Assessment of genetically modified soybean GMB151 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2018‐153)
2021; Wiley; Volume: 19; Issue: 4 Linguagem: Inglês
10.2903/j.efsa.2021.6424
ISSN1831-4732
AutoresHanspeter Naegeli, Jean Bresson, Tamás Dalmay, Ian Crawford Dewhurst, M. Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejátko, F. Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, G. Savoini, Eve Veromann, Fabio Veronesi, Fernando Álvarez, Michele Ardizzone, Silvia Federici, Antonio Fernandez, Andrea Gennaro, José Ángel Gómez Ruiz, Dafni Maria Kagkli, Anna Lanzoni, Franco Maria Neri, Nikoletta Papadopoulou, Konstantinos Paraskevopoulos, Tommaso Raffaello, Franz Streissl, Giacomo De Sanctis,
Tópico(s)Animal Genetics and Reproduction
ResumoEFSA JournalVolume 19, Issue 4 e06424 Scientific OpinionOpen Access Assessment of genetically modified soybean GMB151 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2018-153) EFSA Panel on Genetically Modified Organisms (GMO), Corresponding Author EFSA Panel on Genetically Modified Organisms (GMO) GMO_secretariat_applications@efsa.europa.eu Correspondence:GMO_secretariat_applications@efsa.europa.euSearch for more papers by this authorHanspeter Naegeli, Hanspeter NaegeliSearch for more papers by this authorJean Louis Bresson, Jean Louis BressonSearch for more papers by this authorTamas Dalmay, Tamas DalmaySearch for more papers by this authorIan Crawford Dewhurst, Ian Crawford DewhurstSearch for more papers by this authorMichelle M Epstein, Michelle M EpsteinSearch for more papers by this authorLeslie George Firbank, Leslie George FirbankSearch for more papers by this authorPhilippe Guerche, Philippe GuercheSearch for more papers by this authorJan Hejatko, Jan HejatkoSearch for more papers by this authorFrancisco Javier Moreno, Francisco Javier MorenoSearch for more papers by this authorEwen Mullins, Ewen MullinsSearch for more papers by this authorFabien Nogué, Fabien NoguéSearch for more papers by this authorNils Rostoks, Nils RostoksSearch for more papers by this authorJose Juan Sánchez Serrano, Jose Juan Sánchez SerranoSearch for more papers by this authorGiovanni Savoini, Giovanni SavoiniSearch for more papers by this authorEve Veromann, Eve VeromannSearch for more papers by this authorFabio Veronesi, Fabio VeronesiSearch for more papers by this authorFernando Álvarez, Fernando ÁlvarezSearch for more papers by this authorMichele Ardizzone, Michele ArdizzoneSearch for more papers by this authorSilvia Federici, Silvia FedericiSearch for more papers by this authorAntonio Fernandez, Antonio FernandezSearch for more papers by this authorAndrea Gennaro, Andrea GennaroSearch for more papers by this authorJose Ángel Gómez Ruiz, Jose Ángel Gómez RuizSearch for more papers by this authorDafni Maria Kagkli, Dafni Maria KagkliSearch for more papers by this authorAnna Lanzoni, Anna LanzoniSearch for more papers by this authorFranco Maria Neri, Franco Maria NeriSearch for more papers by this authorNikoletta Papadopoulou, Nikoletta PapadopoulouSearch for more papers by this authorKonstantinos Paraskevopoulos, Konstantinos ParaskevopoulosSearch for more papers by this authorTommaso Raffaello, Tommaso RaffaelloSearch for more papers by this authorFranz Streissl, Franz StreisslSearch for more papers by this authorGiacomo De Sanctis, Giacomo De SanctisSearch for more papers by this author EFSA Panel on Genetically Modified Organisms (GMO), Corresponding Author EFSA Panel on Genetically Modified Organisms (GMO) GMO_secretariat_applications@efsa.europa.eu Correspondence:GMO_secretariat_applications@efsa.europa.euSearch for more papers by this authorHanspeter Naegeli, Hanspeter NaegeliSearch for more papers by this authorJean Louis Bresson, Jean Louis BressonSearch for more papers by this authorTamas Dalmay, Tamas DalmaySearch for more papers by this authorIan Crawford Dewhurst, Ian Crawford DewhurstSearch for more papers by this authorMichelle M Epstein, Michelle M EpsteinSearch for more papers by this authorLeslie George Firbank, Leslie George FirbankSearch for more papers by this authorPhilippe Guerche, Philippe GuercheSearch for more papers by this authorJan Hejatko, Jan HejatkoSearch for more papers by this authorFrancisco Javier Moreno, Francisco Javier MorenoSearch for more papers by this authorEwen Mullins, Ewen MullinsSearch for more papers by this authorFabien Nogué, Fabien NoguéSearch for more papers by this authorNils Rostoks, Nils RostoksSearch for more papers by this authorJose Juan Sánchez Serrano, Jose Juan Sánchez SerranoSearch for more papers