Assessment of genetically modified oilseed rape 73496 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2012-109)

SCIENTIFIC OPINION ADOPTED: 5 May 2021 doi: 10.2903/j.efsa.2021.6610 Assessment of genetically modified oilseed rape 73496 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2012-109) EFSA Panel on Genetically Modified Organisms (GMO), 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 Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann, Fabio Veronesi, Michele Ardizzone, Yann Devos, Silvia Federici, Antonio Fernandez Dumont, Andrea Gennaro, Jose  Angel G omez Ruiz, Franco Maria Neri, Nikoletta Papadopoulou, Konstantinos Paraskevopoulos and Anna Lanzoni Abstract Oilseed rape 73496 was developed to confer tolerance to the herbicidal active substance glyphosate through the expression of the glyphosate acetyltransferase protein GAT4621. The molecular characterisation data and bioinformatic analyses identify no issues requiring food/feed safety assessment. None of the identified differences between oilseed rape 73496 and its conventional counterpart in the agronomic/phenotypic endpoints tested needs further assessment. Differences identified in seed composition of oilseed rape 73496 as compared to its conventional counterpart raise no safety and nutritional concerns in the context of the scope of this application. No safety concerns are identified regarding toxicity and allergenicity of the GAT4621 protein as expressed in oilseed rape 73496. No evidence is found that the genetic modification would change the overall allergenicity of oilseed rape 73496. Based on the outcome of the comparative and nutritional assessments, the consumption of oilseed rape 73496 does not represent any nutritional concern, in the context of the scope of this application. The implementation of a post-market monitoring plan is recommended to confirm the predicted consumption data and to verify that the conditions of use are those considered during the pre-market risk assessment. In the case of accidental release of viable oilseed rape 73496 seeds into the environment, oilseed rape 73496 would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of oilseed rape 73496. The GMO Panel concludes that oilseed rape 73496, as described in this application, is as safe as its conventional counterpart and the non-genetically modified oilseed rape reference varieties tested with respect to potential effects on human and animal health and the environment. ©2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: GMO, oilseed rape, 73496, Regulation (EC) No 1829/2003, GAT4621, N-acetyl amino acids, import and processing Requestor: Competent Authority of The Netherlands Question number: EFSA-Q-2012-00617 Correspondence: GMO_secretariat_appl[email protected] EFSA Journal 2021;19(6):6610www.efsa.europa.eu/efsajournal 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 Nogu e, Nils Rostoks, Jose Juan S anchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Declarations of interest: The declarations of interest of all scientific experts active in EFSA’s work are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch. Acknowledgements: The Panel wishes to thank the members of the Working Groups on Molecular Characterisation, Food and Feed Safety Assessment and Working Group On Comparative Analysis and Environmental Risk Assessment for the preparatory work on this scientific output and EFSA staff members Fernando  Alvarez, Irene Mu~ noz Guajardo, Sylvie Mestdagh and Sonia Hernandez for the support provided to this scientific output. Suggested citation: EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Bresson J-L, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J, Moreno FJ, Mullins E, Nogu e F, Rostoks N, S anchez Serrano JJ, Savoini G, Veromann E, Veronesi F, Ardizzone M, Devos Y, Federici S, Dumont AF, Gennaro A, G omez Ruiz J  A, Neri FM, Papadopoulou N, Paraskevopoulos K and Lanzoni A, 2021. Scientific Opinion on the assessment of genetically modified oilseed rape 73496 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2012-109). EFSA Journal 2021;19(6):6610, 57 pp. https://doi.org/10.2903/j.efsa.2021.6610 ISSN: 1831-4732 ©2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. The EFSA Journal is a publication of the European Food Safety Authority, a European agency funded by the European Union. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 2 EFSA Journal 2021;19(6):6610 Summary The scope of application EFSA-GMO-NL-2012-109 is for food and feed uses, import and processing of the genetically modified (GM) herbicide tolerant oilseed rape 73496 within the European Union (EU). In the present scientific opinion, the scientific Panel on Genetically Modified Organisms of the European Food Safety Authority (EFSA) (hereafter referred to as the ‘GMO Panel’) reports the outcome of its risk assessment of oilseed rape 73496 according to the scope as defined in application EFSAGMO-NL-2012-109. The GMO Panel conducted the assessment of oilseed rape 73496 in line with the principles described in Regulation (EC) No 1829/2003 and its applicable guidelines for the risk assessment of food and feed from GM plants, including their environmental risk assessment. The molecular characterisation data establish that oilseed rape 73496 contains a single insert consisting of one copy of the gat4621 expression cassette, expressing the GAT4621 protein conferring tolerance to the herbicidal active substance glyphosate. Upon transformation, a region of chromosome C02 was potentially inverted and a putative tpt gene interrupted. The relevance of the gene interruption and potential chromosomal inversion for the risk assessment of oilseed rape 73496 is addressed. Bioinformatic analyses of the sequences encoding the newly expressed protein 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 introduced trait is confirmed over several generations. The levels of the GAT4621 protein were obtained and reported adequately. The protein characterisation data of the plantand microbe-produced GAT4621 protein indicate that both proteins are equivalent and thus that the microbial-derived protein (two batches) can be used in safety studies. None of the identified differences between oilseed rape 73496 and its conventional counterpart in the agronomic/phenotypic endpoints tested needs further assessment. Among the differences identified in seed composition between oilseed rape 73496 and its conventional counterpart, the levels of N-acetylaspartate, N-acetylglutamate, N-acetylthreonine, free amino acid glycine, crude fibre, crude fat, acid detergent fibre, neutral detergent fibre, magnesium, pyridoxine, pantothenic acid and 4hydroxyglucobrassicin were further assessed and found to raise no safety and nutritional concerns in the context of the scope of this application. No safety concerns are identified regarding the toxicity and allergenicity of the GAT4621 protein as expressed in oilseed rape 73496. No evidence is found that the genetic modification would change the overall allergenicity of oilseed rape 73496. Based on the outcome of the comparative and nutritional assessments, the consumption of oilseed rape 73496 does not represent any nutritional concern, in the context of the scope of this application. The implementation of a post-market monitoring plan is recommended to confirm the predicted consumption data and to verify that the conditions of use are those considered during the pre-market risk assessment. Considering the introduced trait, the outcome of the agronomic and phenotypic analysis and the routes and levels of exposure, oilseed rape 73496 would not raise safety concerns in the case of accidental release of viable GM oilseed rape seeds into the environment. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of oilseed rape 73496. The GMO Panel concludes that oilseed rape 73496, as described in this application, is as safe as its conventional counterpart and the non-GM oilseed rape reference varieties tested with respect to potential effects on human and animal health and the environment. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 3 EFSA Journal 2021;19(6):6610 Table of contents Abstract................................................................................................................................................... 1 Summary................................................................................................................................................. 3 1. Introduction................................................................................................................................6 1.1. Background ................................................................................................................................6 1.2. Terms of Reference as provided by the requestor .......................................................................... 6 2. Data and methodologies .............................................................................................................. 6 2.1. Data........................................................................................................................................... 6 2.2. Methodologies............................................................................................................................. 7 3. Assessment................................................................................................................................. 7 3.1. Molecular characterisation............................................................................................................ 7 3.1.1. Transformation process and vector constructs ............................................................................... 7 3.1.2. Transgene constructs in the GM plant ........................................................................................... 7 3.1.3. Protein characterisation and equivalence....................................................................................... 8 3.1.4. Information on the expression of the insert................................................................................... 9 3.1.5. Inheritance and stability of inserted DNA ...................................................................................... 9 3.1.6. Conclusion on molecular characterisation ...................................................................................... 9 3.2. Comparative analysis ................................................................................................................... 9 3.2.1. Choice of comparator and production of material for the comparative assessment ........................... 9 3.2.1.1. Statistical analysis of field trials data............................................................................................. 10 3.2.2. Agronomic/phenotypic analysis..................................................................................................... 10 3.2.2.1. Agronomic/phenotypic characteristics tested under field conditions ................................................. 10 3.2.2.2. Agronomic/phenotypic characteristics tested under controlled conditions ......................................... 11 3.2.3. Compositional analysis................................................................................................................. 11 3.2.4. Conclusion on the comparative analysis ........................................................................................ 13 3.3. Food/feed safety assessment ....................................................................................................... 14 3.3.1. Effects of processing ................................................................................................................... 14 3.3.2. Stability of the newly expressed protein ........................................................................................ 16 3.3.3. Toxicology .................................................................................................................................. 17 3.3.3.1. Testing of the newly expressed protein ......................................................................................... 17 3.3.3.2. Assessment of altered levels of endogenous compounds –N-acetylated amino acids........................ 18 3.3.3.3. Assessment of altered levels of compounds other than NAAs.......................................................... 27 3.3.3.4. Testing of the whole genetically modified food/feed ....................................................................... 28 3.3.4. Allergenicity ................................................................................................................................30 3.3.4.1. Assessment of allergenicity of the newly expressed proteins ........................................................... 30 3.3.4.2. Assessment of allergenicity of the whole GM plant ......................................................................... 30 3.3.5. Nutritional assessment................................................................................................................. 30 3.3.5.1. Human nutrition.......................................................................................................................... 30 3.3.5.2. Animal Nutrition .......................................................................................................................... 31 3.3.6. Post-market monitoring of GM food/feed....................................................................................... 32 3.3.7. Conclusion on the food/feed safety assessment ............................................................................. 33 3.4. Environmental risk assessment and monitoring plan....................................................................... 33 3.4.1. Environmental risk assessment ..................................................................................................... 33 3.4.1.1. Persistence and invasiveness of the GM plant ................................................................................ 33 3.4.1.2. Potential for gene transfer ........................................................................................................... 34 3.4.1.3. Interactions of the GM plant with target organisms........................................................................ 35 3.4.1.4. Interactions of the GM plant with non-target organisms ................................................................. 35 3.4.1.5. Interactions with the abiotic environment and biogeochemical cycles .............................................. 35 3.4.2. Post-market environmental monitoring.......................................................................................... 35 3.4.3. Conclusion on the environmental risk assessment and monitoring plan............................................ 36 4. Conclusions................................................................................................................................. 36 5. Documentation as provided to EFSA ............................................................................................. 37 References............................................................................................................................................... 38 Abbreviations ........................................................................................................................................... 45 Appendix A –Statistically significant findings in toxicological studies............................................................. 46 Appendix B –Summary statistics of the baseline dietary intake of NAA (lg/kg bw per day) across European dietary surveys......................................................................................................................................... 