Assessment of genetically modified soybean DBN9004 (application EFSA‐GMO‐BE‐2019‐165)

Abstract

Genetically modified soybean DBN9004 was developed to confer tolerance to glufosinate‐ammonium‐ and glyphosate‐containing herbicides. These properties were achieved by introducing the cp4 epsps and pat expression cassettes. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between soybean DBN9004 and its conventional counterpart needs further assessment. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the CP4 EPSPS and PAT proteins as expressed in soybean DBN9004 and finds no evidence that the genetic modification would change the overall safety of soybean DBN9004, as food and feed. In the context of this application, the consumption of food and feed from soybean DBN9004 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that soybean DBN9004 is as safe as the conventional counterpart and non‐GM soybean varieties tested, and no post‐market monitoring of food/feed is considered necessary. In the case of release of soybean DBN9004 material, including viable grains, into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DBN9004. The GMO Panel concludes that soybean DBN9004 is as safe as its conventional counterpart and the tested non‐GM soybean varieties with respect to potential effects on human and animal health, and the environment.

SUMMARY

Following the submission of application EFSA-GMO-BE-2019-165 under Regulation (EC) No 1829/2003 from Beijing DaBeiNong Biotechnology Co., Ltd. (DBNBC) (referred to hereafter as ‘the applicant’), the Panel on Genetically Modified Organisms of the European Food Safety Authority (referred to hereafter as ‘GMO Panel’) was asked to deliver a scientific opinion on the safety of genetically modified (GM) herbicide tolerant soybean DBN9004 (Unique Identifier DBN-Ø9ØØ4-6) according to Regulation (EU) No 503/2013. The scope of application EFSA-GMO-BE-2019-165 is for import, processing and food and feed uses within the European Union (EU) of soybean DBN9004 and does not include cultivation in the EU.

In this scientific opinion, the GMO Panel reports on the outcome of its risk assessment of soybean DBN9004 according to the scope of the application EFSA-GMO-BE-2019-165. The GMO Panel conducted the assessment of soybean DBN9004 in line with the principles described in Regulation (EU) No 503/2013 and its applicable guidelines for the risk assessment of GM plants. The molecular characterisation data establish that soybean DBN9004 contains a single insert consisting of one copy of the cp4 epsps and pat expression cassettes. Bioinformatic analyses of the sequences encoding the newly expressed proteins (NEPs), the sequences corresponding to open reading frames (ORFs) within the insert or spanning the junctions between the insert and genomic DNA, as well as the flanking regions, do not raise safety concerns. The stability of the inserted DNA and of the introduced trait is confirmed over several generations. The methodology used to quantify the levels of the CP4 EPSPS and PAT proteins is considered adequate. The protein characterisation data comparing the structural, biochemical and functional properties of plant and Escherichia coli-produced CP4 EPSPS and PAT proteins indicate that these proteins are equivalent, and the E. coli-derived proteins can be used in the safety studies.

Considering the selection of test materials, the field trial sites and the associated management practices and the agronomic-phenotypic characterisation as an indicator of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between soybean DBN9004 and its conventional counterpart needs further assessment. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the CP4 EPSPS and PAT proteins as expressed in soybean DBN9004. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of soybean DBN9004, as food and feed. In the context of this application, the consumption of food and feed from soybean DBN9004 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that soybean DBN9004 is as safe as the conventional counterpart and non-GM soybean varieties tested, and no post-market monitoring of food/feed is considered necessary.

Considering the introduced traits, the outcome of the agronomic and phenotypic analysis and the routes and levels of exposure, soybean DBN9004 would not raise safety concerns in the case of accidental release of GM soybean material, including viable seeds, into the environment. The post-market environmental monitoring (PMEM) plan and reporting intervals are in line with the intended uses of soybean DBN9004.

Based on the relevant publication identified through the literature searches, the GMO Panel does not identify any safety issue pertaining to the intended uses of soybean DBN9004.

The GMO Panel concludes that soybean DBN9004, 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.

INTRODUCTION

The scope of the application EFSA-GMO-BE-2019-165 is for food and feed uses, import and processing of soybean DBN9004 but does not include cultivation in the European Union (EU). Soybean DBN9004 was developed to confer tolerance to glufosinate-ammonium- and glyphosate-containing herbicides.

Background

On 20 November 2019, the European Food Safety Authority (EFSA) received from the Competent Authority of Belgium application EFSA-GMO-BE-2019-165 for authorisation of soybean DBN9004 (Unique Identifier DBNØ9ØØ4-6), submitted by Beijing DaBeiNong Biotechnology Co., Ltd. (DBNBC) (hereafter referred to as ‘the applicant’) according to Regulation (EC) No 1829/2003.¹ Following receipt of application EFSA-GMO-BE-2019-165, EFSA informed EU Member States and the European Commission (EC), and made the application available to them. Simultaneously, EFSA published the summary of the application.²

EFSA checked the application for compliance with the relevant requirements of Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013,³ with the EFSA guidance documents, and, when needed, asked the applicant to supplement the initial application. On 12 March 2021, EFSA declared the application valid.

From validity date, EFSA and the Panel on Genetically Modified Organisms of the European Food Safety Authority (referred to hereafter as ‘GMO Panel’) endeavoured to respect a time limit of 6 months to issue a scientific opinion on application EFSA-GMO-BE-2019-165. Such time limit was extended whenever EFSA and/or GMO Panel requested supplementary information to the applicant.

According to Regulation (EC) No 1829/2003, any supplementary information provided by the applicant during the risk assessment was made available to the EU Member States and European Commission (for further details, see the Section 5, below). In accordance with Regulation (EC) No 1829/2003, EFSA consulted the nominated risk assessment bodies of EU Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC.⁴ The EU Member States had 3 months to make their opinion known on application EFSA-GMO-BE-2019-165 as of date of validity.

Terms of Reference as provided by the requestor

According to Articles 6 and 18 of Regulation (EC) No 1829/2003, EFSA and its GMO Panel were requested to carry out a scientific risk assessment of soybean DBN9004 in the context of its scope as defined in application EFSA-GMO-BE-2019-165.

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). In addition to the present scientific opinion, EFSA was also asked to report on the particulars listed under Articles 6(5) and 18(5) of Regulation (EC) No 1829/2003, but not to give an opinion on them because they pertain to risk management.

DATA AND METHODOLOGIES

Data

The GMO Panel based its scientific assessment of soybean DBN9004 on the valid application EFSA-GMO-BE-2019-165, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU Member States and relevant peer reviewed- scientific publications. As part of this comprehensive information package, the GMO Panel received additional unpublished studies submitted by the applicant to comply with the specific provisions of Regulation (EU) No 503/2013. A list of these additional unpublished studies is provided in Appendix A.

Methodologies

The GMO Panel conducted its assessment in line with the principles described in Regulation (EU) No 503/2013, the applicable EFSA guidelines (i.e. EFSA GMO Panel, 2010a, 2011a, 2011b, 2015, 2017; EFSA Scientific Committee, 2011) and explanatory notes and statements (i.e. EFSA, 2010, 2014, 2017a, 2017b, 2018, 2019a, 2019b; EFSA GMO Panel, 2010b, 2018) for the risk assessment of GM plants.

The contractors performed preparatory work for the evaluation of the applicant's literature search (OC/EFSA/GMO/2018/04; OC/EFSA/MESE/2022/03-01-SC17), the completeness and quality of DNA sequencing information (OC/EFSA/GMO/2020/01), the bioinformatic analyses (OC/EFSA/GMO/2021/06) and the analysis of the 90-day toxicity study (EOI/EFSA/2022/01 – CT 17-2024; EOI/EFSA/2022/01 – CT NIF 2023 02).

ASSESSMENT

Introduction

Soybean DBN9004 was genetically modified to confer tolerance to glufosinate-ammonium- and glyphosate-containing herbicides, through expression of the CP4 EPSPS and PAT proteins. The assessment of herbicide residues relevant for this application is in the remit of the EFSA Plant Health and Pesticides Residues Unit (EFSA, 2015, 2019c).

Systematic literature review

The GMO Panel assessed the applicant's literature searches on soybean DBN9004, which include a scoping review, according to the guidelines given in EFSA (2010, 2017a).

A systematic review as referred to in Regulation (EU) No 503/2013 has not been provided in support to the risk assessment of application EFSA-GMO-BE-2019-165. Based on the outcome of the scoping review, the GMO Panel agrees that there is limited value of undertaking a systematic review for soybean DBN9004 at present.

The GMO Panel considered the overall quality of the performed literature searches acceptable. The literature searches identified 16 relevant publications on soybean DBN9004 from electronic databases, and three relevant records from the websites of key organisations. The relevant publications are listed in Appendix B.

None of the relevant records/publications identified through the literature searches reported information pointing to safety issues associated with soybean DBN9004 relevant to the scope of this application.

Molecular characterisation⁵

Transformation process and vector constructs

Soybean DBN9004 was developed by Agrobacterium tumefaciens (also known as Rhizobium radiobacter)-mediated transformation. Soybean cotyledonary node tissue (Jack variety) was co-cultured with a disarmed A. tumefaciens strain EHA101 containing the vector pDBN4003. The plasmid pDBN4003 used for the transformation contains two expression cassettes between the right and left border of the T-DNA, containing the following genetic elements:

• – The cp4 epsps expression cassette consists of the EF-1 polypeptide promoter (Gm17gTsf1) from Glycine max, transit peptide of the ShkG gene (AtCTP2) from Arabidopsis thaliana, the plant codon-optimised sequence of the epsps gene (cp4 epsps) from A. tumefaciens strain CP4, the 3′ untranslated sequence of E9 gene from Pisum sativum.
• – The pat expression cassette consists of the 35S promoter from Cauliflower Mosaic Virus, the plant codon-optimised sequence of the pat gene from Streptomyces viridochromogenes, and the 35S terminator from the Cauliflower mosaic virus.

The vector backbone contained elements necessary for the maintenance and selection of the plasmid in bacteria.

Transgene constructs in the GM plant

Molecular characterisation of soybean DBN9004 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 EFSA GMO Panel assessed the sequencing data and found it compliant with the requirements listed in the EFSA GMO Panel (2018), both in terms of the approach, of the coverage and sensitivity.

Southern analyses indicated that soybean DBN9004 contains a single insert, consisting of a single copy of the T-DNA. The insert and copy number were confirmed by multiple restriction enzyme/probe combinations covering the T-DNA region and flanking regions. PCR and DNA sequence analyses confirmed the results obtained by the Southern analyses. The absence of vector backbone sequences was demonstrated by Southern analysis using five backbone-specific overlapping probes. The nucleotide sequence of the entire insert of soybean DBN9004 together with 992 bp of the 3′ and 1268 bp of the 5′ flanking regions were determined. The insert of 4799 bp is identical to the T-DNA of plasmid pDBN4003, except for the deletion of 25 bp of the right border region and 90 bp of the left border region.