by this authorGiovanni Savoini, Giovanni SavoiniSearch for more papers by this authorEve Veromann, Eve VeromannSearch for more papers by this authorFabio Veronesi, Fabio VeronesiSearch for more papers by this authorFernando Álvarez, Fernando ÁlvarezSearch for more papers by this authorMichele Ardizzone, Michele ArdizzoneSearch for more papers by this authorSilvia Federici, Silvia FedericiSearch for more papers by this authorAntonio Fernandez, Antonio FernandezSearch for more papers by this authorAndrea Gennaro, Andrea GennaroSearch for more papers by this authorJose Ángel Gómez Ruiz, Jose Ángel Gómez RuizSearch for more papers by this authorDafni Maria Kagkli, Dafni Maria KagkliSearch for more papers by this authorAnna Lanzoni, Anna LanzoniSearch for more papers by this authorFranco Maria Neri, Franco Maria NeriSearch for more papers by this authorNikoletta Papadopoulou, Nikoletta PapadopoulouSearch for more papers by this authorKonstantinos Paraskevopoulos, Konstantinos ParaskevopoulosSearch for more papers by this authorTommaso Raffaello, Tommaso RaffaelloSearch for more papers by this authorFranz Streissl, Franz StreisslSearch for more papers by this authorGiacomo De Sanctis, Giacomo De SanctisSearch for more papers by this author First published: 19 April 2021 https://doi.org/10.2903/j.efsa.2021.6424Citations: 1 Requestor: Competent authority of the Netherlands Question number: EFSA-Q-2018-00781 Panel members: Hanspeter Naegeli, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Declarations of interest: The declarations of interest of all scientific experts active in EFSA's work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgments: The Panel wishes to thank the members of the Working Groups on Molecular Characterisation, Food and Feed Safety Assessment and Working Group On Comparative Analysis and Environmental Risk Assessment for the preparatory work on this scientific output and EFSA staff members Irene Muñoz Guajardo, Sylvie Mestdagh, Oberkofler Lorenz, Sonia Hernández Valero and Irina Vlas for the support provided to this scientific output. Adopted: 27 January 2021 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Soybean GMB151 was developed to confer tolerance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides and resistance to nematodes. 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 GMB151 and its conventional counterpart needs further assessment, except for palmitic acid and heptadecenoic acid in seeds and carbohydrate and crude protein in forage, which does not raise nutritional and safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the HPPD-4 and Cry14Ab-1 proteins as expressed in soybean GMB151, and finds no evidence that the genetic modification would change the overall allergenicity of soybean GMB151. In the context of this application, the consumption of food and feed from soybean GMB151 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that soybean GMB151 is as safe as the conventional counterpart and non-GM soybean reference varieties tested, and no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable soybean GMB151 seeds 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 GMB151. The GMO Panel concludes that soybean GMB151 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. Summary The scope of application EFSA-GMO-NL-2018-153 is for food and feed uses, import and processing of the genetically modified (GM) herbicide tolerant and nematode resistant soybean GMB151 in the European Union (EU). In this scientific opinion, the scientific Panel on Genetically Modified Organisms of the European Food Safety Authority (hereafter referred to as the 'GMO Panel') reports on the outcome of its risk assessment of soybean GMB151 according to the scope of the application EFSA-GMO-NL-2018-153. The GMO Panel conducted the assessment of soybean GMB151 in line with the principles described in Regulation (EU) No 503/2013 and its applicable guidelines for the risk assessment of genetically modified (GM) plants. The molecular characterisation data establish that soybean GMB151 contains a single insert consisting of one copy of the hppdPf-4Pa and the cry14Ab-1.b expression cassettes. The quality of the sequencing methodology and data sets was assessed by the EFSA GMO Panel and is in compliance to the requirements listed in the EFSA Technical Note.1 Updated bioinformatics analyses of the sequences encoding the newly expressed proteins and open reading frames (ORFs) present within the insert or spanning the junctions between the insert and genomic DNA, do not raise any 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 Cry14Ab-1 and HPPD-4 proteins is considered adequate. The protein characterisation data comparing the biochemical, structural and functional properties of plant- and microbe-produced Cry14Ab-1 and HPPD-4 proteins indicate that these proteins are equivalent and the microbe-derived proteins can be used in the safety studies. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between soybean GMB151 and its conventional counterpart needs further assessment, except for palmitic acid and heptadecenoic acid in seeds and carbohydrate and crude protein in forage, which does not raise nutritional and safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the HPPD-4 and Cry14Ab-1 proteins as expressed in soybean GMB151, and finds no evidence that the genetic modification would change the overall allergenicity of soybean GMB151. In the context of this application, the consumption of food and feed from soybean GMB151 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that soybean GMB151 is as safe as the conventional counterpart and non-GM soybean reference 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 GMB151 would not raise safety concerns in the case of accidental release of viable GM soybean seeds into the environment. The post-market environmental monitoring (PMEM) plan and reporting intervals are in line with the intended uses of soybean GMB151. The GMO Panel considered the overall quality of the performed literature searches acceptable. The literature searches did not identify any relevant publications on soybean GMB151. The GMO Panel concludes that soybean GMB151 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. 1 Introduction The scope of the application EFSA-GMO-NL-2018-153 is for food and feed uses, import and processing of soybean GMB151 and does not include cultivation in the European Union (EU). Soybean GMB151 was developed to confer tolerance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides such as isoxaflutole and resistance to nematodes. 1.1 Background and Terms of Reference as provided by the requestor On 9 October 2018, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands application EFSA-GMO-NL-2018-153 for authorisation of soybean GMB151 (Unique Identifier BCS-GM151-6), submitted by BASF Agricultural Solutions Seed US LLC (hereafter referred to as 'the applicant') according to Regulation (EC) No 1829/20032. Following receipt of application EFSA-GMO-NL-2018-153, EFSA informed EU Member States and the European Commission, and made the application available to them. Simultaneously, EFSA published summary of the application.3 EFSA checked the application for compliance with the relevant requirements of EFSA guidance documents, and, when needed, asked the applicant to supplement the initial application. On 4 March 2019, EFSA declared the application valid. From validity date, EFSA and its scientific Panel on Genetically Modified Organisms (hereafter referred to as 'the GMO Panel') endeavoured to respect a time limit of six months to issue a scientific opinion on application EFSA-GMO-NL-2018-153. 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 'Documentation', below). In accordance with Regulation (EC) No 1829/2003, EFSA consulted the nominated risk assessment bodies of EU Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC4. The EU Member States had three months to make their opinion known on application EFSA-GMO-NL-2018-153 as of date of validity. 1.2 Terms of Reference as provided by the requestor According to Articles 6 and 18 of Regulation (EC) No 1829/2003, EFSA and its GMO Panel were requested to carry out a scientific risk assessment of soybean GMB151 in the context of its scope as defined in application EFSA-GMO-NL-2018-153. 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). The relevant information is made available in the EFSA Register of Questions including the information required under Annex II to the Cartagena Protocol; a labelling proposal; a Post-Market Environmental Monitoring (PMEM) plan as provided by the applicant; the method(s), validated by the Community reference laboratory, for detection, including sampling, identification of the transformation event in the food-feed and/or foods-feeds produced from it and the appropriate reference materials.5 2 Data and methodologies 2.1 Data The GMO Panel based its scientific risk assessment of soybean GMB151 on the valid application EFSA-GMO-NL-2018-153, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by the Member States and relevant peer-reviewed scientific publications. 