GUIDANCE OF EFSA. European Food Safety Authority 2, 3

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1 GMO Unit version 2 EFSA Journal 2011;9(7):2311 GUIDANCE OF EFSA EFSA guidance on the submission of applications for authorisation of genetically modified food and feed and genetically modified plants for food or feed uses under Regulation (EC) No 1829/ European Food Safety Authority 2, 3 European Food Safety Authority (EFSA), Parma, Italy This guidance, published on 6 July 2012, replaces the earlier version published on 22 July ABSTRACT This document is intended to provide guidance to applicants submitting under Regulation (EC) No 1829/2003 an application for authorisation of genetically modified food and feed and genetically modified plants for food and feed uses including cultivation in the European Union. It complements two recently updated guidance documents of the EFSA Panel on genetically modified organisms, the Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants. This EFSA submission guidance describes the community procedures in the European Union for processing such applications, and provides instructions to applicants on how to prepare and present data in an application to be submitted to EFSA. It is supplemented with ten appendices such as examples of data presentation and a completeness checklist to be filled by the applicants. Instructions described in this EFSA guidance are applicable to applicants submitting under Regulation (EC) No 1829/2003 an application for renewal of authorisation of existing products produced from genetically modified plants. European Food Safety Authority, 2011 KEY WORDS Application, submission, EFSA guidance, genetically modified, GM plants, GMO, Regulation (EC) No 1829/2003, renewal 1 On request from EFSA, Question No EFSA-Q , approved on 7 July Correspondence: gmo@efsa.europa.eu 3 Acknowledgement: EFSA wishes to thank its scientific officers Yi Liu, Nancy Podevin, Christina Ehlert and Karine Lheureux for drafting, its staff, Jaime Aguilera, Per Bergman, Anna Christodoulidou, Yann Devos, Zoltan Diveki, Antonio Fernandez Dumont, Andrea Germini, Luana Giorgini, Ana Gomes, Sonia Hernandez-Valero, Sylvie Mesdagh, Claudia Paoletti, Reinhilde Schoonjans, Mara Todeschi, Ellen Van Haver, Elisabeth Waigmann for providing comments. This guidance has been reviewed by Detlef Bartsch, Gijs Kleter and Huw Jones. 4 Modifications in version 2 include: main text - editorial changes; Appendix B - separate MC requirements for GM plants containing single or stacked events, and editorial changes in FF and ENV parts, Appendix H - replacement; Appendix J newly added. Suggested citation: European Food Safety Authority; Title of the guidance. EFSA Journal 2011;9(7):2311. [27 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2011

2 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 SUMMARY This EFSA guidance is intended for applicants submitting under Regulation (EC) No 1829/2003 an application for authorisation of genetically modified food and feed and genetically modified plants for food and feed uses including cultivation in the European Union (EU). It consists of six chapters. Chapter 1 describes the community procedure for processing such applications in the EU. Chapter 2 provides detailed instructions on how to structure an application and present data in the desired format. Chapter 3 explains specific requirements for different parts of an application, in particular, Parts I, II and VIII. Chapter 4 explains requirements specific to applications concerning GM plants containing stacked events. Chapter 5 specifies the applicability of this EFSA guidance to renewal applications notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003. This EFSA guidance is accompanied by ten appendices: Appendix A describes contact details of applicants for each application; Appendix B is a completeness checklist, which should be filled out by the applicant; Appendix C provides exemplar tables to summarise scientific information and exemplar figures for data presentation; Appendices D-G refer to data provided for the environmental risk assessment; Appendix H is the proof of reception by EU Reference Laboratory for GM Food and Feed; and Appendix I provides the format of the summary of applications. Appendix J outlines the standardised form, which Part II of an application should follow. These appendices should be used by the applicant and by EFSA to ensure: (i) that all required information and all documents, as laid down by EFSA, are provided; and (ii) that an application is in accordance with the recommended structure and format. This EFSA guidance is available on the EFSA website. Like all EFSA guidance documents, this EFSA guidance will be updated in the future in accordance to relevant changes in legislation and/or in accordance with EFSA procedures and guidance documents under Regulation (EC) No 1829/2003. EFSA Journal 2011;9(7):2311 2

3 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 5 Terms of reference as provided by EFSA... 5 Guidance Application procedure in the EU Submission to a national competent authority of the Member States Submission to an institute to develop certified reference materials of the GMO Submission to the European Union Reference Laboratory for GM Food and Feed Receipt of the application by EFSA Completeness check by EFSA Validation of application by EFSA Risk assessment, MS comments and request for additional information Member States comments Request for additional information Adoption of a scientific opinion by the EFSA GMO Panel Networking with Member States on applications with scope seeds and plant propagating material for cultivation in the EU EFSA overall opinion Withdrawal of an application by the applicant Application preparation Structure of an application Submission version Public access version Language Electronic version Format and label of the CD-ROMs File format, size and name Standard units and abbreviations Specifics on the different parts of the application Part I General information Part II Scientific information Content and requirement of Part II Data presentation figures and tables Citations and reference list Part III Cartagena protocol Part IV Labelling Part V Methods of detection, sampling and identification and reference material Part VI Additional information to be provided for GM plants and/or food/feed containing or consisting of GM plants Part VII Summary Part VIII -Administrative documents Letter of consent of access Completeness checklist Applications for GM plants containing stacked events Applications for renewal of authorisations of a GMO plant and/or its derived food and feed under Regulation (EC) No 1829/ Useful websites References Appendices A. Contact details B. Completeness checklist EFSA Journal 2011;9(7):2311 3

4 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 C. Exemplar figures and tables for Part II D. Schematic summary of data for field or greenhouse trial for agronomic and phenotypic characteristics within a season (GM plants containg single or stacked event(s)) E. Schematic summary of information for Insect Resistance Management F. Schematic summary of NTO studies G. Schematic summary of statistical design and analysis for each ERA related study H. Proof of acknowledgement of reception by EURL-GMFF I. Summary of applications J. Template form for Part II scientific information abbreviations EFSA Journal 2011;9(7):2311 4

5 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 BACKGROUND Regulation (EC) No 1829/ lays down community procedures in the EU for the authorisation and supervision of genetically modified (GM) food and feed, as well as for the labelling of such food and feed. Regulation (EC) No 1829/2003 requires that food and feed derived from GM plants should be authorised for placing on the community market only after a scientific evaluation of the highest possible standard, to be undertaken under the responsibility of the European Food Safety Authority (EFSA), of any risk they may pose to human and animal health and to the environment. In accordance with Articles 5(8), 11(6), 17(8) and 23(6) of Regulation (EC) No 1829/2003, EFSA is required to publish detailed guidance to assist applicants in the preparation and the presentation of an application for authorisation. The EFSA GMO Panel published in 2004 and updated in 2006 the Guidance document of the scientific panel on genetically modified organisms (GMO) for the risk assessment of genetically modified plants and derived food and feed, of which Annex I to IV provided instructions on the presentation of applications for the request of authorisation of GM plants and/or derived food and feed (EFSA, 2006b). Recently the EFSA GMO Panel updated two guidance documents, the Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a), recommending scientific criteria necessary for the risk assessment on GM plants and derived products for food and feed uses. To complement these two guidance documents of the EFSA GMO Panel in support to applicants submitting an application, EFSA developed this EFSA guidance to applicants on the preparation and presentation of applications for authorisation of genetically modified plants and derived food and feed under Regulation (EC) No 1829/2003. TERMS OF REFERENCE AS PROVIDED BY EFSA The EFSA guidance provides assistance to applicants for the preparation and presentation of an application for authorisation of GM plants and derived food and feed under Regulation (EC) No 1829/2003 for food and feed uses, import and processing, seeds and plant propagating material for cultivation in the EU. It also provides assistance to applications for renewal of authorisation of existing products produced from GM plants submitted under Regulation (EC) No 1829/2003. It provides information on, for example, the structure of an application, naming of documents, the presentation of reports, data in figures and tables, confidential and non confidential information. The document is complemented by a completeness checklist. The completeness checklist reflects the requirements for applications as outlined in two recently updated guidance documents of the EFSA GMO Panel on genetically modified plants. This completeness checklist will be used by applicants and by EFSA to ensure (i) that the application is in accordance with the recommended structure and formal requirements (ii) that all required information and all documents which are required by EFSA are provided. The applicant should submit a completed checklist upon submission of an application under Regulation (EC) No 1829/2003. SCOPE OF THE EFSA GUIDANCE This EFSA guidance on the submission of applications for authorisation of genetically modified food and feed and genetically modified plants for food or feed uses under Regulation (EC) No 1829/2003 complements with administrative guidelines two recently updated guidance documents of the EFSA GMO Panel, the Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a). 5 Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, , p EFSA Journal 2011;9(7):2311 5

6 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 This EFSA guidance also complements the EFSA GMO Panel s Guidance for renewal of authorisations of existing GMO products lawfully placed on the market, notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003 (EFSA, 2006a). EFSA Journal 2011;9(7):2311 6

7 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 GUIDANCE 1. Application procedure in the EU One of the objectives of Regulation (EC) No 1829/2003 is to lay down community procedures in the EU for the authorisation and supervision of genetically modified food and feed. The process of an application under Regulation (EC) No 1829/2003 is illustrated in Figure 1.The steps are explained in sections 1.1 to Submission to a national competent authority of the Member States In accordance with Articles 5(2) and 17(2) of Regulation (EC) No 1829/2003, an applicant shall submit its application to the national Competent Authority of a Member State (MS CA) (one paper copy and one copy in electronic format [CD-ROM]). In accordance with Articles 5(2) and 17(2), the MS CA shall acknowledge receipt of the application in writing to the applicant within 14 days of its receipt. The acknowledgement shall state the date of receipt of the application. The MS CA shall, without delay, inform EFSA and make the application and any supplementary information supplied by the applicant available to EFSA Submission to an institute to develop certified reference materials of the GMO In accordance to Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003, reference materials must be developed. The applicant shall submit samples of the food and feed and their controls to the institute that will be responsible for the production of certified reference materials (CRM). A statement that the certified reference materials are produced in accordance to Annex II of the Regulation 641/2004 should be mentioned in the application under Part V (see section 3.5) Submission to the European Union Reference Laboratory for GM Food and Feed In accordance with Article 32 and the Annex of the Regulation (EC) No 1829/2003, the European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), formerly named Community reference laboratory, is the Commission s Joint Research Centre, responsible for the validation of methods for sampling, identification and detection of the GM food and feed. After evaluation, the EURL-GMFF submits full evaluation reports to EFSA. The EURL-GMFF examines the completeness of information related to the presence of samples and detection methods. Information and requirements of the EURL-GMFF can be consulted at its website. During the completeness check (see section 1.6), EFSA verifies that a proof of submission of the samples, reagents and methods to the EURL-GMFF is provided in the application. Therefore EFSA recommends the applicant to submit documents and samples to EURL-GMFF before submitting an application to the MS CA, so that the proof of reception by the EURL-GMFF can be immediately included in the application (see Appendix H). EFSA Journal 2011;9(7):2311 7

8 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 Step 1 (a) Applicant submits an application to a MS CA (b) applicant submits samples to an institute for certification (c) Applicant submits detection method and GMO samples to EURL-GMFF Step 2 MS CA forwards application to EFSA 1a MS CA 2 Applicant 1b 1c Assigned Institute EURL-GMFF EC Annex E: Certified reference material report Annex D1: Validity report Annex D2: Validity method report Annex D3: Sampling and extraction report Step 3 (a) EFSA informs EURL-GMFF, EC, all MS CA and public of submission (b) EFSA calls for expression of interest from all ERA MS CA EFSA 4a 3a 3b all MS CA Step 4 (a) EFSA performs completeness check and requests additional information (b) Applicant provides additional information Step 5 EFSA validates application 4a Applicant 4b EFSA all ERA MS CA Completeness check phase Public 5 Risk assessment phase Step 6 Applicant submits validated application to EFSA Applicant 6 Annex B: Cartagena protocol report Annex C: Labelling proposal Annex F: Post-market monitoring plan Step 7 (a) EFSA and its GMO Panel start scientific evaluation (b) EFSA opens 3-months consultation period for all MS CA (c) ERA MS CA starts scientific evaluation 7b all MS CA EFSA 7a 7c Step 8 MS CA provides scientific comments to EFSA and its GMO Panel 8 GMO Panel ERA MS CA Step 9 (a) EFSA GMO Panel requests additional information (b) ERA MS CA requests additional information 9a 9a Applicant 9b 9b Step 10 Applicant provides additional information to EFSA GMO Panel ERA MS CA 11a Annex H: MS CA ERA report Step 11 (a) ERA MS CA delivers its ERA report (b) EFSA GMO Panel adopts scientific opinion 11b Annex A: EFSA GMO Panel scientific opinion Annex G: Answers to all MS CA comments Step 12 (a) EFSA publishes overall opinion (b) EFSA sends overall opinion to EC and EURL-GMFF, informs all MS CA and applicant EFSA 12b EC, EURL-GMFF all MS CA, applicant 12a Risk management phase Overall opinion Figure 1: Overview of steps in processing of an application under Regulation (EC) 1829/2003. This figure is organized in three parts: The left part consists of grey boxes to explain steps in sequential order. The centre flowchart depicts the process with arrows indicating information flow, blue arrows indicating steps for an application, green arrows indicating additional steps for an application with scope seeds and plant propagating material for cultivation in the EU. The right part consists of blue boxes to describe outputs with dashed lines linking the author to a respective output. Note that not all steps are applicable to every application. EC: European Commission, ERA: environmental risk assessment, EURL-GMFF: European Union Reference Laboratory for GM Food and Feed, MS CA: national Competent Authority of a Member State. EFSA Journal 2011;9(7):2311 8