48 Appendix C –Summary statistics of dietary intake of NAA (lg/kg bw per day) across European dietary surveys considering the presence of NAA in conventional foods, and the consumption of protein isolates and oilseed rape powder from oilseed rape 73496............................................................................................. 49 Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 4 EFSA Journal 2021;19(6):6610 Appendix D –Animal dietary exposure to N-acetyl amino acids via oilseed rape 73496 and derived feed ........ 50 Appendix E –NAA risk characterisation by the use of Chemical Specific Adjustment Factors (CSAF) with reference to exposures to N-acetylated amino acids found in oilseed rape 73496.......................................... 53 Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 5 EFSA Journal 2021;19(6):6610 1. Introduction The scope of application EFSA-GMO-NL-2012-109 is for food and feed uses, import and processing of the genetically modified (GM) herbicide tolerant oilseed rape 73496 within the European Union (EU). 1.1. Background On 24 May 2012, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands the application EFSA-GMO-NL-2012-109 for authorisation of herbicide tolerant oilseed rape 73496 (Unique Identifier DP-Ø73496-4), submitted by Pioneer Hi-Bred International (hereafter referred to as ‘the applicant’) within the framework of Regulation (EC) No 1829/2003 1 . Following receipt of application EFSA-GMO-NL-2012-109, EFSA informed EU Member States and the European Commission, and made the application available to them. Simultaneously, EFSA published the summary of the application. 2 EFSA checked the application for compliance with the relevant requirements of its guidance documents (see Section 2.2), and, when needed, asked the applicant to supplement the initial application. On 4 December 2012, 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 6 months to issue a scientific opinion on application EFSA-GMO-NL-2012-109. Such time limit was extended whenever EFSA and/or its GMO Panel requested supplementary information to the applicant. According to Regulation (EC) No 1829/2003, any supplementary information provided by the applicant during the risk assessment was made available to EU Member States and the European Commission (for further details, see the section ‘Documentation as provided to EFSA’). 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 3 . The EU Member States had 3 months to make their opinion known on application EFSAGMO-NL-2012-109 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 oilseed rape 73496 in the context of its scope as defined in application EFSA-GMO-NL-2012-109. 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 OpenEFSA 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; and 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. 4 2. Data and methodologies 2.1. Data The GMO Panel based its scientific risk assessment of oilseed rape 73496 on the valid application EFSA-GMO-NL-2012-109, additional information provided by the applicant during the risk assessment, relevant scientific comments submitted by EU Member States and relevant peer-reviewed scientific publications. 1 Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 18.10.2003, p. 1–23. 2 Available online: https://open.efsa.europa.eu/questions/EFSA-Q-2012-00617 3 Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. OJ L 106, 12.3.2001, p. 1–38. 4 https://open.efsa.europa.eu/questions/EFSA-Q-2012-00617 Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 6 EFSA Journal 2021;19(6):6610 2.2. Methodologies The GMO Panel carried out a scientific risk assessment of oilseed rape 73496 for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003. The GMO Panel took into account the appropriate principles described in its applicable guidelines (i.e. EFSA GMO Panel, 2010a,b,c, 2011a,b) and explanatory notes (i.e. EFSA, 2014, 2017a,b) for the risk assessment of food and feed from GM plants, including their environmental risk assessment. For the assessment of 90-day animal feeding study, the GMO Panel took into account the criteria reported 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 in accordance with the principles 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 and toxicological studies, respectively. 3. Assessment 3.1. Molecular characterisation 3.1.1. Transformation process and vector constructs 5 Oilseed rape 73496 was developed by biolistic transformation of microspores of oilseed rape (Brassica napus L.) line 1822B with a HindIII/NotI fragment named PHP28181A from plasmid PHP28181. The PHP28181A fragment contains the gat4621 (glyphosate acetyl transferase) expression cassette, containing the following genetic elements: the polyubiquitin (UBQ10) promoter of Arabidopsis thaliana; the gat4621 gene; and the 30terminator sequence of a gene encoding the proteinase inhibitor II (pinII terminator) of Solanum tuberosum. The gat4621 gene is a shuffled variant of three gat genes, isolated from Bacillus licheniformis strains 401, B6 and DS3 that has been codon-optimised for expression in plants. The vector backbone sequence contained elements necessary for the maintenance of the plasmid in bacteria. 3.1.2. Transgene constructs in the GM plant 6 Molecular characterisation of oilseed rape 73496 was performed by Southern analysis, polymerase chain reaction (PCR) and DNA sequence analysis, in order to determine copy number, size and organisation of the inserted sequences, and to confirm the absence of plasmid backbone sequences. The approach used was acceptable both in terms of coverage and sensitivity. Southern analyses indicated that oilseed rape 73496 contains a single insert, consisting of a single copy of the PHP28181A fragment used for transformation. The insert and copy number were confirmed by multiple restriction enzyme/probe combinations covering the insert and flanking regions. No signal was observed with probes corresponding to PHP28181 vector backbone sequences. The nucleotide sequence of the entire insert of oilseed rape 73496, together with 2003 nucleotides of the 50and 2038 nucleotides of the 30flanking regions, was determined. The insert of 2109 bp is identical to the fragment of PHP28181A, except for the deletion of the first three base pairs of the 50 end of the PHP28181A fragment. The possible interruption of known endogenous oilseed rape genes by the insertion in event 73496 was evaluated by bioinformatic analyses of the pre-insertion locus and of the genomic sequences flanking the insert. Sequence comparisons of the insert flanking sequences suggest an inversion of a region of chromosome C02. Indeed, the two flanking genomic border sequences were mapped to the reference genome sequence and located about 9.2 Mbp apart, and the 30flanking genomic border sequence in oilseed rape 73496 is on the opposite strand of chromosome C02 compared to the same sequence in the parental line. A SNP marker analysis showed no evidence 5 Part II Scientific Information/Section A.2.1. 6 Part II Scientific Information/Section A.2.2.2. Additional information 9/4/2013, 15/12/2015, 29/4/2016, 3/10/2016, 2/5/2017, 28/9/2018, 17/4/2019 and 9/7/2020. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 7 EFSA Journal 2021;19(6):6610 of deletion of this region during the transformation process. However, the insertion resulted in the disruption of a putative gene, named PGtpt (PredictedGenetpt) showing similarity to a triose phosphate transporter (tpt). Southern blot analyses indicated that there are four copies of the tpt gene in oilseed rape and qRT-PCR analysis revealed a lower overall transcript level of the tpt gene family in leaf from oilseed rape 73496, compared to the control plant. These data suggest that the PGtpt gene has been interrupted in 73496 oilseed rape affecting most probably the level of transcripts for this gene in leaves. Further considerations on the relevance of the gene interruption and potential chromosomal inversion for the risk assessment of oilseed rape 73496 are provided in Sections 3.2.2 and 3.2.3. The results of segregation (see Section 3.1.5) and bioinformatic analyses established that the insert is located in the nuclear genome. Updated bioinformatic analyses of the amino acid sequence of the newly expressed GAT4621 protein revealed no significant similarities to toxins and allergens. In addition, updated bioinformatic analyses of the newly created Open Reading Frames (ORFs) within the insert and spanning the junctions between the insert and genomic DNA did not indicate significant similarities to toxins and allergens. In order to assess the possibility for horizontal gene transfer by homologous recombination (HR), the applicant performed a sequence identity analysis of the inserted regions of bacterial origin in oilseed rape 73496. The likelihood and potential consequences of plant-to-bacteria gene transfer are described in Section 3.4.1.2. 3.1.3. Protein characterisation and equivalence 7 Oilseed rape 73496 expresses one new protein, GAT4621, which is a glyphosate acetyl transferase conferring tolerance to the herbicidal active substance glyphosate. Given the technical restrains in producing large enough quantities from plants, GAT4621 was recombinantly produced in Escherichia coli. A set of biochemical methods was employed to demonstrate the equivalence between oilseed rape and the two batches of E. coli-derived GAT4621 protein used in the different experiments presented in the dossier (see Section 3.3.3). The purified plant protein and E. coli-derived protein (two batches) were characterised and compared in terms of their physico-chemical, structural and functional properties. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis showed that both plantand microbe-produced GAT4621 proteins had the expected molecular weight of ~16.5 kDa and were comparably immunoreactive to GAT4621 protein-specific antibodies. Glycosylation detection analysis demonstrated that none of the GAT4621 proteins were glycosylated. Amino acid sequence analysis of the two GAT4621 proteins by mass spectrometry and N-terminal sequencing methods showed that they matched the deduced sequence as defined by the gat4621 gene. These data also showed that the N-terminal methionine of both the plantand microbialproduced GAT4621 proteins was truncated. Such modifications are common in eukaryotic proteins (e.g. Poledova and Sherman, 2000) and have been previously assessed by the GMO Panel for newly expressed proteins (EFSA GMO Panel, 2017). Due to the purified plant GAT4621 protein being inactive, the activity between the plant and E. coli-derived proteins could not be directly compared. The activity and substrate specificity of the two E. coli-produced GAT4621 was analysed by a biochemical in vitro activity assay. 8 The results from this assay confirmed the acetylation activity of the GAT4621 protein for the intended herbicide as well as for a number of amino acids. The activity of the plant-produced GAT4621 was indirectly demonstrated by the tolerance to the herbicidal active substance glyphosate and compositional analyses (see Section 3.2.3). Based on these data, the GMO Panel accepts the use of the GAT4621 protein produced in bacteria for the safety studies. 7 Dossier Part II Scientific Information/Section A.4.2. Additional information 18/12/2018. 8 The substrate specificity of the E. coli-produced GAT4621 protein has been tested on a range of 21 different agrochemicals, 21 amino acids and 10 antibiotics under in vitro conditions (Annex 22_PHI-2006-184/017). GAT4621 protein has been shown to acetylate certain amino acids, such are aspartate, glutamate and threonine. Increased concentration of the N-acetylated forms of these three amino acids (N-acetylaspartate, N-acetylglutamate and N-acetylthreonine) in oilseed rape 73496 is indicative of an equivalent activity of the plant-produced GAT4621 compared to the E. coli-produced GAT4621 protein. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 8 EFSA Journal 2021;19(6):6610 3.1.4. Information on the expression of the insert 9 Levels of the GAT4621 protein were analysed by enzyme-linked immunosorbent assay (ELISA) in material harvested in a field trial across four locations in the USA and five locations in Canada during 2010 growing season. Samples analysed included whole plants (BBCH15, BBCH33 and BBCH65), roots (BBCH65) and seeds (BBCH90), from plants treated and not treated with the intended herbicide. The mean values, standard deviations and ranges of protein expression levels in seeds for plants treated (n =32) and not treated with the intended herbicide (n =36) are summarised in Table 1. 3.1.5. Inheritance and stability of inserted DNA 10 Genetic stability of the oilseed rape 73496 insert was assessed by Southern analysis of genomic DNA from five generations (T2, T3, T3F2, T3F3, F1) and segregation analysis of the glyphosate tolerance trait of oilseed rape 73496 from five generations (T3F2, BC1F1, BC2F1, BC3F1, F1). 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. The results supported the presence of a single insertion, segregating in a Mendelian fashion. 3.1.6. Conclusion on molecular characterisation The molecular characterisation data establish that oilseed rape 73496 contains a single insert consisting of one copy of the gat4621 expression cassette. Upon transformation, a region of chromosome C02 was potentially inverted and a putative tpt gene interrupted. Further considerations on the relevance of the gene interruption and potential chromosomal inversion for the risk assessment of oilseed rape 73496 are provided in Sections 3.2.2 and 3.2.3. Bioinformatic analyses of the sequences encoding the newly expressed protein and other ORFs within the insert or spanning the junctions between the insert and genomic DNA indicate no significant similarities to toxins and allergens. The stability of the inserted DNA and of the introduced herbicide tolerance trait was confirmed over several generations. The levels of the GAT4621 protein were obtained and reported adequately. The protein characterisation data of the plantand microbial-derived GAT4621 proteins indicate that these proteins are equivalent and thus that the microbial-produced protein (two batches) can be used in the safety studies. 3.2. Comparative analysis 11 3.2.1. Choice of comparator and production of material for the comparative assessment Application EFSA-GMO-NL-2012-109 presents data on agronomic/phenotypic characteristics, as well as seed composition, of oilseed rape 73496 derived from field trials performed in the USA and Canada during the 2010 growing season (Table 2). In addition, seed characteristics of oilseed rape 73496 were evaluated under laboratory (growth chamber) conditions. Oilseed rape 73496 was obtained through the transformation of the double haploid male sterile maintainer line 1822B. The obtained GM oilseed rape, after restoration with line 1822R, was crossed Table 1: Mean values, standard deviations and ranges of the GAT4621 protein in seeds [ng/mg dry weight (dw)] from oilseed rape 73496 Tissue Intended herbicide treatment Not treated Treated Seed (BBCH90) GAT4621 5.6 (a) 1.1 (b) (3.6–8.1) (c) 5.6 1.1 (4.2–8.7) (a): Mean. (b): Standard deviation. (c): Range. 9 Dossier Part II Scientific Information/Section A.2.2.3. 10 Dossier: Part II Scientific information/Section A.2.2.4. 11 Dossier: Part II Scientific information/Section A.3. Additional information: 17/9/2013. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 9 EFSA Journal 2021;19(6):6610 Whey fractions 23 Whey fractions resulting from the production of protein isolates from conventional herbicide-treated oilseed rape 73496 22 were analysed by UPLC-MS/MS for the presence of NAA, NAG, NAGly, NAS and NAT. Three whey fractions are produced during the process, one from the protein precipitation step and the other two from washing the protein isolate pellet. N-acetylated amino acids are predominantly found in whey fraction 1 (see Table 7). 