2.2 Methodologies The GMO Panel conducted its assessment in line with the principles described in Regulation (EU) No 503/2013, its applicable guidelines (i.e. EFSA GMO Panel, 2010a,b, 2011a,b, 2015a,b), explanatory notes and statements (i.e. EFSA, 2017a,b, 2019a) for the risk assessment of GM plants. For the assessment of 90-day animal feeding studies, the GMO Panel took into account the criteria included in the EFSA Scientific Committee guidance on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed (EFSA Scientific Committee, 2011) and the explanatory statement for its applicability (EFSA, 2014). The GMO Panel also assessed the applicant's literature searches, which include a scoping review, in accordance with the recommendations on literature searching outlined in EFSA (2010, 2017a). In the frame of the contracts OC/EFSA/GMO/2013/01 and OC/EFSA/GMO/2014/01, contractors performed preparatory work and delivered reports on the methods applied by the applicant in performing bioinformatic and statistical analyses, respectively. 3 Assessment 3.1 Systematic literature review as requested by Commission Regulation (EU) No 503/2013 The GMO Panel assessed the applicant's literature searches on soybean GMB151, 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-NL-2018-153. Based on the outcome of the scoping review, the GMO Panel agrees that there is limited value of undertaking a systematic review for soybean GMB151 at present. The GMO Panel considered the overall quality of the performed literature searches acceptable. The literature searches did not identify any relevant publications on soybean GMB151. 3.2 Molecular characterisation6 3.2.1 Transformation process and vector constructs Soybean GMB151 was developed by Agrobacterium tumefaciens (also known as Rhizobium radiobacter)-mediated transformation. Explants of soybean variety Thorne were co-cultured with a disarmed A. tumefaciens strain LBA4404 containing the vector pSZ8832. The plasmid pSZ8832 used for the transformation contains two expression cassettes between the right and left border of the T-DNA, containing the following genetic elements: The hppdPf-4Pa expression cassette consists of the P2 × 35S promoter from Cauliflower Mosaic Virus, the TPotpY-1Pf transit peptide containing sequences of the RuBisCO small subunit genes of Zea mays and Helianthus annuus, the hppdPf-4Pa coding sequence of the 4-hydroxyphenylpyruvate dioxygenase gene of Pseudomonas fluorescens and the T35S sequence including the 3′ untranslated region of the 35S transcript of the Cauliflower Mosaic Virus. The cry14Ab-1.b expression cassette consists of the Pubi10At sequence including the promoter region of ubiquitin-10 gene of Arabidopsis thaliana, the cry14Ab-1.b coding sequence of the delta-endotoxin gene of Bacillus thuringiensis and the T35S sequence including the 3′ untranslated region of the 35S transcript of the Cauliflower Mosaic Virus. The vector backbone contained elements necessary for the maintenance and selection of the plasmid in bacteria. 3.2.2 Transgene constructs in the GM plant Molecular characterisation of soybean GMB151 was performed by next generation sequencing (NGS), junction sequence analysis (JSA), polymerase chain reaction (PCR) and DNA sequence analysis, in order to determine insert copy number, size and organisation of the inserted sequences and to confirm the absence of plasmid backbone sequences. The approach used is acceptable in terms of coverage and sensitivity. Overall, the quality of the sequencing methodology and data sets was assessed by the EFSA GMO Panel and is in compliance to the requirements listed in the EFSA Technical Note (EFSA GMO Panel, 2018). NGS/JSA of the whole genome demonstrated that soybean GMB151 contains a single insert, consisting of a single copy of the T-DNA. NGS/JSA also confirmed the absence of plasmid backbone sequences in the soybean genome. Sanger sequencing of PCR-amplified fragments determined the nucleotide sequence of the entire soybean GMB151 event consisting of 7,459 bp of the insert and 1,000 bp of both 5′ and 3′ flanking regions. The Sanger analysis revealed that the insert in soybean GMB151 is characterised by a 482-bp deletion in the promoter P2 × 35S as compared to the T-DNA sequence in the plasmid pSZ8832 used for the transformation. Both NGS/JSA and Sanger sequencing showed that the 3′-end of the event in soybean GMB151 is characterised by a 39-bp filler DNA. Further analysis revealed that one portion of the filler DNA (21 bp) showed identity to ORIpVS1 