9 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/ Receipt of the application by EFSA Correspondence to EFSA concerning GM plant applications should be addressed to: European Food Safety Authority Dr. Karine Lheureux Head of Applications Desk unit Via Carlo Magno 1A Parma Italy Since November 2011, the Applications Desk unit is responsible for the registration of regulated product applications in EFSA, and it is the point of contact for applicants until an application is made valid. In accordance with Articles 5(2) and 17(2) of Regulation (EC) No 1829/2003, once the MS CA forwards an application to EFSA, EFSA acknowledges the receipt of the application to the MS CA. EFSA, without delay, informs the other MS CA and the European Commission (EC). EFSA endeavours to make the summary of the application available to the public through the Register of Questions within two weeks following the reception. EFSA makes the summary available via an electronic secure system called EFSA GMO Extranet to the members of the EFSA GMO Panel and its standing application assessment Working Groups (WGs), European Commission and the MS CA who performs the initial environmental risk assessment (ERA) for applications with scope seeds and plant propagating material for cultivation in the EU Completeness check by EFSA EFSA examines the submitted application in accordance with Regulation (EC) No 1829/2003, in particular Articles 5(3), 5(5), 17(3) and 17(5). The EFSA GMO unit together with the Applications Desk unit check the completeness of an application (Figure 1) and validate the application only if the application fulfils the administrative requirements stipulated in this EFSA guidance, and the scientific requirements and principles stipulated in the EFSA GMO Panel s Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a). To streamline the submission process an applicant is required to fill in a completeness checklist (see section and Appendix B). EFSA endeavours to have the first outcome of the completeness check available within 30 working days from the receipt date of the application. At that moment, the applicant will be informed about an identification code. This identification code should be included in all further correspondence with EFSA, the EURL and European Commission regarding the application. The completeness check process might require further exchange of information between the applicant and EFSA. In such case, EFSA informs the applicant if certain parts of the application need to be modified or completed in order for the application to be validated. After receiving a request for additional information, the applicant should within 30 working days submit the response or inform EFSA by letter by when a comprehensive response will be provided. If the indicated timeline cannot be met, the applicant should inform EFSA as soon as possible of another date. When responding to EFSA questions, the applicant should submit an updated version of the entire application (Parts I to VIII) in the form of CD-ROM(s). EFSA advises that the submission of an updated application is accompanied by a cover letter wherein the applicant precisely describes how each EFSA question was addressed. All missing information must be incorporated in the respective relevant parts of the application. If the applicant wishes to make modifications to the application other than those requested, these additional changes must be indicated in the applicant s cover letter to EFSA. The updated application will replace the previous submission. EFSA endeavours to inform the EFSA Journal 2011;9(7):2311 9

10 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 applicant within 15 working days if the updated application is complete or if certain parts still need to be addressed Validation of application by EFSA Once the application fulfils all requirements, EFSA issues a statement of validity to the applicant. The valid application is made available via an electronic secure EFSA GMO Extranet to all MS CA and the EURL-GMFF. Upon validity, EFSA updates the Summary (Part VII) on the publically accessible EFSA Register of Questions. With this statement, the applicant is requested to submit one paper copy of the valid application and one electronic copy of the public access version (see section 2.2) to EFSA. The applicant shall confirm by letter that this paper copy is identical to the electronic validated version of the application. At this stage, EFSA does not accept any further modifications of the application. EFSA may request additional electronic and paper copies of the valid version. As stated in the validity statement, after validation, the EFSA GMO unit becomes the point of contact for applicants who have questions regarding their applications. All information provided by the applicant is available on the EFSA GMO Extranet. On a weekly basis EFSA informs all registered members of the EFSA GMO Extranet via about updates concerning all applications. This includes all correspondence such as declarations of validity, questions sent to the applicant and responses received, calls for expression of interest to all MS CA designated in accordance with Article 4 of Directive 2001/18/EC 6 for the initial ERA of applications with scope seeds and plant propagating material for cultivation in the EU Risk assessment, MS comments and request for additional information In accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, EFSA shall endeavour to respect a time limit of six months, from the validity date of an application to the publication of the EFSA overall opinion in the EFSA Register of Questions (see section 1.9). From the date of validity, the application enters the risk assessment phase by the EFSA GMO Panel in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003. The scientific assessment of Part II of the application is carried out by three WGs of the EFSA GMO Panel: Molecular Characterisation (MC), Food and Feed (FF), and Environment (ENV). EFSA endeavours to list the application in the agenda of the first or second WG meetings following the statement of validity. The outcomes of the discussions are summarized in the respective WG meeting minutes. EFSA endeavours to send the first questions identified by these three WGs within two and half months after the date of validity Member States comments Within three months following the date of validity, all MS CA can submit to EFSA, via the EFSA GMO Extranet, comments or questions on the valid application under assessment. The three WGs of the EFSA GMO Panel will consider all MS comments submitted during this consultation period and provide a response to each individual comment. These will be published as Annex G of the EFSA overall opinion (see section 1.9) Request for additional information The three WGs of the EFSA GMO Panel may request additional information from the applicant in order to clarify a scientific issue of the risk assessment. The WGs will provide a rationale when sending questions to the applicant. A question from the WGs will not be reiterated. The EFSA GMO Panel will adopt a scientific opinion on the basis of the information available. 6 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 Commission declaration. OJ L 106, , p EFSA Journal 2011;9(7):

11 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 As outlined in Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, the request for additional information will extend the six-month time limit (known as the stop the clock mechanism). After receiving a request for additional information, the applicant should, within 30 working days, submit the response or inform EFSA by letter by when a comprehensive response will be provided. If the indicated timeline cannot be met, the applicant should inform EFSA as soon as possible of another date. Each time an applicant submits additional information, the accompanying cover letter should clearly indicate whether the additional information contains confidential information. The additional information should be provided in electronic form and should contain a public access version if confidential information is included (see section 2.1.2), complying with the requirements stipulated in this guidance document. In addition, the overview tables of studies and relevant figures (e.g. a breeding scheme) should be updated (see Appendix C). A request for additional information may address several parts of an application. The applicant is asked to provide one complete answer in the form of a data package addressing all issues raised. Once EFSA receives the additional information, EFSA endeavours to discuss it in the following WG meeting(s). If the additional information does not raise further questions, the clock will be restarted for the remaining time of the six-month period. EFSA will inform the applicant in writing. Applicants can contact the EFSA GMO unit regarding particular questions, should scientific clarification be needed. In addition to scientific requests from the EFSA GMO Panel, additional information may be requested by EFSA or the EURL-GMFF. EFSA will stop the clock for the clarification on or provision of any elements required under Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003. Requests for additional information may also come from the MS CA carrying out the initial evaluation of the ERA for applications with scope seeds and plant propagating material for cultivation in the EU. In this case, this MS CA asks EFSA to stop the clock with additional questions to the applicant. EFSA then proceeds with the request and informs the applicant in writing and including the letter of this MS CA as an annex Adoption of a scientific opinion by the EFSA GMO Panel During the risk assessment phase of an application, the WGs prepare a scientific opinion which is discussed, amended and adopted by the EFSA GMO Panel at plenary meetings. EFSA endeavours to publish the scientific opinion in the EFSA Journal within three weeks from the date of adoption Networking with Member States on applications with scope seeds and plant propagating material for cultivation in the EU If an application concerns the scope GM plants to be used as seeds or other plant-propagating material for cultivation in the EU, EFSA shall, in accordance to Articles 6(3) and 18(3) of Regulation (EC) No 1829/2003, asks a MS CA to carry out the ERA. For such applications, EFSA will call for expression of interest to all MS CA designated in accordance with Article 4 of Directive 2001/18/EC. EFSA will select a MS CA on the basis of the following criteria: (i) experience in environmental risk assessment; (ii) experience in writing national risk assessment reports; (iii) interest in the crop/trait; and (iv) availability. If no MS CA expresses interest to perform the environmental risk assessment, a formal request will be addressed to the MS CA to which the application was originally submitted. The selected ERA MS CA will carry out the initial ERA and will work in close contact with EFSA staff and with the EFSA GMO Panel. The ERA MS CA is requested to follow the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a). After finalising its evaluation, the ERA MS CA submits its report to EFSA. This report will be carefully considered and integrated during the further assessment by the EFSA GMO Panel before adopting its scientific opinion, and will be the Annex H of the EFSA overall opinion (see section 1.9). EFSA Journal 2011;9(7):

12 1.9. EFSA overall opinion EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 The EFSA overall opinion is available when all parts, as mentioned in Article 6(5) and 18(5) of Regulation (EC) No 1829/2003, are finalised. In accordance with Articles 6(7) and 18(7) of Regulation (EC) No 1829/2003, EFSA makes its overall opinion available to the public through its Register of Questions. This overall opinion includes the scientific opinion of the EFSA GMO Panel, describing its evaluation of the food, feed and/or the environmental risk assessment of the GM plant, stating the rationale for its opinion and the information on which this opinion is based. The overall opinion also includes the comments received from the MS CA and the individual responses provided by the EFSA GMO Panel. The following annexes are included where applicable: Annex A Annex B Annex C Annex D1 Annex D2 Annex D3 Annex E Annex F Annex G Annex H Scientific opinion of the EFSA GMO Panel Compliance report for the Cartagena Protocol (from the applicant) Labelling proposal (from the applicant) Validation report (from EURL-GMFF) Validated method report (from EURL-GMFF) Sampling and extraction report (from EURL-GMFF) Certified Reference Materials report (from the assigned institute) Monitoring plan (from the applicant) Comments from all MS CA and replies of the EFSA GMO Panel on scientific issues MS CA report of the ERA on applications with scope seeds and plant propagating material for cultivation in the EU In accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003, EFSA sends the overall opinion to the European Commission and EURL-GMFF, and informs all MS CA and the applicant. The application now enters the risk management phase including EU authorisation. Status of the decision on authorisation can be found in the EU register of genetically modified food and feed Withdrawal of an application by the applicant If an applicant wishes to withdraw an application during completeness check phase or risk assessment phase, the applicant should send a letter to EFSA with the request for withdrawal, copying the European Commission and the MS CA to which the application was originally submitted. This letter will be made available to the public in the EFSA Register of Questions. 2. Application preparation 2.1. Structure of an application An application for the authorisation of GM food and feed and GM plants for food and feed uses in the EU should be submitted as a paper and an electronic (CD-ROM) copy. An application should consist of eight parts, as defined by Regulation (EC) No 1829/2003 (see Table 1). Documents should be named and organised as the structure illustrated in Table 2. Please note that EFSA does not accept parts of an application submitted by different applicants, nor does EFSA compile information submitted by different applicants to obtain one complete document or application. EFSA Journal 2011;9(7):

13 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 Table 1: Overview with cross-references between the different parts of the application and the Regulation (EC) No 1829/2003 Part I: General information Part II: Scientific information (Technical Dossier) Part III: Cartagena Protocol Part IV: Labelling proposal Part V: Methods of detection, sampling and identification and reference material Part VI: Additional information Part VII: Summary Part VIII: Administrative documents Parts Regulation (EC) No 1829/2003 Articles 5&17 (3)(a)(b)(d)(e)(f)(h)(k); Articles 5&17 (5)(a)(b) Articles 5&17 (3)(c) Articles 5&17 (3)(f) (g); Articles and Articles Articles 5&17 (3)(i)(j) Articles 5&17 (5), more specifically Annex IV of Directive 2001/18/EC Articles 5&17 (3)(l) Table 2: Overview of the required structure and folder/file names Folder name File name and sub-folder name Part_I_General_info General_info.pdf Part_II_Scientific_info Main_text_Application_identification code.pdf PMEM_Plan.pdf References 1 Part_III_Cartagena_Protocol Appendices 2,3 Part_IV_Labelling Labelling.pdf Part_V_Sampling and Detection Part_VI_Additional_info ERA Appendices D to G Cartagena.pdf Sampling and Detection.pdf CI Additional info.pdf Part_VII_Summary Summary_Application_number.pdf Part_VIII_Administrative_doc See section All published documents cited in the main text of the application shall be present in subfolder References and formatted as indicated in section All unpublished documents provided by the applicant and cited in the main text of the application shall be present in the subfolder Appendices and formatted as indicated in section In case unpublished studies of the applicant are classified as CI and non-ci, two sub-folders should be provided: Appendices (CI) and Appendices (non-ci). If the Appendices folder is not labelled with CI or non-ci, all documents within that folder will be considered being non-ci Submission version An application received by EFSA should consist of one paper and one electronic copy (see section 2.4). The electronic copy should contain all information, and documents be structured as indicated in Table 2. The applicant can choose to divide confidential (CI) and non-confidential (non-ci) information to separate CD-ROMs, or to include them in the same CD-ROM. Each CD-ROM containing confidential information should be labelled as described in section 2.4. The folders should be named and organized as indicated in Table 2. In case CD-ROM is password protected, the password(s) can be provided separately. For the paper copy the applicant can choose not to print legal references (e.g. Directive 2001/18/EC, Regulation (EC) No 1829/2003, etc.), consensus documents (e.g. Organisation for Economic Cooperation and Development (OECD) consensus documents, Codex alimentarius, etc.), EFSA outputs EFSA Journal 2011;9(7):