A comparison with the pre-insertion locus indicated that 1545 bp were deleted from the soybean genomic DNA. The possible interruption of known endogenous soybean genes by the insertion in soybean DBN9004 was evaluated by bioinformatics analyses of the pre-insertion locus and of the genomic sequences flanking the insert. The results of these analyses do not indicate the interruption of any known endogenous gene in soybean DBN9004. The results of segregation (see Section 3.3.5) and bioinformatics analyses are compatible with a single insertion in the nuclear genome.

Bioinformatics analyses of the amino acid sequence of the newly expressed CP4 EPSPS and PAT proteins reveal no significant similarities to either toxins or allergens. In addition, bioinformatic analyses of the newly created open reading frames (ORFs) within the insert and spanning the junctions between the insert and genomic DNA also do not indicate significant similarities to toxins and allergens.

In order to assess the possibility for horizontal gene transfer (HGT) by homologous recombination (HR), the applicant performed a sequence identity analysis of the inserted DNA in soybean DBN9004, which includes two expression cassettes containing plant codon optimised NEP coding sequences, with microbial DNA. The likelihood and potential consequences of plant-to-bacteria gene transfer are described in Section 3.5.1.2.

Protein characterisation and equivalence

Soybean DBN9004 expresses two new proteins: CP4 EPSPS and PAT. Given the technical restraints in producing large enough quantities from plants, these proteins were recombinantly produced in Escherichia coli. A set of biochemical methods was employed to demonstrate the equivalence between the soybean DBN9004 and E. coli-derived CP4 EPSPS and PAT. Purified proteins from these two sources were characterised and compared in terms of their biochemical, structural and functional properties.

CP4 EPSPS protein characterisation and equivalence

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that both plant and E. coli-produced CP4 EPSPS proteins had the expected molecular weight of ~47 kDa and were comparably immunoreactive to CP4 EPSPS protein specific antibodies. Glycosylation detection analysis demonstrated that none of the two CP4 EPSPS proteins were glycosylated. Amino acid sequence analysis of the plant-derived CP4 EPSPS protein by mass spectrometry (MS) methods showed that the protein matched the deduced sequence as defined by the cp4 epsps gene. Functional equivalence was demonstrated by a biochemical in vitro activity assay which shows that both proteins have comparable activity for the intended herbicide.

PAT protein characterisation and equivalence

SDS-PAGE and Western blot analysis showed that both plant and E. coli-produced PAT proteins had the expected molecular weight of ~21 kDa and were comparably immunoreactive to PAT protein specific antibodies. Glycosylation detection analysis demonstrated that neither of the two PAT proteins was glycosylated. Amino acid sequence analysis of the plant-derived PAT protein by MS methods showed that the protein matched the deduced sequence as defined by the pat gene. In addition, the MS data showed that the N-terminal methionine of the plant-derived PAT protein was truncated, while the N-terminal methionine of the E. coli-derived PAT protein was oxidised. N-terminal methionine removal is a co-translational enzymatic event common in eukaryotic proteins (Polevoda & Sherman, 2000), whereas methionine oxidation also can be introduced artifactually during purification or sample handling (Wingfield, 2017). Functional equivalence was demonstrated by a biochemical in vitro activity assay which showed that both proteins have comparable activity for the intended herbicide.

The protein characterisation data comparing the biochemical, structural and functional properties of plant and microbe-produced CP4 EPSPS and PAT proteins, indicate that these proteins are equivalent and the E. coli-derived proteins can be used in the safety studies.

Information on the expression of the insert

Protein levels of CP4 EPSPS and PAT were analysed by enzyme-linked immunosorbent assay (ELISA) in material harvested in a field trial across five locations in China during the 2020 growing season. Samples analysed included leaves [V5, R3 and R6], root [R6], forage [R6] and mature seeds [R8], from plants treated with the intended herbicides, as well as forage and seeds from plants not treated with the glufosinate and glyphosate herbicides. The mean values, standard deviations and ranges of protein expression levels in seeds (n = 5) and forage (n = 5) of the CP4 EPSPS and PAT proteins used to estimate human and animal dietary exposure (see Section 3.5.4) are reported in Table 1.

TABLE 1 Mean values (n = 5) and standard deviation of newly expressed protein in seeds [μg/g dry weight (dw) and μg/g fresh weight (fw)] and forage (μg/g dw) from soybean DBN9004.

Tissues	Glufosinate and glyphosate treatment
Not treated	Treated
μg/g dry weight (dw)	μg/g fresh weight (fw)	μg/g dry weight (dw)	μg/g fresh weight (fw)
Seed (R8)
CP4 EPSPS	192.39a ± 40.95b (108.2–266.19)c	172.32 ± 21.06 (91.66–241.4)	191.85 ± 40.53 (96.15–277.07)	171.81 ± 24.49 (87.2–246.24)
PAT	1.14 ± 0.27 (0.59–1.71)	1.03 ± 0.09 (0.5–1.57)	1.17 ± 0.34 (0.45–2.04)	1.05 ± 0.16 (0.38–1.73)
Forage (R6)
CP4 EPSPS	492.02 ± 80.91 (348.85–702.06)	142.69 ± 23.47 (101.17–203.6)	489.86 ± 77.14 (358.97–661.37)	142.06 ± 22.37 (104.1–191.8)
PAT	19.5 ± 7.46 (10.74–41.46)	5.65 ± 2.17 (3.11–12.02)	18.6 ± 5.39 (10.06–36.05)	5.39 ± 1.56 (2.91–10.46)

Inheritance and stability of inserted DNA

Genetic stability of soybean DBN9004 insert was assessed by Southern analysis of genomic DNA from three generations (T3, T5, T7) and PCR-based segregation analysis of both glyphosate and glufosinate tolerance traits of soybean DBN9004 in two progenies (F2, F3). For the Southern analysis, 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 assessed by observing tolerance to glyphosate and glufosinate in T2-T6 and BC3F3 generations of DBN9004. Tolerance to glyphosate and glufosinate was demonstrated across all tested generations. The results support the presence of a single insertion, segregating in a Mendelian fashion.

Conclusion on molecular characterisation

The molecular characterisation data establish that soybean DBN9004 contains a single insert consisting of one copy of the cp4 epsps and pat expression cassettes. Bioinformatic analyses of the sequences encoding the NEPs, the sequences corresponding to ORFs within the insert or spanning the junctions between the insert and genomic DNA, as well as the flanking regions, do not raise safety concerns. The stability of the inserted DNA and of the introduced trait was confirmed over several generations. The methodology used to quantify the levels of the CP4 EPSPS and PAT proteins is considered adequate. The protein characterisation data comparing the structural, biochemical and functional properties of plant and E. coli-derived CP4 EPSPS and PAT proteins indicate that these proteins are equivalent and that the E. coli-derived proteins can be used in the safety studies.

Comparative analysis⁶

Overview of studies conducted for the comparative analysis

Application EFSA-GMO-BE-2019-165 presents data on agronomic and phenotypic characteristics, as well as on forage and seed composition of soybean DBN9004 (Table 2). In addition, the application contains data on characteristics of seed from soybean DBN9004.

TABLE 2 Main comparative analysis studies to characterise soybean DBN9004 provided in the application EFSA-GMO-BE-2019-165.

Study focus	Study details	Comparator	Non-GM reference varieties
Agronomic and phenotypic analysis	Field study, China, 2020, 8 sitesa	Jack	6b
Compositional analysis

Experimental field trial design and statistical analysis

At each field trial site, the following materials were grown in a randomised complete block design with four replicates: soybean DBN9004 not exposed to the intended herbicides, soybean DBN9004 exposed to the intended herbicides, the conventional counterpart Jack and three non-GM reference varieties.

The agronomic, phenotypic and compositional data were analysed as specified by EFSA GMO Panel (2010b, 2011a). This includes, for each of the two treatments of soybean DBN9004, the application of a difference test (between the GM soybean and the conventional counterpart) and an equivalence test (between the GM soybean and the set of non-GM commercial reference varieties). The results of the equivalence test are categorised into four possible outcomes (I–IV, ranging from equivalence to non-equivalence).⁷

Suitability of selected test materials

Selection of the test materials

The comparator used in the field trials is the non-GM soybean variety Jack, which has the same genetic background as soybean DBN9004 (as documented by the pedigree and by the additional information) and is considered to be the conventional counterpart.

Jack is a soybean variety that was developed at the Illinois Agricultural Experiment Station and was not specifically selected to be grown in China. However, the typical growing latitudes for Jack are compatible with the selected field trial sites (best adapted to latitudes ranging between 40 and 42° North, Nickell et al., 1990). As reported in Table 2, six commercial non-GM reference varieties with registered days to maturity ranging from 100 to 135 were selected by the applicant and, at each selected site, three reference varieties were tested. Despite the selection of non-GM reference varieties was not justified a priori (EFSA, 2015), on the basis of the provided information on days to maturity, year of commercialisation and growth type, the GMO Panel considers the selected non-GM reference varieties acceptable for the comparative assessment.

Seed production and quality

Seeds of soybean DBN9004 and the conventional counterpart (Jack) used in the 2020 field trials were produced from plants free of diseases, harvested and stored under similar conditions, before being sown in the field trial sites. The seed lots were verified for their identity via event-specific PCR analysis.

The seeds were tested for their germination capacity under warm temperature conditions.⁸ Germination capacity of the soybean DBN9004 was compared with the one of its comparator and the results⁹ of these studies indicate that the seed germination of soybean DBN9004 was not different than that of its comparator.

Conclusion on suitability

The GMO Panel concludes that the soybean DBN9004, the conventional counterpart and the non-GM soybean reference varieties as well as the test materials are acceptable for the comparative analysis.

Representativeness of the receiving environments

Selection of field trial sites

The selected field trials sites were located in commercial soybean-growing regions of China. The soil and climatic characteristics of the selected fields were diverse,¹⁰ corresponding to optimal, near-optimal and sub-optimal conditions for soybean cultivation (Sys et al., 1993).

The GMO Panel considers that the selected sites reflect commercial soybean-growing regions in which the test materials are likely to be grown.

Meteorological conditions

Maximum and minimum mean temperatures and sums of precipitation were provided for each site on a daily basis. No exceptional weather conditions were reported at any of the selected sites; therefore, the GMO Panel considers that the meteorological dataset falls within the historical range of climatic conditions normally occurring at these sites.

Management practices

The field trials included plots containing soybean DBN9004, plots with the conventional counterpart and plots with non-GM soybean reference varieties, managed according to local agricultural practices. In addition, the field trials included plots containing soybean DBN9004 managed following the same agricultural practices and exposed to the intended glufosinate-ammonium- and glyphosate-containing herbicides. Glufosinate-ammonium-containing herbicide was applied at BBCH 15–16¹¹ growth stage and after 7 days glyphosate-containing-herbicide was applied to the same plots.