3.3.2. Stability of the newly expressed protein 23 Protein stability is one of several relevant parameters to consider in the weight-of-evidence approach in protein safety (EFSA GMO Panel, 2010c, EFSA GMO Panel, 2011a, EFSA GMO Panel, 2017, EFSA GMO Panel, 2021). The term protein stability encompasses several properties such as thermal stability, pH-dependent stability, proteolytic stability and physical stability (e.g. tendency to aggregate), among others (Li et al., 2019). It has been shown, e.g. that when characteristics of known food allergens are examined, one of the most prominent traits attributed to food allergens is protein stability (Helm, 2001; Breiteneder and Mills, 2005; Costa et al., 2021). Effects of temperature and pH on the newly expressed protein The effects of temperature and pH on the GAT4621 protein have been previously evaluated by the GMO Panel (EFSA GMO Panel, 2013). The GAT4621 lost most of its activity at temperatures greater than 53°C. In vitro protein degradation by proteolytic enzymes The resistance to degradation by pepsin of a microbial GAT4621 protein in solutions at pH ~1.2 has been previously assessed by the GMO Panel (EFSA GMO Panel, 2013). As described, the GAT4621 protein was degraded within the first 30 seconds of incubation, while less intensely staining bands corresponding to low-molecular weight fragments (≤3 kDa) were still visible throughout the incubation period. (b): One sample above the LOQ (0.0125 lg/g fw) and three samples below the LOQ; for samples below LOQ half the value of the LOQ value was used to calculate the mean. (c): LOQ =0.0250 lg/g fw. (d): LOQ =0.0125 lg/g fw. Table 7: Mean levels of N-acetylated amino acids (four technical replicates of one sample) in herbicide-treated oilseed rape 7496 seeds, in protein isolates and in the whey fractions obtained during the production of the protein isolates 23 Oilseed rape 73496 seeds Protein isolate Whey fraction 1 Whey fraction 2 Whey fraction 3 (lg/g) (lg/mL) NAA 1,323 13.01 140.8 20.40 2.919 NAG 49.57 0.4329 5.492 0.7435 0.1061 NAT 1.436 0.006753 (a) 0.1301 0.01940 0.002391 NAGly 0.1533 <LOQ (b) 0.01407 0.001529 <LOQ (c) NAS 1.961 0.007375 (a) 0.1984 0.02584 0.002747 NAA: N-acetylaspartate; NAG: N-acetylglutamate; NAT: N-acetylthreonine; NAGly: N-acetylglycine; NAT: N-acetylserine. (a): One sample above the LOQ (0.0125 lg/g fw) and three samples below the LOQ; for samples below LOQ half the value of the LOQ value was used to calculate the mean. (b): LOQ =0.0125 lg/g fw. (c): LOQ =0.001250 lg/g fw. 23 Dossier Part II Scientific information/Sections 4.2 and 5.1. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 16 EFSA Journal 2021;19(6):6610 3.3.3. Toxicology 3.3.3.1. Testing of the newly expressed protein 24 Oilseed rape 73496 expresses the new protein GAT4621, a glyphosate acetyltransferase conferring tolerance to the herbicidal active substance glyphosate. The GMO Panel has previously assessed this protein (EFSA GMO Panel, 2013), but was unable to conclude on its safety due to the lack of an adequate 28-day toxicity study. In the context of this application, the GMO Panel assessed the safety of the GAT4621 protein considering bioinformatic analyses (Section 3.1.2), protein characterisation (Section 3.1.3), in vitro studies (Section 3.3.2) and a new 28-day toxicity study spontaneously provided by the applicant. Bioinformatics Bioinformatic analysis of the amino acid sequence of the GAT4621 protein revealed no significant similarities to known toxins (Section 3.1.2). 28-day repeated dose toxicity study in the rat The new 28-day repeated-dose toxicity study provided in the application was conducted in accordance with OECD TG 407 (2008) and the principles of Good Laboratory Practice (GLP). Five groups of Crl:CD1(ICR) mice (10 per sex per group, individually housed, approximately 9-week old at study start dosing) were given 1) a standard diet (control group); 2) a diet containing the GAT4621 protein at the target dose of 100, 300 or 1,000 mg/kg body weight (bw) per day (low, intermediate and high dose test diet groups); and 3) a diet containing bovine serum albumin (BSA) protein at the target dose of 1,000 mg/kg bw per day (BSA control group). These groups are hereafter defined as main study groups. Ten additional animals/sex per group were dedicated for coagulation analysis and are mentioned hereafter as satellite study groups. The test and BSA diets were prepared by mixing to a standard rodent diet the test substance at 0.5, 1.5 or 5 g/kg diet, or the BSA at 5 g/kg diet. The GMO Panel noted that at the start of dosing, the age of the animals and the variation in body weights among animals were slightly outside the OECD TG 407 (2008) requirements. These were considered minor deviations with no impact on the study results. The GAT4621 protein used in this study was produced by a recombinant system (E. coli, lot PCF0041) equivalent to the protein newly expressed in oilseed rape 7496 (Section 3.1.3) since it was demonstrated to have the expected molecular weight and N-terminal sequence and a 98% coverage of the expected protein sequence at MALDI–MS. 25 The test substance used in this study contained 0.82 mg GAT4621/mg lyophilised powder. The test substance was stored frozen (–80°C) and considered stable for long storage. Levels of the GAT4621 or BSA protein were measured by ELISA in the diets at the time of mixing (Day 0) to assess their concentration and homogeneity; on Day 1, 6, 28 and 46 (high dose test diet only) to evaluate their stability. During the treatment period, all animals were given approximately 14 g/day (7 g twice a day/mouse) of control or test diets. Water was provided ad libitum. In-life procedures and observations and terminal procedures were conducted in accordance with OECD TG 407 (2008). Ophthalmoscopy examinations, functional observational batteries (FOBs) and motor activities were recorded on main study groups only. Haematological and clinical chemistry analyses were performed on main study groups, while coagulation analysis was performed on satellite groups. Detailed necropsy examination, organ weight and histopathological examination (controls, BSA controls and high dose group) were conducted on main study groups only. The results of the diet analyses revealed that the test diets met the expected GAT4621 concentrations at the time of diet formulation, 26 that these were homogenous and that all test diets were stable up to 28 days in terms of GAT4621 content. In-life data endpoints, with the exception of total and ambulatory motor activity counts, were analysed by sex, using a two-sided analysis of variance (ANOVA) model; in case a statistically significant does effect was identified, pairwise comparisons (Dunnett’s test) were done between the test groups and the control group and the BSA group, and between the BSA group and the control 24 Dossier Part II Scientific information/Section A. 4.2. Spontaneous information 29/8/2017 and additional information 20/6/ 2018, 18/12/2018. 25 17 kDa by Western blot and 16.5 kDa by MALDI-MS. 26 An anomalous value for concentration was noted on Day 28 in the high-dose diet but not confirmed on Day 46, therefore considered not relevant. Achieved concentrations for all the three diets were 84% of the nominal value. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 17 EFSA Journal 2021;19(6):6610 group. Total and ambulatory motor activity counts were recorded prior to the initiation of the study and near the end of the feeding period for control, BSA and test diet groups and were analysed by sex and session, using a repeated measures ANOVA model (factors: treatment, time interval and time-bytreatment interaction), followed by pairwise group comparisons across the pooled time intervals if the main effect of treatment was significant; if the time-by-treatment interaction was significant, the pairwise comparisons were also conducted for each individual time interval. Categorical FOBs data were analysed using Fisher’s test. Based on feed consumption, the average GAT4621 consumption was 59.4, 183.6 and 595.5 mg/kg/ bw per day in males and 75.1, 214.5 and 740.1 mg/kg/bw per day in females (low-, intermediateand high-dose groups, respectively). 27 Animals from the BSA control group consumed 716.4 (males) and 832.7 (females) mg BSA/kg bw per day (based on nominal value). There were no deaths. Isolated clinical findings observed in females from the BSA and low dose test group were considered incidental. The GMO Panel assessed the statistically significant findings observed in the treated groups and concluded that these are not adverse effects of the treatment with GAT4621 protein (see Appendix A, Table A.1). No gross pathological findings related to the treatment with GAT4621 protein were seen at necropsy. At microscopic examinations of selected organs and tissues, an increased incidence of mononuclear cell infiltrate was noted in the kidneys of males given the high dose test diet, as compared to controls. This finding was described as minimal; it is compatible with background microscopic findings in mice of this strain and age and considered not an adverse effect related to treatment with GAT4621 protein. No other relevant differences in the incidences and severity of the histopathological findings were noted between high dose test dose group and those given the control diets. The GMO Panel concluded that no adverse effects related to the treatment were observed in mice exposed by diet to 595.5 (males) and 740.1 (females) mg GAT4621/kg bw per day for 28 days. 3.3.3.2. Assessment of altered levels of endogenous compounds –N-acetylated amino acids 28 The GMO Panel assessed the altered levels of N-acetylated amino acids (NAA, NAG and NAT) observed in oilseed rape 73496 as compared to its conventional counterpart (Section 3.2.4) with regard to their relevance for food and feed safety taking into account available toxicological studies, other relevant information on the biological role and metabolism of N-acetylated amino acids and dietary exposure assessment. Toxicological studies on N-acetyl amino acids Rodent studies The applicant provided toxicological studies on NAA, NAG and NAT, which were already assessed by the GMO Panel in the context of previous applications (EFSA GMO Panel, 2011c, 2013). A summary of these studies and the outcome of the GMO Panel assessment are presented in Table 8. Although the applicant set the no observed adverse effect level (NOAEL) for NAA at 500 mg/kg bw per day (based on the 90-day repeated dose toxicity and in the two-generation reproductive toxicity dietary studies in rats), the GMO Panel had previously concluded that it is appropriate to use the intermediate dose level as the reference value for risk assessment considerations (see Table 8and EFSA GMO Panel, 2011c, 2013). In particular, the most conservative NOAEL was chosen for risk characterisation (229.5 mg/kg per bw per day from 90-day repeated dose toxicity dietary study in male rats, see Table 8). 27 Dietary exposures include a correction for the purity of the test item (84%). 28 Dossier Part II Scientific information/Section A. 4.4. Additional information 28/4/2016, 29/4/2016, 28/8/2017 and 29/8/2017. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 18 EFSA Journal 2021;19(6):6610 42-day feeding study in broiler on NAA 29 To further support the safety evaluation of dietary oilseed rape 73496 in chickens, the applicant provided a 42-day feeding study on chicken for fattening with graded supplementation levels of pure NAA in the standard diets, to investigate growth performance and toxicological endpoints. Necropsy with major organ weights, haematology and routine clinical blood chemistry, histopathology of the salivary glands 30 and measurement of the content of NAA and aspartic acid in liver and muscle tissues were assessed. A total of 240 male chickens for fattening (day-old Ross 708) were randomly allocated to five dietary treatment groups with 48 chicks per treatment (four pens per treatment, 12 birds per pen) and fed standard diets 31 alone (carrier control group) or supplemented with NAA at three different levels (test groups), or with L-aspartic acid (Asp comparative control group) at 100 mg/kg bw per day (Asp comparative control group). Test diets were formulated, manufactured and characterised to provide target exposure to NAA of 25, 50 and 100 mg/kg bw per day, corresponding to an approximate supplemental 10%, 20% and 40% incorporation of oilseed rape 73496 meal in diets. Diets and water were offered ad libitum. Table 8: Toxicological studies on N-acetylated amino acids provided by the applicant and outcome of the previous assessment by the GMO Panel Compound Study Target dose Outcome of previous GMO Panel assessments NAA Rat acute toxicity 2,000; 5,000 (a) No adverse effects at 2,000 (a) ; toxicity at 5,000 (a) Rat 28-day repeated dose toxicity (dietary) 10/100, 100/500, 1,000 (b) NOAEL (b) : 852.3 (males); 890.1 (females) Rat 90-day repeated dose toxicity (dietary) 100, 250, 500 (b) NOAEL (b),(c) : 229.5 (males); 253.2 (females) Rat two-generation reproductive toxicity (dietary) 100, 250, 500 (b) NOAEL (b),(c) : 245.7(males F1), 269.1(females F1); 237.2 (males F2), 500 (females F2) Bacterial reverse mutation test (Ames test) 333, 667, 1,000, 3,333, 5,000 (d) Negative Mouse Bone Marrow Erythrocyte Micronucleus Test 500, 1,000, 2,000 (a) Negative NAG Rat acute toxicity 2,000 (a) No adverse effects at 2,000 (a) Rat 28-day repeated dose toxicity dietary 100, 500, 1,000 (b) NOAEL (b) : 914.2 (males); 1,006.6 (females) Bacterial reverse mutation test (Ames test) 333, 667, 1,000, 3,333, 5,000 (d) Negative Mouse Bone Marrow Erythrocyte Micronucleus Test 333, 1,000, 2,000 (a) Negative NAT Rat acute toxicity 2,000 (a) No adverse effects at 2,000 (a) Rat 28-day repeated dose toxicity dietary 100, 500, 1,000 (b) NOAEL (b) : 848.5 (males); 913.6 (females) Bacterial reverse mutation test (Ames test) 333, 667, 1,000, 3,333, 5,000 (d) Negative Mouse Bone Marrow Erythrocyte Micronucleus Test 500, 1,000, 2,000 (a) Negative (a): mg/kg body weight. (b): mg/kg body weight per day. (c): The GMO Panel has previously concluded that it is appropriate to use the intermediate dose as the reference value for risk assessment consideration (EFSA GMO Panel, 2011a–c, 2013). (d): lg/plate. 29 Additional information 29/4/2016; 29/8/2017. 30 Acinar cells hypertrophy of the salivary glands in both male and female rats orally exposed to 500 mg NAA/kg bw was reported in a 90-day rat study (see Table 8). 31 Starter (0–21 days), grower (22–35 days) and finisher (36–42 days) diets formulated to meet the nutrient requirements of a commercial broiler in accordance with the National Research Council’s Nutrient requirements for Poultry (NRC, 1994). Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 19 EFSA Journal 2021;19(6):6610 Statistical analysis on mortality, performance and toxicology endpoints was conducted. Differences were considered significant at a p-value of ≤0.05 and adjustment (FDR) was made across all endpoints within each pairwise comparison between diet groups. For mortality data, Fisher’s exact test was conducted. For all continuous endpoints, if <50% of non-missing data values were at a uniform value, a mixed model analysis was applied. For endpoints that were measured on a per pen basis, statistical modelling was conducted on a response variable at the pen level. For endpoints that were measured on an individual broiler basis, statistical modelling was conducted on a response variable at the individual broiler level. For mortality data, Fisher’s exact test was conducted. The target exposures to NAA were met, and even exceeded, during the entire phases of the study. The mean actual exposures were at least 28.4, 62.5 and 121.6 mg/kg bw per day, respectively, in the low, mean and high-dose test groups for the entire duration of the study, corresponding to an approximate supplemental 11%, 25%, 48% incorporation of 73496 oilseed rape meal in diets. Overall mortality was low (4%) with no significant difference between the groups, and no adverse clinical signs were reported throughout the study. No statistically significant difference was seen in body weights and body weight gains, feed consumption and feed conversion, absolute and relative kidney and liver weights (pre-chilled), dressed carcass weights (post-chilled), breast, thigh, wing, leg and abdominal fat weights, when the NAA test groups and Asp-positive control group were compared to the negative control group. Moreover, there were no statistically significant differences in absolute and relative selected organ weights, or test substance-related effects on haematology, coagulation or clinical chemistry examined parameters when the NAA test groups, and Asp-positive control group were compared to the negative control group. The microscopic examination of salivary glands showed the absence or attenuation of secretory units of one or more lobules in all groups, with a slightly higher incidence and severity in the lingual and sublingual glands of animals fed diets with NAA at the highest dose (test group). The lack of association of these findings with NAA consumption was confirmed by the outcome of an expert scientific opinion provided by the applicant, 32 based on the comparison of the histology of salivary glands of broilers from the present study with that of strain, age and gender-matched control broilers from other studies, fed either similar diets or standard commercial poultry diets (using a series of parasagittal step sections of the lingual salivary glands); all animals were sourced from the same breeder and housed under similar environmental conditions at the same test facility. Variability with respect to the severity score of ‘decreased glands’was observed among animals within the same treatment groups, and there were no differences in nuclear cytology or evidence of significant cellular pathology or other morphologic changes (e.g. inflammation, degeneration, necrosis) in any of the test animals that would suggest an adverse effect. Furthermore, the composition and cellularity of the salivary glands were highly dependent on the plane of section. Therefore, although subtle quantitative histological intergroup differences were observed in the salivary tissue of broiler chickens, they were considered a consequence of the normal variability of salivary gland histology and the variation in the plane of section of a dispersed gland, and no pathological alterations were observed to indicate a treatment-related effect of NAA on the salivary glands of broiler chickens under the conditions of this study. There were no statistically significant differences in NAA or Asp concentrations in the breast or liver tissues of fasted or non-fasted broilers when the NAA test groups or the Asp comparative control group were compared to the carrier control group. Based on the results of this study, the EFSA GMO Panel concludes that administration of diets containing NAA for 42 consecutive days at an average overall dose level of 121.6 mg/kg bw per day (highest dose evaluated) to broilers, did not cause adverse effects on mortality, growth performance or clinical and anatomic pathology variables and no effects on NAA and Asp tissue concentrations of male Ross 708 broiler chickens. The mean actual dose of 121.6 NAA mg/kg bw per day corresponds to an approximate supplemental 48% incorporation of 73496 oilseed rape meal in diets that exceed the standard incorporation of conventional oilseed rape into commercial animal’s diets. Therefore, the GMO Panel considers the use of oilseed rape 73496 as feed material safe for the broiler and that the incorporation of this GM oilseed rape into animal’s diet has no limitations other than those of conventional oilseed rape. 32 Additional information 29/8/2017. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 20 EFSA Journal 2021;19(6):6610 Other information on N-acetylated amino acids. 33 NAA and NAG are produced by the mammalian metabolism and are normal constituents of many foods and feedstuff (Hession et al., 2008), with NAA being detected in human bio-fluids as reported in the Human Metabolome (https://hmdb.ca/) and Chemical Entities of Biological Interest (https:// www.ebi.ac.uk/chebi/) databases. N-acetylation and de-acetylation of cellular proteins are widespread processes with a regulatory function in metabolism (Perrier et al., 2005; Smith and Denu, 2007; Hwang et al., 2010). A number of both specific and unspecific N-acetyltransferases acetylate free amino acids, amines and drugs. The synthesis of NAA is known to occur in brain neurons and has important roles in the function of the central nervous system, following release into the extracellular fluid and uptake by glial cells, where it is hydrolysed to aspartic and acetic acid (Baslow, 2003; Baslow, 2010). Hydrolase enzymes from the aminoacylase family catalyse the hydrolysis of acylated L-amino acids to their constituent L-amino acids and an acyl group. Aminoacylases genes (ACY1 and ASPA) are nearly ubiquitously present in organs and tissues, with sequences moderately to highly conserved across animal species (Yates et al., 2020). Acylase I and acylase II enzymes catalyse the stereospecific hydrolysis of N-acetylated amino acids, including NAA, and are identified in multiple tissues from various species including intestine, liver and kidneys, presumably mediating the catabolism of ingested N-acetylated amino acids under normal dietary conditions (Birnbaum et al., 1952; Birnbaum, 1955; Nadler and Cooper, 1972; D’Adamo et al., 1973; Endo, 1978; Endo, 1980; Daabees et al., 1984; Giardina et al., 1997; Giardina et al., 1999; Lindner et al., 2000; Arnaud et al., 2004; Hershfield et al., 2006; Surendran et al., 2006; Mersmann et al., 2011; Luna et al., 2013). Deacetylation of NAA was shown to be rapid in studies in mice using radiolabelled NAA and L-aspartic acid; after intraperitoneal injection, both substances were metabolised at a similar rate (as determined by measurement of expired radioactive CO2) indicating a rapid hydrolysis of the N-acetyl group (Berlinguet and Lalibert e, 1966). Studies in premature infants, rats, dogs and pigs with enterally or parenterally administered N-acetylated amino acids (cysteine, tryptophan, tyrosine, methionine, threonine, glutamine) have shown that the nutritional value of the N-acetylated amino acids was comparable to that of free amino acids, also suggesting an efficient de-acetylation (Boggs, 1978; Neuh€ auser-Berthold et al., 1988; Gouttebel et al., 1992; van Goudoever et al., 1994; Arnaud et al., 2004; L opez-Pedrosa et al., 2007). Dietary exposure assessment to N-acetyl amino acids Human dietary exposure to N-acetyl amino acids Humans are habitually exposed to N-acetyl amino acids since they are natural constituents of different foods; the presence of NAA and NAG has been described and quantified in a broad range of foods including meat, fish, eggs, brewed coffee, vegetables and fruits (Hession et al., 2008). Likewise, other N-acetyl amino acids, among them NAS and NAT, are described as frequent components of dietary proteins although concentration levels have not been reported (Persson et al., 1985; Van de Mortel et al., 2010a; Van de Mortel et al., 2010b). Table 9shows a selection of foods with quantified levels of NAA and NAG; it can be seen that in several cases, the levels are similar or higher than those measured in the protein isolates from oilseed rape 73496. Table 9: Selection of different foods with reported levels of NAA and NAG (a),(b) (complete food list in Hession et al., 2008), and levels of NAA and NAG as reported in seeds and protein isolates from oilseed rape 73496 NAA (lg/g) NAG (lg/g) Soybean 0.3–0.7 (c) 0.7–1.2 Stout beer 0.14 0.21 Brewed coffee 3.8 0.3 Brewed espresso coffee 15.4 1.8 Cocoa powder 26.8 62.2 Dark chocolate 4.5 10.2 Broccoli 0.09 0.8 33 Dossier Part II Scientific information/Section A4.4. Additional information 10/9/2020 and 10/11/2020. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 21 EFSA Journal 2021;19(6):6610 Dietary intake of NAG and NAT Even though the levels of NAG and NAT in oilseed rape 73496 were significantly higher than those present in the conventional counterpart, no dietary intake estimations were considered needed for these N-acetyl amino acids. This decision was based on 1) the relatively low levels of these N-acetyl amino acids as compared to those of NAA, 2) their very low levels or absence in oilseed processed commodities, e.g. protein isolates and RBD oil, 3) their presence in conventional foods, in particular NAG, at similar or higher levels than those present in the seeds, 4) toxicological information (see Table 8). Dietary intake of NAA Based on the levels of NAA described for the different conventional foods (see Table 9), the baseline intake of NAA was estimated across different ages classes in the European population using individual consumption data from the EFSA Comprehensive European Food Consumption database (EFSA consumption database). 34 In the young population (including adolescents) and in the adult population, the maximum dietary intake estimates (95th percentile) were 47.3 and 99.0 lg/kg bw per day, respectively (see Appendix B). As today, oil is almost the only food commodity derived from oilseed rape regularly consumed by the European population, 35 although other food commodities derived from oilseed rape have been approved as novel food in recent years, e.g. protein isolates (EFSA NDA Panel, 2013) and rapeseed powder (EFSA NDA Panel, 2020). Different preparations of protein isolates from oilseed rape meal are found under commercial names (Puratein ® , Supertein TM , etc.). However, protein isolates from oilseed rape are literally absent in the European market as verified in Mintel’s Global New Products Database. 36 This was further confirmed by the absence of consumption data for protein isolates/meat imitates from oilseed rape in the EFSA consumption database. The protein meal resulting from oil extraction is currently almost exclusively used as animal feed since the food industry is still often encountering diverse challenges, e.g. undesirable flavour/colour, functional properties of the proteins, etc., when using the protein fraction (Wanasundara et al., 2016; Chmielewska et al., 2020; Fetzer et al., 2020). Different dietary intake scenarios for NAA were conducted taking into account the food commodities from oilseed rape available today in the market: RBD oil, protein isolates and oilseed rape powder: 1) N-acetyl amino acids are not present in RBD oil from oilseed rape 73496 (Section 3.3.1); therefore, consumption of RBD oil from the GM oilseed rape is not expected to contribute to the dietary intake of N-acetyl amino acids. 2) A conservative intake scenario was conducted assuming that protein isolates from oilseed rape 73496 could be used as protein supplements by the adult population. Using the highest concentration of NAA reported in protein isolates (18 lg/g, see Section 3.3.1.) and a daily consumption of 30 grams of protein isolate, this would result in an additional intake NAA (lg/g) NAG (lg/g) Spinach 0.04 1.8 Whole egg 1.5 0.05 Ground chicken 4.7 0.07 Ground turkey 7.4 0.09 Canned sardines 10.2 0.2 Seeds from oilseed rape 73496 (b) 1,670 (c) 28.9 Protein isolate from oilseed rape 73496 13–18 0.43–0.61 NAA: N-acetyl aspartate; NAG: N-acetylglutamate. (a): Levels of NAA and NAG are the result of two determinations. (b): Seeds treated with the intended herbicide. (c): Result expressed in dry weight. 34 Accessed on January 2021. Consumption data from the UK were included in the EFSA Comprehensive European Food Consumption Database when the UK was a member of the European Union. 35 From double low cultivars, i.e. varieties with a low content of erucic acid (<2% expressed as percentage of total fatty acids) and reduced content of glucosinolates (<25 mmol/kg at a moisture content of 9%) under Regulation (EC) No 2316/1999. 36 The Mintel’s GNPD is an online database which monitors new introductions of packaged goods in the market worldwide. It contains information of over 2.5 million food and beverage products of which more than 1,000,000 are or have been available on the European food market. Mintel started covering EU’s food markets in 1996, currently having 25 out of its 28 member countries and Norway presented in the Mintel GNPD. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 22 EFSA Journal 2021;19(6):6610 of ~ 8 lg/kg bw per day, considering a default body weight of 70 kg in adults (EFSA Scientific Committee, 2012). This additional intake based on the consumption of protein isolates from oilseed rape 73496 represents less than 10% of the maximum baseline dietary intake of NAA observed in adults. 3) No data were available on the presence of NAA in the recently approved novel food oilseed rape powder. In a worst-case scenario (overly conservative), it was assumed that oilseed rape powder might have a similar concentration of NAA to that in the seeds (~ 1,500 lg/g), i.e. no losses of NAA occur during the production of the oilseed rape powder. A dietary intake scenario was conducted considering the described proposed uses of oilseed rape powder in food products (EFSA NDA Panel, 2020), the concentrations of NAA analysed in the conventional foods (see Table 9) and the consumption of protein isolates as protein supplements. The EFSA consumption database was used as a source of individual consumption data. The maximum dietary intake estimates of NAA (95th percentile) were 992.9 and 444.8 lg/kg bw per day in the young population (including adolescents) and in the adult population, respectively (see Appendix C). When using this dietary intake scenario in the risk assessment of NAA, one should take into account the overly conservative nature of the intake estimations. This mainly refers to the assumption that all NAA present in the seeds will also be present in the oilseed rape powder, with no losses during a production process that includes different washing and extraction steps (EFSA NDA Panel, 2020). Animal dietary exposure to N-acetyl amino acids Dietary exposure to N-Acetylaspartate (NAA), N-Acetylglutamate (NAG) and N-Acetylthreonine (NAT) in oilseed rape 73496 was estimated by the applicant across different animal species, as summarised below (for details, refer to Appendix D) following conservative approaches. Estimations of exposure to NAA, NAG and NAT are based on the assumption that the totality of oilseed rape products fed to animals is derived from oilseed rape 73496 (100% replacement scenario). Moreover, in the absence of a feed consumption database for animals (EFSA, 2019), estimations are based on default values for theoretical maximal inclusion rates of feed materials in diets, selected from the literature. Dietary exposure to NAA, NAG and NAT in poultry, swine, cattle and sheep was estimated based on the consumption of oilseed rape 73496 meal. 37 1) Background exposures to NAA in poultry, swine, cattle, sheep, salmon, dog and cat were estimated based on the consumption of simple diets (not nutritionally balanced) consisting of the combination of two conventional feed materials (i.e. maize grains and distillers grain with solubles, forage/silage from maize, alfalfa and grass, soybean, oilseed rape and fish meal) with known concentration of NAA; a comparison was also made with the exposures based on the consumption of simple diets containing oilseed rape meal 73496 as one of the combined feed materials in order to determine whether a safe comparative consumption could be established. 