14 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 (e.g. scientific opinions and statements published previously by the EFSA GMO Panel), and scientific articles published in peer-reviewed journals. Confidential information: The applicant shall clearly state which parts of the application are claimed to be confidential in accordance with Article 2(3) of Regulation (EC) No 641/2004, together with a verifiable justification in accordance with Article 30 of Regulation (EC) No 1829/2003. In accordance with Article 30 of Regulation (EC) No 1829/2003, after consultation with the applicant, the European Commission will determine which information can be kept confidential and inform the applicant and EFSA about the confidentiality decision. The main text of the application itself cannot contain any confidential information. Sections or studies considered as confidential by the applicant should be identified by including CI in brackets in the file name, e.g. Appendix x (CI).pdf and CONFIDENTIAL on the corresponding pages. If the author s names are claimed as confidential, they should not be included in the file name and citation (see section 3.2.3). Each time an applicant submits additional information, it should be clearly indicated in an accompanying cover letter whether the additional information contains confidential information Public access version In accordance with Regulation (EC) No 1049/ from the date of validity, EFSA will, on request, grant public access to the non-confidential parts of an application without prior consultation of the applicant. Therefore, upon validation of the application, the applicant shall provide EFSA with a CD- ROM containing the public access version of the application. The recommended name for the CD-ROM is Public_Access_Application identification code. The public access version of the application must follow the same structure as the original application (see Table 2). The public access version should not contain confidential information, and should, otherwise, be identical to the validated electronic version. During the EFSA risk assessment phase, when submitting additional information to EFSA, if the additional information contains confidential information, a public access version of the additional information shall be submitted. Upon the confidentiality decision by the European Commission, the applicant shall provide EFSA with a CD-ROM containing the final public access version of the application. The CD-ROM should indicate the date of the confidentiality decision Language An application, including technical reports and scientific publications, shall be written in idiomatic English. The text should be carefully checked for errors. Peer-reviewed articles and published reports in languages other than English should be accompanied by translations of the relevant parts Electronic version Format and label of the CD-ROMs The provided CD-ROM(s) should be clearly labelled and include the following information: name of the GM plant event and plant species; EFSA application identification code (once provided) name of company; 7 Regulation (EC) No 1049/2001 of the European Parliament and of the Council of 30 May 2001 regarding public access to European Parliament, Council and Commission documents. OJ L 145, , p EFSA Journal 2011;9(7):

15 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 date of submission; submission type: o first submission (CC1) o updated versions (CC2, CCx) o valid version o additional information; CI, non-ci, or public access version; CD-ROM number (applicable only if more than one CD-ROM is submitted per application, e.g. CD-ROM 1 of 2 ) File format, size and name All documents cited in Part I and Part II should be provided preferably as portable document format (PDF), should be accessible to allow reading, printing, word searching and copying of text from the file using Adobe Acrobat Standard (version 7.0 or later) software. Text and figures of all parts of an application should be fully legible. Other software format types, such as Word, Excel and GenBank, are acceptable for specific files. In all cases, they should fulfil the same criteria as required for PDF files. Sequence information is preferably submitted in GenBank format and includes the annotation information. The size of single documents should be limited to 25 MB. In case a study report combines multiple analyses with extensive data, making it exceed 1000 pages, the applicant should consider separating the outcome of the different analyses into several independent documents. If this is not possible the applicant can, for the paper version, submit the study report without appendices that contain e.g. raw data. When needed, EFSA will request the applicant to submit a paper copy for the whole study report. The documents should be formatted for standard DIN A4 (210 x 297 mm) paper. The recommended font of text is Times New Roman or Arial, points for normal text and 9-10 points for footnotes. All fonts used in the document should be embedded in the PDF files to ensure that they are always readable and searchable. The standardised naming of the CI and non-ci folders and files of an application is recommended in Table 2. When no standard name is recommended, the file name should be concise and informative of its content including the name of the event, and contain no more than 40 characters including spaces. File and folder names should not include the following special characters: \ / : *? \" < > #. All documents should be well structured and include a table of content. On each page of the application, the file name, company name, GM plant event name, and page number should be included in the header or footer. To improve navigation through PDF documents the use of bookmarks and hyperlinks is encouraged Standard units and abbreviations The International System of Units (SI) 8 must be used. For the naming of chemical compounds and for chemical quantities, units and symbols, the applicants should follow the International Union of Pure and Applied Chemistry (IUPAC) nomenclature 9. Acronyms and abbreviations should be defined the first time they are mentioned. A list of abbreviations should be included at the beginning of Part II of the application. Gene and protein names should respect nomenclature and style of the relevant species EFSA Journal 2011;9(7):

16 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 For a herbicide or other chemical substances, both trade name and the active substance should be indicated. For a GM event it is advisable to use only the event name in the Part II Scientific information, and to include both the event name and its trade name in the Part VII Summary (see Appendix I). 3. Specifics on the different parts of the application 3.1. Part I General information Part I of an application should contain the following information: 1. Name and address of the applicant (company or institute); 2. Name and qualification of the responsible scientist(s) and contact details 10 of the responsible person for all dealings with EFSA; 3. Designation and specification of the GM plant and derived product; 4. Scope of the application (a) GM food Food containing or consisting of GM plants Food produced from GM plants or containing ingredients produced from GM plants (b) GM feed Feed containing or consisting of GM plants Feed produced from GM plants (c) GM plants for food or feed uses Products other than food and feed containing of consisting of GM plants with the exception of cultivation Seeds and plant propagating material for cultivation in the EU Where an application is limited to either food or feed use, it shall contain a verifiable justification explaining why the authorisation shall not cover both uses in accordance with Article 27 of Regulation (EC) No 1829/2003. For an application of a GM plant containing stacked events the applicant should seek confirmation with the European Commission regarding the scope of the application concerning the subcombinations. 5. Unique identifier A proposal for a unique identifier for the GM plant and derived products in question, developed in accordance with Commission Regulation (EC) No 65/ Where applicable and where relevant to the risk assessment, a detailed description of the method of production and manufacturing. 10 The applicant is responsible to notify EFSA any change regarding the contact person or any of the contact details during the evaluation period. EFSA Journal 2011;9(7):

17 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 This would include, for example, a description of methods used to process the GM plant materials during the preparation of food/feed, food/feed ingredients, food/feed additives or food flavourings. 7. Where appropriate, the conditions for placing on the market the food(s) or feed(s) produced from it, including specific conditions for use and handling Part II Scientific information Content and requirement of Part II The scientific content of chapters and sections in the document Main_text_Application_identification code.pdf (see Table 2) should comply with the requirements of the EFSA GMO Panel s Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a). Scientific guidance on specific topics can be consulted in the following scientific opinions of the EFSA GMO Panel: Guidance on selection of comparators for the risk assessment of genetically modified plants and derived food and feed (EFSA, 2011b). Guidance on the Post Market Environmental Monitoring (PMEM) of genetically modified plants (EFSA, 2011c). Statistical considerations for the safety evaluation of GMOs (EFSA, 2010b). Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed (EFSA, 2010c). Scientific opinion on the assessment of potential impacts of genetically modified plants on non-target organisms (EFSA, 2010d). Scientific opinion on guidance for the risk assessment of genetically modified plants used for non-food or non-feed purposes (EFSA, 2009). Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials (EFSA, 2008). The numbering of chapters and sections should follow the one indicated in the template form (see Appendix J). At the beginning of the main text of the application, the applicant should include an overview table of all studies and reports carried out in support to the submitted application. An example of such an overview table is provided in Tables 1 of Appendix C. Part II of an application shall contain all scientific data pertinent to the investigated GM plant and/or its derived food and feed (published and unpublished). The assessment of the GM plant, provided in the application, forms the basis for the evaluation by the EFSA GMO Panel. Care should be taken that all the information presented in main text, appendices, tables and figures is coherent. If a requirement of the guidance document(s) does not apply for certain part(s) of an application, the applicant should justify the omission of such data. For GM plants containing single events, the Part II should be a complete stand-alone document containing all information required for a full risk assessment of the product(s) in question. In accordance with Articles 5(3)(e) and 17(3)(e) of Regulation (EC) No 1829/2003, all published and unpublished studies shall be provided in Part II and clearly cited in the main text. For GM plants containing stacked events, all relevant information related to its single events should be summarised in each section of the main text, and clear reference to the study report of the single event(s) underpinning the conclusion should be included. EFSA Journal 2011;9(7):

18 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 The applicants are requested to complement an application with relevant scientific findings that are published in peer-reviewed journals during the risk assessment phase by EFSA. Assessors should not be required to undertake any additional literature reviews, to assemble or process data in order to evaluate an application. Data provided in support of an application should have the same quality as those submitted to peerreviewed journals. Particular attention should be paid to the sensitivity and specificity of methods employed and to the adequacy and appropriateness of controls or reference points in the comparative assessment. To ensure the quality of the studies it is advisable, where available, to use internationally agreed protocols and test methods (EFSA, 2011a). Compliance to international quality standard (e.g. Good Laboratory Practice) and international guidelines (e.g. OECD) should be stated where applicable. Detailed descriptions of all studies provided by the applicant, including experimental methods and study design, should be provided. The detailed information can be appended to the study report or provided as a separate report. The raw data and the programming code used for the statistical analysis for the comparative assessment should be submitted as part of the application dossier and should be given in an editable form (EFSA, 2011a). For the output of the bioinformatic searches of the flanking sequences it is acceptable that only the alignments for the best hundred hits are displayed if the query, algorithm, database is chosen in such a way that all relevant similarities for risk assessment are within the first hundred hits. In addition, when the number of hits is greater than one hundred, the applicant should indicate that all hits were screened to confirm that all relevant hits for risk assessment are displayed. Appendices D to G of this document refer to the data in support of the environmental risk assessment of a GM plant. The level of detail required in the ERA will depend upon the characteristics of the GM plant and the scope of the application and routes of environmental exposure. Appendix D1 or D2 should be compiled for each field trial conducted for the comparative analysis of agronomic and phenotypic characteristics. Appendix E is requested for applications of GM insect resistant plants with the scope seeds and plant propagating material for cultivation in the EU. In Appendix F, for each functional group, a table should be compiled summarising the NTO studies submitted in support of the application. Appendix G should be compiled for each experimental study submitted in support of the environmental risk assessment of a GM plant. All compiled appendices D to G should be saved in the folder Appendices, subfolder ERA_Appendices D to G (see Table 2) Data presentation figures and tables Illustrations, including figures and tables, are often convenient and helpful to present and clarify information. Applicants are encouraged to effectively use figures and tables to illustrate experimental data including physical map(s) of the functional elements, comparison of protein expression, three dimensional image reconstruction, study overview, field trial design, results of the compositional analysis, graphical presentations of statistical analysis and ERA related information. The resolution and quality of images of gels, blots and other analyses should be sufficient to enable the non-equivocal interpretation of the data. Examples of MC and FF data overview tables related to Part II can be found in Tables 2-4 of Appendix C. Examples of tables and figures in a specific MC or FF section can be found in Figures 1-2 and Tables 5-6 of Appendix C. Schematic summaries of ERA data are explained in Appendices D-G. Figure preparation: Each figure should have a self-explanatory title and a legend, be numbered according to its appearance, and be cited in the text. No specific feature within an image (e.g., a Western blot) may be enhanced, obscured, moved, removed or introduced. The grouping or consolidation of images from multiple sources must be made explicit by the arrangement of the figure and in the figure legend. Adjustment of brightness, contrast or colour balance is acceptable if it is applied to the whole image and if it does not obscure, eliminate or miss-represent any information present in the original, including background. EFSA Journal 2011;9(7):

19 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 Table preparation: Each table should have a self-explanatory title, be numbered according to the order of its appearance, and be cited in the text. Tables should have legends when appropriate. The text should not fully duplicate the material in the tables Citations and reference list The applicant should cite all provided published and unpublished studies. Citations should be presented in an alphabetical reference list at the end of the document for all provided documents Published studies, proceedings, reports, guidelines, and legislation The citation should be derived from the file name. EFSA recommends citing a published study such as (Johnson et al., 2010) or (Johnson and van Cauwelaert, 2009). Examples for the formatting of references in the reference list can be found in section EndNote style files are available for use upon request. The following format should be applied to the reference list: no full stops after author initials and no commas between author last name and initial(s); and between the penultimate and final author; when the last name starts with van, de, etc., alphabetise the names according to the preposition (e.g. van Cauwelaert comes under v ); comma between the end of the author(s) name(s) and the year, and full stop after the year. journal names are preferably written in full and in regular font (no italics, no underline, etc.). abbreviated journal names should be avoided; the volume number (where applicable) shall be followed by a comma; the issue or band number shall not be provided unless necessary to identify the publication. If included it shall be followed by a comma; a page range shall be inserted (e.g ), for certain references the total number of pages (pp.) are indicated (e.g. 75 pp.), or for single page references the page (p.) where the reference is found (e.g. p. 18); full stop at the end of each reference; two or more works by the same author(s) cited at the same time (in alphabetical order), the author(s) surname(s) should not be repeated and the years be separated by a comma, from the oldest to the most recent (Smith et al., 2007, 2008) or (Johnson, 2006, 2007; Smith et al., 2007a, b) Unpublished studies The citation should be derived from the file name. EFSA recommends citing an unpublished study such as (Appendix x), or (Johnson et al., 2010), or (Johnson and van Cauwelaert, 2009). If the authors names are claimed as confidential, they should not be included in the citation. These unpublished studies should be listed in an overview table. Examples are given in Tables 1-3 of the Appendix C Examples for the formatting of references in the reference list Journal articles: Icoz I and Stotzky G, Fate and effects of insect-resistant Bt crops in soil ecosystems. Soil Biology and Biochemistry 40, Unpublished studies carried out by applicants: (if authors names not claimed to be confidential): Smith DK and Cramer JL, Updated bioinformatics evaluation of the CP4 EPSPS protein. [Applicant name] Technical Report, [Report number], Appendix 4, Updated bioinformatics evaluation of the CP4 EPSPS protein. [Applicant name] Technical Report, [Report number], EFSA Journal 2011;9(7):

