dose group was compared to the control group using Dunnett's test. For body weight gains, globulin, albumin/globulin ratio, red blood cell (RBC) indices, and differential white blood cell (WBC) counts only descriptive statistics were reported.
The results of the dose confirmation analyses revealed that the average recoveries for AAD-12 and BSA protein in 0.5% METHOCEL TM were 71.7% and 73.2%, respectively, based on nominal dosing suspensions at 110 mg/mL. The average recoveries were therefore within the acceptable experimental variation (70-120%). The GMO Panel noted that based on the measured concentration of AAD-12 protein in the dosing suspension, the actual dose administered was 789 mg/kg bw per day. The results of the homogeneity analyses indicated that the preparations were homogeneously mixed.
The few statistically significant differences between the BSA control and vehicle control groups in the examined parameters were considered by the GMO Panel to be within normal biological variability; therefore, both the vehicle control and the BSA control groups were considered suitable to be used as the control groups for the comparison and evaluation of data from the AAD-12 protein group.
No mortality occurred during the treatment period. The GMO Panel considered that the isolated clinical findings and the few ophthalmic changes observed at the end of the study in the AAD-12 protein group were not treatment-related.
No statistically significant differences in body weight or body weight gain were observed in the AAD-12 protein group compared to the BSA control group.
In comparison with the BSA control group, males of the AAD-12 protein group showed statistically significantly lower feed consumption during specific time periods (days 1-2, 2-3, 15-22, 22-29); these differences were not considered as an adverse treatment-related effect by the GMO Panel as there were no statistically significant differences in body weights and body weight gains in animals of the AAD-12 protein group when compared with the BSA control group.
Haematology analysis showed statistically significantly higher WBC counts in males of the AAD-12 protein group when compared with the BSA control group. This finding was largely due to changes in two individual animals (one with an abscess in the neck region and a granulomatous inflammation around the oesophagus; the other showed chronic inflammation of the mediastinal tissue). The changes which largely resulted from (not significantly) higher values in neutrophil counts 43 were attributed by the applicant to inadvertent trauma associated with repeated oral gavage and not to treatment with the AAD-12 protein. The GMO Panel agrees with the interpretation by the applicant.
The platelet (PLT) count in males treated with the AAD-12 protein was statistically significantly higher compared with the BSA control group. This finding was, according to the applicant, 43 attributed to one animal with inadvertent trauma (abscess in the neck region and a granulomatous inflammation around the oesophagus) related to repeated oral gavaging procedure and not related to the treatment with the AAD-12 protein. After removing this animal from the statistical analysis, the increase in the PLT count showed no statistical significance. The increase was considered by the applicant not to be related to the treatment with the AAD-12 protein. The GMO Panel noted that even after removing this single animal with the highest PLT count (1,836 9 10 3 /lL), the mean PLT count in the male AAD-12 protein group (1,499 9 10 3 /lL, versus 1,555 9 10 3 /lL) was still higher compared to both the BSA protein group (1,321 9 10 3 /lL) and the vehicle control group (1,302 9 10 3 /lL). The GMO Panel could not exclude that the increased PLT count was related to the treatment with the AAD-12 protein for the following reasons: (1) the PLT counts for the individual animals in the male AAD-12 protein group (range 1,386-1,836 9 10 3 /lL) were either comparable to or higher than the two highest PLT counts for individual animals in the BSA protein group (1,454 and 1,510 9 10 3 /lL) and the vehicle control group (1,398 and 1,511 9 10 3 /lL); (2) the SD (87) for the AAD-12 protein group after removing the single animal with the highest PLT count was lower than that for the control groups (BSA protein: 168, vehicle control: 143); and (3) the PLT counts for five of six of the individual animals in the male AAD-12 protein group (range 1,466-1,836 9 10 3 /lL) were higher than the historical control range (1,205-1,417 9 10 3 /lL) presented in the study report (five studies between 2012 and 2016). However, as the increase was slight and not statistically significant after removing the single animal with the highest PLT count, the GMO Panel considered that this difference was not toxicologically relevant.
In females treated with the AAD-12 protein, the reticulocyte (RET) count was slightly, but statistically significantly lower compared with the BSA control group; the mean value was very close to that of the vehicle control group and within the historical control range. This difference was not considered to be toxicologically relevant by the GMO Panel as there were no differences in related haematological parameters.
There were no other significant differences in haematological and clinical chemistry parameters, or in the prothrombin time. However, the GMO Panel noted that the haematological, clinical chemistry and coagulation examinations were only performed on six or five animals per gender per group and thus, the GMO Panel recommendation to use a higher number of animals (EFSA GMO Panel, 2011a) was not fulfilled for the examination of these parameters.
Organ weight determinations showed no statistically significant differences except for a lower absolute (but not relative) epididymides weight (7.6%) in males treated with the AAD-12 protein compared with the BSA control group. This difference was not considered as toxicologically relevant by the GMO Panel as there was no difference in the relative epididymides weight.
Macroscopic examinations at necropsy revealed no gross pathological findings related to the treatment with the AAD-12 protein. Microscopic examinations of selected organs and tissues identified no treatment-related differences in the incidence and severity of the histopathological findings between the groups.
The GMO Panel noted that the haematological, clinical chemistry and coagulation examinations were only performed on six or five animals per gender per group (in line with OECD TG 407 minimum requirements) and thus, the GMO Panel recommendation to use a higher number of animals to ensure appropriate statistical power (EFSA GMO Panel, 2011a) was not fulfilled for the examination of these parameters. Nevertheless, the GMO Panel concluded that no adverse effects were observed in this study after a 28-day administration of the AAD-12 protein to mice at the dose tested (789 mg/kg bw per day).