The GMO Panel considers that the management practices, including sowing, harvesting and application of plant protection products were appropriate for the selected receiving environments.

Conclusion on representativeness

The GMO Panel concludes that the geographical locations, soil and climatic characteristics, meteorological conditions and management practices of the field trial sites are typical for receiving environments where the tested materials could be grown.

Agronomic and phenotypic analysis

Thirteen agronomic and phenotypic endpoints¹² plus information on abiotic stressors, disease incidence and arthropod damage were collected from the field trial sites (see Table 2).

The statistical analysis (Section 3.4.2) was applied to all these endpoints, with the following results:

• For soybean DBN9004 (not treated with the intended herbicides), the test of difference identified statistically significant differences with the conventional counterpart for 1000 seed weight, which fell under equivalence category II.
• For soybean DBN9004 (treated with the intended herbicides), the test of difference identified statistically significant differences with the conventional counterpart for final plant stand and 1000 seed weight. These endpoints fell under equivalence category I or II.

Compositional analysis

Soybean DBN9004 seeds and forage harvested from eight sites (Table 2) were analysed for 65 constituents (7 in forage and 58 in seeds), including those recommended by OECD (OECD, 2012). The statistical analysis was not applied to one constituent in seeds, because its concentration in all the samples was below the limit of quantification.¹³

The statistical analysis was applied to a total of 64 constituents (57 in seeds¹⁴ and 7 in forage¹⁵); a summary of the outcome of the test of difference and the test of equivalence is presented in Table 3:

• For soybean DBN9004 not treated with the intended herbicides, statistically significant differences with the conventional counterpart were found for three endpoints in seeds. All these endpoints for which significant differences were found between the soybean DBN9004 and the conventional counterpart fell under equivalence category I or II.
• For soybean DBN9004 treated with the intended herbicides, statistically significant differences with the conventional counterpart were found for two endpoints in seeds. All these endpoints for which significant differences were found between the soybean DBN9004 and the conventional counterpart fell under equivalence category I or II.

TABLE 3 Outcome of the comparative compositional analysis in seeds and forage for soybean DBN9004. The table shows the number of endpoints in each category.

	Test of differencea
Not treatedb	Treatedb
Not different	Significantly different	Not different	Significantly different
Test of equivalence c	Category I/II	52	3d	53	2d
Category III/IV	–	–	–	–
Not categorised	9e	–	9e	–
Total endpoints	64	64

The GMO Panel assessed all the significant differences between the soybean DBN9004 and the conventional counterpart, taking into account the potential impact on plant metabolism and the natural variability observed for the set of non-GM reference varieties. No endpoints with outcomes under category III/IV were identified.

Conclusion on comparative analysis

Considering the selection of test materials, the field trial sites and the associated management practices, as well as the agronomic-phenotypic characterisation as an indicator of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis.

Taking into account the natural variability observed for the set of non-GM reference varieties, the GMO Panel concludes that:

• None of the differences identified in agronomic and phenotypic characteristics between soybean DBN9004 and the conventional counterpart need further assessment regarding their potential environmental impact.
• None of the differences identified in forage and seed composition between soybean DBN9004 and the conventional counterpart need further assessment regarding food and feed safety.

Food/feed safety assessment¹⁶

Overview of overarching information for food/feed assessment

Compositional analysis

The compositional analysis of soybean DBN9004 and the conventional counterpart provided by the applicant and assessed by the GMO Panel is described in Section 3.4.6.

Newly expressed proteins

The safety of CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004 have been previously assessed in several events¹⁷ and in the scientific literature (Harrison et al., 1996; Hérouet et al., 2005), and no safety concerns for humans, farmed and companion animals were identified by the GMO Panel.

Molecular characterisation

The protein characterisation of the newly expressed CP4 EPSPS and PAT proteins provided by the applicant and assessed by the GMO Panel is described in Section 3.3.3. Furthermore, the equivalence between the soybean and the E. coli-derived proteins was demonstrated.

Substrate specificity

The GMO Panel has assessed the substrate specificity of other EPSPS proteins in the past (e.g. EFSA GMO Panel, 2022). The mechanism of action of EPSPS proteins is a biochemical reaction involving conversion of shikimate-3-phosphate and phosphoenolpyruvate to 5-enolpyruvylshikimate-3-phosphate.

The PAT protein has been previously assessed by the GMO Panel (e.g. EFSA GMO Panel, 2021, 2024a, 2024b). PAT enzyme activity is limited to the acetylation of the glufosinate-ammonium substrate (Hérouet et al., 2005).

The GMO Panel is not aware of any additional information that would change its previous assessments.

Stability of the NEPs

The effects of temperature and pH on the CP4 EPSPS and PAT proteins, along with their in vitro degradation, have been extensively evaluated in previous assessments by the GMO Panel. As part of this application, the applicant has submitted additional experimental data supporting these previous assessments.

Synergistic or antagonistic interactions

The potential for a functional interaction among the CP4 EPSPS and PAT proteins has been assessed with regard to human and animal health. Based on current scientific knowledge on the biological function of the two proteins (Table 4), no synergistic or antagonistic interactions between these two proteins, which could raise safety concerns for food and feed from soybean DBN9004 are expected.

TABLE 4 Intended effects of the NEPs in soybean DBN9004.

Protein	Intended effect in GM plant
CP4 EPSPS	The CP4 EPSPS protein confers tolerance to glyphosate-containing herbicides acting on the shikimic acid pathway for the biosynthesis of aromatic amino acids in plants
PAT	The PAT protein confers tolerance to glufosinate-ammonium-containing herbicides acting by acetylation of glufosinate ammonium

Effect of processing

Soybean DBN9004 will undergo existing production processes used for conventional soybean. Based on the outcome of the comparative assessment, processing of the GM soybean into food and feed products is not expected to result in products being different from those of conventional non-GM soybean varieties currently in the EU market.¹⁸

Toxicology

The strategies to assess the toxicological impact of any changes on the GM soybean DBN9004 food and feed resulting from the genetic modification focus on the assessment of (i) NEPs; (ii) new constituents other than NEPs; (iii) altered levels of food and feed constituents; and (iv) the whole genetically modified food and feed.

Assessment of NEPs

The safety of CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004 have been previously assessed in several events¹⁹ and in the scientific literature (Harrison et al., 1996; Hérouet et al., 2005), and no safety concerns for humans, farmed and companion animals were identified by the GMO Panel. Updated bioinformatics analyses revealed no similarities of the CP4 EPSPS and PAT proteins with known toxins. The GMO Panel is not aware of any new information that would change the previous conclusion on the safety of these proteins. Based on the information provided by the applicant, information retrieved from the scientific literature and the experience gained from the previous assessment of CP4 EPSPS and PAT proteins, the GMO Panel considers that there are no toxicological concerns for the CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004.

Assessment of new constituents other than proteins

Based on the outcome of the studies considered in the comparative analysis and molecular characterisation, no new constituents other than the NEPs have been identified in seed and forage from soybean DBN9004. Therefore, no further food and feed safety assessment of components other than the NEPs is required.

Assessment of altered levels of food and feed constituents

Based on the outcome of the studies considered in the comparative analysis and molecular characterisation, none of the differences identified between soybean DBN9004 and its non-GM comparator in seed and forage composition require further assessment.

Testing of the whole genetically modified food and feed

Based on the outcome of the molecular characterisation, comparative analysis and toxicological assessment, no indications of findings relevant to food and feed safety have been identified for soybean DBN9004 related to the stability and expression of the insert, and to modifications of toxicological concern in the composition of soybean DBN9004 (see Sections 3.3, 3.4.6 and 3.5.2). Therefore, animal studies with food/feed derived from soybean DBN9004 are not considered necessary by the GMO Panel (EFSA GMO Panel, 2011a). In accordance with Regulation (EU) No 503/2013, the applicant provided a 90-day feeding study in rats fed with diets containing meal (toasted and defatted) derived from soybean DBN9004.

In this study, pair-housed Crl:CD (SD) rats (16 per sex per group; two rats per cage) were allocated to six groups using a randomised complete block design with eight replications per sex.

Groups were fed diets containing full fat soybean DBN9004 grains from plants treated with the intended herbicides (glyphosate and glufosinate-ammonium) at 20% and 10% of inclusion level (the latter supplemented with 10% of the non-GM comparator soybean), the non-GM comparator-Jack soybean (inclusion level 20%) and the three reference varieties (Heinong 84, Jidou 12 and Zhonghuang 35) (inclusion level 20%).

The study was adapted from OECD test guideline 408 (OECD, 2018), aligned with EFSA Scientific Committee guidance (EFSA Scientific Committee, 2011) and EFSA Explanatory statement (EFSA, 2014) and complied with the principles of good laboratory practice (GLP)²⁰ with some minor deviations not impacting the study results and interpretation.

The stability of the test and control materials was not verified; however, in accordance with product expiration declared by the diet manufacturer, the constituents of the diets were considered stable for the duration of the treatment. The GMO Panel considered this justification acceptable. Diet preparation procedures and regular evaluations of the mixing methods guaranteed the homogeneity and the proper concentration of the test or control substances in them.

Event-specific PCR analysis confirmed the presence of the event DBN9004 in both the GM grains and diets and excluded the presence of the event in the respective controls. ELISA analyses also confirmed the presence of the event DBN9004 in the GM soybean grains and GM diets.

Both the GM grains and diets were analysed for nutrients, antinutrients and potential contaminants. Balanced diets were formulated based on the specifications for Certified Rodent Diet.

Feed and water were provided ad libitum. In-life procedures and observations and terminal procedures were conducted in accordance with OECD TG 408 (2018).

An appropriate range of statistical tests were performed on the results of the study. Detailed description of the methodology and of statistically significant findings identified in rats given diets containing grains derived from soybean DBN9004 is reported in Appendix C.

There were no test diet-related incidents of mortality or clinical signs. No test diet-related adverse findings were identified in any of the investigated parameters. A small number of statistically significant findings were noted but these were not considered adverse effects of treatment for one or more of the following reasons:

• were within the normal variation for the parameter in rats of this age;
• were of small magnitude;
• were identified at only a small number of time intervals with no impact on the overall value;
• exhibited no consistent pattern with related parameters or endpoints;
• exhibited no consistency with increasing incorporation levels.

No gross pathology findings related to the administration of the test diet were observed at necropsy, and the microscopic examinations of a wide range of organs and tissues did not identify relevant differences in the incidence or severity of the histopathological findings related to the administration of the test diet compared to the control group.

The GMO Panel concludes that this study is in line with the requirements of Regulation (EU) No 503/2013 and that no treatment-related adverse effects were observed in rats after feeding diets containing soybean DBN9004 grains at 10% or 20% for 90 days.