20 Book: EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 Gregory N and Grandin T, Animal welfare and the meat market. CABI, Wallingford, UK, 185 pp. Book section: Bookers E, Heutinck L, van Reened C and Wolthuis-Fillerup M, Application of risk assessment to animal welfare. In: Animal welfare and the meat market. Eds Gregory NG and Grandin T. CABI, Wallingford, UK, Proceedings/Conference paper: Bookers E, Heutinck L, van Reened C and Wolthuis-Fillerup M, Veal calves generalize their response across familiar and unfamiliar persons in a repeatable on-farm fear of humans test. Proceedings of the 4th International Workshop on the Assessment of Animal Welfare at Farm and Group Level (WAFL), Ghent, Belgium, Thesis: Lund V, Ethics and animal welfare in organic animal husbandry: An interdisciplinary approach. Thesis (PhD), Swedish University of Agricultural Sciences, Uppsala, Sweden. 79 pp. Online document: BAS (Bristol Aquarists Society), online. Background information about goldfish. available at Brosowski J, 1999, online. Animal Diversity Web. Dicentrarchus labrax. University of Michigan, available at Part III Cartagena protocol The application shall provide information required under Article 5(3)(c) and Article 17(3)(c) of Regulation (EC) No 1829/2003 for the purpose of complying with the Cartagena Protocol. Depending on the scope of the application, the provided information shall contain as a minimum the information specified in Annexes II or III to Regulation (EC) No 1946/ Part IV Labelling The application shall include: (a) A proposal for labelling in all official Community languages, where a proposal for specific labelling is required in accordance with Articles 5(3)(f) and 17(3)(f) of Regulation (EC) No 1829/2003; (b) Either a reasoned statement that the food/feed does not give rise to ethical or religious concerns or a proposal for labelling in all official Community languages as required by Articles 5(3)(g) and 17(3)(g) of Regulation (EC) No 1829/2003; and, (c) When appropriate a proposal for labelling complying with the requirements of point A(8) of Annex IV to Directive 2001/18/EC Part V Methods of detection, sampling and identification and reference material The applicant shall provide methods for detection, sampling and identification, as well as samples of the food or feed and their controls samples to the European Union Reference Laboratory (EURL) as referred to in Article 32 of Regulation (EC) 1829/2003. The applicant shall follow the instructions for the preparation and the sending of the samples provided by the EU Reference laboratory (EURL) as 11 OJ L 287, , p. 1. EFSA Journal 2011;9(7):

21 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 referred to in Article 32 of Regulation (EC) 1829/200. Information and requirements of the EURL- GMFF can be consulted at its website. The application shall include a proof of submission of the samples, reagents and methods to the EURL-GMFF. The application shall include information as to the place where the reference material can be accessed Part VI Additional information to be provided for GM plants and/or food/feed containing or consisting of GM plants The information required in the notification as set out in Annex III to Directive 2001/18/EC shall be provided where it is not covered by the requirements of other parts of the application Part VII Summary The summary of an application should follow a standardised form (see Appendix I). Depending on the scope of the application, some of the requested information may not be applicable. The summary shall not contain parts considered to be confidential in accordance with Article 30 of Regulation (EC) No 1829/2003. The summary will be published on the EFSA website (see sections 1.4 and 1.6) Part VIII -Administrative documents Part VIII of the application shall contain all administrative documents related to the application. The list of documents and the standardised naming for the files are listed in Table 3. Table 3: List of administrative documents and their recommended file names File name 01-Letter_to_MS_submission.pdf 02-Confidentiality_Data_protection.pdf 03a-Access_letter_event1.pdf 03b-Access_letter_event2.pdf 03c.etc. 04-CClist.exl 05-EURL-GMFF.pdf 06-DoConformity.pdf 07-Contact.doc File content Cover letter accompanying the submission of the application Agreement to confidentiality and data protection For GM plants containing stacked events: Letter(s) of consent of access for all GM plants containing the single events (see section 3.8.1). Therefore, letters are needed both in case the authorisation is given for applications from the same and other applicant. Completeness checklist: filled in by the applicant (Appendix B) Proof of reception of samples, reagents and methods by EURL-GMFF (Appendix H) Declaration of Conformity between the paper and electronic versions of the application Contact details of the person for all correspondence with EFSA (Appendix A) Letter of consent of access If, in an application, the applicant refers to data provided in other applications that have been submitted previously to EFSA (e.g. in the case of GM plants containing stacked events for which applications of GM plants containing the single events have already been submitted), a letter of consent of access is required from the applicant. This letter will authorise EFSA and all MS CA to refer and use those data that have been provided previously in one specific application. If this authorisation concerns multiple applications, several letters of consent of access will be needed. 12 The applicant should notify EFSA, European Commission and the EURL-GMFF of changes in the contact details during the evaluation process. EFSA Journal 2011;9(7):

22 Completeness checklist EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 The completeness checklist (see Appendix B), an administrative document, will be used first by applicants then by EFSA to verify the completeness of the application and to ensure that: (i) all information and all documents, as required by the Regulation (EC) No 1829/2003 and EFSA, are provided; (ii) the application is consistent with the requested structure; (iii) the information is presented according to the formatting rules required by EFSA. The applicant shall fill in the checklist to indicate which information: (i) is provided; (ii) is not provided (including justification); (iii) is not applicable or relevant (including justification). EFSA will use the completeness checklist submitted by the applicant when performing its completeness check. 4. Applications for GM plants containing stacked events In accordance with the approach described in the guidance documents of the EFSA GMO Panel, the risk assessment of GM plants containing stacked events requires the risk assessment of the GM plants containing these events independently (i.e. GM plants containing single events) (EFSA, 2011a), and the ERA of the single events is a pre-requisite for the risk assessment of stacked events (EFSA, 2010a). This approach is based on the following considerations: the information included in applications for GM plants containing single events may change during the risk assessment because of submission of supplementary information. The evaluation of applications for GM plants containing stacked events is built on the knowledge acquired during the risk assessment of all the involved single events. Therefore, EFSA considers that the risk assessment of an application for GM plants containing stacked events can only start after the risk assessment of the respective single events is completed and no outstanding issues remain. In line with this approach, the EFSA GMO Panel will start the risk assessment of an application for a GM plant containing stacked events once the risk assessments of all applications for the respective single events are finalised by the EFSA GMO Panel. Applications for GM plants containing stacked events are assessed as stand-alone, complete and consolidated applications and should be prepared in accordance with this EFSA administrative guidance and the EFSA GMO Panel s Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010a). If an application for GM plants containing stacked events includes references to previously submitted applications for the corresponding GM plants containing single or GM plants containing lower combinations of stacked events under Regulation (EC) 1829/2003, such references must be precise and detail the section, page number, appendix, figure, name of the relevant report, etc. For GM plants containing stacked events the primary concern for risk assessment is to establish that the combination of events is stable and that no interactions between the stacked events, that may raise safety concerns compared to the single events, occur (EFSA, 2011a & 2011b). In order to allow for the assessment of and conclusions on interactions between multiple events, the applicant is asked to include in the application, either in the Part II or as a separate appendix, a table comprising compositional data (combined site results) including mean values and ranges for all materials analysed EFSA Journal 2011;9(7):

23 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 for all single events stacked in the GM plant, the GM plant containing stacked events and all controls. If the events were not included in the same set of field trials, the applicant is asked to include indications on the origin of the data on the single events [references to previous applications and e.g. number of field sites, season(s), location(s), number(s) of replicates]. For the preparation of an application for a GM plant containing stacked events, the applicant should use the completeness checklist specifically developed for such type of applications (Appendix B). 5. Applications for renewal of authorisations of a GMO plant and/or its derived food and feed under Regulation (EC) No 1829/2003 Authorisations for GM food and feed under Regulation (EC) No 1829/2003 are valid for a maximum of 10 years and may be renewed for successive 10-year periods. In accordance with Articles 11(1) and 23(1) of Regulation (EC) No 1829/2003, an application for renewal shall be sent to the European Commission a minimum of one year before the expiry date of the authorisation. Applicants should consult the guidance for renewal by the EFSA GMO Panel (EFSA, 2006a) for submitting an application of renewal to European Commission. Upon receipt, EFSA performs the completeness check and risk assessment in the same manner as described in sections 1.5 to Instructions described in sections 2 and 3 of this EFSA guidance replace section 4 of the Guidance for renewal of authorisations of existing GMO products lawfully placed on the market, notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003 by the EFSA GMO Panel (EFSA, 2006a). Administrative documents for a renewal application as listed in Table 3 (see subsection 3.8) should be submitted, except that the submission letter should be named as 01-Letter_to_EC_submission.pdf, and 04-cc checklist is not required. USEFUL WEBSITES Applicants can access the status of their application at EFSA Register of Questions: Community Reference Laboratory for GM food and feed: Decision of authorisation can be followed at EU register of genetically modified food and feed: EFSA GMO Extranet: EFSA Journal: Minutes of EFSA GMO Panel plenary meetings: Minutes of EFSA GMO Panel WG meetings: REFERENCES EFSA, 2006a. Guidance document of the Scientific Panel on Genetically Modified Organisms for renewal of authorisations of existing GMO products lawfully placed on the market, notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003. The EFSA Journal 435, 1-4. EFSA, 2006b. Guidance document for the risk assessment of genetically modified plants and derived food and feed by the Scientific Panel on Genetically Modified Organisms. The EFSA Journal 374, EFSA, EFSA Panel on Genetically Modified Organisms (GMO); Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials. Food and Chemical Toxicology 46: S2 S70. EFSA Journal 2011;9(7):

24 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 EFSA, EFSA Panel on Genetically Modified Organisms (GMO); Scientific opinion on guidance for the risk assessment of genetically modified plants used for non-food or non-feed purposes. EFSA Journal 1164:1-42. EFSA, 2010a. EFSA Panel on Genetically Modified Organisms (GMO); Guidance on the environmental risk assessment of genetically modified plants. EFSA Journal 8(11):1879. EFSA, 2010b. EFSA Panel on Genetically Modified Organisms (GMO); Statistical considerations for the safety evaluation of GMOs. EFSA Journal 8(1):1250. EFSA, 2010c. EFSA Panel on Genetically Modified Organisms (GMO); Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed. EFSA Journal 8(7):1700. EFSA, 2010d. EFSA Panel on Genetically Modified Organisms (GMO); Scientific Opinion on the assessment of potential impacts of genetically modified plants on non-target organisms. EFSA Journal 8(11):1877. EFSA, 2011a. EFSA Panel on Genetically Modified Organisms (GMO); Guidance for risk assessment of food and feed from genetically modified plants. EFSA Journal 9(5):2150. ERSA, 2011b. EFSA Panel on Genetically Modified Organisms (GMO); Guidance on selection of comparators for the risk assessment of genetically modified plants and derived food and feed. EFSA Journal 9(5):2149. EFSA, 2011c. Guidance of the Post Market Environmental Monitoring (PMEM) of genetically modified plants. EFSA Journal 9(8): EFSA Journal 2011;9(7):

25 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 APPENDICES All appendices can also be downloaded from the EFSA website, here. A. CONTACT DETAILS The filled document should be submitted preferably in pdf format and included in Part VIII (see Table 3). B. COMPLETENESS CHECKLIST This checklist strives to list scientific requirements specified in the two recently updated guidance documents of the EFSA Panel on genetically modified organisms, the Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011a) and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2011a). It needs to be clearly said that not every scientific requirement described in one individual cell of the column of the spreadsheet Part II scientific information is mandatory. The filled document should be submitted in Excel format and included in Part VIII (see Table 3). C. EXEMPLAR FIGURES AND TABLES FOR PART II This appendix contains examples of figures and tables to present data on molecular characterisation and food and feed risk assessment. Figures and tables are useful to provide an overview of studies in the application and snap-shots of each study, to add clarity to parts of a study with illustrations, and to streamline the risk assessment process. These figures and tables should not be viewed as precise templates as the data in each application differs. Other formats of these figures and tables will be accepted, provided that the same aim is achieved. They should be included in Part II (see Table 2). D. SCHEMATIC SUMMARY OF DATA FOR FIELD OR GREENHOUSE TRIAL FOR AGRONOMIC AND PHENOTYPIC CHARACTERISTICS WITHIN A SEASON (GM PLANTS CONTAING SINGLE OR STACKED EVENT(S)) Appendix D, a schematic summary, should be provided for each field trial conducted for the comparative analysis of agronomic and phenotypic characteristics. It should be stored in the folder Appendices, subfolder ERA_Appendices D to G (see Table 2). E. SCHEMATIC SUMMARY OF INFORMATION FOR INSECT RESISTANCE MANAGEMENT Appendix E is requested for applications of GM insect resistant plants with the scope seeds and plant propagating material for cultivation in the EU. The applicant should include it in the folder Appendices, subfolder ERA_Appendices D to G (see Table 2). F. SCHEMATIC SUMMARY OF NTO STUDIES In Appendix F, for each functional group, a table should be compiled summarising the NTO studies submitted in support of the application The complied appendix should be included in the folder Appendices, subfolder ERA_Appendices D to G (see Table 2). G. SCHEMATIC SUMMARY OF STATISTICAL DESIGN AND ANALYSIS FOR EACH ERA RELATED STUDY EFSA Journal 2011;9(7):