Toxicological assessment of components other than newly expressed proteins
No new constituents other than the AAD-12, PAT and 2mEPSPS proteins are expressed in soybean DAS-44406-6.
With the exception of increased lectin activity (up to 31%), no relevant changes in the composition of the GM soybean were detected in the comparative compositional analysis (Table 3).
Lectins are a superfamily of proteins selectively binding carbohydrates and they function as recognition molecules in cell-molecule and cell-cell interactions in a variety of biological systems (Sharon and Lis, 2004;Miyake et al., 2007). Lectins are natural components in plants used for food and feed (Nachbar and Oppenheim, 1980;Peumans and Van Damme, 1996;Vasconcelos and Oliveira, 2004) and are widely distributed among Leguminosae, including soybean (Gupta, 1987). The ingestion of legumes containing high levels of certain lectins may be associated with gastrointestinal effects in humans and animals (Noah et al., 1980;Rodhouse et al., 1990;Bardocz et al., 1995;Grant et al., 1995).
In its toxicological assessment of the increase in lectin activity, the GMO Panel took into account the following: 1) The toxicity of raw soybean lectins is low compared to other commonly consumed legumes (Nasi et al., 2009), consisting of reduced growth performance and transient small intestine hypertrophy in experimental feeding studies . 2) Current industrial and traditional home processing practices are known to considerably reduce lectin content and/or activity in legumes, including soybean (Liener, 1994;Duranti and Gius, 1997;OECD 2012), and the safe use of soybean depends on such practices (K € onig et al., 2004). However, it cannot be excluded that residual lectin activity is still present in processed soybean products (Peumans and Van Damme, 1996;Rizzi et al., 2003;Vasconcelos and Oliveira, 2004). 3) The observed increase was considered in the context of the high variability reported for lectin activity and lectin protein content in raw soybean (Becker-Ritt et al., 2004;OECD, 2012;Maria John et al., 2017). Regarding any possible impact of the observed increase on the levels of residual activity in processed products, it can be considered that (measurable) residual activity is also characterised by high variability, as observed across different products (Calderon de la Barca et al., 1991) and between different samples of the same product (Maenz et al., 1999).
Based on these considerations, the GMO Panel is of the opinion that the observed increase (up to 31%) in lectin activity in raw soybean DAS-44406-6 is unlikely to raise additional toxicological concerns for soybean DAS-44406-6 with respect to conventional soybean varieties.
The nutritional impact of the increase in lectin activity in soybean DAS-44406-4 will be discussed in Section 3.3.1.5.