Allergenicity

The strategies to assess the potential risk of allergenicity focus: (i) on the source of the recombinant protein; (ii) on the potential of the NEP to induce sensitisation or to elicit allergic reactions in already sensitised persons; and (iii) on whether the transformation may have altered the allergenic properties of the modified plant. Furthermore, the assessment also takes into account potential adjuvant properties of the NEPs, which is defined as the ability to enhance an allergic reaction.

Assessment of allergenicity of the newly expressed proteins

A weight-of-evidence approach was followed, taking into account all of the information obtained on the NEP, as no single piece of information or experimental method yielded sufficient evidence to predict allergenicity (Codex Alimentarius, 2009; EFSA GMO Panel, 2011a, 2017; Regulation (EU)No 503/2013).

The cp4 epsps and pat genes originate from A. tumefaciens and S. viridochromogenes, respectively, none of which are considered common allergenic sources. The safety of CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004 have been previously assessed in several events²¹ and in the scientific literature (Harrison et al., 1996; Hérouet et al., 2005), and no safety concerns for humans, farmed and companion animals were identified by the GMO Panel.

Updated bioinformatic analyses of the amino acid sequences of the CP4 EPSPS and PAT proteins, using the criterion of more than 35% identity in a sliding window of 80 amino acids, revealed no relevant similarities to known allergens.

The studies on protein stability of the CP4 EPSPS and PAT proteins have been described in Section 3.5.1. In addition, the GMO Panel did not find an indication that the NEPs CP4 EPSPS and PAT at the levels expressed in soybean DBN9004 might be adjuvants.

Furthermore, the applicant provided information on the safety of the CP4 EPSPS and PAT proteins regarding their potential hazard to cause a celiac disease response.²² For such assessment, the applicant followed the principles described in the EFSA GMO Panel guidance document (EFSA GMO Panel, 2017). The assessment of the CP4 EPSPS protein identified no perfect or relevant partial matches with known celiac disease peptide sequences. The assessment of the PAT protein revealed partial matches containing the Q/E-X1-P-X2 motif and required further investigations. These partial matches have been previously assessed by the EFSA GMO Panel (EFSA GMO Panel, 2022). Briefly, based on additional considerations on the position and nature of amino acids flanking the motif (EFSA GMO Panel, 2017), the relevant peptides containing the motif do not raise concern as they fail to mimic gluten sequences. Therefore, no indications of safety concerns were identified by the GMO Panel.

Based on experienced gain on the assessment of CP4 EPSPS and PAT proteins and information retrieved from the scientific literature, the GMO Panel considers that the information provided by the applicant is sufficient to conclude on the safety of these proteins. The GMO Panel considers that there are no indications that the newly expressed CP4 EPSPS and/or PAT proteins in soybean DBN9004 may be allergenic.

Assessment of allergenicity of the whole GM plant or crop

Soybean is considered a common allergenic food²³ (OECD, 2012). Therefore, any potential change in the endogenous allergenicity of the GM plant should be assessed (Regulation (EU) No 503/2013). For such assessment, the applicant included in the comparative analysis specific allergens relevant for soybean (Section 3.4.6) quantified using liquid chromatography with tandem MS, which has been previously considered acceptable (EFSA GMO Panel, 2010c, 2017; Fernandez et al., 2013; Selb et al., 2017). These allergens were selected based on the list of potential soybean allergens described in the pertinent OECD document (OECD, 2012) and a scientific rational supporting their selection was provided by the applicant and considered acceptable by the GMO Panel. No changes in the levels of endogenous allergens raising concern are identified by the GMO Panel.

In the context of this application, the GMO Panel considers that there is no evidence that the genetic modification might substantially change the overall allergenicity of soybean DBN9004 when compared with that of the conventional counterpart and the non-GM reference varieties tested.

Dietary exposure assessment to new constituents

In line with Regulation (EU) No 503/2013 the applicant provided dietary exposure estimates to CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004. Dietary exposure was estimated based on protein expression levels reported in this application for soybean DBN9004 treated with the intended herbicides (glufosinate and glyphosate herbicides), the currently available consumption data and feed practices, the foods and feeds currently available on the market and the described processing conditions.

For the purpose of estimating dietary exposure, the levels of the NEPs in soybean DBN9004 seeds and forage were derived from material harvested in a field trial across five locations in China during the 2020 growing season (Table 1, Section 3.3.4).

Human dietary exposure

Chronic and acute estimations of dietary exposure to CP4 EPSPS and PAT proteins newly expressed in soybean DBN9004 were provided. The applicant followed the methodology described in the EFSA Statement ‘Human dietary exposure assessment to NEP in GM foods’ to anticipate human dietary exposure making use of summary statistics of consumption (EFSA, 2019a).

Human dietary exposure was estimated across European countries for different population groups: young population (infants, toddlers, ‘other children’), adolescents, adult population (adults, elderly and very elderly) and special populations (pregnant and lactating women). Since no specific consumption data were available on commodities containing, consisting of or obtained from soybean DBN9004 seeds, a conservative scenario with 100% replacement of conventional soybean by the GM soybean was considered. Consumption figures for all relevant commodities (e.g. soya bread, textured soy protein, soya drink, tofu, etc.) were retrieved from the EFSA Comprehensive European Food Consumption Database (EFSA consumption database).²⁴

Mean protein expression values on fresh weight basis are considered as the most adequate to estimate human dietary exposure (both acute and chronic) when working with raw primary commodities that are commonly consumed as processed blended commodities (EFSA, 2019a). Different recipes and factors were considered to estimate the amount of soybean seeds in the consumed commodities before assigning NEP levels to the relevant commodities.²⁵ No losses in the NEPs during processing were considered.

The highest anticipated acute dietary exposure (highly exposed population) was in the age class ‘Infants’ with estimates of 2.3 mg/kg bw per day and 0.014 mg/kg bw per day for CP4 EPSPS and PAT proteins, respectively. The main contributor to the exposure in the dietary survey with the highest estimates would be ‘Follow-on formula, soya-based, powder’. In the dietary exposure scenario anticipating acute consumption of soybean-derived protein supplements, the highest estimates would range between 0.018 mg/kg bw per day for PAT and 3.0 mg/kg bw per day for CP4 EPSPS (in the adult population, high consumers).

The highest anticipated chronic dietary exposure (highly exposed population) was in the age class ‘Infants’ with estimates of 0.8 mg/kg bw per day and 0.005 mg/kg bw per day for CP4 EPSPS and PAT proteins, respectively. The main contributor to the exposure in the dietary survey with the highest estimates would be follow-on formula, soya-based, powder. In the dietary exposure scenario anticipating chronic consumption of soybean-derived protein supplements, the highest estimates would range between 0.012 mg/kg bw per day for PAT and 1.9 mg/kg bw per day for CP4 EPSPS (in the adult population, high consumers).

The GMO Panel considers pollen supplements as a possible contributor to the dietary exposure to CP4 EPSPS and PAT proteins, under the assumption that these supplements might be made of pollen from soybean DBN9004. Consumption data on pollen supplements are available for few consumers across seven different European countries.²⁴ However, since no expression data for CP4 EPSPS and PAT in pollen were available, the potential dietary exposure to these NEPs via the consumption of pollen supplements could not be estimated.

Animal dietary exposure

Anticipated dietary exposure to PAT and CP4 EPSPS proteins in soybean DBN9004 was estimated across different animal species, as below described, assuming the consumption of soybean products commonly entering the feed supply chain (i.e. soybean meal and forage). A conservative scenario with 100% replacement of conventional soybean products by the soybean DBN9004 products was considered.

Mean levels (dry weight) of the NEPs in seeds and forage from the soybean DBN9004 treated with the intended herbicide used for animal dietary exposure are listed in Table 1.

Mean levels (dry weight) of the NEPs in soybean meal were estimated to be 1.33-fold higher than in seed, based on conversion factors that take into account the protein content in this feed material relative to soybean seed (OECD, 2012), and assuming that no protein is lost during their processing.

The applicant estimated dietary exposure to PAT and CP4 EPSPS proteins via the consumption of soybean meal in cattle (beef and dairy), sheep (ram/ewe and lamb), pigs (breeding and finishing), poultry (layer and turkey), based on default values for animal body weight, daily feed intake and inclusion rates (percentage) of soybean meal in diets/rations, as provided for the EU by OECD (2013). In addition, dietary exposure was also estimated via the consumption of soybean forage in cattle (beef and dairy) and sheep (ram/ewe and lamb), based on default values for animal body weight and daily feed intake as provided for the EU by OECD (2013). References for inclusion rates (percentage) of soybean forage in diets or rations were based on those provided by OECD (2012) for lactating dairy cattle and used for all four species.

Estimated dietary exposure in the relevant animals is reported in Appendix D.

Nutritional assessment of endogenous constituents

The intended trait of soybean DBN9004 is herbicide tolerance, with no intention to alter nutritional parameters. Comparison of the composition of the soybean DBN9004 with its conventional counterpart and the non-GM reference varieties did not identify differences that would require further safety assessment. From these data, the GMO Panel concludes that soybean DBN9004 is nutritionally equivalent to its conventional counterpart and the non-GM reference varieties used.

Post-market monitoring of GM food/feed

Soybean DBN9004, as described in this application, does not raise any nutritional concern and is as safe as its conventional counterpart and the non-GM reference varieties tested. The GMO Panel concludes that based on the information considered in its safety assessment, a post-market monitoring plan for food and feed is not necessary.

Conclusions on the food/feed safety assessment

The newly expressed CP4 EPSPS and PAT proteins in soybean DBN9004 do not raise safety concerns for human and animal health. No interactions between the newly expressed CP4 EPSPS and PAT proteins relevant for food and feed safety were identified. Moreover, the GMO Panel did not identify indications of safety concerns regarding allergenicity or adjuvanticity related to the presence of the newly expressed CP4 EPSPS and PAT. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of soybean DBN9004, as food and feed. Based on the outcome of the comparative assessment and the nutritional assessment, the GMO Panel concludes that the consumption of soybean DBN9004 does not represent any nutritional concern, in the context of the scope of this application. The GMO Panel concludes that soybean DBN9004, as described in this application, is as safe as the conventional counterpart and the non-GM reference varieties tested, and no post-market monitoring of food/feed is considered necessary.

Environmental risk assessment and monitoring plan

Environmental risk assessment²⁶

Considering the scope of this application, which excludes cultivation, the environmental risk assessment (ERA) mainly takes into account: (i) the exposure of microorganisms to recombinant DNA in the gastrointestinal tract of animals fed with GM material and of microorganisms present in environments exposed to faecal material of these animals (manure and faeces); (ii) the deliberate or accidental release into the environment of the GM material, including viable soybean DBN9004 seeds during transportation and/or processing (EFSA GMO Panel, 2010a).