26 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 For each experimental study submitted in support of the environmental risk assessment, the applicant should compile a separate Appendix G. All complied appendices should be included in the folder Appendices, subfolder ERA_Appendices D to G (see table 2). H. PROOF OF ACKNOWLEDGEMENT OF RECEPTION BY EURL-GMFF This appendix contains an Acknowledgement of reception of samples, reagents and methods used by EURL-GMFF. A copy of such document issued by EURL-GMFF for a specific GM event should be included in Part VIII (see Table 3). I. SUMMARY OF APPLICATIONS This appendix specifies the standardised form, which the summary of an application should follow. Depending on the scope of the application, some of the requested information may not be applicable. The summary shall not contain parts considered to be confidential in accordance to Article 30 of Regulation (EC) No 1829/2003. The filled document should be submitted preferably in pdf format and included in Part VII (see Table 2). Please be reminded that during the completeness check phase, an updated version should be included when a revised application is sent to EFSA. J. TEMPLATE FORM FOR PART II SCIENTIFIC INFORMATION This appendix specifies the standardised form, which Part II of an application should follow. The titles are in line with the titles as listed in the completeness checklist. EFSA Journal 2011;9(7):

27 EFSA submission guidance to applicants on the preparation and presentation of applications under Regulation (EC) No 1829/2003 ABBREVIATIONS CI: Confidential Information CA: National Competent Authority CC: Completeness Check CD-ROM: Compact Disk - Read Only Memory EURL-GMFF: European Union Reference Laboratory for GM Food and Feed EC: European Commission EU: European Union EFSA: European Food Safety Authority ENV: Environment ERA: Environmental Risk Assessment FF: Food and Feed GM: Genetically Modified GMO: Genetically Modified Organisms MC: Molecular Characterization MS: Member State MS CA national Competent Authority of a Member State nonci: non-confidential Information WG: Working Group EFSA Journal 2011;9(7):

28 Appendix A APPLICATION IDENTIFICATION CODE (EVENT NAME) APPENDIX A: CONTACT DETAILS OF APPLICANT 1- Applicant (company or person 1 ) a. Name of the applicant or company: b. Address i. street, number: ii. post code: iii. city/town: iv. country: c. Telephone: d. Fax: e Contact Person 2 (for all correspondence with EFSA, EC and EURL-GMFF) a. Name of the contact person: b. Company (if different than that of the applicant): c. Position: d. Address i. street, number: ii. post code: iii. city/town: iv. country: e. Telephone: f. Fax: g. 1 According to Articles 4(6) and 16(6) of Regulation (EC) No 1829/ The applicant is responsible to notify EFSA in case the contact person or any of the contact details change during the evaluation period. 1

29 General requirements For EFSA use only General requirements as outlined in the EFSA submission guidance 2011 Yes, provided No, not provided (justification needed) Not applicable EFSA agrees EFSA comments/questions to applicants Structure of the application and file names 1 electronic copy 1 paper copy The following eight parts are provided Part I - Genernal information Part II Scientific information including main text and all cited references Post-market environmental monitoring plan Part III Cartagena Protocol Part IV Labelling proposal Part V Detection and validation methods Part VI Additional information Part VII Summary Part VIII Administrative documents Cover letter accompanying the submission of the application Agreement to confidentiality and data protection Letter(s) of consent of access for all single events Completeness checklist: filled in by the applicant (Appendix B) Proof of sending the samples and control samples to the EURL-GMFF (Appendix H) Declaration of Conformity between the paper and electronic versions of the application

30 General requirements Information on passwords of files (if applicable) which can be provided separately Contact details of the person for all correspondence with EFSA (Appendix A) One file per part (except for Part II, Part V and Part VIII where references or documents should be provided as separate documents) Confidential information Files containing confidential information contain "CI" in the file names (e.g. "Appendix_5_CI.pdf") Language Application in English language Electronic version CD(s) labelled as described in section 2.4.1of the Admin guidance File format, size and name All references cited in Part I and II provided as separate pdf files allowing reading, printing, word searching and copying of text from the pdf file DNA sequence information in Gen Bank format including annotation information Raw data provided for all experimental studies carried out by the applicant (e.g. compositional, agronomic, protein expression, bioinformatic data) Raw data for comparative assessment submitted in editable format Programming code used for the statistical analysis for the comparative assessment provided File size smaller than 25 MB Files are word searchable All files named as described in section 2.1 of the Administrative guidance Files names shorter than 40 characters Files names do not reveal confidetial information (e.g. authors' names protected by confidentiality claims) All documents contain a table of content Standard units and abbreviations Units are expressed in International System of Units

31 General requirements List of abbreviations provided at the beginning of Part II For the specific requirements of Parts I to VII, please refer to the different worksheets Comments (up to 500 characters) Please insert your comments here

32 Part I - General Info For EFSA use only Part I - General information Yes, provided No, not provided (justification required in Part I) Not applicable (justification EFSA agrees required in Part I) EFSA comments/quest ions to applicants 1. Name and address of the applicant (company or institute) 2. Name, qualification and experience of the responsible scientist(s) and contact details of the responsible person for all 3. Designation and specification of the GM plant and derived product Results of the transformation (newly expressed proteins, traits) For GM plants containing stacked transformation events ABCD: Description of how the GM plant containing stacked transformation events was produced Description of how the single events were produced EFSA scientific opinion finalised for single event A EFSA scientific opinion finalised for single event B EFSA scientific opinion finalised for single event C EFSA scientific opinion finalised for single event D EFSA scientific opinion finalised for single event E 4. Scope of the application

33 Part I - General Info The risk assessment of a GM plant containing stacked events should address all sub-combinations occurring by natural segregation from the GM plant (EFSA, 2011a). Confirmation of the scope of the application by the European Commission List of all sub-combinations addressed in the application Where an application is limited to either food or feed use, it shall contain a verifiable justification explaining why the authorisation shall not cover both uses in accordance with Article 27 of Regulation (EC) No 1829/ Unique identifier for the GM plant and derived products in question, developed in accordance with Regulation (EC) No 65/ Where applicable and where relevant to the risk assessment, a detailed description of the method of production and manufacturing. For example, a description of methods used to process the GM plant materials during the preparation of food/feed, food/feed ingredients, food/feed additives or food flavourings. 7. Where appropriate, the conditions for placing on the market of the food(s) or feed(s) produced from it, including specific conditions for use and handling Comments (up to 500 characters) Please insert your comments here

34 Part II - Sci info (single) Part II - Scientific Information Applicants should submit a completed completeness checklist when submitting a new application. EFSA will use this completed list during the completeness check and double check if the information was provided. For each line of this checklist, applicants should check one box only per line. Yes Information provided OR study carried out according to principles of EFSA guidances No Information not provided OR study does not follow requirements of EFSA guidances In either case, a justification is provided in Part II Not applicable The information is not applicable due to the nature of the GM plant and or the derived food/feed. A justification is included in Part II. For EFSA use only EFSA agrees EFSA checks if i) the information provided is in line with the EFSA guidances on food, feed or environmental risk assessment, ii) the justification for omission of data/information is acceptable, iii) and if the presentation of the application is in line with the EFSA submission i guidance. EFSA comments/questions to applicants General requirements for Part II (as outlined in the EFSA Submission Guidance) Overview table of all Appendices and key references provided in support of this application - Please refer to the example in Table 1 of Appendix C of the EFSA Submission Guidance Detailed description of all studies (including experimental methods, analysis of data) Data presentation - Figures and Tables High quality images of gels, blots and other analyses For each table: number, title and legend (see example in Appendix C). For each figure: number, title and legend.(see example in Appendix C).

35 Part II - Sci info (single) Citations and References Alphabetical reference list at the end of Part II Citations of published studies in line with the formatting requirements of the EFSA Submission guidance section Citations of unpublished studies in line with the formatting requirements of the EFSA Submission guidance section If authors' names are claimed as confidential, they are not included in the citation Requirements for the Molecular Characterisation and Food and Feed risk assessment as outlined in the EFSA guidance for risk assessment of food and feed from genetically modified plants 2011 The food and feed risk assessment considers the principles of the EFSA guidance on risk assessment of food and feed from GM plants (2011) and - follows a comparative approach - follows a stepwise approach (hazard identification, hazard characterisation, exposure assessment, risk characterisation) Reference to relevant chapters of the EFSA guidance for risk assessment of food and feed from genetically modified plants is performed on a case-by-case basis Comments (up to 500 characters) Please insert your comments here A. Hazard identification and characterisation Study overview table - Please refer to the example in Table 2 of Appendix C of the EFSA Submission Guidance 1. Information relating to the recipient or (where appropriate) parental plants a) Complete name: (a) family name

36 Part II - Sci info (single) (b) genus (c) species (d) subspecies (e) cultivar/breeding line or strain (f) common name b) Geographical distribution and cultivation of the plant, including the distribution within the Union c) Information on the recipient or parental plants relevant to their safety, including any known toxicity and/or allergenicity of constituents and the plant d) Data on the past and present use of the recipient organism. This information should include: i) the history of safe use for consumption as food and/or feed ii) information on how the plant is typically cultivated, transported and stored iii) whether special processing is required to make the plant safe to eat iv) the description of the normal role of the plant in the diet (e.g. which part of the plant is used as a food source, whether its consumption is important in particular subgroups of the population, what important macro- or micro-nutrients it contributes to the diet) Comments (up to 500 characters) Please insert your comments here 2. Molecular Characterisation for GM plants containing single events 2.1 Information relating to the genetic modification Description of the methods used for the genetic modification Information provided on the following elements: a) method of genetic transformation including relevant references b) the recipient plant material c) strain of Agrobacterium or other microbes used d) helper plasmids

37 Part II - Sci info (single) e) source of carrier DNA Source of nucleic acid used for transformation: Information on the donor organism(s) and on the nucleic acid sequence(s) intended to be inserted comprising the following elements: a) the complete sequence of the nucleic acid intended to be inserted and indication of any alteration(s) to sequences b) the history of safe use of the gene product(s) arising from the regions intended for insertion

38 Part II - Sci info (single) c) data on the possible relationship of the gene products to known toxins, antinutrients and allergens d) information regarding each donor organism including its taxonomic classification and its history of use regarding food and feed safety Nature and source of vector(s) used including nucleotide sequences intended for insertion The following information is provided: a) a physical map of the functional elements and other plasmid/vector components together with the relevant information needed for the interpretation of the molecular analyses - indication of the region intended for insertion b) a table identifying each component of the plasmid/vector, its size, its origin and its intended function Comments (up to 500 characters) Please insert your comments here 2.2 Information relating to the GM plant General description of the trait(s) and characteristics which have been introduced or modified - description of the trait(s), of the resulting changes on phenotype and metabolism of the plant, and of its intended use - description of the mode of action Information on the sequences actually inserted or deleted including the following elements: a) size and copy number of all detectable inserts, both complete and partial. This is typically determined by Southern analysis.

39 Part II - Sci info (single) Southern analyses should - provide complete coverage of sequences that could be inserted into the host plant, such as any part of the plasmid/vector, or any remaining carrier or foreign nucleic acid introduced in the GM plant. - span the entire transgenic locus/loci as well as the flanking sequences - include appropriate controls b) organisation and sequence of the inserted genetic material at each insertion site c) in the case of deletion(s), size and function of the deleted region(s) d) sub-cellular location(s) of insert(s) and methods for its determination e) sequence information for both 5 and 3 flanking regions at each insertion site, with the aim of identifying interruptions of known genes - bioinformatic analyses using up-to-date databases with the aim of performing both intra-species and inter-species similarity searches f) identification of ORFs within the insert and spanning the junction sites - ORFs analysed between stop codons, not limiting their lengths - bioinformatic analysis of ORFs present within the insert using up-to-date databases to determine similarity with known toxin(s) or allergen(s) - bioinformatic analysis of ORFs spanning the junction sites using up-to-date databases to determine similarity with known toxin(s) or allergen(s) bioinformatic overview table (Appendix C Table 3) Information on the expression of the inserted/modified sequence Overview table - Field trial for protein expression analyses (Appendix C Table 4) The following elements should be provided: - data from plants grown under representative conditions including a rationale for the selection of sites

40 Part II - Sci info (single) - information on specific treatments linked to the trait (e.g. use of herbicides) - rationale for selection of analysed parts - rationale for selection of samples during development - data from minimum three growing sites or one site over three seasons a) methods used - information on specificity of methods used - raw data of protein expression analysis (including reference in main text) b) the range and mean values for the levels of the newly produced protein(s) (or levels of the relevant RNA(s), protein(s) or metabolite(s)) For GM plants containing single events for food, feed, import and processing purposes - rationale for selection of comparator - assessment of the impact of specific treatments linked to the trait (e.g. use of herbicides) For GM plants containing single events, which include cultivation in the scope - Depending on the trait, information for the assessment of impacts on target and non-target organisms - data for plants grown under conditions representative of typical cultivation practices in Europe Genetic stability of the inserted/modified sequence and phenotypic stability of the GM plant - information on safety implications of the loss of function of specific genes from multi-gene expression cassettes after their insertion into the plant For GM plants containing single events - data from 5 generations or vegetative cycles - rationale if providing less than 5 generations or vegetative cycles - information on the source of material, the sampling design and the number of plants used for the analysis - data on inheritance patterns (including statistical methods applied)