90-day feeding study in rats
The applicant provided a sub-chronic (90-day) feeding study, which was conducted in accordance with OECD TG 408 44 and in compliance with the principles of GLP. Five groups of singly caged Crl:CD (SD) rats (12 per sex per group, approximately 8-week old at study start) were fed balanced diets (formulated in accordance with specifications of the Purina Mills Inc. Certified Rodent LabDiet â 5002) containing approximately 20% (weight/weight) dehulled/defatted toasted soybean meal derived from soybean DAS-44406-6 (test diet), from the conventional counterpart Maverick 45 (control diet) or from one of three non-GM commercial soybean varieties (Dairyland 99915, Porter 75148, and Williams 82) (reference diets) for at least 90 days.
Prior to diet preparations, analyses for presence or absence of the three newly expressed proteins AAD-12, PAT, and 2mEPSPS were performed by ELISA in dehulled/defatted toasted meal from soybean DAS-44406-6 and from the conventional counterpart materials. Because the proteins were not detected, PCR was performed to confirm that the event was present only in the dehulled/defatted toasted meal from soybean DAS-44406-6.
Feed and water were provided ad libitum. All animals were observed daily for mortality and general clinical signs. Detailed clinical observations (DCO) were conducted on all animals pre-treatment and then weekly. Ophthalmoscopy was carried out before the start and at the end of the treatment period. Body weights and feed consumption were recorded weekly. At the end of the treatment period, blood samples were taken for coagulation; haematology and clinical chemistry and urine samples were taken for urinalysis. At necropsy, all animals underwent a detailed necropsy and selected organs were weighed. A comprehensive histological examination was performed on selected organs and tissues from all animals fed the test and the control diet, as well as on gross lesions from all animals fed the reference diets.
The GMO Panel noted that functional testing was not performed. According to the study report 'No functional testing was performed in this study because previous observations in testing of transgenic soybean products did not reveal any neurotoxicity deficits either in this laboratory (. . .) or in similar studies conducted by others reported in the literature Delaney et al., 2008;Chukwudebe et al., 2011).' According to the OECD TG 408 text 'Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits'. The GMO Panel interprets the OECD TG 408 text as functional testing can be omitted if data on functional observations are available from other studies with the test material under evaluation. As no treatmentrelated clinical signs were observed in this study (see below), the GMO Panel considered that the deviation from the OECD TG 408 is not a major deviation compromising the 90-day study.
The GMO Panel noted the following deviations from the recommendations in the EFSA Guidance document on conducting 90-day studies in rodent (EFSA Scientific Committee, 2011): (1) animals were housed individually whereas EFSA recommends that animals of the same sex should be housed in 44 OECD (Organisation for Economic Co-operation and Development), Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents. OECD Guidelines for the Testing of Chemicals, Section 4. 45 Dossier: Part II -Section A4.5; additional information received on 2/12/2014. pairs; (2) only one dose level was used in the study whereas EFSA recommends two dose levels; and (3) a power analysis was not performed.
Statistical analyses were run separately for each sex. The group fed the test diet was independently compared with the group fed the control diet. The groups fed diets containing the reference varieties were used to provide reference ranges. Means and standard deviations were reported for all continuous data and for body weight gains, globulin, albumin/globulin ratio, RBC indices and differential WBC counts. Statistical outliers were excluded from feed consumption analyses. Body weights, feed consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation and appropriate haematological data were evaluated by Bartlett's test for equality of variances and, on the basis of these outcomes, a t-test or Wilcoxon rank sum test were performed. DCO incidence data were statistically analysed by a z-test of proportions comparing soybean DAS-44406-6 group versus conventional counterpart group. No correction method for multiple comparisons in the same group was applied.
No mortalities occurred during the treatment period. The GMO Panel considered that the isolated clinical findings and the few ophthalmic changes observed at the end of the study in the test diet group were not treatment-related.
No statistically significant differences in the mean terminal body weight or body weight gain were observed in the test diet group compared to those given the control diet. No statistically significant changes were observed in feed consumption, except for two instances (days 8-15 and days 78-85) of higher values for females fed the test diet compared to female rats given the control diet. The GMO Panel considered that, in the absence of changes in the mean terminal body weight and body weight gain, these differences were not toxicologically relevant. Statistically significant differences in some haematological and clinical chemistry parameters were observed between animals fed the test diet and the control diet.
The total WBC count was lower in males fed the test diet; the GMO Panel considered that, in the absence of effects in the other immune-related parameters assessed, this difference was not toxicologically relevant.
The RET count was lower in males fed the test diet, and the RBC count, haemoglobin concentration and haematocrit were slightly higher in females fed the test diet. Adverse haematological effects, e.g. anaemia, would have been associated with decreases in RBC parameters and often also with an increase in the RET count. Therefore, the GMO Panel considered that the decrease in the RET count and the increases in RBC parameters were not toxicologically relevant.
The serum aspartate aminotransferase (AST) level was higher, and the cholesterol and triglyceride levels were lower in males fed the test diet. The GMO Panel considered that, in the absence of other effects in the liver, these differences were not toxicologically relevant.
There were no other significant differences in haematological and clinical chemistry parameters, or in the prothrombin time and urinalysis.
Organ weight determinations showed no statistically significant differences between animals fed the test diet and those given the control diet.
Macroscopic examinations at necropsy revealed no gross pathological findings related to the consumption of the test diet. Microscopic examinations of selected organs and tissues identified no treatment-related differences in the incidence and severity of the histopathological findings between the test diet group and the control group.
The GMO Panel noted some limitations in the study (i.e. functional testing was not performed; animals housed individually; only one dose level in the study; and a power analysis was not performed).
The GMO Panel concluded that no adverse effects were observed in this study after a 90-day administration to rats of a diet formulated with 20% dehulled/defatted toasted soybean meal derived from soybean DAS-44406-6.

42-day feeding study in broiler
The applicant provided a 42-day feeding study with a total of 600 (equal numbers of male and female) chickens for fattening (Ross 708, day-old at start). 28 The birds were randomly allocated to five dietary treatment groups with 120 chicks per treatment (12 pens per treatment, six pens for male and six for female, 10 birds per pen). Diets containing soybean DAS-44406-6 (verified by PCR) treated with 2,4-D and glyphosate were compared to diets containing its conventional counterpart or one of three non-GM commercial soybean varieties (Dairyland 99915, Porter 75148 or Williams 82 soybean). Meal was prepared from soybean grains and the formulated isocaloric diets were analysed for proximates, fibres, isoflavones, minerals, amino acids, anti-nutrients, mycotoxins and pesticides. The chickens were fed starter, grower and finisher diets in mash form containing 39%, 35% and 31% soybean meal, respectively. The nutrient content of the diets was adjusted according to the standards of the National Research Council (NRC, 1994). AAD-12, EPSPS and PAT proteins were detected in grain, but not found in the meals prepared from the grain. Feed and water were provided ad libitum.
Chickens were observed twice daily for clinical signs. Body weight and feed intake were measured at day 1, 14, 28 and 42. At day 42, three birds per pen were euthanized and processed for carcass evaluation (yield, dressing percentage, weight of thighs, breast, wings, legs, abdominal fat and whole liver). ANOVA (pen was considered as the experimental unit) was applied to determine statistical differences between groups; pair-wise comparison was made by Dunnett's test.
Mortality was low (1.8%), with no significant difference between groups. No significant treatment 9 sex interactions was detected for growth parameters. Overall no significant differences between soybean DAS-44406-6 and its conventional counterpart were found for final body weight (ca. 2.4 kg), daily feed intake (ca. 90 g) and feed:gain ratio (1.57). In comparisons made with individual commercial varieties, no relevant differences were found. Carcass parameters did not differ between soybean DAS-44406-6 and the conventional counterpart, except for thigh weight.
The GMO Panel concludes that administration of diets containing up to 39% soybean DAS-44406-6 to broilers, up to 42 days, did not cause adverse effects. Moreover, the measured performance endpoints were similar between groups fed balanced diets containing GM and non-GM soybeans.