Persistence and invasiveness of the GM plant

Cultivated soybean (Glycine 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, generally unable to survive in the environment without proper management (Lu, 2005).

Occasional feral GM soybean plants may occur outside cultivation areas, but survival is limited mainly by a combination of low competitiveness, absence of a dormancy phase and susceptibility to plant pathogens (OECD, 2000). Additionally, soybean is a sub-tropical species susceptible to cold climatic conditions (Bramlage et al., 1978; Staniak et al., 2021; Szczerba et al., 2021; Tyagi & Tripathi, 1983), although cold tolerance varies across maturity groups and among cultivars (Alsajri et al., 2019; Wang et al., 2023). Soybean can grow as volunteers and 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, as for the same reasons mentioned above, soybean seeds usually do not survive during cold winters (Matsushita et al., 2020; Owen, 2005) and any soybean volunteers can be effectively controlled by mechanical methods or appropriate chemical control (Bond & Walker, 2009; Jhala et al., 2013; Soltani et al., 2019). Owing to this, soybean plants are often not considered a problematic volunteer in temperate climates (Jhala et al., 2021). Thus, the establishment and survival of feral and volunteer soybean in the EU is currently limited and transient.

The applicant provided an additional study on seed survival and competitiveness (Study DBNBC-FLD-2019013). The results of this study do not point to an increased risk of persistence and invasiveness of soybean DBN9004 compared to its conventional counterpart.

It is unlikely that the intended traits of soybean DBN9004 will provide a selective advantage to soybean plants, except when they are exposed to glufosinate-ammonium- and/or glyphosate-containing herbicides. However, if this was to occur this fitness advantage will not allow the GM plant to overcome other biological and abiotic factors (described above) limiting plant's persistence and invasiveness. Therefore, the presence of the intended traits will not affect the persistence and invasiveness of the GM plant.

In conclusion, the GMO Panel considers that it is very unlikely that soybean DBN9004 will differ from conventional soybean varieties in their ability to survive until subsequent seasons, or to establish occasional feral plants under European environmental conditions in case of accidental release into the environment of viable soybean DBN9004 seeds.

Potential for gene transfer

A prerequisite for any gene transfer is the availability of pathways for the transfer of genetic material, either through HGT of DNA, or through vertical gene flow via cross-pollination from feral plants originating from spilled grains.

Plant-to-microorganism gene transfer

Genomic DNA can be a component of food and feed products derived from soybean. It is well documented that such DNA becomes substantially degraded during processing and digestion in the human or animal gastrointestinal tract. However, bacteria in the digestive tract of humans and animals, and in other environments, may be exposed to fragments of DNA, including the recombinant fraction of such DNA.

Current scientific knowledge of recombination processes in bacteria suggests 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, 2009).

Homologous recombination is known to facilitate horizontal transfer of non-mobile, chromosomal DNA fragments to bacterial genomes. This requires the presence of at least two stretches of DNA sequences that are similar in the recombining DNA molecules. In the case of sequence identity with the transgene itself, recombination would result in gene replacement. In the case of identity with two or more regions flanking recombinant DNA, recombination could result in the insertion of additional DNA sequences in bacteria and thus confer the potential for new properties.

In addition to homology-based recombination processes, at a lower transformation rate, the non-homologous end joining and microhomology-mediated end joining are theoretically possible (EFSA, 2009; Hülter & Wackernagel, 2008). Independently of the transfer mechanism, the GMO Panel did not identify a selective advantage that a theoretical HGT would provide to bacterial recipients in the environment.

Bioinformatic analysis of event DBN9004 revealed that the genetic elements encoding for PAT and CP4 EPSPS proteins were plant codon-optimised and did not provide sufficient sequence identity to bacterial DNA which would facilitate homologous recombination.

In summary, there is no indication for an increased likelihood of horizontal transfer of DNA from soybean DBN9004 to bacteria. Given the nature of the recombinant DNA, the GMO Panel identified no safety concern linked to an unlikely but theoretically possible HGT.

Plant-to-plant gene transfer

The potential for occasional feral soybean DBN9004 plants originating from seed import spills to transfer recombinant DNA to sexually compatible plants and the environmental consequences of this transfer were considered.

For plant-to-plant gene transfer to occur, imported GM soybean seeds need to germinate and develop into plants in areas containing sympatric wild relatives and/or cultivated soybean with synchronous flowering and environmental conditions favouring cross-pollination. It must be noted that most soybean DBN9004 seeds are processed in the countries of production or in ports of importation.

Vertical gene transfer from soybean (G. max) is limited to the species of the subgenus Soja to which G. max belongs to, as well as the wild relatives G. soja and G. gracilis (Zhang et al., 2023). Although wild relatives exist elsewhere, no wild relatives of the subgenus Soja have been reported in Europe so far (Dorokhov et al., 2004; Lu, 2005). Therefore, vertical gene transfer from GM soybean is restricted to cultivated soybean (G. max).

Soybean is an annual, almost completely self-pollinating crop with a percentage of cross-pollination usually below 1% (Abud et al., 2007; Lu, 2005; OECD, 2000; Ray et al., 2003; Yoshimura et al., 2006), although 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 (Ahrent & Caviness, 1994; Caviness, 1966; Gumisiriza & Rubaihayo, 1978; Kikuchi et al., 1993; Lu, 2005; Ray et al., 2003).

The potential of spilled soybean seeds to establish, grow and produce pollen is extremely low and transient (see Section 3.6.1.1). Therefore, the likelihood/frequency of cross-pollination between occasional feral GM soybean plants resulting from seed spillage, and weedy or cultivated soybean plants is also considered extremely low. 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 for the reasons given in Section 3.5.1.1, even if exposed to the intended herbicides.

Interactions of the GM plant with target organisms

Taking the scope of application EFSA-GMO-BE-2019-165 (no cultivation) and the absence of target organisms into account, potential interactions of occasional feral soybean DBN9004 plants arising from seed import spills with target organisms are not considered a relevant issue.

Interactions of the GM plant with non-target organisms

The environmental risk assessment considers potential effects of the GM plant on populations of non-target organisms, defined as all those species directly or indirectly exposed to the GM plant and which are not targets of the newly expressed metabolite(s) it expresses. The GMO Panel evaluated the potential hazards of the NEPs and considered that the environmental exposure of non-target organisms to spilled GM soybean material or occasional feral GM soybean plants arising from spilled soybean DBN9004 grains will be limited. Additionally, ingested proteins are typically degraded before entering the environment through faecal material of animals fed with GM soybean (Harmon & Swanson, 2020; Miner-Williams et al., 2014; Mok & Urschel, 2020; Santos-Hernández et al., 2018; van Bruchem et al., 1985), and the data provided for the assessment of protein stability (see Section 3.5.1.2.3) supports that also the NEPs will be degraded. As compared to non-GM soybean, the GMO Panel considers that potential interactions of soybean DBN9004 with non-target organisms do not raise any safety concern.

Interactions with biogeochemical cycles

Biogeochemical cycles encompass the microbiologically mediated movement, transformation and storage of carbon, nitrogen and other compounds. The GMO Panel evaluated the potential hazards of the NEPs and considered that the environmental exposure to spilled GM soybean material or occasional feral GM soybean plants arising from spilled soybean DBN9004 grains will be limited. Additionally, proteins are typically degraded before entering the environment through faecal material of animals fed with GM soybean (Harmon & Swanson, 2020; Miner-Williams et al., 2014; Mok & Urschel, 2020; Santos-Hernández et al., 2018; van Bruchem et al., 1985), and the data provided for the assessment of protein stability (see Section 3.5.1.2.3) supports that also the NEPs will be degraded. As compared to non-GM soybean, the GMO Panel considers that potential interactions of soybean DBN9004 with and biogeochemical cycles do not raise any environmental safety concern.

Post-market environmental monitoring²⁷

The objectives of a post-market environmental monitoring (PMEM) plan, according to Annex VII of Directive 2001/18/EC, are: (i) 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 (ii) to identify the occurrence of adverse effects of the GMO, or its use, on human health or the environment that were not anticipated in the ERA.

Monitoring is related to risk management, and thus a final adoption of the PMEM plan falls outside the mandate of EFSA. However, the GMO Panel gives its opinion on the scientific rationale of the PMEM plan provided by the applicant (EFSA GMO Panel, 2011b).

As the ERA did not identify potential adverse environmental effects from soybean DBN9004, no case-specific monitoring is required.

The PMEM plan proposed by the applicant for soybean DBN9004 includes: (i) the description of a monitoring approach involving operators (federations involved in import and processing), reporting to the applicant, via a centralised system, any observed adverse effect(s) of GMOs on human health and the environment; (ii) a collaboration between the applicant and other industry members of CropLife Europe for the implementation of a common general surveillance methodology that includes collection of information recorded by the various operators; and (3) the review of relevant scientific publications retrieved from literature searches (Lecoq et al., 2007; Windels et al., 2009). The applicant proposes to submit a PMEM report on an annual basis for the duration of the authorisation period.

The GMO Panel considers that the scope of the PMEM plan provided by the applicant is consistent with the intended uses of soybean DBN9004. The GMO Panel agrees with the reporting intervals proposed by the applicant in its PMEM plan.

Conclusion of the environmental risk assessment and monitoring plan

The GMO Panel concludes that it is unlikely that soybean DBN9004 would differ from conventional soybean varieties in its ability to persist under European environmental conditions. Considering the scope of application EFSA-GMO-BE-2019-165, interactions of occasional feral soybean DBN9004 plants with the biotic and abiotic environment are not considered to be relevant issues. The analysis of HGT from soybean DBN9004 to bacteria does not indicate a safety concern. Therefore, considering the introduced traits, the outcome of the agronomic and phenotypic analysis, and the routes and levels of exposure, the GMO Panel concludes that soybean DBN9004 would not raise safety concerns in the event of release of GM material, including 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 DBN9004.

OVERALL CONCLUSIONS

The GMO Panel was asked to carry out a scientific assessment of soybean DBN9004 for import, processing and food and feed uses in accordance with Regulation (EC) No 1829/2003.

The molecular characterisation data establish that soybean DBN9004 contains a single insert consisting of one copy of the cp4 epsps and pat expression cassettes. The quality of the sequencing methodology and datasets was assessed by the EFSA GMO Panel and is in compliance with the requirements listed in the EFSA Technical Note. Bioinformatic analyses of the sequences encoding the newly expressed proteins, the sequences corresponding to ORFs within the insert or spanning the junctions between the insert and genomic DNA do not raise safety concerns. The stability of the inserted DNA and of the introduced trait has been confirmed over several generations. The methodology used to quantify the levels of the CP4 EPSPS and PAT proteins is considered adequate. The protein characterisation data comparing the structural, biochemical and functional properties of plant and E. coli-derived CP4 EPSPS and PAT proteins indicate that these proteins are equivalent, and the E. coli-derived proteins can be used in the safety studies.