41 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 2.3 Conclusion - information on structure of the insert - information on expression of the insert - information on stability - conclusion if molecular characterisation raises a safety concern - conclusion if genetic modification(s) raise(s) any issues regarding the potential for producing new toxins or allergens conclusion if unintended changes were identified Comments (up to 500 characters) Please insert your comments here 3. Comparative assessment 3.1 Criteria for the selection of comparator(s) Information on the breeding scheme (pedigree) in relation to the GM plant, the conventional counterpart and/or other comparator(s) used in the risk assessment (Appendix C) Details on the nature of the conventional counterpart and/or other comparator(s) Justification for the selection of the conventional counterpart and/or other comparator(s) (NB it is recommended that this selection is done in accordance to chapter of the EFSA guidance document) Justification for the selection of non-gm reference varieties used to estimate the variability required to set equivalence limits (as explained in chapter of the FF guidance)

42 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 3.2 Field trials: experimental design and statistical analysis Experimental design Test materials included in field trials - GM plant - conventional counterpart and/or other comparator(s) - non-gm reference varieties - For herbicide tolerant GM plants: the GM plant exposed to the intended herbicide(s), the comparator treated with conventional herbicide management regimes and the GM plant treated with the same conventional herbicide management regimes (please refer the EFSA comparator guidance) Description of field trials including information on - geographical locations (including indication on a map) - crop rotation(s)/crops grown in previous seasons in the same plots - management of the field before sowing - date of sowing - soil type - agronomic practices during cultivation including, but not limited to, the following examples: --- herbicide use and other weed management practices --- pest management practices --- fertilizer regime --- irrigation - climatic and other cultivation/environmental conditions during growth - time of harvest - conditions during harvest and storage of the harvested material

43 Part II - Sci info (single) If the field trial includes one GM plant only, information on the following items should be provided: - Description of the field trial design, including, but not limited to, the following elements: -- Blocking (e.g. completely randomised or randomised block experimental design) -- Rationale underlying choice of locations (NB the different sites selected for the trials should be representative of the range of receiving environments where the crop will be grown) -- Choice of reference lines in each location (NB at each site, there should be at least three appropriate non-gm reference varieties of the crop that have a known history of safe use, which must also be identical between all replicates) -- Identity of the GM plant, conventional counterpart, other comparator(s) and references within the different replicates in each location (NB within each site the GM plant, the conventional counterpart (and additional comparators, where appropriate) should be identical for all replicates) The field trial design respects the following requirements: - At each site: at least three appropriate non-gm reference varieties of the crop that have a known history of safe use - At least 4 replicates for each test material in the particular locations - If less than three non-gm references have been included at a particular site, at least six replicates per site - Each field trial should be replicated at a minimum of eight sites - At least six different reference varieties are used over the entire set of trials - all test materials are randomised to plots within a single field at each site If the field trial includes more than one GM plant of the same species, the following two criteria should be met: i. Occurrence of each of the comparator(s) in the same block as the particular GM plant ii. Randomization of the test materials within each block If the number of plots per block required for such a trial were to exceed 16, a description of the following items: - Description of whether a partially balanced incomplete block design has been used. If so, the following two criteria have been met:

44 Part II - Sci info (single) i. Each of the comparator(s) always occurs together with its respective GM plant in the same block. ii. All of the non-gm reference varieties appear in each of the incomplete blocks and are fully randomised with the GM plants and their comparator(s). Statistical analysis Raw data in an editable form The programming code used for the statistical analysis in an editable form For each endpoint, the following two tests are performed: - difference test - equivalence test Statistical models used to derive confidence and equivalence limits: - Model used for the calculation of confidence limits for both tests (a linear mixed model is recommended) - Model used for the calculation of equivalence limits for the equivalence test (a mixed model, slightly different from the previous model, is recommended) For each endpoint analysed, the following details should be given: (a) the assumptions underlying the analysis

45 Part II - Sci info (single) (b) full specification of the mixed models chosen, including fixed and random effects (c) results of any test of interaction between the test materials and sites (d) fixed effects, together with the appropriate estimated residual variation with which it is compared, and variance components for the random factors (e) estimated degrees of freedom (f) any other relevant statistics Discussion of the likely impact of other growing conditions not tested in the trial Graphical display for all endpoints Discussion of all significant differences observed between the GM plant, its comparator and, where applicable, any other test material, focussing on their biological relevance Comments (up to 500 characters) Please insert your comments here 3.3 Compositional analysis Description of the quality standards applied for sampling, analysis and preparation of the material tested Description of the samples analysed, including but not limited to the following: - raw agricultural commodity/ies - processed products (food and feed, food ingredients, feed materials, food and feed additives or food flavourings) Description of the compounds analysed The compounds selection has been in line with OECD consensus documents Description of the categories of parameters that have been analyzed, including but not limited to key macro- and micro-nutrients and other parameters falling in the following categories: - proximates (including moisture and total ash)

46 Part II - Sci info (single) - anti-nutritional compounds - natural toxins - other secondary plant metabolites characteristic for specific plant species - vitamins and minerals - for oil-rich plants and/or plants used for oil production: fatty acid profile - for plants used as an important protein source: amino acid profile For compounds not listed by OECD, justification for their selection, including the following - metabolites and additional compounds likely to be affected/formed as a result of the particular genetic modification but not mentioned by OECD, to be selected on a case-by-case basis Comments (up to 500 characters) Please insert your comments here 3.4 Agronomic and phenotypic characteristics Protocols for these field trials and a description of whether they follow the specifications made in chapter of the EFSA MC/FF guidance document Raw data on agronomic and phenotypic characteristics Description of whether these field trials are different from those for the compositional analysis

47 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 3.5 Effects of processing Detailed description of the different processing technologies Assessment whether the processing and/or preserving technologies applied are likely to modify the characteristics of the GM end-products compared with their non-gm counterparts Justification for not performing toxicological tests with the processed products For GM plants with modified metabolism: Scientific rationale for the selected approach to assess processed products For GM plants containing new compounds: - evaluation of effects of processing on these new compounds - evaluation of need for specific processing measures - evaluation whether the processing results in formation of new compounds Comments (up to 500 characters) Please insert your comments here Conclusions The conclusions should contain the following statements a) Concluding statement on the agronomic and phenotypic characteristics of the GM plant being, except for the introduced trait(s), different or not to those of its comparator and/or equivalent to the non-gm reference varieties, taking into account natural variation

48 Part II - Sci info (single) b) Concluding statement on compositional characteristics of the GM plant and derived food and feed being, except for the introduced trait(s), different or not to those of its comparator and/or equivalent to the non-gm reference varieties, taking into account natural variation c) Concluding statement on whether further investigation is needed for those characteristics showing differences between the GM plants and derived food and feed and the comparator, or showing lack of equivalence with respect to non-gm reference varieties, taking into account natural variation Comments (up to 500 characters) Please insert your comments here 4. Toxicological assessment Justification for performing toxicological tests Justification for not performing toxicological tests The toxicological assessment considers a) presence and levels of newly expressed proteins b) potential presence of other new constituents c) possible changes in the levels of endogenous constituents beyond normal variation d) impact of other changes in composition due to the genetic modification 4.1 Standardised Guidelines for Toxicity Tests For each toxicity test: Description of the use of test methods (NB internationally agreed test methods described by the OECD or by the European Commission should be used for toxicity testing) For each toxicity test: Justification for the selection of a specific test protocol For each toxicity test: Explanation and justification of any adaptation of these protocols, or use of any methods that differ from these protocols

49 Part II - Sci info (single) For each toxicity test: If performed in EU, the test follows principles of good laboratory practice as laid down by Directive 2004/10/EC For each toxicity test: If performed outside EU, the test follows the OECD Principles of Good Laboratory Practice (GLP) For each feeding study: - description of experimental design - description of statistical approach - endpoints measurements including feed intake, body weight, animal performance and, if applicable, bioavailability of nutrients For studies other than toxicological studies: A description of the standards and quality systems under which they have been conducted (NB they should be performed under ISO or GLP standards or other appropriate quality assurance systems) Comments (up to 500 characters) Please insert your comments here 4.2 Assessment of newly expressed proteins All newly expressed proteins are assessed Documentation of a proper use and safe consumption as food and feed of both the plant and the newly expressed proteins If a protein produced by microorganisms is used, the following information should be provided "de minimis" so as to be able to sufficiently demonstrate equivalence of the tested protein to the newly expressed protein as expressed in the GM plant: - comparisons of the molecular weight, - comparison of amino acid sequence, - comparison of post-translational modification, - comparison of immunological reactivity - comparison of functional characteristics and biochemical activity

50 Part II - Sci info (single) Evaluation of significance of differences between the GM plant expressed protein and its microbial substitute with respect to their potential safety impact Demonstration of safety of newly expressed proteins including a) molecular and biochemical characterisation of the newly expressed proteins including - the molecular weight - the amino acid sequence - post-translational modifications - a description of the function - functional characteristics and biochemical activity. In the case of newly expressed enzymes, information on the enzyme activities including the temperature and ph range for optimum activity, substrate specificity and possible reaction products Evaluation of potential interactions between the newly expressed proteins and other plant constituents with respect to safety impact b) up-to-date bioinformatic search for homology - to toxins - to proteins exerting a normal metabolic or structural function - bioinformatic i overview table (Appendix D Table 3) and reference to it in the main text c) information on the stability of the protein under the relevant processing and storage conditions for the food and feed derived from the GM plant - examination of influences of temperature and ph changes - characterisation of potential modification(s) of the proteins (e.g. denaturation) and/or production of stable protein fragments generated through processing and storage d) data concerning the resistance of the newly expressed protein to proteolytic enzymes - e.g. in vitro investigations using appropriate and standardised tests - characterisation and evaluation of stable breakdown products with regard to the potential risks associated with their biological activity e) repeated dose toxicity studies using laboratory animals Repeated dose toxicity study OR Information demonstrating the safety of the newly expressed protein (including its mode of action) AND

51 Part II - Sci info (single) Information demonstrating that the protein is not structurally and functionally related to proteins adversely affecting human or animal health Repeated dose 28-day oral toxicity study in rodents with the newly expressed protein in line with the following requirements: - carried out according to principles specified in OECD guideline sufficient number of animals per group e.g. 10/sex (EFSA recommendation) in order to obtain an adequate statistical power Discussion of the outcome of the 28-day toxicity study, and justify whether further targeted investigations are needed When two or more proteins are newly expressed in the GM plant - assessment of possible synergistic or antagonistic interactions between two or more newly expressed proteins and their relevance to human or animal health Comments (up to 500 characters) Please insert your comments here 4.3. Assessment of new constituents other than proteins Safety assessment of identified new constituents including an evaluation of their toxic potency 4.4 Assessment of altered levels of food and feed constituents Risk assessment (if the genetic modification altered the content of the food and feed constituents beyond the levels expected for the non-gm counterpart), based on- and including a description of - the following items: - physiological function and/or toxic properties of these constituents as well as the anticipated changes in intake levels. Justification for performing tests to assess altered levels of food and feed constituents Justification for not performing tests to assess altered levels of food and feed constituents

52 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 4.5 Assessment of the whole food and/or feed derived from GM plants Justification for performing whole food and/or feed testing Justification for not performing whole food and/or feed testing 90-day feeding study in rodents (if the composition of the food and/or feed derived from GM plant is substantially modified or there are any indications for the potential occurrence of unintended effects based on the preceding molecular, compositional or phenotypic analyses) A description of the design and performance of 90-day feeding study in rodents, in particular a description if and how the following principles have been respected: - performance according to the principles of OECD guideline 408 following an adapted protocol - description and justification of adaptations of guideline test groups (NB normally a minimum of two test doses and a negative control are used) - statement explaining as to whether the highest dose is the maximum achievable without causing nutritional imbalance - statement explaining as to whether the lowest dose contains the tested food and/or feed in an amount at least equivalent to the one consumed by humans or animals - information on how the natural variation of test parameters is derived, e.g. from historical background data and/or from the inclusion of reference varieties in the experiments? - statistical analysis (NB this should focus on the detection of possible differences between the test material and its control) Discussion of the outcome of the 90-day feeding study including a justification if further tests are needed Description of other studies than a 90-feeding study that have been provided If so, a justification for this/these study/studies

53 Part II - Sci info (single) Interpretation of animal studies Evaluation of changes in test parameters, in particular with respect to the following issues: (i) relationship with the applied doses, (ii) possible correlations with changes in other biologically related parameters, (iii) incidental occurrence, (iv) gender specificity and (v) normal biological variation When a difference is noted at the highest dose applied, consideration of other factors to determine whether there is a relationship with treatment. Comments (up to 500 characters) Please insert your comments here 4.6 Conclusions The conclusions of the toxicological assessment should include - but need not be limited to - the following items: a) A conclusion on whether the available information on the newly expressed protein(s) and other new constituents resulting from the genetic modification gives indications, or not, of adverse effects in particular, whether and at which dose levels adverse effects were identified in specific studies b) A conclusion on whether the information on natural constituents, with levels different from those in the comparator, provides indications of adverse effects, in particular, whether and at which dose levels adverse effects were identified in specific studies c) A conclusion on whether toxicologically relevant effects have been identified from the animal studies made on the whole food and feed derived from the GM plant, compared to its comparators Evaluation of the results of the toxicological assessment in the light of anticipated intake of the food and feed derived from GM plants