Allergenicity
The strategies to assess the potential risk of allergenicity focus on the source of the recombinant protein, on the potential of the newly expressed protein to induce sensitisation or to elicit allergic reactions in already sensitised persons and on whether the transformation may have altered the allergenic properties of the modified plant.

Assessment of allergenicity of the newly expressed proteins 46
A weight-of-evidence approach was followed, taking into account all of the information obtained on the newly expressed protein, as no single piece of information or experimental method yields sufficient evidence to predict allergenicity (EFSA, 2006;Codex Alimentarius 2009;EFSA GMO Panel, 2011a).
The 2mepsps gene originates from Zea mays L., which is not considered to be a common allergenic food. The aad-12 gene originates from D. acidovorans and the pat gene originates from S. viridochromogenes, microorganisms which are not considered to be allergenic sources.
Updated bioinformatic analyses 5 of the amino acid sequences of the 2mEPSPS, AAD-12 and PAT proteins, using the criterion of 35% identity in a sliding window of 80 amino acids, revealed no significant similarities to known allergens. In addition, the applicant also performed analyses searching for matches of eight contiguous identical amino acid sequences between the 2mEPSPS, AAD-12 and PAT proteins and known allergens, which confirmed the outcome of the previous bioinformatic analysis.
The study on resistance to degradation of the 2mEPSPS, AAD-12 and the PAT proteins by pepsin has been described in Section 3.3.1.2.a.
The GMO Panel has previously evaluated the safety of the 2mEPSPS and PAT proteins in the context of several applications and no concerns on allergenicity were identified (e.g. EFSA, 2007EFSA, , 2009bEFSA GMO Panel, 2011c.
There is no information available on the structure or function of the newly expressed AAD-12, 2mEPSPS and PAT proteins that would suggest an adjuvant effect of the individual proteins or their simultaneous presence in soybean DAS 44406-6 resulting in or increasing an eventual IgE response to a bystander protein.
In the context of the present application, the GMO Panel considers that there are no indications that the newly expressed 2mEPSPS, AAD-12 and PAT proteins, individually or their simultaneous presence, in soybean DAS 44406-6 may be allergenic.

Assessment of allergenicity of the whole GM plant or crop 47
Soybean is considered to be a common allergenic food 48 (OECD, 2012). Therefore, any potential change in the endogenous allergenicity of the GM plant when compared with that of its appropriate comparator(s) should be assessed in line with the applicable EFSA guidance document (EFSA GMO Panel, 2011a). The applicant performed in vitro allergenicity studies and a comparative analysis using extracts from soybean DAS 44406-6, its conventional counterpart and non-GM reference soybean varieties.
Initially, the applicant provided a one-dimensional electrophoresis followed by Western blot and an ELISA inhibition studies using pooled sera from 10 individuals allergic to soybean. In addition, the applicant provided a two-dimensional electrophoresis followed by Western blot or Coomassie blue, and ELISA inhibition studies using single serum from six individuals allergic to soybean. On request from the GMO Panel, the applicant performed a qualitative and quantitative comparative analysis of the twodimensional Western blots which was carried out by densitometry. According to the applicant, minor variations in spot intensities, which are commonly seen in such studies, were observed between soybean DAS 44406-6 and its conventional counterpart. The GMO Panel did not identify indications of safety concern.
Subsequently, the applicant spontaneously provided a measurement of specific known allergens in soybean by mass spectrometry approaches (i.e. liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)), which has previously been considered as an alternative/complementary acceptable approach for the assessment of endogenous allergenicity in GM plants (EFSA GMO Panel, 2010b;Fernandez et al., 2013). The allergens measured by LC-MS/MS were Gly m 1, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28 K, Gly m Bd 30 K and Gly m 8 in soybean seeds. The applicant selected these allergens based on a list of potential soybean allergens described in the OECD consensus document on soybean (2012). The comparative analysis was performed with the combination of difference and equivalence testing recommended by the EFSA GMO Panel (2011a). The outcome of such analysis showed that the genetic modification did not induce relevant changes in the levels of any of the natural endogenous allergens tested.
In the context of this application and considering the information above, the GMO Panel is of the opinion that there is no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS 44406-6, when compared with that of its conventional counterpart and non-GM commercial reference soybean varieties.