38 Among the different outcomes, the most conservative exposures were selected for further risk characterisation (see below), as reported in Table 10: Table 10: Conservative Dietary Exposures to NAA Animal species Simple diet: oilseed rape 73496 meal +conventional feed material Daily feed intake (kg DM animal/kg body weight) Dietary exposure mg/kg bw IR% Cattle Cattle for fattening (8/400) 14.5 Oilseed rape 73496 meal (20%) +grass silage (50%) Swine Pig for fattening (2.20/60) 25.9 Oilseed rape 73496 meal (20%) +maize DDGS (75%) Comp. animals Cat (0.06/3) 14.0 Oilseed rape 73496 meal (20%) +maize grain (25%) 37 Dossier Part II Scientific information/Section B. 38 Additional information: 6/12/2017. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 23 EFSA Journal 2021;19(6):6610 2) Dietary exposure to NAA and NAG in calf was estimated based on the consumption of milk replacer, making the conservative assumption that 100% of the protein in milk replacer would be from oilseed rape protein isolates. 20 3) Simulation of dietary exposure to NAA, NAG and NAT in ruminants (i.e. cattle for fattening, dairy cow and sheep/goat) was estimated based on the consumption of oilseed rape solubles (whey), alone or combined with oilseed rape meal. As oilseed rape protein isolate production is not a common industrial practice, soy protein isolate and the corresponding whey fraction productions were examined as a surrogate; theoretical inclusion rates for oilseed rape were derived from the literature, considering the reporting of adverse nutritional impact of soy solubles at experimental inclusion rates above 10% in diets. 22 The GMO Panel notes that the incorporation in the diet of oilseed rape 73496 meal in substitution of conventional oilseed rape meal determines an increased exposure to NAA, NAG and NAT in all the animal species investigated and in all the proposed scenarios. Feed products other than meal for incorporation into the diet would include oilseed rape protein isolates and its by-product whey. However, these products, at the current status of knowledge, do not represent common ingredients currently used in animal rations and diets. To date, there is very little or no consumption of oilseed rape protein isolates as feed, due to relatively low protein yield and high costs of production compared to available alternatives (Campbell et al., 2016); however, their future use as feed cannot be excluded (e.g. as milk replacer for calves, but also in piglets). Whey from protein isolate production is highly diluted and most often considered a waste stream of the process; soluble products from whey could be produced and used as feed ingredient; however, this process requires substantial energy and therefore is not economical. 39 Therefore, the products were not considered for risk characterisation in the context of this application. The GMO Panel notes that these human and animal intake estimates of N-acetyl amino acids only considers the food and feed commodities from oilseed rape that can currently be used in the European market, making use of consumption/feeding data of food and feed assumed to be replaced by food and feed from oilseed rape 73496. In the future, the intakes to N-acetyl amino acids might vary due to changes in consumption/feeding patterns and, above all, by the introduction in the market of new products from oilseed rape (e.g. seeds, whey fraction, etc.). RISK CHARACTERISATION Human risk characterisation Human dietary exposure to NAA was estimated considering the presence of NAA in conventional foods and the processed foods from oilseed rape that can currently be in the European market (RBD oil, protein isolates and oilseed rape powder). The maximum dietary exposure to NAA combining the consumption of conventional food and considering the processed foods from oilseed rape 73496 was estimated in the age class ‘Other children’(992.9 lg/kg bw day, 95th percentile dietary intake). In the adult population the maximum 95th percentile dietary intake estimate was 444.8 lg/kg bw day (age class ‘Adults’). A NOAEL of 229.5 mg/kg bw day was derived for salivary glands hypertrophy in male rats in a 90-day repeated dose toxicity study with NAA (see Table 8, Section 3.3.3). The highest dietary exposure estimates in the age classes ‘Other children’and ‘Adults’provide Margin of Exposures (MoE) to the NOAEL of ca. 225 and ca. 500, respectively. These MoE are considered acceptable as they exceed the default 100 fold factor applied when extrapolating from animal data to humans and noting the extent of the information currently available on NAA (see Section 3.3.3.2). The GMO Panel concludes that the human dietary exposure to NAA as estimated from the combined consumption of conventional food and considering the processed food from oilseed rape 73496 is unlikely to present a risk to health in humans. Animal risk characterisation The levels of NAG and NAT were significantly higher in seeds from oilseed rape 73496 than in those the conventional counterpart (see Section 3.2.3) and noted to achieve even higher concentration in 39 This waste stream is typically disposed of as municipal waste. In a limited number of cases where this is not possible due to both municipal restrictions and the inability to treat waste on site, a soy soluble product can be produced which could be used in animal diets (personal communication from Solae LLC, St Louis, Missouri, USA). The soy soluble product is produced by concentrating the soy whey via drying. Consumption of soy solubles produced from whey is not currently authorised as a feed ingredient in the EU, and no pending EU applications for either soy or oilseed rape solubles produced from whey fractions are known). Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 24 EFSA Journal 2021;19(6):6610 the defatted toasted meal from the GM oilseed rape, as compared to the non-GM one (Section 3.3.1). Based on the 28-day toxicity studies on NAG and NAT (Table 8) and the dietary intake estimates provided by the applicant (see Animal dietary exposure to N-acetyl amino acids above and Appendix D), the GMO Panel notes that the margin of exposure for NAG and NAT is at least 1,000 fold for all animal species. The GMO Panel concludes therefore that exposures to NAG and NAT via animal feed from oilseed rape 73496, as described in this application, pose no concerns to animal health. The initial estimated exposures provided by the applicant indicates that NAA in animals via feed result in low MoEs (less than 100) to the most conservative NOAEL (229.5 in male rats from the 90day toxicity study, see Table 8) for most species. The GMO Panel concluded that a potential concern on animal health could not be excluded and requested additional information from the applicant to address exposures to NAA from feed from oilseed rape 73496. A 42-day study on NAA in the broiler to provide target exposure to NAA of 25, 50 and 100 mg/kg bw per day, corresponding to an approximate supplemental 10%, 20% and 40% incorporation of oilseed rape 73496 meal into commercial poultry’s diets confirmed that there are no limitations in the use of oilseed rape 73496 as feed material at the currently used inclusion rate for poultry (around 20%). The GMO Panel did not consider it necessary to elaborate further the risk assessment on fish (no salivary glands are present in fish). The applicant provided further exposure estimates in animal species other than poultry (see Appendix D), as well as generic information on the normal occurrence and metabolism of NAA in animals and on the function and physiology of salivary glands. Moreover, the applicant provided studies on the toxicokinetics of NAA in rats, goats and pigs, together with a proposal to base the assessment on a Compound Specific Assessment Factor (CSAF) approach. Description of the CSAF based approach proposed, information on the toxicokinetics provided to support the exercise and the actual CSAF based assessment of NAA in feed from oilseed rape 73496 are summarised below. Additional details are given in Appendix E. CSAF based approach (see also Appendix E) The default assessment factor used when deriving an acceptable human exposure level from the no-observed adverse effect levels (NOAEL) in animal studies is 100. This factor accounts for differences in sensitivity between the experimental animal and the average human and for variations in sensitivity within the human population to protect sensitive sub-groups. This factor of 100 has also been utilised in the assessment of feed additives as an indicator of the expected margin between the NOAELs in laboratory animal studies and intakes in farm and domestic animals (MoE) (EFSA FEEDAP Panel, 2017a,b). Where specific data are available, it is possible to derive Chemical Specific Adjustment Factors (also known as Chemical Specific Assessment Factors and Data Derived Evaluation Factors) to replace the default 100-fold assessment factor. The overall CSAF can be lower or higher than the default of 100. The concept was developed by comparing the findings seen in humans and experimental animals exposed to pharmaceuticals and was described in detail by the World Health Organisation (IPCS 2005). The CSAF approach splits the default factor of 100 into four separate factors addressing differences in toxicokinetics (how a compound is absorbed, metabolised, distributed and excreted) and toxicodynamics (how a specific level of exposure affects the target tissue). Each individual factor can be modified, if suitable data are available, and then combined to give the overall CSAF. CSAFs have been referenced by EFSA in the Scientific Opinions on Default values (EFSA Scientific Committee, 2012) and Uncertainty Analysis (EFSA Scientific Committee, 2018). A CSAF based approach has been used by EFSA in the re-evaluation of phosphates (EFSA FAF Panel, 2019). Information on toxicokinetics submitted to EFSA (see also Appendix E) To address the low margins between estimated exposures and the NOAEL of 229.4 mg/kg bw per day (see Section 3.3.3.2) and to support a CSAF approach, the applicant performed studies in goats (representative ruminant), pigs (representative monogastric animal) and rats (the species used in the toxicity studies) to investigate the toxicokinetics of NAA. The results have been used to develop a CSAF based assessment to determine if exposures to NAA from animal feed derived from oilseed rape 73496 present an acceptable risk. Details of the studies are presented in Appendix C. The key results from the studies relevant to the CSAF approach are described below: –NAA is a normal component of the blood plasma, present at similar levels in all three tested species; Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 25 EFSA Journal 2021;19(6):6610 of research also in feed. 51 Therefore, their future application cannot be excluded. One of the largest potential use of rape protein isolates for livestock could be reasonably as milk replacer, which is primarily used for calves and in piglets. The potential use of whey as a feed ingredient must be assessed together with the prevalence of protein isolate production. If oilseed rape protein isolate were to be produced, there is a possibility for oilseed rape solubles (whey) to be used as a feed ingredient. Although it is very unlikely that oilseed rape protein isolate and the corresponding whey fraction will be available as commercial feed products, this might be kept under monitoring in the next future. 4-Hydroxyglucobrassicin is a derivative of glucobrassicin, one of the several glucosinolates that can be found in Brassicaceae. The GMO Panel considers that the increased level reported in seeds of oilseed rape 73496 does not represent an issue for animal nutrition. The maximum reported levels of total glucosinolates in oilseed rape 73496, significantly higher than those in its conventional counterpart, fall within the natural variability represented by non-GM oilseed rape reference varieties, and are well below the maximum glucosinolate content set-out for double-zero rapeseed varieties under Regulation (EC) No 2316/1999 (see Section 3.3.5.1). Moreover, Mejicanos et al. (2016) reports 4-hydroxyglucobrassicin content of 1.2 and 0.3 lmol/g in oilseed rape meal from Brassica napus and Brassica juncea, showing a certain variability of these compounds across varieties which can be fed to animals. Glycine is considered a non-essential amino acid, even though Wu, 2014 suggests that adequate provision of all amino acid is important to improve efficiency of animal production. The magnitude of the decrease observed in oilseed rape 73496 (treated and non-treated) as compared to the non-GM comparator does not constitute an issue for animal nutrition. Dietary fibre (crude fibre, NDF, ADF) is considered essential for animal health due to its influence on gastrointestinal tract physiology in animals. The observed increase of crude fibre, ADF, NDF in oilseed rape 73496 (treated and not treated), as compared to the non-GM comparator does not constitute an issue for animal nutrition. The observed increase of crude fat in treated oilseed rape 73496 as compared to the non-GM comparator does not constitute an issue for animal nutrition. Magnesium is an essential mineral in animal nutrition, and the diet must supply the adequate amount to satisfy the requirement. Many feeds are a good source of magnesium, and several magnesium sources, among which magnesium oxide is the most used, are included in the diet when the content in feeds is not sufficient, or when antagonists to magnesium absorption are present, i.e. high level of potassium. The lower level of magnesium found in oilseed rape 73496 (treated and not treated with the intended herbicide) as compared to non-GM counterpart does not represent an issue for animal nutrition. Pyridoxine, a form of vitamin B6, plays an essential role mainly in amino acid metabolism. Vitamin B6 is produced by microorganisms in intestinal tracts of animals, but whether significant quantities are absorbed and utilised is in doubt. Muscle, liver, vegetables, whole grain cereals and their by-products, are among the best sources of pyridoxine. The bioavailability of two common feed ingredients is 65% for soybean meal with corn varying from 45% to 56% (McDowell and Ward, 2008). The level of vitamin B6, as other vitamins, contained in all feeds is affected by processing, subsequent storage and presence of antagonist in some feeds, i.e. hydrazic acid in linseed meal. In ruminants, vitamin B6 is mainly obtained by microbial synthesis in the rumen. The decrease observed in the non-treated oilseed rape as compared to the non-GM comparator does not constitute an issue for animal nutrition, considering also that hydrosoluble vitamins can be added in the diet of animals. Pantothenic acid is a component of enzymes involved in carbohydrate, fat and protein metabolism. This vitamin is found in several feeds, i.e. wheat and rice bran, yeast, but the quantity present is generally insufficient to satisfy nutrient requirements for most monogastric species, so as many other micronutrients is added to the diet of animals. Pantothenic acid, as many other hydrosoluble vitamins, is synthesised in the rumen. The decrease observed in the non-treated oilseed rape as compared to the non-GM comparator does not constitute an issue for animal nutrition. 3.3.6. Post-market monitoring of GM food/feed In accordance with Article 6(5)(e) of Regulation (EC) No 1829/2003, based on the outcome of the risk assessment of oilseed rape 73496 and, in particular, on the safety assessment of NAA, EFSA 51 http://www.canproingredients.ca/research_development.php Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 32 EFSA Journal 2021;19(6):6610 recommends to implement a PMM plan. This PMM plan should initially focus on the collection of import data to Europe of oilseed rape 73496 and/or its products, entering the food and feed supply chains. If imports are identified, consumption data should be collected for humans and animals (e.g. through dietary surveys) on oilseed rape 73496 and/or its food and feed products to confirm the predicted consumption data and to verify that the conditions of use are those considered during the pre-market risk assessment. 