54 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 5. Allergenicity assessment 5.1 Assessment of allergenicity of the newly expressed protein Indication whether the source of the transgene is allergenic Assessment of the newly expressed proteins for a possible role in the elicitation of gluten-sensitive enteropathy or other enteropathies which are not IgE-mediated (if the introduced genetic material is obtained from wheat, rye, barley, oats or related cereal grains) Weight of evidence approach for the assessment of possible allergenicity of the newly expressed protein(s) considering the following: a) Amino acid sequence homology comparison between the newly expressed protein and known allergens using a comprehensive database - amino acid sequence homology - structural similarities - bioinformatic overview table (Appendix C Figure 3) and reference to it in the main text Description of the algorithms applied Alignment uses the following criterion: 35% sequence identity to a known allergen over a window of at least 80 amino acids (minimum requirement) - Sequence alignment parameters used in the analysis [all parameters, including calculation of percent identity (PID), are provided] b) Specific serum screening Justification for not performing specific serum screening Justification for performing specific serum screening Specific serum screening should be performed in the following cases: 1) the source of the introduced gene is considered allergenic (select "yes" if applicable); 2) there is any indication of relationship between the newly expressed protein and a known allergen, based on sequence homology or structure similarity (select "yes" if applicable)

55 Specific serum screening study report Part II - Sci info (single)

56 Part II - Sci info (single) c) Pepsin resistance and in-vitro digestibility tests if these data have not yet been provided as part of the assessment of potential toxicity of the newly expressed proteins, data on pepsin resistance and in-vitro digestibility should address the following issues: - Pepsin resistance test performed under standardized conditions (e.g. simulated gastric fluid, low ph, fixed pepsin:protein ratio) - Additional in-vitro digestibility tests using different conditions (e.g. pepsin incubations with varying ph values) so as to assess the digestibility of the newly expressed protein in individuals with impaired digestive function - Additional in-vitro digestibility tests in the presence of matrix components, if applicable, that can influence the newly expressed protein's digestibility - Additional in-vitro digestibility tests with the newly expressed protein following conditions representative to food and feed processing, if applicable, that can influence the newly expressed protein's digestibility d) based on outcome of a to c, additional tests for - and data on - potential allergenicity of the newly expressed proteins (optional, e.g. in-vitro and in-vivo data) other tests Comments (up to 500 characters) Please insert your comments here 5.2 Assessment of the allergenicity of the whole GM plant Description of whether the recipient plant species is a major allergenic organism in its own right Justification for not performing allergenicity tests with the whole GM plant Justification for performing allergenicity tests with the whole GM plant Test of any potential change in the allergenicity of the GM plant by comparison of the allergen repertoire with that of its appropriate comparator (e.g. 2-dimensional immunoblots using individual patients' sera) (if the recipient of the introduced gene is known to be allergenic)

57 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 5.3 Adjuvanticity Consideration of possible role of the newly expressed proteins as adjuvants (if known functional aspects of the newly expressed protein or structural similarity to known strong adjuvants may indicate possible adjuvant activity) Comments (up to 500 characters) Please insert your comments here Conclusions Conclusions indicating whether - the novel protein(s) is (are) likely to be allergenic - the food derived from GM plant is likely to be more allergenic than the comparator Further characterisation of the food derived from the GM plant in the light of anticipated intake and appropriate conditions for placing on the market (if there is a likelihood of allergenicity in one of the above mentioned cases) Labelling proposal (if there is a likelihood of allergenicity in one of the above mentioned cases)

58 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 6. Nutritional assessment Nutritional evaluation to demonstrate that the introduction of food and feed derived from a GM plant into the market is not nutritionally disadvantageous to humans and animals, respectively. This evaluation should include an assessment of: (i) the nutritional relevance of newly expressed proteins and other new constituents; (ii) the changes in the levels of endogenous constituents in the GM plant and derived food and feed; (iii) the potential alterations in the total diet for the consumers/animals. 6.1 Nutritional assessment of food derived from GM plants Justification for not performing nutritional tests Justification for performing nutritional tests based on a nutritional assessment of food derived from GM plants considering a) the composition of the food with regard to the levels of nutrients and anti-nutrients b) the bioavailability and biological efficacy of nutrients in the food, also taking into account the potential influences of transport, storage and expected treatment of the foods c) the anticipated dietary intake of the food and resulting nutritional impact Comments (up to 500 characters) Please insert your comments here

59 Part II - Sci info (single) 6.2. Nutritional assessment of feed derived from GM plants Justification for not performing nutritional tests Justification for performing nutritional tests based on a nutritional assessment of feed derived from GM plants considering - Nutritional assessment using target animal species (if compositional characteristics of the feed derived from a GM plant are different and/or not equivalent to those of its comparator) For nutritionally modified GM plants: Livestock study using an appropriate control diet with similar nutrient profile formulated with a non-gm control supplemented with the specific nutrient as present in the GM plant (if the GM plant contains introduced traits, such as altered nutrient density or levels, affecting animal performance) Target animal feeding studies should span: (i) from the growing and/or the finishing period to slaughter for chickens, pigs, and cattle; (ii) the major part of the lactation cycle for dairy cows; and (iii) the laying cycle for laying hens or quails For each feeding study: - description of experimental design - description of statistical approach - endpoints measurements always including feed intake, body weight, animal performance and bioavailability of nutrients Comments (up to 500 characters) Please insert your comments here Conclusions Conclusions of the nutritional assessment indicating: - if the food and feed is nutritionally equivalent to its comparator, taking into account natural variation - in case of lack of equivalence, if the identified changes have an impact on the anticipated intake of the food and feed

60 Part II - Sci info (single) - if the unintended effects of the genetic modification, either identified during hazard identification or assumed based on the preceding molecular, compositional and phenotypic analyses, have affected the nutritional value of the food and feed Comments (up to 500 characters) Please insert your comments here B. Exposure assessment - Anticipated intake/extent of use Information on - intended function - dietary role - expected level of use of the food and feed derived from GM plants Determination of the concentrations of the newly expressed proteins, other new constituents and endogenous constituents with levels altered as a result of the genetic modification (e.g. due to changes in metabolic pathways) in those parts of the GM plant intended for food or feed use. 3.2 Estimation of intake taking into account influences of processing storage, and expected treatment of the food and feed Assessment of the anticipated change in total intake of a constituent realistic as well as worst intake scenarios (if the genetic modification has resulted in an altered level of this constituent, or if a new constituent occurs naturally in other food and feed products) Estimation of the anticipated average and maximum intake levels of the food and feed based on representative recent consumption data for products derived from the respective conventional plants. Identification of particular groups of the population with an expected high exposure and should consider them within the risk assessment. Description of any assumption made on the exposure assessment

61 Part II - Sci info (single) Data on anticipated import and production quantities Information on known or anticipated human/animal intake considering all possible routes of exposure Report of any adverse effect(s) of exposure due to individuals professional activities (e.g. farming, seed processing) and studies to further characterise these adverse effects (for this point refer to section of the guidance document) Comments (up to 500 characters) Please insert your comments here C. Risk characterisation Comprehensive risk characterisation considering all available evidence from analyses including - molecular characterisation - phenotypic and agronomic analyses - compositional analyses - toxicity assessment - allergenicity assessment - exposure assessment Description and quantification of uncertainties Demonstration that a) the consumption of food and feed derived from GM plants is as safe as the respective comparators b) the food derived from a GM plant is not nutritionally disadvantageous for the consumer compared to the food which is intended to replace c) the feed derived from a GM plant does not nutritionally disadvantageous for animals compared to the feed which is intended to replace d) the feed derived from a GM plant does not harm or mislead the consumer by impairing distinctive features of the animal products compared to conventionally produced feed 3.3 Indication of assumptions during risk assessment

62 Part II - Sci info (single) Information justifying the inclusion or non inclusion of a proposal for labelling in accordance with Articles 5(3)(f) and 17(3)(f) of Reg. (EC) No 1829/2003 Comments (up to 500 characters) Please insert your comments here D. Post Market Monitoring (PMM) of food and feed derived from GM plants Justification for performing or not a post market monitoring Monitoring plan, if applicable Comments (up to 500 characters) Please insert your comments here 3.4 E. ERA Requirements for the ERA as outlined in the EFSA guidance on the ERA of genetically modified plants 2010 Reference to relevant section of the EFSA guidance on the environmental risk assessment (ERA) of genetically modified plants 2010

63 Part II - Sci info (single) 1. Introduction The issues outlined in chapters 2.3.1, 2.3.2, and of the ERA guidance document should be considered throughout the ERA. EFSA does not expect a dedicated section on these chapters in the submitted application 2. General approach of the ERA The ERA considers the principles of the EFSA guidance on ERA of GM plants (2010) and - is science-based, transparent and performed on a caseby-case basis - follows a systematic approach (6 steps, 7 areas of risks) - follows a comparative approach - addresses uncertainties The ERA considers - immediate and/delayed, direct and indirect effects - intended effects - unintended effects (event-specific ) taking into account the data collected/generated from i) the molecular characterisation ii) the compositional analysis iii) the agronomic and phenotypic characterisation iv) the GM plant-environment interactions taking into account in planta data The ERA considers the scope of the application and the different levels and routes of exposure to the GM plant 2.1 Choice of comparators Description of comparator(s) For single events Assessment of similarities and differences in the interaction of the GM plant and the environment in relation to conventional counterpart (where feasible and appropriate) For vegetatively propagated crops, the conventional counterpart shall, in principle, be the non-gm near-isogenic line 2.1 and

64 Part II - Sci info (single) For sexually reproducing crops, the conventional counterpart shall have a genetic background as close as possible to the GM plant under assessment The following information is provided - Breeding scheme of the GM plant - Breeding scheme of all chosen comparator(s) - Justification for the selection of the comparator(s) - Details and justification of treatments and management regimes 2.2 Receiving environments The relevant receiving environment(s) is/are described including the following: - characteristics of the receiving environments - representative management systems - range of relevant biotic and abiotic interactions Justification of representativeness of the receiving environments Justification of representativeness of the selected management systems Consideration of a worst-case scenario Consideration of the presence of other GM plants in the same receiving environments Justification that the generated data are relevant for other receiving environments and risk conclusions are valid for other receiving environments 2.3 General statistical principles An overview of statistical design and analysis for each study presented in the ERA part of the application For each ERA related study, Appendix G -ERA-statistical design and analysis is compiled Consideration of uncertainties Discussion of the level of uncertainty in the ERA in comparison with the current uncertainties displayed in the scientific literature Description of the types of uncertainties encountered and considered during the different risk assessment steps (steps 1 to 5) Description of the relative importance of these types of uncertainties and their influence on the assessment outcome Highlight and quantification as far as possible of uncertainties inherent in the different steps of the ERA

65 Part II - Sci info (single) Definition as precisely as possible of the terms for the expression of risks and associated uncertainties 2.4 Long-term effects Potential long-term effect(s) are identified and described by a desk study in the 7 areas of risk (chapters of the ERA guidance document) and classified according to - category 1 of long-term effects: result of chronic exposure - category 2 of long-term effects: result of increase in spatial and temporal complexity The long-term effects are addressed in each specific area of risk including - methods, approaches and data sets used to reach conclusions - the basis of and justification for the conclusions - cross-link to parts of the post-market environmental monitoring (PMEM) plan designed to observe possible longterm effects Comments (up to 500 characters) Please insert your comments here Specific areas of risks 3.1. Persistence and invasiveness including plant-to-plant gene flow Step 1: Problem formulation A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) information on the conditions of the production systems and relevant semi-natural and natural habitats Step 2: Hazard characterisation 3 3.1

66 Part II - Sci info (single) Species-specific background information Description of the parental species including information on - the reproductive biology - the characteristics associated with weediness and invasiveness - the factors limiting persistence and invasiveness - the hybridisation and introgression potential with any sympatric compatible relatives Stage 1: Event-specific information on - the seed germination characteristics (see Appendix D ERA agronomic characteristics) - the phenotype under agronomic conditions For each field trial the following Appendices are compiled: Appendix D ERA agronomic characteristics Appendix G ERA statistical design and analysis - the reproductive biology of the GM plant - the potential for seed persistence leading to volunteer occurrence Conclusions of stage 1 assessment Potential unintended effects, resulting from the transformation process, have been shown not to alter the fitness of the GM plant compared to the conventional counterpart in stage 1? if YES, then GM trait specific information can be used in the subsequent stages For plants that can either reproduce or overwinter in the EU consideration of stage 2 For plants that can either reproduce or overwinter: Stage 2: Trait-specific information The applicant has addressed the following questions (see Figure 4 of the ERA guidance document 2010) a) Will the GM plant be more persistent than conventional counterpart under agricultural conditions? b) Will the GM trait increase the fitness of the GM plant or compatible relative under agricultural conditions? c) Can the GM plant form feral populations under EU conditions? d) Can the GM plant hybridise with sympatric compatible relatives outside production systems? Conclusions of stage 2 assessment

67 Part II - Sci info (single) If feral populations are likely and/or if hybridisation is plausible: Stage 3: Trait-specific information The applicant has addressed the following questions (see Figure 4 of the ERA guidance document 2010) a) Will the GM trait alter the fitness of feral plants or compatible relatives in semi-natural habitats? b) Will the GM trait alter the range of feral plants or populations of compatible relatives? Conclusions of stage 3 assessment If altered fitness or the ability to occupy new niches are demonstrated: Stage 4: Trait-specific information The applicant has addressed the following question (see Figure 4 of the ERA guidance document 2010) a) Will the GM trait caused populations of feral plants or compatible relatives to change in size? Conclusions of stage 4 assessment Step 3: Exposure characterisation Exposure characterisation for each hazard identified in step and Identification and description of pathway(s) of exposure 3.1.4: Step 4: Risk characterisation Risk characterisation is provided for all identified risks Information on the acceptability of the characterised risk(s) (within the range defined as acceptable during the problem formulation) 3.1.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies 3.1.6: Step 6: Conclusions Conclusions on - the impact of the GM plant and/or hybridising relatives in the production systems - the impact of the GM plant and/or hybridising relatives in semi-natural and natural habitats