Nutritional assessment of GM food/feed
Comparison of the composition of soybean DAS-44406-6 with its conventional counterpart and non-GM reference varieties identified differences in the lectin activity in seeds (up to 31% increase in the GM soybean, Table 3). Lectins are known to be antinutritional factors for humans and animals (Section 3.3.1.2).
The presence of lectins in common plant foods such as Leguminosae is well known (Peumans and Van Damme, 1996;Vasconcelos and Oliveira, 2004). Current industrial and traditional home processing practices are used to considerably reduce lectin content and/or activity in legumes, including soybean (Liener, 1994;Duranti and Gius, 1997;OECD, 2012). It cannot be excluded that residual lectin activity is still present in processed soybean food products (Calderon de la Barca et al., 1991;Peumans and Van Damme, 1996;Rizzi et al., 2003); hence, dietary exposure to functionally active lectins at residual levels in processed products is considered a common event (Nachbar and Oppenheim, 1980;Vasconcelos and Oliveira, 2004).
Soybean oil is the predominant soybean product for human consumption. The oil is obtained by fractionation of soybean seeds and is almost entirely composed of fat, with a negligible non-fat fraction (0.3%) consisting of moisture, insoluble and volatile matter (Gandhi, 2009). Even in the worst-case scenario, assuming that the entire non-fat fraction of the oil derived from soybean DAS-44406-6 are lectins, the observed increase in lectin activity in soybean DAS-44406-6 (up to 31%) would not raise concerns compared to oil from non-GM soybeans. Other processing procedures (e.g. soaking, heating and fermentation) are known to considerably reduce active lectin content in soybean (Gupta, 1987, Codex Alimentarius 1989Reddy and Pearson, 1994;Duranti and Gius, 1997;Lajolo and Genovese, 2002;OECD, 2012). Soybean sprouting is also known to be accompanied by a decrease in active lectin content (Rizzi et al., 2003). In the context of this application, it was also demonstrated that toasting is effective in lowering lectin activity of DAS-44406-6 soybean seeds to very low levels. 28 The GMO Panel considers that the additional intake of active lectins from soybean DAS-44406-6 food products (deriving from the 31% increase in the raw material) is likely to be negligible compared to the habitual dietary intake of lectins from non-GM soybean food products.
Soybean meal is the by-product of the extraction of soybean oil and it is the most important protein source used to feed farm animals. In the solvent extraction process, soybeans are cracked, heated, flaked and the oil is extracted by solvent (usually hexane). The obtained by-product is then subjected to treatments that improve its nutritional value by a decrease in the activity of antinutritional factors (e.g. lectins and enzyme inhibitors). Current conditions during the commercial processing of soybean into commodity oil, meal and other products, and on-farm processing conditions (e.g. soaking, roasting, extrusion and micronisation) have consistently shown the ability to reduce the presence and/ or activity of lectins and enzyme inhibitors to the extent that they can be consumed by monogastric animals. Quality processes and testing such as urease activity are in place, mainly in commercial production, to assure that many of these commercial products have undergone the proper processing procedures for use in feed applications. 49 Urease activity is highly positively related to lectin and trypsin inhibitor activity (Fasina et al., 2003). The GMO Panel considers that, within the frame of good farming practice described above and considering current processing practices, the observed increase in lectin activity is unlikely to influence animal nutrition.
The GMO Panel concluded that food and feed derived from soybean DAS-44406-6 are expected to have no adverse nutritional impact, as compared to those from its conventional counterpart and commercial non-GM reference varieties.

Post-market monitoring of GM food/feed
There was no indication that food/feed products derived from soybean DAS-44406-6 are less safe or nutritious than those derived from its conventional counterpart or the non-GM commercial varieties (Sections 3.2.1.3, 3.3.1.2 and 3.3.1.5). Therefore, in line with EFSA (2006) and EFSA GMO Panel (2011a), the GMO Panel is of the opinion that post-market monitoring of the GM food/feed is unnecessary.

Conclusion on the food/feed safety assessment
The safety assessment identified no concerns regarding the potential toxicity of the 2mEPSPS, AAD-12 and PAT proteins newly expressed in soybean DAS-44406-6, considering their structural and functional properties, the results of bioinformatic analyses and the results of a sub-acute 28-day toxicity study on AAD-12. The GMO Panel did not identify indications of safety concerns regarding allergenicity or adjuvanticity with the 2mEPSPS, AAD-12 and PAT proteins or regarding the overall allergenicity of DAS-44406-6. The observed increase in lectin activity in raw soybean DAS-44406-6 is unlikely to raise additional concerns for food/feed safety and nutrition for the GM soybean as compared to its conventional counterpart and the non-GM commercial varieties. No other changes in soybean DAS-44406-6 composition relevant for food/feed safety and nutrition were identified. Considering current soybean processing, soybean DAS-44406-6 is as safe and nutritious as its conventional counterpart and the non-GM commercial varieties.
to faecal material (manure and faeces); and (2) the accidental release into the environment of viable seeds of soybean DAS-44406-6 during transportation and/or processing (EFSA GMO Panel, 2010c).