sequence from plasmid pSZ8832 while another portion (17 bp) showed identity to a sequence of the soybean genomic 3′-flanking region. A comparison with the sequenced pre-insertion locus indicated that 63 bp were deleted from the soybean genomic DNA. The possible interruption of known endogenous soybean genes by the insertion in soybean GMB151 was evaluated by bioinformatics analyses of the pre-insertion locus and of the genomic sequences flanking the insert. The results of these analyses indicate that the insertion may have occurred in the 3′ UTR of the predicted gene for BON1-associated protein 1-like protein (NCBI accession number XM_006583276). The coding region of the gene is not affected and since the soybean genome is a partially diploidised tetraploid, paralogues (multiple copies of the gene) are expected. The function of the predicted gene for BON1-associated protein 1-like protein has not been characterised in soybean. In Arabidopsis, interruption of BAP1 gene (encoding BON1-associated protein 1) leads to constitutively active defence response and results in a dwarf phenotype (Yang et al., 2007) that has not been observed in soybean GMB151. Overall, these analyses indicate that the insertion of the T-DNA in the 3′UTR of the predicted gene for BON1-associated protein 1-like protein does not lead to unintended effects in soybean GMB151; this is also confirmed by compositional, agronomic and phenotypic characteristics (see Section 3.3). The results of segregation (see Section 3.2.5) and bioinformatics analyses establish that the insert is located in the nuclear genome. In addition, updated bioinformatics analyses of the amino acid sequence of the newly expressed HPPD-4 protein reveal no significant similarities to toxins and allergens. Initial bioinformatic analyses of the amino acid sequences of the Cry14Ab-1 revealed no significant similarities to known toxins but similarity of this protein with an allergen was detected. This hit was further assessed by the applicant and it is discussed in Section 3.4.4.1. Subsequently, updated bioinformatic analysis did not reveal any relevant hits. The updated bioinformatic analyses of the newly created open reading frames (ORFs) within the insert and spanning the junctions between the insert and genomic DNA indicated the presence of an eight amino acid exact match between an ORF and a putative chitinase (Cas s 5) from chestnut (Castanea sativa). This ORF is found within the transcriptional unit of the Cry14Ab-1.b coding sequence but in a different reading frame and without any translational start codons (ATG). In conclusion, this analysis indicates that the expression of an ORF showing significant similarities to toxins or allergens is highly unlikely. In order to assess the possibility for horizontal gene transfer (HGT) by homologous recombination (HR), the applicant performed a sequence identity analysis for soybean GMB151 to microbial DNA. The likelihood and potential consequences of plant-to-bacteria gene transfer are described in Section 3.5.1.2. 3.2.3 Protein characterisation and equivalence Soybean GMB151 expresses two new proteins: Cry14Ab-1 and HPPD-4. HPPD-4 is a modified 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas fluorescens, conferring tolerance to HPPD inhibiting herbicides. The GMO Panel has previously evaluated other HPPD proteins (EFSA GMO Panel, 2015a,b, 2020). Protein Cry14Ab-1 belongs to the Cry (crystal)-type protein family, in particular to the 'nematicidal branch' of Cry proteins from B. thuringiensis (Sanahuja et al., 2011). Given the technical restraints in producing large enough quantities for safety testing from plants, these proteins were recombinantly produced in Escherichia coli. A set of biochemical methods was employed to demonstrate the equivalence between the GMB151 and E. coli-produced Cry14Ab-1 and HPPD-4. Purified proteins from these two sources were characterised and compared in terms of their biochemical, structural and functional properties. Cry14Ab-1 protein characterisation and equivalence Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis showed that both plant- and microbe-produced Cry14Ab-1 proteins had the expected molecular weight of ~ 131.1 kDa and were comparably immunoreactive to Cry14Ab-1 protein specific antibodies. Glycosylation detection analysis demonstrated that none of the Cry14Ab-1 proteins were glycosylated. Amino acid sequence analysis of the plant-derived Cry14Ab-1 protein by mass spectrometry (MS) methods showed that the protein matched the deduced sequence as defined by the