Considering the selection of test materials, the field trial sites and the associated management practices and the agronomic-phenotypic characterisation as an indicator of the overall field trial quality, the GMO Panel concludes that the field trials are appropriate to support the comparative analysis. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between soybean DBN9004 and its conventional counterpart needs further assessment.

The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the CP4 EPSPS and PAT proteins as expressed in soybean DBN9004. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of soybean DBN9004, as food and feed. In the context of this application, the consumption of food and feed from soybean DBN9004 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that soybean DBN9004, as described in this application, is as safe as the conventional counterpart and non-GM soybean varieties tested, and no post-market monitoring of food/feed is considered necessary.

In the case of accidental release of soybean DBN9004 material into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean DBN9004.

Based on the relevant publication identified through the literature searches, the GMO Panel does not identify any safety issue pertaining to the intended uses of soybean DBN9004.

The GMO Panel concludes that soybean DBN9004, 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.

DOCUMENTATION AS PROVIDED TO EFSA

The documentation as provided to EFSA is available on Open EFSA.²⁸

ABBREVIATIONS

• ADF
• acid detergent fibre
• BBCH
• Biologische Bundesanstalt, Bundessortenamt and CHemical industry
• bp
• base pair
• bw
• body weight
• DM
• dry matter
• dw
• dry weight
• ELISA
• Enzyme-Linked Immunosorbent Assay
• EPSPS
• 5-enolpyruvylshikimate-3-phosphate synthase
• ERA
• environmental risk assessment
• FOB
• functional observational battery
• fw
• fresh weight
• GLP
• good laboratory practice
• GM
• genetically modified
• GMO Panel
• Panel on Genetically Modified Organisms
• GMO
• genetically modified organisms
• HGT
• horizontal gene transfer
• HR
• homologous recombination
• MS
• mass spectrometry
• NA
• not applicable
• NDF
• neutral detergent fibre
• NEP
• newly expressed protein
• OECD
• Organisation for Economic Co-operation and Development
• ORFs
• open reading frames
• PAT
• phosphinothricin acetyltransferase
• PCR
• polymerase chain reaction
• PMEM
• post-market environmental monitoring
• SDS-PAGE
• sodium dodecyl sulphate polyacrylamide gel electrophoresis
• SES
• standardised effect sizes
• T-DNA
• transfer-deoxyribonucleic acid

ACKNOWLEDGEMENTS

The Panel wishes to thank the members of the Working Groups on molecular characterisation, food and feed safety assessment and the working group on comparative analysis and environmental risk assessment for the preparatory work on this scientific output and EFSA staff member Reinhilde Schoonjans for the support provided to this scientific output.

REQUESTOR

Competent Authority of Belgium

QUESTION NUMBER

EFSA-Q-2020-00013

COPYRIGHT FOR NON-EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

Josep Casacuberta, Francisco Barro, Albert Braeuning, Ruud de Maagd, Michelle M Epstein, Thomas Frenzel, Jean-Luc Gallois, Frits Koning, Antoine Messéan, F Javier Moreno, Fabien Nogué, Giovanni Savoini, Alan H Schulman, Christoph Tebbe and Eve Veromann.

Appendix

APPENDIX A - Additional studies

List of additional studies performed by or on behalf of the applicant with regard to the evaluation of the safety of soybean DBN9004 for humans, animals or the environment

Study identification	Title
DBNBC-IRA-2016004	Germination and dormancy characteristics of DBN9004
DBNBC-FLD-2019013	Survival and competitiveness assessment of DBN9004
DBNBC-IRA-2016007	Germination and Dormancy Characteristics of DBN9004

Appendix

APPENDIX B - List of relevant publications identified by the applicant through literature searches (January 2010–January 2025)

Reference
Bell, E., Nakai, S., & Burzio, L. A. (2018). Stacked genetically engineered trait products produced by conventional breeding reflect the compositional profiles of their component single trait products. Journal of Agricultural and Food Chemistry, 66, 7794–7804
Costa, F. R., Rech, R., Duke, S. O., & Carvalho, L. B. (2018). Lack of effects of glyphosate and glufosinate on growth, mineral content, and yield of glyphosate- and glufosinate-resistant maize. Gm Crops & Food-Biotechnology in Agriculture and the Food Chain, 9, 189–198
CTNBio—Comissão Técnica Nacional De Biossegurança—Brazil Diário Oficial da União N°219 January 31, 2025 Technical Opinion: 8777/2023
de Araújo, G. V., Albrecht, A. J. P., Albrecht, L. P., de Carvalho, H. W. P., Migliavacca, R. A., & Silva, A. F. M. (2021). Effect of glyphosate and glufosinate on nutritional content and agronomic performance of maize possessing cp4epsps and pat transgenes. Australian Journal of Crop Science, 15(5), 773–779
de Cerqueira, D. T. R., Schafer, A. C., Fast, B. J., & Herman, R. A. (2017). Agronomic performance of insect-protected and herbicide-tolerant MON 89034 x TC1507 × NK603 × DAS-40278-9 corn is equivalent to that of conventional corn. Gm Crops & Food-Biotechnology in Agriculture and the Food Chain, 8, 149–155
Fast, B. J., Galan, M. P., & Schafer, A. C. (2016). Event DAS-444Ø6-6 soybean grown in Brazil is compositionally equivalent to non-transgenic soybean. GM Crops & Food, 7, 79–83
He, H. P., Ren, Z. T., Shen, W. J., Liu, B., & Xue, K. (2018). Effects of transgenic herbicide-tolerate maize on biodiversity of arthropod communities in the fields. Journal of Ecology and Rural Environment, 34, 333–341
Lepping, M. D., Herman, R. A., & Potts, B. L. (2013). Compositional equivalence of DAS-444Ø6-6 (AAD-12 + 2mEPSPS + PAT) herbicide-tolerant soybean and nontransgenic soybean. Journal of Agricultural and Food Chemistry, 61, 11180–11190
Liu, W., Li, L., Zhang, Z., Dong, M., & Jin, W. (2020). iTRAQ-based quantitative proteomic analysis of transgenic and non-transgenic maize seeds. Journal of Food Composition and Analysis, 92
Liu, W. X., Dong, M., Meng, L. X., Wan, Y. S., & Jin, W. J. (2021). Metabolic profiling of maize seeds with both insect- and herbicide-resistance genes (cry1Ab and epsps), dual herbicide-resistance genes (epsps and pat), and natural genotypic varieties. Chemical and Biological Technologies in Agriculture, 8(1), article 57
Lundry, D. R., Burns, J. A., Nemeth, M. A., & Riordan, S. G. (2013). Composition of grain and forage from insect-protected and herbicide-tolerant corn, MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (SmartStax), is equivalent to that of conventional corn (Zea mays L.). Journal of Agricultural and Food Chemistry, 61, 1991–1998
McNaughton, J., Roberts, M., Rice, D., Smith, B., Hinds, M., Delaney, B., Iiams, C., & Sauber, T. (2011). Evaluation of broiler performance and carcass yields when fed diets containing corn grain from transgenic stacked-trait product DAS-Ø15Ø7-1xdas-59122-7xmon-ØØ81Ø-6xmon-ØØ6Ø3-6. Journal of Applied Poultry Research, 20, 542–553
Ministerio de Agricultura, Ganadería y Pesca, Argentina. Resolución-2019-17-APN-SAYBIMPYT.
Ministry of Agriculture and Rural Affairs of the People's Republic of China.
Papineni, S., Fletcher, D. W., Cromwell, G. L., & Ekmay, R. D. (2017a). Comparative performance of broilers fed diets containing DAS-44406–6 and non-transgenic soybean meal. Poultry Science, 96, 1244–1249
Papineni, S., Murray, J. A., Ricardo, E., Dunville, C. M., Sura, R. K., & Thomas, J. (2017b). Evaluation of the safety of a genetically modified DAS-444Ø6-6 soybean meal and hulls in a 90-day dietary toxicity study in rats. Food and Chemical Toxicology, 109, 245–252
Papineni, S., Passage, J. K., Ekmay, R. D., & Thomas, J. (2018). Evaluation of 30% DAS-444Ø6-6 soybean meal in a subchronic rat toxicity study. Regulatory Toxicology and Pharmacology, 94, 57–69
Qian, Z. Y., Bultman, J., Papineni, S., He, N., Lanigan, L., Herman, R. A., & Hard, G. C. (2018). Safety evaluation of DAS-44406-6 soybeans in Wistar rats. Regulatory Toxicology and Pharmacology, 92, 152–164
Xiang, D., Luo, M., Jiang, F., Wen, Z., Chen, X., Wang, X., Xu, X., Wei, W., & Xu, J. (2023). Safety assessment of subchronic feeding of insect-resistant and herbicide-resistant transgenic soybeans to juvenile channel catfish (Ictalurus punctatus). Scientific Reports, 13(1), 5445.

Appendix

APPENDIX C - Statistical analysis and statistically significant findings in the 90-day toxicity study in rats on soybean DBN9004

C.1

Statistical analysis of the 90-day study on soybean DBN9004 in rats

The following endpoints were statistically analysed: Clinical Observation, Functional Observational Battery (FOB), Body Weight, Food Consumption, Haematology, Clinical Chemistry, Hormone, Urinalysis, Ophthalmic Examination, Bone Marrow Smears, Gross Necropsy and Histopathology. For all continuous endpoints, mean, standard deviation in terms of the standardised effect sizes (SES) of each dose group for each sex, variable and period or time interval were reported. The main statistical analysis compared each of the test diet groups (10% W1534 (DBN-Ø8ØØ2-3) dietary group, 20% W1534 (DBN-Ø8ØØ2-3) dietary group, 10% W0664 (DBN-Ø9ØØ4-6) dietary group, 20% W0664 (DBNØ9ØØ4-6) dietary group) separately with the control group (20% W0664-C01 (Jack Soybean)). Continuous data were analysed separately for each variable and period or time interval, according to a Linear Mixed Model (factor: Diet, sex and interaction ‘dose-sex’); then, pairwise comparisons, between each test and control group (separately for each sex) were performed using a t-test (at the 5% level of significance). Ordinal multi-category data were analysed using a Kruskal–Wallis (K–W) H test. Binomial category data and unordered multi-category data were analysed by Fisher's exact probabilities test. Missing data were considered by the Panel and found not to have an impact on the results (Table C.1).

TABLE C.1 Statistically significant findings in 90-day study on soybean DBN9004 in rats.