Persistence and invasiveness of the GM plant 50
Cultivated soybean (G. max (L.) Merr.) is a species in the subgenus Soja of the genus Glycine. The species originated from eastern Asia and is a highly domesticated crop (Lu, 2005). Cultivated soybean seeds rarely display any dormancy characteristics and can grow as volunteers in the year after cultivation only under certain environmental conditions. If volunteers occur, they do not compete well with the succeeding crop, and can easily be controlled mechanically or chemically (OECD, 2000). The presence of volunteers of G. max was occasionally reported in some areas of Italy where soybean is intensively cultivated (Celesti-Grapow et al., 2010). However, soybean seeds usually do not survive during the winter owing to herbivory, rotting and germination, or owing to management practices prior to planting the subsequent crop (Owen, 2005). Also, survival of soybean plants outside cultivation areas is limited mainly by a combination of low competitiveness, absence of a dormancy phase, and susceptibility to plant pathogens and cold climatic conditions.
The applicant presented agronomic and phenotypic data on soybean DAS-44406-6 gathered from field trials conducted in soybean growing areas in the United States (Section 3.2.1.2). Soybean DAS-44406-6 (both treated and untreated with the intended herbicides) had lower yields in some comparisons with the conventional counterpart, and glyphosate-treated soybean DAS-44406-6 had higher pod count (+12%) and seed count (+10%) than the conventional counterpart. Considering the observed differences all together, it is unlikely that these will change the fitness (e.g. survival, fecundity, competitiveness) or invasiveness characteristics of soybean DAS-44406-6 plants, given the low survival capacity of soybean.
Further, it is considered unlikely that herbicide tolerance will provide soybean DAS-44406-6 a selective advantage outside cultivation. The application of 2,4-D-, glyphosate-and/or glufosinate-based herbicides is not expected to improve their ability to survive over seasons because of soybean vulnerability to several abiotic and biotic factors. Therefore, it is considered very unlikely that soybean DAS-44406-6 will differ from conventional soybean varieties in its ability to survive until subsequent seasons or to establish feral populations under European environmental conditions. The GMO Panel is not aware of any scientific report of increased survival capacity, including overwintering, of existing GM soybean varieties (Dorokhov et al., 2004;Owen, 2005;Bagavathiannan and Van Acker, 2008;Lee et al., 2009). Therefore, the GMO Panel is of the opinion that the likelihood of environmental effects of soybean DAS-44406-6 in Europe will not be different from that of conventional soybean varieties.

Effects of gene transfer 51
A prerequisite for any gene transfer is the availability of pathways for the transfer of genetic material, either through horizontal gene transfer of DNA, or through vertical gene flow via crosspollination from feral plants originating from spilled seed.

1) Plant-to-bacteria gene transfer
Genomic plant DNA is a component of many food and feed products derived from soybean. It is well documented that DNA present in food and feed becomes substantially degraded during processing and digestion in the human or animal gastrointestinal tract. However, a low level of exposure of fragments of ingested DNA, including the recombinant fraction of such DNA, to bacteria in the digestive tract of humans, domesticated animals and other environments exposed to the GM plant or plant material is expected.
Current scientific knowledge of recombination processes in bacteria indicates that horizontal transfer of non-mobile, chromosomally located DNA fragments between unrelated organisms (such as from plants to bacteria) is not likely to occur at detectable frequencies under natural conditions (for further details, see EFSA, 2009a).
A successful horizontal gene transfer would require stable insertion of the recombinant DNA sequences into a bacterial genome and conferring a selective advantage to the transformed host. The only mechanism known to facilitate horizontal transfer of non-mobile, chromosomal DNA fragments to bacterial genomes is HR. This requires the presence of stretches of DNA sequences that are similar in the recombining DNA molecules. In addition to substitutive gene replacement, the insertion of nonhomologous DNA sequences is facilitated if their flanking regions share sequence similarity with bacterial sequences in the recipient.
Soybean DAS-44406-6 contains some genetic elements derived from bacterial genome origin. These are (1) the coding sequence of the aad-12 gene from D. acidovorans; (2) the coding sequence of the pat gene from S. viridochromogenes; (3) two UTRs comprising the transcriptional terminator and polyadenylation site of ORF23 and for ORF1 from plasmid pTI5955 from A. tumefaciens (Barker et al., 1983); and (4) an intervening sequence of 228 bp from the Ti plasmid C58 of A. tumefaciens, respectively.
Both aad-12 and pat genes were optimized to modify the G+C codon bias to a level more typical for plant expression and thereby the DNA sequences changed to levels (< 90% sequence identity) which would not facilitate HR with native sequences as present in D. acidovorans or S. viridochromogenes, respectively. Sequence identity sufficient to facilitate HR, however, was found for ORF23 and ORF1 of pTI5955. Furthermore, sequence identity was found for the intervening sequence originating from the A. tumefaciens plasmid C58. The similarities between the abovementioned genes and bacterial genes were confirmed by bioinformatic analyses provided by the applicant.
Theoretically, the sequence identities found between the plant-inserted DNA sequences and sequences of the A. tumefaciens plasmid C58 could facilitate homologous recombination and with pTI5955 double HR, respectively. Recombination between the intervening sequences with those plasmids present on plasmid C58 would only replace identical or nearly DNA sequences and thus adds no new property. Equally this would be the case for HR between ORF23 and ORF1. However, double HR between both sequences in soybean DAS-44406-6 and pTI5955 in A. tumefaciens could result in the insertion of the plant codon-optimized pat gene. However, such a recombination and gain of the pat gene would occur at the cost of replacing a large part of the T-DNA region (Barker et al., 1983) and thus producing a plasmid which would have lost its capacity to infect plants.
Agrobacterium tumefaciens or close relatives from the genus Rhizobium occur in soil, water and in the plant rhizosphere, but are not typical constituents of the microbial communities as they occur in the main receiving microbial habitat, i.e. the gastrointestinal tract of humans or animals. However, occurrence of the recombinant genes outside the immediate receiving environment (through faecal material) in soils cannot be ruled out (Hart et al., 2009) and is therefore also taken into account for assessing the risks associated with a horizontal gene transfer.
In case that, despite a very low exposure, a recombinant modified pTI5955 plasmid with an inserted pat gene, as described above, would be generated in an A. tumefaciens strain, it is unlikely that this pat gene would provide a selective advantage in the given environment: The gene is optimized for plant expression and regulated by the CsVMV promoter. The expression of such a promoter-gene construct in bacteria is unknown, but generally the expression level of eukaryotic promoters in bacteria is inefficient (Warren et al., 2008). Furthermore, natural variants of pat genes have been found in different soil-inhabiting bacteria and thus pat genes would not confer a novel trait to soil microbial communities.
In addition to homology-based recombination processes, non-homologous (illegitimate) recombination that does not require the presence of DNA similarity between the recombining DNA molecules is theoretically possible. However, illegitimate recombination events have not been detected in studies that have exposed bacteria to high concentrations of GM plant DNA (EFSA, 2009a). Thus, this process, in comparison with HR, is not considered to contribute significantly to horizontal gene transfer events.
In conclusion, the GMO Panel identified a theoretical possibility of a facilitated double HR between genes from soybean DAS-44406-6 and bacteria which may occur in soil, which however, in addition to a high unlikelihood due to limited exposure and biological restrictions, would not provide a selective advantage to a possible bacterial recipient. Therefore, the GMO Panel concludes that the recombinant DNA in soybean DAS-44406-6 does not represent an environmental risk in relation to its potential for horizontal transfer to bacteria.