3.3.7. Conclusion on the food/feed safety assessment The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the GAT4621 protein as expressed in oilseed rape 73496 and finds no evidence that genetic modification would change the overall allergenicity of oilseed rape 73496. No safety concerns are identified with regards to the increased levels of the N-acetyl amino acids NAA, NAG and NAT in food and feed derived from oilseed rape 73496 as considered during this risk assessment. The GMO Panel recommends to implement a PMM plan to confirm the predicted consumption of oilseed rape 73496 and/or its food and feed products and the application of conditions of uses considered during the pre-market risk assessment. Based on the outcome of the comparative assessment and the nutritional assessment, the GMO Panel concludes that the consumption of oilseed rape 73496 does not represent any nutritional concern, in the context of the scope of this application. The GMO Panel concludes that oilseed rape 73496, as described in this application, is as safe as its conventional counterpart and the non-GM reference varieties tested. 3.4. Environmental risk assessment and monitoring plan 52 3.4.1. Environmental risk assessment Considering the scope of application EFSA-GMO-NL-2012-109, which excludes cultivation, the environmental risk assessment (ERA) of oilseed rape 73496 mainly takes into account: 1) the exposure of microorganisms to recombinant DNA in the gastrointestinal tract of animals fed GM material and of microorganisms present in environments exposed to faecal material of these animals (manure and faeces); and 2) the accidental release into the environment of viable oilseed rape 73496 seeds during transportation and/or processing (EFSA GMO Panel, 2010b). 3.4.1.1. Persistence and invasiveness of the GM plant Oilseed rape (Brassica napus AACC) is an annual allotetraploid species (2n =38, genome constitution AACC), which has probably evolved through hybridisation and polyploidisation between the two diploid species Brassica rapa (2n =20, AA) and Brassica oleracea (2n =18, CC). Oilseed rape seeds have the ability to survive in soils for more than 10 years (Hails et al., 1997; Begg et al., 2006; Lutman et al., 2004; Lutman et al., 2005; Lutman et al., 2008; Mess ean et al., 2007; D’Hertefeldt et al., 2008; Gruber et al., 2008; Beckie and Warwick, 2010; Peltonen-Sainio et al., 2014; Belter, 2016) and demographic studies and surveys have shown the ability of oilseed rape (B. napus) seed to establish self-perpetuating populations outside agricultural areas, mainly in semi-natural and ruderal habitats in different countries (e.g. Crawley et al., 1993; Pascher et al., 2010, 2017; Devos et al., 2012; Bauer-Panskus et al., 2013; Hecht et al., 2014; Schulze et al., 2014; Katsuta et al., 2015; Bailleul et al., 2016; Busi and Powles, 2016; Franzaring et al., 2016; Nishizawa et al., 2016). Oilseed rape is generally regarded as an opportunistic species, which can take advantage of disturbed sites (e.g. mowed areas, semi-natural habitats) to germinate and capture resources rapidly. In undisturbed natural habitats, oilseed rape lacks the ability to establish stable populations over successive years, possibly due to the absence of competition-free germination sites (Crawley et al., 1993, 2001; Meffin et al., 2015) and exposure to biological and abiotic stressors likely limiting fitness (COGEM, 2013; Busi and Powles, 2016). Once established in competition-free germination sites, feral populations decline over a period of years (Crawley and Brown, 1995, 2004; Knispel et al., 2008; Squire et al., 2011; Banks, 2014; Busi and Powles, 2016). However, if habitats are disturbed on a regular basis, then feral populations can persist for longer periods (Pessel et al., 2001; Claessen et al., 2005a, 2005b; Garnier et al., 2006; Elling et al., 2009; Pascher et al., 2010; Banks, 2014) and can have the characteristics of a weed or ruderal (Banks, 2014). The persistence or recurrence of a population in one location is 52 Dossier Part II Scientific information/Sections E3 and E4. Additional information: 28/1/2014, 3/10/2016 and 28/9/2018. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 33 EFSA Journal 2021;19(6):6610 variously attributed to replenishment with fresh seed spills, to recruitment from seed emerging from the soil seedbank or shed by resident feral adult plants or to redistribution of feral seed from one location to another (Pivard et al., 2008a, 2008b; Banks, 2014; Bailleul et al., 2016). Banks (2014) showed that the substantial increase in small and large (100–1,000 plants) feral populations occurred throughout the studied area during study years in Scotland. It is unlikely that the intended trait of oilseed rape 73496 will provide a selective advantage to oilseed rape plants, except when they are exposed to glyphosate-containing herbicides. Should these plants be exposed to such herbicides, their abundance may increase locally (Londo et al., 2010, 2011; Watrud et al., 2011), allowing the establishment of transient populations. However, the likelihood of such an event will be restricted to managed environments, which may occasionally be treated with such herbicides. Moreover, this fitness advantage will not allow oilseed rape 73496 to overcome other biological and abiotic factors (described above) limiting plant’s persistence and invasiveness. In conclusion, the GMO Panel considers it unlikely that oilseed rape 73496 will differ from conventional oilseed rape varieties in its ability to survive and establish feral populations under European environmental conditions in case of accidental release into the environment of viable oilseed rape 73496 seeds. 3.4.1.2. 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 seeds. Plant-to-microorganism gene transfer Genomic DNA can be a component of food and feed products derived from oilseed rape. 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). The only mechanism known to facilitate horizontal transfer of non-mobile, chromosomal DNA fragments to bacterial genomes is homologous recombination. 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 nonhomologous end joining and microhomology-mediated end joining are theoretically possible (H€ ulter and Wackernagel, 2008; EFSA, 2009). 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. The updated bioinformatic analysis of the inserted DNA did not identify sufficient sequence identity with bacterial DNA (including the gat4621 gene, which was originally derived from B. licheniformis, but which has been codon-optimised for expression in plants) that would facilitate homologous recombination-mediated gene transfer between plants and bacteria. In summary, there is no indication for an increased likelihood of horizontal transfer of DNA from oilseed rape 73496 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 For plant-to-plant gene transfer to occur, imported GM oilseed rape seeds need to germinate and develop into plants in areas containing sympatric wild relatives and/or cultivated oilseed rape with synchronous flowering and environmental conditions favouring cross-pollination. Oilseed rape is an open pollinating crop plant capable of cross-pollinating with other Brassica crops (Eastham and Sweet, 2002). It can also spontaneously hybridise with sexually compatible feral and wild relatives. Several hybrids between oilseed rape and wild relatives have been reported in the scientific Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 34 EFSA Journal 2021;19(6):6610 literature. Evidence suggests that transgenes could readily introgress into B. rapa,B. juncea and B. oleracea, and is expected to be rare with B. nigra,Hirschfeldia incana,Raphanus raphanistrum and Sinapis arvensis (reviewed by Liu et al., 2013; Ellstrand et al., 1999, Ellstrand et al., 2013; FitzJohn et al., 2007; Devos et al., 2009; Tang et al., 2018). Under field conditions, transgene introgression has only been confirmed for B. rapa (Hansen et al., 2001, 2003; Jørgensen et al., 2004; Norris et al., 2004; Warwick et al., 2003, 2008; Jørgensen, 2007). For transgene introgression to occur, feral GM oilseed rape must require some overlap in flowering in time and space with compatible relatives. Subsequently, transgenes must be transmitted through successive backcross generations or selfing, so that they become stabilised into the genome of the recipient (de Jong and Rong, 2013; Garnier et al., 2014). Because of these barriers (Luijten et al., 2015), reported incidences of hybrids and backcrosses with B. rapa were found to be low in fields (Jørgensen et al., 2004; Norris et al., 2004; Warwick et al., 2008; Elling et al., 2009), or at ports, along roadsides and riverbanks (Saji et al., 2005; Aono et al., 2006, 2011; Yoshimura et al., 2006; Elling et al., 2009; Katsuta et al., 2015; Luijten et al., 2015). The GMO Panel does not consider the occurrence of feral oilseed rape 73496 plants, pollen dispersal and consequent cross-pollination as environmental harm in itself, as there is no evidence that the intended trait will enhance the vertical gene flow potential, or fitness, persistence or invasiveness of feral oilseed rape 73496, or cross-compatible plants such as hybridising wild relatives. However, when exposed to glyphosate-containing herbicides, occasional cross-compatible plants that acquired the herbicide tolerance trait through vertical gene flow are likely to exhibit a selective advantage, which may lead to their increased abundance. The likelihood of such an event to happen will be restricted to managed environments, which may occasionally be treated with such herbicides, so that environmental impacts will be minimal. Therefore, the GMO Panel considers that the acquisition of the herbicide tolerance trait by cross-compatible plants would not create additional environmental impacts. In conclusion, the GMO Panel considers that the likelihood of environmental effects because of the spread of genes from oilseed 73496 rape in Europe will not differ from that of conventional oilseed rape varieties. 3.4.1.3. Interactions of the GM plant with target organisms Taking the scope of application EFSA-GMO-NL-2012-109 (no cultivation) and thus the absence of target organisms into account, potential interactions of feral oilseed rape 73496 plants arising from seed import spills with target organisms are not considered a relevant issue. 3.4.1.4. Interactions of the GM plant with non-target organisms Given that environmental exposure of non-target organisms to spilled GM seeds or feral GM oilseed rape plants arising from spilled oilseed rape 73496 seeds is limited, and because ingested proteins are degraded to a great extent before entering the environment through faecal material of animals fed GM oilseed rape, potential interactions of oilseed rape 73496 with non-target organisms are not considered by the GMO Panel to raise any environmental safety concern. 3.4.1.5. Interactions with the abiotic environment and biogeochemical cycles Given that environmental exposure to spilled seeds or feral oilseed rape 73496 plants arising from seed import spills is limited, and because most proteins are degraded before entering the environment through faecal material of animals fed GM oilseed rape, potential interactions with the abiotic environment and biogeochemical cycles are not considered by the GMO Panel to raise any environmental safety concern. 3.4.2. Post-market environmental monitoring The objectives of a post-market environmental monitoring (PMEM) plan, according to Annex VII of Directive 2001/18/EC, are to: 1) confirm that any assumption regarding the occurrence and impact of potential adverse effects of the GMO, or its use, in the ERA are correct; and 2) identify the occurrence of adverse effects of the GMO, or its use, on human health or the environment that were not anticipated in the ERA. Monitoring is related to risk management, and thus, a final adoption of the PMEM plan falls outside the mandate of EFSA. However, the GMO Panel gives its opinion on the scientific rationale of the PMEM plan provided by the applicant (EFSA GMO Panel, 2011b). As the ERA does not identify potential adverse environmental effects from oilseed rape 73496, no case-specific monitoring is required. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 35 EFSA Journal 2021;19(6):6610 The PMEM plan proposed by the applicant for oilseed rape 73496 includes: 1) the description of an 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; 2) a coordinating system established by EuropaBio for the collection of information recorded by the various operators; and 3) the review of relevant scientific publications retrieved from literature searches (Lecoq et al., 2007; Windels et al., 2008). The applicant proposes to submit a PMEM report on an annual basis and a final report at the end of the authorisation period. The scope of the PMEM plan provided by the applicant is consistent with the intended uses of oilseed rape 73496. The GMO Panel agrees with the reporting intervals proposed by the applicant in its PMEM plan. 3.4.3. Conclusion on the environmental risk assessment and monitoring plan It is unlikely that oilseed rape 73496 would differ from conventional oilseed rape varieties in its ability to persist under European environmental conditions. Considering the scope of the application EFSA-GMO-NL-2012-109, interactions of feral oilseed rape 73496 plants with the biotic and abiotic environment are not considered to be relevant issues. The analysis of HGT from oilseed rape 73496 to bacteria does not indicate a safety concern. Therefore, considering the introduced trait, the outcome of the agronomic and phenotypic analysis, and the routes and levels of exposure, the GMO Panel concludes that oilseed rape 73496 would not raise safety concerns in the event of accidental release of viable GM oilseed rape 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 oilseed rape 73496. 4. Conclusions The GMO Panel was asked to carry out a scientific assessment of oilseed rape 73496 for import, processing and food and feed uses in accordance with Regulation (EC) No 1829/2003. The molecular characterisation data establish that oilseed rape 73496 contains a single insert consisting of one copy of the gat4621 expression cassette. Upon transformation, a region of chromosome C02 was potentially inverted and a putative tpt gene interrupted. The relevance of the gene interruption and potential chromosomal inversion for the risk assessment of oilseed rape 73496 is addressed. Bioinformatic analyses of the sequences encoding the newly expressed protein and other ORFs 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 introduced trait is confirmed over several generations. The levels of the GAT4621 protein were obtained and reported adequately. The protein characterisation data of the plantand microbial-derived GAT4621 proteins indicate that both proteins are equivalent, and thus, that the microbial-produced protein (2 batches) can be used in the safety studies. None of the identified differences in the agronomic/phenotypic endpoints between oilseed rape 73496 and its conventional counterpart needs further assessment. Among the differences identified in seed composition between oilseed rape 73496 and its conventional counterpart the levels of NAA, NAG and NAT, the free amino acid glycine, crude fibre, crude fat, ADF, NDF, magnesium, pyridoxine, pantothenic acid and 4-hydroxyglucobrassicin were further assessed and found not to raise nutritional and safety concerns. No safety concerns are identified regarding toxicity and allergenicity of the GAT4621 protein as expressed in oilseed rape 73496. No evidence is found that the genetic modification would change the overall allergenicity of oilseed rape 73496. Based on the outcome of the comparative and nutritional assessments, the consumption of oilseed rape 73496 does not represent any nutritional concern, in the context of the scope of this application. The implementation of a PMM plan is recommended to confirm the predicted consumption of oilseed rape 73496 and/or its food and feed products; and the application of conditions of uses considered during the pre-market risk assessment. There is a low likelihood of environmental effects resulting from the accidental release of viable seeds from oilseed rape 73496 into the environment. The PMEM plan and reporting intervals are in line with the intended uses of oilseed rape 73496. The GMO Panel concludes that oilseed rape 73496, as described in this application, is as safe as its conventional counterpart and the non-GM oilseed rape reference varieties tested with respect to potential effects on human and animal health and the environment. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 36 EFSA Journal 2021;19(6):6610 5. Documentation as provided to EFSA •Letter from the Competent Authority of The Netherlands received on 24 May 2012 concerning a request for authorization of the placing on the market of oilseed rape 73496 submitted in accordance with Regulation (EC) No 1829/2003 by Pioneer Overseas Corporation. •Application EFSA-GMO-NL-2012-109 validated by EFSA, 4 December 2012. •Request for supplementary information to the applicant, 20 February 2013. •Request for supplementary information to the applicant on behalf of EURL-GMFF, 2 April 2013. •Receipt of supplementary information from the applicant, 9 April 2013. •Receipt of supplementary information, from the applicant to EURL-GMFF, 26 April 2013. •Request for supplementary information, from EURL-GMFF to the applicant, 22 May 2013. •Receipt of supplementary information, from the applicant to EURL-GMFF, 4 June 2013. •Request for supplementary information to the applicant, 13 August 2013. •Receipt of supplementary information from the applicant, 17 September 2013. •Request for supplementary information to the applicant, 12 December 2013. •Receipt of supplementary information from the applicant, 28 January 2014. •Request for supplementary information to the applicant, 11 July 2014. •Request for supplementary information to the applicant, 8 December 2014. •Receipt of supplementary information from the applicant, 23 January 2015. •Request for supplementary information to the applicant, 11 March 2015. •Receipt of supplementary information from the applicant, 16 April 2015. •Receipt of supplementary information from the applicant, 27 April 2015. •Receipt of supplementary information from the applicant, 29 September 2015. •Request for supplementary information to the applicant, 2 December 2015. •Receipt of supplementary information from the applicant, 15 December 2015 •Request for supplementary information to the applicant, 29 February 2016. •Receipt of supplementary information from the applicant, 29 April 2016. •Request for supplementary information to the applicant, 19 May 2016. •Receipt of supplementary information from the applicant, 3 October 2016. •Receipt of supplementary information submitted spontaneously by the applicant, 22 November 2016. •Request for supplementary information to the applicant, 2 December 2016. •Request for supplementary information to the applicant, 16 December 2016. •Receipt of supplementary information from the applicant, 2 May 2017. •Receipt of supplementary and spontaneous information from the applicant, 29 August 2017. •Request for supplementary information to the applicant, 6 October 2017. •Receipt of supplementary information from the applicant, 6 December 2017. •Request for supplementary information to the applicant, 19 December 2017. •Receipt of supplementary information from the applicant, 20 June 2018. •Request for supplementary information to the applicant, 3 July 2018. •Receipt of supplementary information from the applicant, 28 September 2018. •Request for supplementary information to the applicant, 29 October 2018. •Request for supplementary information to the applicant, 13 November 2018. •Receipt of supplementary information from the applicant, 18 December 2018. •Receipt of supplementary information from the applicant, 10 January 2019. •Receipt of supplementary information submitted spontaneously by the applicant, 16 April 2019. •Request for supplementary information to the applicant, 27 May 2019. •Request for supplementary information to the applicant, 23 April 2020. •Request for supplementary information to the applicant, 18 May 2020. •Receipt of supplementary information from the applicant, 9 July 2020. •Receipt of supplementary information from the applicant, 10 September 2020. •Request for supplementary information to the applicant, 7 October 2020. •Receipt of supplementary information from the applicant, 8 October 2020. •Request for supplementary information to the applicant, 14 October 2020. •Receipt of supplementary information from the applicant, 10 November 2020. 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Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 41 EFSA Journal 2021;19(6):6610 Appendix B –Summary statistics of the baseline dietary intake of NAA (lg/kg bw per day) across European dietary surveys Dietary intake (lg/kg bw per day) N Mean dietary intake 95th percentile dietary intake Min Median Max Min Median Max Infants 13 1.1 3.2 15.3 8.7 16.4 47.3 Toddlers 20 3.2 6.9 15.5 10.8 22.7 45.3 Other children 30 3.7 7.2 12.6 11.8 20.3 34.8 Adolescents 30 2.7 5.5 7.6 9.1 17.7 23.7 Adults 35 2.8 16.7 39.9 11.5 42.0 99.0 Elderly 25 2.8 15.7 46.0 9.4 35.6 92.9 Very elderly 17 4.3 14.9 43.3 12.2 36.4 52.7 Pregnant women 5 5.9 7.9 8.8 13.4 21.8 28.3 Lactating women 2 4.6 –15.2 12.9 –35.8 NAA: N-acetylaspartate. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 48 EFSA Journal 2021;19(6):6610 Appendix C –Summary statistics of dietary intake of NAA (lg/kg bw per day) across European dietary surveys considering the presence of NAA in conventional foods, and the consumption of protein isolates and oilseed rape powder from oilseed rape 73496 Dietary intake (lg/kg bw per day) N Mean dietary intake 95th percentile dietary intake Min Median Max Min Median Max Infants 13 1.1 22.6 144.8 9.0 114.4 651.5 Toddlers 20 6.9 104.4 375.7 36.6 461.9 933.8 Other children 30 17.8 90.9 412.6 55.3 428.6 992.9 Adolescents 30 13.3 56.6 244.6 51.3 252.2 674.1 Adults 35 13.4 52.2 217.5 49.4 211.4 444.8 Elderly 25 11.2 47.9 210.2 32.9 175.3 418.6 Very elderly 17 14.8 44.7 201.3 54.2 193.5 305.1 Pregnant women 5 12.1 40.5 57.1 53.4 164.1 214.9 Lactating women 2 40.0 –75.6 214.3 –236.7 NAA: N-acetylaspartate. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 49 EFSA Journal 2021;19(6):6610 Appendix D –Animal dietary exposure to N-acetyl amino acids via oilseed rape 73496 and derived feed a) Technical Dossier: Part II, section B Dietary exposure to N-Acetylaspartate (NAA), N-Acetylglutamate (NAG) and N-Acetylthreonine (NAT) in oilseed rape 73496 was estimated by the applicant across different animal species (i.e. poultry, swine, cattle and sheep), assuming the consumption of oilseed rape meal, the main rapeseed by-product entering the feed supply chain. A conservative scenario with 100% replacement of conventional oilseed rape meal by oilseed rape 73496 meal was considered. Mean levels (dry weight) of NAA, NAG and NAT in un-hulled toasted meal processed from oilseed rape 73496 seeds treated with the intended herbicide (i.e. glyphosate) were used as occurrence data (see Table D.1). Dietary exposure was based on estimates for animal body weight, daily feed intake and inclusion rates (percentage) of oilseed rape meal in diets (OECD, 2009). Estimated dietary exposures in livestock animals is reported in Table D.1. b) Additional information: 6/12/2017 The applicant provided estimations of background exposures to NAA in poultry, swine, cattle, sheep, salmon, dog and cat, based on the theoretical consumption of simple diets (not nutritionally balanced) consisting of the combination of two selected conventional feed materials (i.e. maize grains and distillers grain with solubles, forage/silage from maize, alfalfa and grass, soybean, oilseed rape and fish meal) with known concentration of NAA; a comparison was made with the exposures based on the theoretical consumption of simple diets containing oilseed rape meal 73496 as one of the combined feed materials in order to determine whether a safe comparative consumption could be established. Concentration of NAA for selected feedstuffs and for dog and cat foods were used as occurrence (see Table D.2). Dietary exposure was based on estimates for body weight and daily feed intake obtained from EFSA’s guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), combined with feedstuff inclusion rates obtained from OECD Guidance Document on Residues in Livestock (OECD, 2013), Canola Council of Canada (2015), Food and Agriculture Organization of the United Nations (FAO, 2017), Advisory Committee on Animal Feedingstuffs (ACAF Secretariat, 2001), Atti et al. (2007), Mandell et al. (1997), Windsor (2001) and Purina (personal communication). Estimated dietary exposures in farmed and companion animals is reported in Table D.2. Table D.1: Dietary exposure (DE) to NAA, NAG and NAT (mg/kg bw per day) in livestock animals based on the consumption of oilseed rape meal Animal species Body weight (kg) Daily feed intake (kg DM/Animal) Inclusion rate (%) NAA (a) NAG (b) NAT (c) mg/kg bw per day mg/kg bw per day mg/kg bw per day Poultry Broiler 1.7 0.12 18 38.99 0.68 0.04 Layer 1.9 0.13 10 21 0.37 0.02 Turkey 7 0.50 20 43.90 0.77 0.04 Swine Breeding 260 6 20 14.18 0.25 0.01 Finishing 100 3 20 18.42 0.32 0.02 Cattle Beef 500 12 –––– Dairy 650 25 10 13.43 0.23 0.01 Sheep Ram/ewe 75 2.5 –––– Lamb 40 1.7 –––– (a): NAA concentration in meal: as-is for poultry and swine (3070 mg/kg); dry weight basis for cattle and sheep (3,489 mg/kg). (b): NAG concentration in meal: as-is for poultry and swine (53.7 mg/kg); dry weight basis for cattle and sheep (61 mg/kg). (c): NAT concentration in meal: as-is for poultry and swine (2.90 mg/kg); dry weight basis for cattle and sheep (3.30 mg/kg). Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 50 EFSA Journal 2021;19(6):6610 Table D.2: Dietary exposure (DE) to NAA (mg/kg bw per day) in food-producing and non-foodproducing animals based on the consumption of simple diets consisting of the combination of two conventional feed materials Simple diets Animal daily feed intake (kg DM animal/kg body weight) Inclusion rate (%)/NAA (mg/kg bw per day) Maize grain Maize forage/ silage Alfalfa silage Grass silage Maize DDGS Soybean meal Fish meal Oilseed rape meal Oilseed rape 73496 meal Maize Grain Chicken for fattening 0.158/2 70% 0.058 NA NA NA 60% 0.585 40% 0.103 10% 0.274 18% 0.292 18% 49.7 Laying hen 0.106/2 70% 0.039 10% 0.111 NA NA 50% 0.333 25% 0.058 10% 0.184 10% 0.126 10% 18.5 Turkey for fattening 0.176/3 50% 0.031 NA NA NA 50% 0.357 45% 0.068 10% 0.191 20% 0.224 20% 41.0 Sow lactating 5.28/175 70% 0.022 20% 0.104 NA 20% 0.355 75% 0.274 30% 0.035 10% 0.105 20% 0.121 20% 21.1 Pig for fattening 2.20/60 70% 0.027 NA NA NA 75% 0.333 30% 0.042 10% 0.127 20% 0.148 20% 25.6 Cattle for fattening 8/400 80% 0.017 80% 0.235 25% 0.333 50% 0.569 30% 0.083 20% 0.022 5% 0.044 20% 0.083 20% 14.0 Dog 0.25/15 45% 0.008 NA NA NA NA 15% 0.011 NA 20% 0.063 20% 11.6 Cat 0.06/3 25% 0.005 NA NA NA NA 30% 0.014 NA 20% 0.071 20% 14.0 Maize DDGS Chicken for fattening 0158/2 70% 0.585 NA NA NA 60% 0.527 40% 0.572 10% 0.743 18% 0.761 18% 50.1 Laying hen 0.106/2 70% 0.333 10% 0.367 NA NA 50% 0.295 25% 0.314 10% 0.440 10% 0.382 10% 18.8 Turkey for fattening 0.176/3 50% 0.357 NA NA NA 50% 0.326 45% 0.364 10% 0.487 20% 0.520 20% 41.3 Sow lactating 5.28/175 70% 0.274 20% 0.334 NA 20% 0.585 75% 0.252 30% 0.265 10% 0.334 20% 0.351 20% 21.3 Pig for fattening 2.20/60 80% 0.333 NA NA NA 75% 0.306 30% 0.321 10% 0.406 20% 0.427 20% 25.9 Maize Forage/ Silage Cattle for fattening 8/400 80% 0.235 80% 0.218 25% 0.535 50% 0.771 30% 0.285 20% 0.224 5% 0.245 20% 0.284 20% 14.2 Dairy cow 20/650 30% 0.261 60% 0.252 40% 1.03 60% 1.27 30% 0.354 25% 0.262 5% 0.294 10% 0.302 10% 11.0 Grass Silage Cattle for fattening 8/400 80% 0.569 80% 0.771 25% 0.869 50% 0.553 30% 0.619 20% 0.558 5% 0.580 20% 0.618 20% 14.5 Dairy cow 20/650 30% 1.03 60% 1.27 40% 1.80 60% 1.02 30% 1.12 25% 1.03 5% 1.06 10% 1.07 10% 11.8 Sheep/goat 1.2/60 30% 1.00 NA 40% 1.50 90% 0.995 30% 1.06 25% 1.00 10% 1.05 15% 1.04 15% 11.5 Fish Meal Salmon 0.0021/012 NA NA NA NA NA 12% 0.156 32% 0.153 20% 0.211 20% 12.4 Note: NA: not applicable. Concentrations (lg/g) of NAA for select feedstuffs: maize grain 1.04; maize forage/silage 13.6; alfalfa silage 63.3; grass silage 55.3; maize DDGS 11.1; soybean meal 1.42; fish meal 27.4; oilseed rape meal 16.5; oilseed rape 73496 meal 3489; dog food 2.81; cat food 9.50. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 51 EFSA Journal 2021;19(6):6610 c) Additional information: 29/8/2017 The applicant provided estimations of exposure in calves based on the consumption of milk replacer, making the conservative assumption that 100% of the protein in milk replacer would be from oilseed rape protein isolate. Concentration of NAA and NAG in oilseed rape protein isolates were used as occurrence (see Table D.3). Estimated dietary exposures in calves are reported in Table D.3. d) Additional information: 20/6/2018 The applicant simulated estimations of exposure in ruminants (i.e. cattle for fattening, dairy cow and sheep/goat) based on the consumption of oilseed rape solubles (whey), alone or combined with oilseed rape meal. Dietary exposure was based on estimates for body weight and daily feed intake obtained from EFSA’s guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), combined with oilseed rape inclusion rates obtained from OECD Guidance Document on Residues in Livestock (OECD, 2013). Since oilseed rape protein isolate production is not a common industrial practice, soy protein isolate and the corresponding whey fraction productions were examined as a surrogate; theoretical inclusion rates of 10% were indeed derived from the literature, considering the reporting of adverse nutritional impact of soy solubles (whey) at experimental inclusion rates higher than 10% in diets (Perry et al., 1976; van Eys, 2012). Concentration of NAA, NAG and NAT, in oilseed rape solubles (whey) were used as occurrence (see Table D.4). Table D.3: Dietary exposure to NAA and NAG (mg/kg bw per day) in calves based on the consumption of milk replacer (protein isolates) Animal species NAA (a) NAG (b) mg/kg bw per day mg/kg bw per day Calf (c) 0.072 0.0024 (a): NAA concentration in oilseed rape protein isolates (18 mg/kg). (b): NAG concentration in oilseed rape protein isolates (0.611 mg/kg). (c): The total protein intake from milk replacer was estimated by multiplying the content of protein in milk replacer powder (EFSA GMO Panel, 2011c; BAMN, 2008) by the solids content in prepared liquid milk replacer (Krishnamoorthy and Moran, 2011) to determine the total protein content in the prepared liquid milk replacer. This was multiplied by consumption (high end value) of liquid milk replacer by a calf at 5 days of age (Krishnamoorthy and Moran, 2011) and divided by the average calf birth weight of a Jersey calf, one of the smaller breeds of cattle (Queensland Government, 2012) to determine the approximate total protein intake, 4 g/kg BW per day by a 5-day old calf. Table D.4: Dietary exposure to NAA, NAG and NAT (mg/kg bw per day) in ruminants based on the ‘theoretical’consumption of whey from oilseed rape protein isolate and oilseed rape meal, alone or combined Animal daily feed intake (kg DM animal/ kg body weight) Oilseed rape by-products (IR%) NAA (a) NAG (b) NAT (c) mg/kg bw per day mg/kg bw per day mg/kg bw per day Cattle for fattening 8/400 Oilseed rape meal (20%) 14.0 0.244 0.0132 Oilseed rape whey (10%) 9.71 0.379 0.00897 Oilseed rape meal +whey (20%) +10%) 23.7 0.623 0.0222 Dairy cow 20/650 Oilseed rape meal (20%) 10.7 0.188 0.0101 Oilseed rape whey (10%) 14.9 0.583 0.0138 Oilseed rape meal +whey (20%) +10%) 25.7 0.770 0.0239 Sheep/goat 1.2/60 Oilseed rape meal (20%) 10.5 0.183 0.00989 Oilseed rape whey (10%) 9.71 0.379 0.00897 Oilseed rape meal +whey (20%) +10%) 20.2 0.562 0.0189 (a): NAA concentration in oilseed rape meal (3070 lg/g) and in Whey 1 Fraction of oilseed rape protein isolate production (140.8 lg/g). (b): NAG concentration in oilseed rape meal (53.7 lg/g) and in Whey 1 Fraction of oilseed rape protein isolate production (5.49 lg/g). (c): NAT concentration in oilseed rape meal (2.90 lg/g) and in Whey 1 Fraction of oilseed rape protein isolate production (0.13 lg/g). Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 52 EFSA Journal 2021;19(6):6610 Appendix E –NAA risk characterisation by the use of Chemical Specific Adjustment Factors (CSAF) with reference to exposures to N-acetylated amino acids found in oilseed rape 73496 Background •The default assessment factor used when deriving an acceptable human exposure level from the no-observed adverse effect levels (NOAEL) in animal studies is 100. This factor accounts for differences in sensitivity between the experimental animal and the average human and for variations in sensitivity within the human population, to protect sensitive sub-groups. This factor of 100 has also been utilised an indicator of the expected margin between the NOAELs in laboratory animal studies and intakes in farm and domestic animals (EFSA FEEDAP Panel, 2017a,b). •Where specific data are available, it is possible to derive Chemical Specific Adjustment Factors (also known as Chemical Specific Assessment Factors and Data Derived Evaluation Factors) to replace the default 100-fold assessment factor. The overall CSAF can be lower or higher than the default of 100. The concept was developed by comparing the findings seen in humans and experimental animals exposed to pharmaceuticals and was described in detail by the World Health Organisation (IPCS, 2005). The CSAF approach splits the default factor of 100 into four separate factors addressing differences in toxicokinetics (how a compound is absorbed, metabolised, distributed and excreted) and toxicodynamics (how a given exposure affects the target tissue) –see Figure E.1. Each individual factor can be modified, if suitable data are available, and then combined to give the overall CSAF. •CSAFs have been referenced by EFSA in the Scientific Opinions on Default values (EFSA Scientific Committee, 2012) and on Uncertainty Analysis (EFSA Scientific Committee et al., 2018). A CSAF based approach has been used by EFSA in the re-evaluation of phosphates (EFSA FAF Panel et al., 2019). A CSAF based approach has been proposed by the applicant for the assessment of N-acetyl aspartate (NAA) present in feed derived from GM 73496 oilseed rape in the context of this application and this is described below. •The CSAF approach as above described was developed for assessments of human safety extrapolating from experimental animal data. In the context of this dossier the extrapolation is conducted from experimental animals to a representative animal for an order or sub-order. In this evaluation the interspecies factors are considered to apply between rats and goats/pigs and the interindividual factors apply between pigs/goats and all respective other relevant species and life stages. The finding of concern (i.e. salivary gland hypertrophy) was seen in some but not all of the rats exposed at the effect dose in the relevant studies (see Table 8in Section 3.3.3.2 of the scientific opinion) and can be considered as including some conservatism in affecting the more sensitive individuals. In addition, rats exposed during gestation, lactation and post-weaning and through maturity showed no greater sensitivity to the salivary gland Figure E.1: Sub-division of the default 100-fold assessment factor (from IPCS, 2005) Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 53 EFSA Journal 2021;19(6):6610 effects than those exposed as young adults, as noted in a rat two-generation reproductive toxicity study (see Table 8in Section 3.3.3.2 of the scientific opinion). Data submitted in support of a CSAF •The applicant has performed comparison studies in goats (representative ruminants), pigs (representative monogastric) and rats (the test species) to investigate the toxicokinetics of NAA, and results are presented below. Goats: groups of Boer goats (3/sex per group) received NAA (99.9% pure) in gelatine capsules at a nominal dose of 25 mg/kg bw per day for 14 days. Blood samples were taken regularly pre-dosing and from 5 min to 12 h after dosing on days 1 and 14. Samples of blood plasma were analysed by UHPLC and LC/MS/MS for NAA and aspartic acid. 53 Pigs: groups of Landrace cross pigs (3/sex/group) received NAA (99.9% pure) in a small volume of feed at a nominal dose of 25 mg/kg bw per day for 14 days (actual dose 26 mg/kg bw per day). Blood samples were taken and analysed as described above for goats. 53 Rats: groups of Sprague Dawley rats (6/sex/group) received NAA (99.9% pure in water) by gavage at doses of 10, 25, 75, 250 or 500 mg/kg bw per day for 14 days. Blood samples were taken and analysed as described above for goats. 53 •In addition to the toxicokinetic studies, relevant information was presented on: ○the physiology and anatomy of salivary glands, ○the background exposure to NAA in the diet, ○the presence and function of NAA in animals, ○the metabolism of NAA and on the expression levels of the main metabolising enzymes across species. The information confirmed that NAA was a natural component of the diet/feed but at levels much lower than those associated with oilseed rape 73496; that NAA was naturally present in the body of animals, with a function in the CNS; the initial step in the metabolism is a simple transamination to give aspartic acid and that the primary metabolising enzymes are present in a wide range of species (see also Section 3.3.3.2 of the Scientific Opinion). •The key results from the toxicokinetic studies are presented in Table E.1 below and a summary comparison between rats, goats and pigs is presented in Table E.2 below. The were no notable differences between males and females of any species. The derivation of the relevant CSAF is outlined in Table E.3. Data are available only for the toxicokinetics of NAA across species in blood plasma with no data on levels in the salivary gland, therefore no adjustment of the toxicodynamic factors can be performed. Potential modes of action a) Direct mode of action in the mouth When performing a CSAF based assessment it is valuable if the mode of action underlying the adverse effect is well understood. The mode of action underlying the salivary gland hypertrophy seen in some rats exposed to NAA (see Section 3.3.3.2) has not been investigated in detail, but generic information is available. The GMO Panel considered that given the physiological mechanisms controlling the production of saliva and mechanisms leading to hypertrophy it is likely that the findings in rats exposed to NAA were due to a direct mode of action of NAA in the mouth. Data supporting this include: •Compensatory or adaptative hypertrophy is considered to represent a physiological response to a repeated/high stimulus (King, 2007; Mastorides & Maronpot, 2002). •Factors resulting in stimulation of saliva excretion include taste and presence of food in the mouth. (https://www.britannica.com/science/human-digestive-system/Salivary-glands). •Saliva is alkaline and buffers acid food, acid compounds stimulate secretion (vinegar/lemon juice). NAA is acidic –pKa ~ 3.5. (https://hmdb.ca/metabolites/HMDB0000812) •Some studies indicated that substances may produce salivary gland hypertrophy when given in the diet but not by gavage. This effect was suggested to be an outcome of the exposure to the 53 Additional information 10/9/2020. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 54 EFSA Journal 2021;19(6):6610 test substance in the oral cavity rather than being a systemic effect by a limited number of studies (Wells and Voelkel, 1963; Burdock et al., 2000). In their studies, Wells and Voelkel and Burdock et al., administered the test substances in question (RP-1 and pancreatin), known to cause salivary gland enlargement when administered through the diet, both by diet and by gavage. •Some chemicals can act systemically to increase saliva secretion (e.g. pesticides which result in increased levels of acetylcholine) but salivary gland hypertrophy is not a common finding in toxicity studies with these types of compounds. No reports of NAA having a similar activity likely to increase acetylcholine were identified. On the basis that NAA acts on the salivary glands via a direct action in the mouth, the effects would be essentially independent of absorption, distribution, metabolism and excretion. Therefore, both toxicokinetic factors can be removed leaving a residual CSAF of 8 based on the toxicodynamic factors. This would apply when extrapolating from rats to any other species and life-stage (see Table E.3 below). b) Systemic MoA As the mode of action of NAA on salivary glands has not been investigated in detail, a supplementary CSAF based approach assuming a systemic mode of action was performed to see if any significant risks might be missed by adopting a direct mode of action approach. Using the data from the submitted toxicokinetic studies and noting that in a rat two-generation study there was no evidence of any sensitive life-stages and that expression data on the main enzymes metabolising Nacetylated amino acids are widely distributed across species (Yates et al., 2020), adjustment of the CSAFs by modifying the toxicokinetic factors was evaluated for the various species but retaining the default toxicodynamic factors (see below and Table E.3). Systemic CSAF for goats/ruminants The toxicokinetic data on NAA (Table E.1 below) show that the peak plasma concentration in goats is over 20 times lower than in rats administered the same dose (25 mg/kg bw per day). The Area Under the Curve (AUC) is sixfold lower in goats than rats on day 1, and 33-fold lower on day 14. These data support a reduction in the interspecies toxicokinetic factor to 0.17 using the most conservative comparator of AUC on day 1 (see Table E.2 below). The low systemic exposure to NAA in goats appears to be due to its degradation in the ruminant digestive tract as there is no initial peak and being a small molecule extensive absorption of NAA would be expected. The ruminant digestive tract is reported to be consistent across ruminant species and degradation in the digestive tract is independent of absorption and distribution. Therefore, there would be expected to be little variation across the ruminant species. Suckling ruminants might be outliers in terms of the toxicokinetics of NAA as they have a less developed ruminant digestive system, but as NAA is not lipophilic and is unlikely to concentrate in milk, exposures to NAA via milk are not considered to be significant compared to those from direct consumption of feed. An interindividual toxicokinetic factor of 1 would be supported. In conclusion, an overall CSAF in ruminants would be 1.3 (0.17 32.5 3133.16). Table E.1: Results of the toxicokinetics of NAA in the plasma of goats, pigs and rats Compound Time point (h) Species, route of exposure, dose (mg/kg bw per day) Goat Pig Rat Capsule Feed (small amount) Gavage 25 25 25 500 Day 1 NAA (ng/mL) 0* 45 47 71 74 0.5 53 3,795 8,300 40,300 1 58 4,930 4,525 77,400 2 208 1,840 766 98,150 12 51 42 65 296 Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 55 EFSA Journal 2021;19(6):6610 Systemic CSAF for pigs/swine The toxicokinetic data on NAA (Table E.1) show that the peak concentration in pigs is lower, by a factor of <2, on days 1 and 14 than that in rats administered the same dose (25 mg/kg bw per day). The AUC is lower in pigs than rats on day 1, but higher (1.2-fold) on day 14. These data indicate that the toxicokinetics of NAA in pigs is essentially the same in pigs and rats and support a reduction in the interspecies toxicokinetic factor to 1. The toxicokinetics in pigs (assuming a body weight 30 kg) and Compound Time point (h) Species, route of exposure, dose (mg/kg bw per day) Goat Pig Rat Capsule Feed (small amount) Gavage 25 25 25 500 Cmax (ng/mL) –410 4,930 8,550 98,100 AUC 0–12 h (h.ng/mL) –1,700 8,545 10,160 314,335 Tmax (h) –4 1 0.25–0.5 2 Aspartic acid (ng/mL) $ 0* 1,245 1,555 6,900 3,970 0.5 993 1,415 4,530 4,680 1 855 1,460 5,120 8,080 2 1050 2,120 4,000 5,950 12 970 1,650 4,300 3,830 Day 14 NAA (ng/mL) 0* 52 51 70 112 0.5 53 4,700 9,915 52,300 1 58 7,240 3,650 97,000 2 130 2,570 4,400 98,500 12 56 58 74 400 Cmax (ng/mL) –171 7,200 13,000 98,500 AUC 0–12 h (h.ng/mL) –625 12,430 10,300 318,350 Tmax (h) –2–4 1 0.25–0.5 2 Aspartic acid (ng/mL) 0* 1,415 1,550 4,730 1,260 0.5 914 1,170 5,500 3,420 1 952 2,520 6,060 5,430 2 1,015 2,000 5,690 3,140 12 1,150 2,115 5,620 1,720 *: Sample taken prior to dosing, representing the background concentration.. $: Aspartic acid is reported to be the primary metabolite of NAA and is itself rapidly metabolised. AUC –Area Under the Curve –an integration of the concentration in plasma over time. Cmax –The peak concentration in plasma –modelled to cover changes between sampling times. Tmax –The time at which Cmax occurs. Table E.2: Summary comparison of the mean toxicokinetic values of NAA on rats, goats and swine Species Dose (mg/kg/bw per day) Goat Pig Rat 25 25 25 500 Day 1 NAA Cmax (ng/mL) 410 (0.05)* 4,930 (0.6) 8,550 98,100 NAA AUC 0–12 h (h.ng/mL) 1,700 (0.17) 8,545 (0.84) 10,160 314,335 Day 14 NAA Cmax (ng/mL) 171 (0.013) 7,200 (0.55) 13,000 98,500 NAA AUC 0–12 h (h.ng/mL) 625 (0.06) 12,430 (1.2) 10,300 318,350 *: Expressed as proportion of rat value at 25 mg/kg bw per day. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 56 EFSA Journal 2021;19(6):6610 rats (assuming a body weight 200 g) are similar and the key metabolic step(s) in the degradation of NAA is likely to be simple, as it is water soluble and would not require conjugation for excretion. NAA is a normal component of the blood of rats, goats and pigs and it is considered reasonable to assume that there will be no significant differences in the toxicokinetics of NAA between pigs and other swine. Suckling animals might be outliers in terms of the toxicokinetics of NAA, but as NAA is not lipophilic and is unlikely to concentrate in milk, exposures to NAA via milk are not considered to be significant compared to those from direct consumption of feed. A reduction in the interindividual toxicokinetic factor to 1 is proposed. In conclusion, an overall CSAF for swine would be 8 (1 92.5 9193.16). Other monogastric animals As data are available only from two monogastric animals (rats and pigs), it is considered that any change from the default values for extrapolating from pigs to different species of monogastric animals is not appropriate, as it is not supported by any specific data. Therefore, an overall CSAF for other monogastric animals would be 25 (2.5 93.16 9193.16). Table E.3: Outline of the derivation of the overall CSAFs based on the toxicokinetic studies and potential modes of action behind the effects of NAA on salivary glands Rat to standard species* Standard species to different life stages or related species Overall CSAF Toxicokinetic Toxicodynamic Toxicokinetic Toxicodynamic Default 4 2.5 3.16 3.16 100 Direct Action $ 1 2.5 1 3.16 8 Systemic action Ruminant 0.17 2.5 1 3.16 1.3 Swine 1 2.5 1 3.16 8 Other monogastric 1 2.5 3.16 3.16 25 *: Standard species are those used in the toxicokinetic studies i.e. goat for ruminants; pigs for swine and other monogastric animals. $: Considered to be the more likely mode of action for the salivary gland hypertrophy. Assessment of GM oilseed rape 73496 www.efsa.europa.eu/efsajournal 57 EFSA Journal 2021;19(6):6610