Statistically significant parameter/endpoint	Finding	GMO panel interpretation
Mortality, signs, FoB, organ wts, gross or microscopic pathology	No deaths; no significant or biologically relevant findings
Body weight, weight-gain, food consumption.	Increases and decreases during some phases of the study	No consistent pattern. Overall, body weight gains and food consumption similar to controls. Within normal variation. Not an adverse effect of treatment
Haemoglobin	Decreased in low dose males (2%)	Small magnitude. Not seen at the top dose. Not an adverse effect of treatment
Creatinine	Increased in both sexes and both dose level (25%)	No dose response. Consistent with values in secondary control diets. Within normal variation. Not an adverse effect of treatment
Sodium	Increased in both male groups (1%)	Low magnitude. Within normal variation. Not an adverse effect of treatment
Chloride	Increased in low dose males (1%)	Low magnitude. Within normal variation. No dose response. Not an adverse effect of treatment
Low Density Lipoprotein (LDL)	Decreased in top dose males (25%)	Decrease not adverse in isolation. Consistent with values in secondary control diets. Within normal variation. Not and adverse effect of treatment
T3 & T4	Increased in both male groups (15%–25%)	Consistent with values in secondary control diets. Within normal variation. Not and adverse effect of treatment
Urine volume	Decreased in top dose females (50%)	Also evident in top dose males (30%) but not statistically significant. In females the specific gravity (SG) of the urine was also decreased indicating a possible effect on kidney function, whereas in males the SG was increased indicating reduced water consumption. In top dose females there was no consistent pattern of changes in endpoints associated with kidney function (blood urea nitrogen, electrolytes, creatinine and no pathological changes to the kidney or bladder). Overall, it is considered that the decreased urinary volume is not an adverse effect of treatment

Appendix

APPENDIX D - Animal dietary exposure (Table D.1)

TABLE D.1 Dietary exposure to PAT and CP4 EPSPS proteins (mg/kg bw per day) in livestock, based on the consumption of soybean meal and forage.

Animal category	BW (kg)	TDI feed (kg DM/animal)	IR (%)	Dietary exposure (mg/kg bw per day)
Soybean meal	Forage	Soybean meal	Forage
PAT
Beef	500	12	20	20	0.007	0.089
Dairy	650	25	25	20	0.014	0.143
Rams/ewes	75	2.5	25	20	0.012	0.124
Lambs	40	1.7	25	20	0.016	0.158
Breeding swine	260	6	30	–	0.010	–
Finishing swine	100	3	30	–	0.0140	–
Broiler	1.7	0.12	40	–	0.0439	–
Layer	1.9	0.13	25	–	0.026	–
Turkeys	7	0.5	45	–	0.050	–
CP4 EPSPS
Beef	500	12	20	20	1.224	2.351
Dairy	650	25	25	20	2.453	3.768
Rams/ewes	75	2.5	25	20	2.126	3.265
Lambs	40	1.7	25	20	2.711	4.163
Breeding swine	260	6	30	–	1.766	–
Finishing swine	100	3	30	–	2.296	–
Broiler	1.7	0.12	40	–	7.204	–
Layer	1.9	0.13	25	–	4.364	–
Turkeys	7	0.5	45	–	8.201	–

References

Abud, S., de Souza, P. I. M., Vianna, G. R., Leonardecz, E., Moreira, C. T., Faleiro, F. G., Júnior, J. N., Monteiro, P. M. F. O., Rech, E. L., & Aragão, F. J. L. (2007). Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil. Genetics and Molecular Research, 6, 445–452.

Ahrent, D. K., & Caviness, C. E. (1994). Natural cross‐pollination of 12 soybean cultivars in Arkansas. Crop Science, 34, 376–378.

Alimentarius, C. (2009). Foods derived from modern biotechnology. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme. https://www.fao.org/docrep/011/a1554e/a1554e00.htm

Alsajri, F. A., Singh, B., Wijewardana, C., Irby, J. T., Gao, W., & Reddy, K. R. (2019). Evaluating soybean cultivars for low‐ and high‐temperature tolerance during the seedling growth stage. Agronomy, 9, 13. https://doi.org/10.3390/agronomy9010013

Bond, J. A., & Walker, T. W. (2009). Control of volunteer glyphosate‐resistant soybean in rice. Weed Technology, 23, 225–230. https://doi.org/10.1614/WT‐08‐156.1

Bramlage, W. J., Leopold, A. C., & Parrish, D. J. (1978). Chilling stress to soybeans during imbibition. Plant Physiology, 61, 525–529.

Caviness, C. E. (1966). Estimates of natural cross‐pollination in Jackson soybeans in Arkansas. Crop Science, 6, 211–212.

Celesti‐Grapow, L., Pretto, F., Carli, E., & Blasi, C. (Eds.). (2010). Flora vascolare alloctona e invasiva delle regioni d'Italia. Casa Editrice Università La Sapienza.

Dorokhov, D., Ignatov, A., Deineko, E., Serjapin, A., Ala, A., & Skryabin, K. (2004). In H. C. M. den Nijs, D. Bartsch, & J. Sweet (Eds.), Introgression from genetically modified plants into wild relatives (pp. 151–161). CAB International.

EFSA (European Food Safety Authority). (2009). Consolidated presentation of the joint Scientific Opinion of the GMO and BIOHAZ Panels on the “use of antibiotic resistance genes as marker genes in genetically modified plants” and the Scientific Opinion of the GMO Panel on “consequences of the opinion on the use of antibiotic resistance genes as marker genes in genetically modified plants on previous EFSA assessments of individual GM plants”. EFSA Journal, 7(6), 1108. https://doi.org/10.2903/j.efsa.2009.1108

EFSA (European Food Safety Authority). (2010). Application of systematic review methodology to food and feed safety assessments to support decision making. EFSA Journal, 8(6), 1637. https://doi.org/10.2903/j.efsa.2010.1637

EFSA (European Food Safety Authority). (2014). Explanatory statement for the applicability of the guidance of the EFSA Scientific Committee on conducting repeated‐dose 90‐day oral toxicity study in rodents on whole food/ feed for GMO risk assessment. EFSA Journal, 12(10), 3871. https://doi.org/10.2903/j.efsa.2014.3871

EFSA (European Food Safety Authority). (2015). Reasoned opinion on the review of the existing maximum residue levels (MRLs) for glufosinate according to article 12 of Regulation (EC) No 396/2005. EFSA Journal, 13(1), 3950. https://doi.org/10.2903/j.efsa.2015.3950

EFSA (European Food Safety Authority), Devos, Y., Guajardo, I. M., Glanville, J., & Waigmann, E. (2017a). Explanatory note on literature searching conducted in the context of GMO applications for (renewed) market authorisation and annual post‐market environmental monitoring reports on GMOs authorised in the EU market. EFSA Supporting Publications, 14(4), EN‐1207. https://doi.org/10.2903/sp.efsa.2017.en‐1207

EFSA (European Food Safety Authority), Gennaro, A., Gomes, A., Herman, L., Nogue, F., Papadopoulou, N., & Tebbe, C. (2017b). Technical report on the explanatory note on DNA sequence similarity searches in the context of the assessment of horizontal gene transfer from plants to microorganisms. EFSA Supporting Publications, 14(7), EN‐1273. https://doi.org/10.2903/sp.efsa.2017.en‐1273

EFSA (European Food Safety Authority), Paraskevopoulos, K., Ramon, M., Dalmay, T., du Jardin, P., Casacuberta, J., Guerche, P., Jones, H., Nogué, F., Robaglia, C., & Rostoks, N. (2018). Explanatory note on the determination of newly expressed protein levels in the context of genetically modified plant applications for EU market authorisation. EFSA Supporting Publications, 15(8), EN‐1466. https://doi.org/10.2903/sp.efsa.2018.EN‐1466

EFSA (European Food Safety Authority), Gomez Ruiz, J. A., Bresson, J.‐L., Frenzel, T., & Paoletti, C. (2019a). Statement on the human dietary exposure assessment to newly expressed proteins in GM foods. EFSA Journal, 17(7), 5802. https://doi.org/10.2903/j.efsa.2019.5802

EFSA (European Food Safety Authority), Devos, Y., Guajardo, I. M., Alvarez, F., & Glanville, J. (2019b). Explanatory note on literature searching conducted in the context of GMO applications for (renewed) market authorisation and annual post‐market environmental monitoring reports on GMOs authorised in the EU market. EFSA Supporting Publications, 16(4), EN‐1614. https://doi.org/10.2903/sp.efsa.2019.en‐1614

EFSA (European Food Safety Authority). (2019c). Review of the existing maximum residue levels for glyphosate according to Article 12 of regulation (EC) No 396/2005 – Revised version to take into account omitted data. EFSA Journal, 17(10), 5862. https://doi.org/10.2903/j.efsa.2019.5862

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2010a). Guidance on the environmental risk assessment of genetically modified plants. EFSA Journal, 8(11), 1879. https://doi.org/10.2903/j.efsa.2010.1879

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2010b). Statistical considerations for the safety evaluation of GMOs. EFSA Journal, 8(1), 1250. https://doi.org/10.2903/j.efsa.2010.1250

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2010c). Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed. EFSA Journal, 8(7), 1700. https://doi.org/10.2903/j.efsa.2010.1700

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2011a). Scientific Opinion on guidance for risk assessment of food and feed from genetically modified plants. EFSA Journal, 9(5), 2150. https://doi.org/10.2903/j.efsa.2011.2150

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2011b). Scientific Opinion on guidance on the post‐market environmental monitoring (PMEM) of genetically modified plants. EFSA Journal, 9(8), 2316. https://doi.org/10.2903/j.efsa.2011.2316

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2015). Guidance on the agronomic and phenotypic characterisation of genetically modified plants. EFSA Journal, 13(6), 4128. https://doi.org/10.2903/j.efsa.2015.4128

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2017). Guidance on allergenicity assessment of genetically modified plants. EFSA Journal, 15(5), 4862. https://doi.org/10.2903/j.efsa.2017.4862

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Casacuberta, J., Nogué, F., Naegeli, H., Birch, A. N., De Schrijver, A., Gralak, M. A., Guerche, P., Manachini, B., Messéan, A., Nielsen, E. E., Robaglia, C., Rostoks, N., Sweet, J., Tebbe, C., Visioli, F., Wal, J.‐M., Moxon, S., Schneeberger, K., … Jones, H. (2018). Scientific Opinion on the technical note on the quality of DNA sequencing for the molecular characterisation of genetically modified plants. EFSA Journal, 16(7), 5345. https://doi.org/10.2903/j.efsa.2018.5345

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dalmay, T., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Naegeli, H., Moreno, F. J., Nogué, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Ardizzone, M., De Sanctis, G., … Raffaello, T. (2021). Scientific Opinion on the assessment of genetically modified maize NK603 × T25 × DAS‐40278‐9 and subcombinations, for food and feed uses, under regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2019‐164). EFSA Journal, 19(12), 6942. https://doi.org/10.2903/j.efsa.2021.6942