2) Plant-to-plant gene transfer
Considering the scope of application EFSA-GMO-NL-2012-106 and the biology of soybean, the potential of occasional feral GM soybean plants originating from seed import spills to transfer recombinant DNA to sexually cross-compatible plants is assessed.
The genus Glycine is divided into two distinct subgenera: Glycine and Soja. The subgenus Glycine contains 16 perennial wild species, whereas the cultivated soybean, G. max, and its wild and semi-wild annual relatives, G. soja and G. gracilis, are classified in the subgenus Soja (OECD, 2000). Owing to the low level of genomic similarity among species of the genus Glycine, G. max can cross with only other members of the Glycine subgenus Soja under natural conditions (Singh et al., 1987;Hymowitz et al., 1998;Lu, 2005). Hence, the three species of the subgenus Soja are capable of cross-pollination and the hybrid seed that is produced can germinate normally and produce plants with fertile pollen and seed (Abe et al., 1999;Nakayama and Yamaguchi, 2002). As G. soja and G. gracilis are indigenous to China, Taiwan, Korea, Japan, the far-east region of Russia, Australia, the Philippines and the South Pacific, and as they have not been reported in other parts of the world where the cultivated soybean is grown (Dorokhov et al., 2004;Lu, 2005), the plant-to-plant gene transfer from soybean is restricted to cultivated areas and occasional soybean plants resulting from seed spillage in the EU.
Soybean is an annual, almost completely self-pollinating crop with a percentage of cross-pollination usually below 1% (OECD, 2000;Ray et al., 2003;Lu, 2005;Yoshimura et al., 2006;Abud et al., 2007). Soybean pollen dispersal is limited because the anthers mature in the bud and directly pollinate the stigma of the same flower (OECD, 2000).
However, cross-pollination rates as high as 6.3% have been reported for closely spaced plants (Ray et al., 2003), suggesting the potential for some within-crop gene flow in soybean. These results indicate that natural cross-pollination rates can fluctuate significantly among different soybean varieties under particular environmental conditions, such as favourable climate for pollination and an abundance of pollinators (Gumisiriza and Rubaihayo, 1978;Kikuchi et al., 1993;Ahrent and Caviness, 1994;Ray et al., 2003;Lu, 2005).
For plant-to-plant gene transfer to occur, imported soybean DAS-44406-6 seeds need to be processed outside the importing ports, transported into regions of soybean production in Europe, spilled during transportation, germinate and develop into plants in the very close vicinity of soybean fields, and there needs to be an overlap of flowering periods and environmental conditions favouring cross-pollination. It must be noted that most soybean DAS-44406-6 seeds are processed in the countries of production or in ports of importation. The overall likelihood of cross-pollination between feral GM soybean plants and cultivated soybean is therefore extremely low.
In conclusion, even if cross-pollination would occur, the GMO Panel is of the opinion that the likelihood of environmental effects as a consequence of the spread of genes from occasional feral GM soybean plants in Europe will not differ from that of conventional soybean varieties (see previous Section 3.4.1.1).