cry14Ab-1 gene. These sequence analysis data were consistent with the previously analysed microbe-produced Cry14Ab-1. In addition, the MS data showed that the N-terminal methionine of the plant-produced Cry14Ab-1 protein was truncated and aspartic acid-two was acetylated. Such modifications are common in eukaryotic proteins (e.g. Polevoda and Sherman, 2000). Functional equivalence was demonstrated by an insect feeding bioassay which showed that plant- and microbe-derived Cry14Ab-1 proteins had comparable insecticidal activity. HPPD-4 protein characterisation and equivalence SDS–PAGE and western blot analysis showed that both plant- and microbe-produced HPPD-4 proteins had the expected molecular weight of ~ 40.3 kDa and were comparably immunoreactive to HPPD-4 protein specific antibodies. Glycosylation detection analysis demonstrated that none of the HPPD-4 proteins were glycosylated. Amino acid sequence and intact mass analysis of the plant-derived HPPD-4 protein by MS methods showed that the protein matched the deduced sequence and molecular weight as defined by the hppd-4 gene. These sequence analysis data were consistent with the previously analysed microbe-produced HPPD-4. In addition, the MS data showed that the N-terminal methionine was truncated from both HPPD-4 proteins and the four C-terminal amino acids of the plant-produced HPPD-4 protein were also truncated. These data also indicated the presence of another two variants of the plant-produced HPPD-4; one with an additional N-terminal cysteinic sulfinic acid and the third variant with an N-terminal cysteinic sulfinic acid and the four C-terminal amino acids truncated. Such modifications are common in eukaryotic proteins (e.g. Polevoda and Sherman, 2000). N-terminal sequence analysis by Edman degradation of the plant- and microbe-derived HPPD-4 proteins produced data consistent with those by MS. Functional equivalence was demonstrated by an in vitro assay which showed that plant- and microbe-derived HPPD-4 proteins had comparable enzymatic activity. Microbially produced HPPD-4 protein was also screened for its ability to utilise certain endogenous plant substrates. A number of compounds that could be substrates of this enzyme and potentially present in plants in addition to the intended substrate were tested. Although some catalysis was observed at a slow rate and with high protein amount for 3,4-dHPP, none of the compounds is likely to be a genuine in vivo substrate. The data demonstrated that it is unlikely that HPPD-4 has a metabolic impact within soybean GMB151 different from that of the native (endogenous) enzyme. The protein characterisation data comparing the biochemical, structural and functional properties of plant- and microbe-produced Cry14Ab-1 and HPPD-4 proteins, indicate that these proteins are equivalent, and the microbial derived proteins can be used in the safety studies. 3.2.4 Information on the expression of the insert Protein levels of Cry14Ab-1 and HPPD-4 were analysed by an enzyme-linked immunosorbent assay (ELISA) in material harvested in a field trial across three locations in the USA during the 2016 growing season. Samples analysed included leaves (BBCH 13-14, BBCH 16-18, BBCH 60-66, BBCH 76-79), root (BBCH 13-14, BBCH 16-18, BBCH 60-66, BBCH 76-79), flower (BBCH 60-66), forage (BBCH 76-79), whole plant (BBCH 89-99) and seed (BBCH 89-99) from plants treated and not treated with the intended herbicide. The mean values and standard deviations of protein expression levels in seeds (n = 12), forage (n = 12) and flowers (n = 12) of the Cry14Ab-1 and HPPD-4 proteins used to estimate human and animal dietary exposure (see Section 3.4.5) are reported in Table 1. Table 1. Mean values (n = 12) and standard deviation of newly expressed protein in seeds [μg/g dry weight (dw) and μg/g fresh weight (fw)] flowers and forage (μg/g dw) from soybean GMB151 Tissue Isoxaflutole 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 (BBCH 89-99) Cry14Ab-1 101.82a ± 45.77b (45.78–176.46)c 90.23 ± 41.04 (40.82–157.00) 88.25 ± 40.01 (16.10–153.73) 78.49 ± 35.94 (14.33–137.81) HPPD-4 4.50 ± 2.93 (1.25–9.92) 3.99 ± 2.62 (1.12–8.83) 4.50 ± 3.60 (1.33–12.84) 4.01 ± 3.23 (1.19–11.51) Flowersc (BBCH 60-66) Cry14Ab-1 53.37 ± 11.47 (37.06–71.59) 51.81 ± 18.42 (33.13–77.77) HPPD-4 57.08 ± 14.43 (43.06–79.62) 74.52 ± 27.82 (41.63–127.86) Forage (BBCH 76-79) Cry14Ab-1 55.56 ± 10.01 (39.95–69.31) 52.73 ± 10.15 (41.40–72.40) HPPD-4 120.18 ± 42.47 (78.96–203.83) 129.03 ± 45.32 (87.61–196.27) a Mean value. b :Standard deviation. c :Range.
Referência(s)