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dalmay, T., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Moreno, F. J., Naegeli, H., Nogué, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Ardizzone, M., Camargo, A. M., … Streissl, F. (2022). Scientific Opinion on the assessment of genetically modified Maize MON 87429 for food and feed uses, under regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2019‐161). EFSA Journal, 20(11), 7589. https://doi.org/10.2903/j.efsa.2022.7589

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dalmay, T., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Moreno, F. J., Naegeli, H., Nogue, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Ardizzone, M., Camargo, A. M., … Raffaello, T. (2024a). Assessment of genetically modified maize DP915635 for food and feed uses, under regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2020‐172). EFSA Journal, 22(1), 8490. https://doi.org/10.2903/j.efsa.2024.8490

EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dalmay, T., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Naegeli, H., Moreno, F. J., Nogué, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Ardizzone, M., De Sanctis, G., … Xiftou, K. (2024b). Assessment of genetically modified maize DP910521 (application GMFF‐2021‐2473). EFSA Journal, 22(8), 8887. https://doi.org/10.2903/j.efsa.2024.8887

EFSA Scientific Committee. (2011). EFSA guidance on conducting repeated‐dose 90‐day oral toxicity study in rodents on whole food/feed. EFSA Journal, 9(12), 2438. https://doi.org/10.2903/j.efsa.2011.2438

Fernandez, A., Mills, E. N., Lovik, M., Spoek, A., Germini, A., Mikalsen, A., & Wal, J. M. (2013). Endogenous allergens and compositional analysis in the allergenicity assessment of genetically modified plants. Food and Chemical Toxicology, 62, 1–6. https://doi.org/10.1016/j.fct.2013.08.023

Gumisiriza, G., & Rubaihayo, P. R. (1978). Factors that influence outcrossing in soybean. Zeitschrift für Acker‐ und Pflanzenbau/Journal of Agronomy and Crop Science, 147, 129–133.

Harmon, D. L., & Swanson, K. C. (2020). Review: Nutritional regulation of intestinal starch and protein assimilation in ruminants. Animal, 14, S17–S28. https://doi.org/10.1017/S1751731119003136

Harrison, L. A., Bailey, M. R., Naylor, M. W., Ream, J. E., Hammond, B. G., Nida, D. L., Burnette, B. L., Nickson, T. E., Mitsky, T. A., Taylor, M. L., & Fuchs, R. L. (1996). The expressed protein in glyphosate‐tolerant soybean, 5‐enolypyruvylshikimate‐3‐phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. The Journal of Nutrition, 126(3), 728–740. https://doi.org/10.1093/jn/126.3.728

Hérouet, C., Esdaile, D. J., Mallyon, B. A., Debruyne, E., Schulz, A., Currier, T., Hendrickx, K., van der Klis, R. J., & Rouan, D. (2005). Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate‐ammonium herbicide in transgenic plants. Regulatory Toxicology and Pharmacology, 41(2), 134–149. https://doi.org/10.1016/j.yrtph.2004.11.002

Hülter, N., & Wackernagel, W. (2008). Double illegitimate recombination events integrate DNA segments through two different mechanisms during natural transformation of Acinetobacter baylyi. Molecular Microbiology, 67, 984–995. https://doi.org/10.1111/j.1365‐2958.2007.06096.x

Jhala, A. J., Beckie, H. J., Peters, T. J., Culpepper, A. S., & Norsworthy, J. K. (2021). Interference and management of herbicide‐resistant crop volunteers. Weed Science, 69, 257–273.

Jhala, A. J., Sandell, L., Kruger, G., Wilson, R., & Knezevic, Z. (2013). Control of glyphosate‐resistant volunteer soybean in corn. Crop watch. University of Nebraska–Lincoln.

Kikuchi, A., Murata, K., Tabuchi, K., & Sakai, S. (1993). Inheritance of seed embryo color and investigation of degree of natural cross‐pollination in soybeans. Breeding Science, 43(Suppl. 2), 112.

Lecoq, E., Holt, K., Janssens, J., Legris, G., Pleysier, A., Tinland, B., & Wandelt, C. (2007). General surveillance: Roles and responsibilities the industry view. Journal Für Verbraucherschutz Und Lebensmittelsicherheit‐Journal of Consumer Protection and Food Safety, 2(S1), 25–28. https://doi.org/10.1007/s00003‐007‐0244‐1

Lu, B. R. (2005). Multidirectional gene flow among wild, weedy, and cultivated soybeans. In J. Gressel (Ed.), Crop Ferality and Volunteerism (pp. 137–147). OECD + Taylor & Francis.

Matsushita, A., Goto, H., Takahashi, Y., Tsuda, M., & Ohsawa, R. (2020). Consideration of familiarity accumulated in the confined field trials for environmental risk assessment of genetically modified soybean (Glycine max) in Japan. Transgenic Research, 29, 229–242. https://doi.org/10.1007/s11248‐020‐00193‐z

Meier, U. (2001). Growth stages of mono‐and dicotyledonous plants (2nd ed.). BBCH Monograph. Federal Biological Research Centre for Agriculture and Forestry.

Miner‐Williams, W. M., Stevens, B. R., & Moughan, P. J. (2014). Are intact peptides absorbed from the healthy gut in the adult human? Nutrition Research Reviews, 27, 308–329. https://doi.org/10.1017/S0954422414000225

Mok, C. H., & Urschel, K. L. (2020). Invited review – Amino acid requirements in horses. Asian‐Australasian Journal of Animal Sciences, 33(5), 679–695. https://doi.org/10.5713/ajas.20.0050

Nickell, C. D., Noel, G. R., Thomas, D. J., & Waller, R. (1990). Registration of ‘Jack’ soybean. Crop Science, 30, 1365. https://doi.org/10.2135/cropsci1990.0011183X003000060059x

OECD (Organisation for Economic Co‐operation and Development). (2000). Consensus document on the biology of Glycine max (L.) Merr. (soybean). ENV/JM/MONO(2000)9. Series on harmonization of regulatory oversight in biotechnology No. 15. Organisation for Economic Co‐operation and Development.

OECD (Organisation for Economic Co‐operation and Development). (2012). Revised consensus document on compositional considerations for new varieties of soybean [Glycine max (L.) Merr.]: Key food and feed nutrients, Antinutrients, toxicants and allergens. Organisation for Economic Cooperation and Development.

OECD (Organisation for Economic Co‐operation and Development). (2013). Guidance document on residues in livestock. In: Series on pesticides No 73ENV/JM/MONO(2013), 8, 4 September 2013.

OECD (Organisation for Economic Co‐operation and Development). (2018). Test No. 408: Repeated dose 90‐day oral toxicity study in rodents, OECD guidelines for the testing of chemicals, section 4. OECD Publishing.

Owen, M. D. K. (2005). Maize and soybeans – Controllable volunteerism without ferality? In J. Grassel (Ed.), Crop Ferality and volunteerism (pp. 149–165). CRC Press.

Polevoda, B., & Sherman, F. (2000). Na‐terminal acetylation of eukaryotic proteins. Journal of Biological Chemistry, 275, 36479–36482. https://doi.org/10.1074/jbc.R000023200

Ray, J. D., Kilen, T. C., Abel, C. A., & Paris, R. L. (2003). Soybean natural cross‐pollination rates under field conditions. Environmental Biosafety Research, 2, 133–138. https://doi.org/10.1051/ebr:2003005

Santos‐Hernández, M., Miralles, B., Amigo, L., & Recio, I. (2018). Intestinal signaling of proteins and digestion‐derived products relevant to satiety. Journal of Agricultural and Food Chemistry, 66(39), 10123–10131. https://doi.org/10.1021/acs.jafc.8b02355

Selb, R., Wal, J. M., Moreno, F. J., Lovik, M., Mills, C., Hoffmann‐Sommergruber, K., & Fernandez, A. (2017). Assessment of endogenous allergenicity of genetically modified plants exemplified by soybean – Where do we stand? Food and Chemical Toxicology, 101, 139–148.

Soltani, N., Shropshure, C., & Sikkema, P. H. (2019). Control of volunteer adzuki bean and soybean in white bean with halosulfuron. Canadian Journal of Plant Science, 99, 375–378. https://doi.org/10.1139/cjps‐2018‐0261

Staniak, M., Stępień‐Warda, A., Czopek, K., Kocira, A., & Baca, E. (2021). Seeds quality and quantity of soybean [Glycine max (L.) Merr.] cultivars in response to cold stress. Agronomy, 11, 520. https://doi.org/10.3390/agronomy11030520

Sys, C., Van Ranst, E., Debaveye, J., & Beernaert, F. (1993). Land evaluation. Part III: Crop requirements. Agricultural publication No. 7. General Administration for Development Cooperation.

Szczerba, A., Płażek, A., Pastuszak, J., Kopeć, P., Hornyák, M., & Dubert, F. (2021). Effect of low temperature on germination, growth, and seed yield of four soybean (Glycine max L.) cultivars. Agronomy, 11, 800. https://doi.org/10.3390/agronomy11040800

Tyagi, S. K., & Tripathi, R. P. (1983). Effect of temperature on soybean germination. Plant and Soil, 74(2), 273–280. https://doi.org/10.1007/BF02143617

van Bruchem, J., Rouwers, S. M. G., Bangma, G. A., Leffering, C. P., & van Adrichem, P. W. M. (1985). Digestion of proteins of varying degradability in sheep. 1. Fermentation in and rate of passage from the reticulorumen. Netherlands Journal of Agricultural Science, 33, 263–272.

Wang, X., Li, X., Zhou, Q., Song, S., & Dong, S. (2023). Comparison and evaluation of low‐temperature tolerance of different soybean cultivars during the early‐growth stage. Agronomy, 13, 1716. https://doi.org/10.3390/agronomy13071716

Windels, P., Alcalde, E., Lecoq, E., Legris, G., Pleysier, A., Tinland, B., & Wandelt, C. (2009). General surveillance for import and processing: The EuropaBio approach. Journal of Consumer Protection and Food Safety, 3(Suppl. 2), 14–16. https://doi.org/10.1007/s00003‐009‐0359‐7

Wingfield, P. T. (2017). N‐terminal methionine processing. Current Protocols in Protein Science, 88, 6–14. https://doi.org/10.1002/cpps.29

Yoshimura, Y., Matsuo, K., & Yasuda, K. (2006). Gene flow from GM glyphosate‐tolerant to conventional soybeans under field conditions in Japan. Environmental Biosafety Research, 5, 169–173. https://doi.org/10.1051/ebr:2007003

Zhang, L., Liu, L., Fang, Z., Shen, W., Dai, Y., Jia, R., Liang, J., & Liu, B. (2023). Fitness changes in wild soybean caused by gene flow from genetically modified soybean. BMC Plant Biology, 23, 424. https://doi.org/10.1186/s12870‐023‐04398‐2