Interactions of the GM plant with target organisms 52
Considering the scope of application EFSA-GMO-NL-2012-106, and the absence of target organisms, potential interactions of feral soybean DAS-44406-6 plants arising from seed import spills with target organisms are not considered a relevant issue by the GMO Panel.

Interactions of the GM plant with non-target organisms 53
Considering the scope of application EFSA-GMO-NL-2012-106, and the low level of exposure to the environment, potential interactions of spilled seeds or occasional feral soybean DAS-44406-6 plants arising from seed import spills with non-target organisms are not considered a relevant issue by the GMO Panel.

Interactions with the abiotic environment and biochemical cycles 54
Considering the scope of application EFSA-GMO-NL-2012-106, and the low level of exposure to the environment, potential interactions of occasional feral soybean DAS-44406-6 plants arising from seed import spills with the abiotic environment and biogeochemical cycles are not considered a relevant issue by the GMO Panel.

Post-market environmental monitoring 55
The objectives of a 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 content of the PMEM plan provided by the applicant (EFSA GMO Panel, 2011b).
The PMEM plan proposed by the applicant for soybean DAS-44406-6 includes: (1) the description of an approach involving operators (federations involved in soybean import and processing), reporting to the applicant, via a centralised system, any observed adverse effect(s) of GMOs on human health and the environment; (2) a coordinating system established by EuropaBio for the collection of information recorded by the various operators; and (3) the use of networks of existing surveillance systems (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 consent period.
The GMO Panel considers the scope of the PMEM plan provided by the applicant is consistent with the scope of soybean DAS-44406-6. As the ERA does not cover cultivation and did not identify potential adverse environmental effects from soybean DAS-44406-6, no case-specific monitoring is necessary. The GMO Panel agrees with the reporting intervals proposed by the applicant in its PMEM plan.

Conclusion on the environmental risk assessment and monitoring plan
In the case of accidental release into the environment of viable seeds of soybean DAS-44406-6, there are no indications of an increased likelihood of establishment and spread of occasional feral soybean DAS-44406-6 plants, unless these plants are exposed to the intended herbicides. The GMO Panel is of the opinion that this will not result in different environmental impacts compared to conventional soybean.
Considering the scope of application EFSA-GMO-NL-2012-106, interactions of soybean DAS-44406-6 with the biotic and abiotic environment are not considered to be relevant issues. Bioinformatic analysis of the inserted DNA identified a theoretical possibility of a facilitated double HR between genes from DAS-44406-6 and bacteria which may occur in soil. The GMO Panel did not identify a concern in relation to the theoretically possible horizontal gene transfer to bacteria owing to the lack of a selective advantage.
Therefore, considering the introduced traits, the outcome of the comparative analysis, the routes of exposure and the limited exposure levels, the GMO Panel concludes that soybean DAS-44406-6 would not raise safety concerns in the event of accidental release of viable GM soybean seeds into the environment.
The scope of the PMEM plan provided by the applicant and the reporting intervals are in line with the intended uses of soybean DAS-44406-6 and the GMO Panel guidelines on the PMEM of GM plants (GEFSA GMO Panel, 2011b).

Conclusions
The GMO Panel was asked to carry out a scientific assessment of soybean DAS-44406-6 for import, processing, and food and feed uses in accordance with Regulation (EC) No 1829/2003. The molecular characterisation data and bioinformatics analyses did not identify issues requiring further assessment for food/feed safety.
The agronomic and phenotypic characteristics of soybean DAS-44406-6 tested revealed no relevant differences between soybean DAS-44406-6 and its conventional counterpart, except for pod count, seed count and yield for soybean DAS-44406-6 treated with glyphosate. No differences in composition requiring further assessment for food/feed safety were found between soybean DAS-44406-6 and its conventional counterpart, except for a higher lectin activity (increased up to 31%) in soybean DAS-44406-6. The increase in lectin activity is unlikely to raise additional concerns for food/feed safety and nutrition for soybean DAS-44406-6 as compared to its conventional counterpart and the non-GM commercial varieties. No concerns regarding the potential toxicity or allergenicity of the newly expressed 2mEPSPS, PAT and AAD-12 proteins were identified, and no evidence that the genetic modification might significantly change the overall allergenicity of soybean DAS-44406-6 was found. The GMO Panel concludes that soybean DAS-44406-6, assessed in this application, is as safe and as nutritious as its conventional counterpart and the non-GM soybean reference varieties tested. The GMO Panel considers that post-market monitoring of food/feed derived from soybean DAS-44406-6 is not necessary, given the absence of safety concerns identified.
The GMO Panel concluded that there is a very low likelihood of environmental effects resulting from the accidental release of viable seeds from soybean DAS-44406-6 into the environment. Considering the scope of the application with regard to food and feed use, interactions with the biotic and abiotic environment were not considered an issue. Risks associated with an unlikely, but theoretically possible, horizontal gene transfer from soybean DAS-44406-6 to bacteria have not been identified. The scope of the PMEM plan provided by the applicant is in line with the intended uses of soybean DAS-44406-6.
In conclusion, the GMO Panel considers that the information available for soybean DAS-44406-6 addresses the scientific comments raised by the Member States and that soybean DAS-44406-6, as described in this application, is as safe as the conventional counterpart and other non-GM soybean varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application.