68 Part II - Sci info (single) - the acceptability of the anticipated harm - the risk management strategies needed to mitigate any harm Comments (up to 500 characters) Please insert your comments here 3.2. Plant to micro-organisms gene transfer Step 1: Problem formulation A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) The problem formulation should focus on - the molecular characterization of the DNA sequence inserted, including promoters is given - the presence of antibiotic marker gene (ARM) - the homologies between inserted plant DNA sequences and DNA sequences from relevant microbial recipients - the presence of recipient micro-organisms for transgenic DNA in the receiving environment(s) - Selective conditions enhancing the probability of dissemination and maintenance of the genetic material from the GM plant in natural microbial communities - the persistence of the GM plant material after harvest the potential for long-term establishment of the genetic material from the GM plants in natural microbial communities Hazard characterisation Characterisation of each hazard identified in step Assessment of prevalence and distribution of genes

69 Part II - Sci info (single) Step 3: Exposure characterisation Exposure characterisation for each hazard identified in step and Exposure characterisation is taking into account - the sub-cellular location and copy number of the recombinant DNA - the environmental routes of exposure of the GM plants and the recombinant DNA - the stability of the DNA in the relevant environment(s) The exposure characterisation is considering the different routes of exposure in the receiving environment(s) - the plant production system - the food and feed chain - the gastro-intestinal system 3.2.4: Step 4: Risk characterisation Risk characterisation is provided for each identified risk, e.g. by estimating - the estimated probability of occurrence - any positive selection pressure in receiving environment - the magnitude of the consequences of the adverse effect(s) 3.2.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies 3.2.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s) The potential impacts are also evaluated for indirect effects on biogeochemical cycles

70 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 3.3 Interactions between the GM plant and target organisms Step 1: Problem formulation Description of the target organisms A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) Step 2: Hazard characterisation Evaluation of the potential hazards identified in step 1 e.g. for the target organisms to develop resistance Background information on - the biology, life cycle, ecology and/or behaviour of the target organisms - the resistance mechanisms - the heritability and linkages to virulence, fitness and selective advantage - the distribution of the target organism and its resistant populations in European environments - the host range of the target organism - the population genetics and epidemiology of susceptible and resistant target organisms - the frequency of resistant individuals or resistance allele(s) - the mode of action of the transgenic products towards the target organisms - the baseline susceptibility of the target organisms to the transgenic products Various scenarios are considered, including a worst case scenario 3.3

71 Part II - Sci info (single) Step 3: Exposure characterisation Data characterising the exposure of target organisms to the GM plants should include - expression level of the transgenic products in plant tissues consumed by TO - estimation of the levels of intake of the transgenic product(s) at various development stages of the target organisms - influence of the expression level and its variability on the interaction between the GM plant and the target organism - proportion of the population of the target organisms exposed to the GM plant in the receiving environment(s) - baseline frequency of resistant individuals or resistance/virulence alleles - deployment of other GM plants expressing similar traits in the receiving environment 3.3.4: Step 4: Risk characterisation Risk characterisation is provided for each identified risk identified, e.g. - evolving resistance - developing undesired changes in the interaction between the target plant pathogens and the GM plants in the receiving environment(s) 3.3.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies An IRM plan is presented Annex II-ERA-IRM is compiled Cross link with PMEM taking into account risk management strategies 3.3.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s)

72 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 3.4 Interactions of the GM plant with non-target organisms (NTOs) Step 1: Problem formulation The following elements are considered - the plant and the objective of the inserted trait(s) are clearly described - the receiving environments are clearly described - the selected NTO focal species are clearly described - the selected NTO focal species are commonly present in European environments - if the NTOs are NOT commonly present in European environments, are justifications provided? A problem formulation is given including identification of potential ti hazards identification of pathways of exposure of NTOs to plant/plant products) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) Was the stepwise approach (Figure 5 of the ERA GD 2010) followed to select focal NTO species to be tested? According to this stepwise approach, did you address the following questions step 1: identification of NT functional groups likely to be exposed to the GM plant step 2:categorisation of NT species from identified functional groups did you also consider endangered NT species or species of economic/cultural value? step 3: ranking species based on the ecological criteria step 4: final selection of focal species 3.4

73 Part II - Sci info (single) Step 2: Hazard characterisation Was a tiered approach followed to assess effects on NTO? Did you test at least one focal NTO species per functional group identified? Did you provide tier 1a studies? Did you provide tier 1b (in planta) studies? Did you provide tier 2 studies? If not, justification provided Did you provide tier 3 studies? If not, justification provided For each tier study provided, please indicate the selected assessment and measurements endpoints, the experimental details of the study and the trigger values to move between tiers (see Appendix F) Appendix F - ERA NTO Appendix G - ERA statistical design and analysis Did you also assess unintended effects based on a weight-ofevidence approach? Did you consider the following data? (1) molecular data (2) compositional data (3) data from agronomic & phenotypic field trials (4) GM plant-environment interactions taking into account in planta data Did you provide field-generated data from outside EU? Step 3: Exposure characterisation The exposure of NTO to the newly inserted product(s)/gm plant is evaluated, considering the (0) scope of the application, (1) characteristics of the NTO (e.g. spatial distribution, trophic levels, feeding habits) (2) characteristics of the GM plant, its transgene(s) and the products thereof (e.g. spatial distribution, pollen dispersal & deposition, time/location of pollen, shed, product concentration in the various parts of the plant over the growing season) (3) characteristics of the host plant(s) (e.g. range and spatial distribution of host plants) (4) and other external factors (e.g. rainfall, agricultural management practices) 3.4.4: Step 4: Risk characterisation

74 Part II - Sci info (single) Risk characterisation is provided for each identified risk. Specific characterization and quantification of the identified risk(s) for each selected endpoint (1) in the production site of the GM plant? (2) outside the production site in different habitats where relevant exposure of sensitive NTO may occur? 3.4.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies Were the management strategies designed for worstcase scenario of high exposure? Do they comply with common principles of good agricultural practices like crop rotations, integrated pest management? 3.4.6: 346 Step 6: Conclusions The conclusions are provided, taking into account any proposed risk management strategie(s) (1) in the production site of the GM plant (2) outside the production site in different habitats where relevant exposure of sensitive NTO may occur? Conclusion on intended effects on NTOs Conclusion on unintended effects on NTOs Comments (up to 500 characters) Please insert your comments here 3.5 Impacts of the specific cultivation, management and harvesting techniques Step 1: Problem formulation A problem formulation is given including 3.5

75 Part II - Sci info (single) identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) Identification of the various representative management and production systems in which the GM plant might be introduced Identification of potential changes of receiving environment(s) and management and production systems which are foreseeable in the near future Description how the introduction of the GM plant might alter the existing management and production systems, taking into consideration direct and indirect effects Identification of relevant assessment endpoints representing the aspects of the environment(s) that need to be protected from adverse effects due to changes in cultivation, management and harvesting techniques. Identification of the potential adverse effects that may result from the changes in management and production systems in a range of different environments, taking account of anticipated future changes in agriculture associated with other drivers Step 2: Hazard characterisation For each representative management and production system: Identification of the possible environmental adverse effects due to the change in management practices and cultivation practices, including the cultivation of other plants - Consideration of the potential impact of the GM plant on the cultivation of other plants and of its consequences. - Consequences of risk management measures identified in other chapter sections are being considered ; Information on the potential long-term and indirect environmental impacts of the management and production systems in countries where the GM plant is/has been grown (even outside EU) Models are used to support the risk assessment Step 3: Exposure characterisation 3 scenarios for exposure characterisation are considered

76 Part II - Sci info (single) - a "field level" or "substitution" scenario is described - a "landscape scenario" or "typical" scenario is described - a "worst-case" scenario is described A "fourth" scenario is described considering the potential adoption of other GM plants in the receiving environment Models are used to support the scenario analysis 3.5.4: Step 4: Risk characterisation Risk characterisation is provided for each scenario analysis, e.g. assessment as to whether the specific GM management practices cause greater, similar or lower adverse environmental effects than the current management and production systems they are likely to replace Models are used to complement applicant's statement and clarify uncertainties 3.5.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies Information on whether - the proposed management and production systems are consistent with the environmental protection goals and - the strategies proposed do not pose more harm than non- GM management strategies Models are used to complement applicant's statement and clarify uncertainties 3.5.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s) The conclusions are taking into account effects of further potential changes in the receiving environment(s) and farming systems

77 Part II - Sci info (single) Comments (up to 500 characters) Please insert your comments here 3.6 Effects on biogeochemical processes Step 1: Problem formulation A problem formulation is given including identify potential hazards identify pathways of exposure (plant / environment) identify aspect of the environment to be protected (protection goals) formulate risk hypothesis to be tested define assessment & measurement endpoints define acceptable effect size (limits of concern) Identify if GM plants and their associated management have potential adverse effects on biogeochemical processes compared to the effects of a range of current production systems (link to 3.5) - at production site - in the wider environment Step 2: Hazard characterisation An assessment is provided whether the hazard identified in step 1 would have additional adverse effects relative to current production practice Step 3: Exposure characterisation The exposure of the hazard characterised in step 2 are discussed The assessment of the GM plant and its management affecting biogeochemical processes in the production site is provided The assessment of the GM plant and its management affecting biogeochemical processes in the wider environment is provided 3.6

78 Part II - Sci info (single) The assess the potential exposure to GM plant products through manure or organic plant matter, (imported as fertilizer or soil amendment derived from faeces animal fed GMO) or derived from other bioproducts of industrial processes is provided 3.6.4: Step 4: Risk characterisation Risk characterisation is provided for each risk identified and is carried out both at the production site and in the wider environment The risk characterisation demonstrates that the GM plant and its management do not have more adverse effects on biogeochemical cycles than any present system 3.6.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If yes, the management strategies are proposed and defined The efficacy and reliability of each management strategy are discussed The final level of risk, after applying the management strategies, is provided Cross link with PMEM taking into account risk management strategies Step 6: Conclusions The conclusions are provided, taking into account any risk management strategies The conclusions consider in both production site and the wider environment The conclusions consider long-term effects of adverse changes in biogeochemical processes and address indirect effects on biogeochemical processes as a consequences of altered production practices related to GM plant Comments (up to 500 characters) Please insert your comments here 3.7. Effects on human and animal health The issue is considered in the application 3.7

79 Part II - Sci info (single) Reference is given to the MC-FF GM plant guidance document and to the allergenicity guidance document If the applicant is for non-food or non-feed purposes, reference is given to the EFSA GMO Panel guidance document (EFSA, 2009) 3.8. Overall risk evaluation and conclusions The overall evaluation of the risk of the GM plant in the receiving environment(s) is provided The overall evaluation is taking into account - the risk characterisation - any risk management strategies proposed - assumptions made during the ERA - nature and magnitude of the uncertainties associated Cross link with PMEM taking into account risk management strategies 4. PMEM Plan for General Surveillance (GS) Consideration of the scope of the application and the level of exposure The GS plan relies on the following tools: - GMO-focused systems like farmer questionnaires - existing monitoring networks - literature review Identification of risk(s) or critical uncertainty during the ERA A Case-Specific Monitoring (CSM) plan is provided considering the risk(s) identified during the ERA including any uncertainty on risk management measures An Insect-Resistant Management (IRM) plan is provided If yes, - Appendix E - ERA IRM is compiled - A strategy for managing resistance (e.g. High dose/refuge) is provided - A proposal to train and educate the farmers (e.g. to implement refuges) is provided - A proposal to monitor the implementation of resistance management measures is provided - A proposal to monitor the change in susceptibility of target pests is provided Information on data quality, management and statistical analyses Reporting the results of monitoring on an annual basis 3.8 4

80 Review and adaptation proposed Comments (up to 500 characters) Please insert your comments here Part II - Sci info (single)

81 Part II - Sci info (stack) Part II - Scientific Information Applicants should submit a completed completeness checklist when submitting a new application. EFSA will use this completed list during the completeness check and double check if the information was provided. For each line of this checklist, applicants should check one box only per line. Yes Information provided OR study carried out according to principles of EFSA guidances No Information not provided OR study does not follow requirements of EFSA guidances In either case, a justification is provided in Part II Not applicable The information is not applicable due to the nature of the GM plant and or the derived food/feed. A justification is included in Part II. For EFSA use EFSA agrees EFSA checks if i) the information provided is in line with the EFSA guidances on food, feed or environmental risk assessment, ii) the justification for omission of data/information is acceptable, iii) and if the presentation of the application is in line with the EFSA submission guidance. EFSA comments/questions to applicants General requirements for Part II (as outlined in the EFSA Submission Guidance) For GM plants containing stacked events: summary of relevant information for single events in each section with clear references to previous applications / studies Detailed description of all studies (including experimental methods, analysis of data) Data presentation - Figures and Tables High quality images of gels, blots and other analyses For each table: number, title and legend (see example in Appendix C). For each figure: number, title and legend.(see example in Appendix C). Citations and References Alphabetical reference list at the end of Part II Citations of published studies in line with the formatting requirements of the EFSA Submission guidance section Citations of unpublished studies in line with the formatting requirements of the EFSA Submission guidance section If authors' names are claimed as confidential, they are not included in the citation

82 The food and feed risk assessment considers the principles of the EFSA guidance on risk assessment of food and feed from GM plants (2011) and - follows a comparative approach - follows a stepwise approach (hazard identification, hazard characterisation, exposure assessment, risk characterisation) - is performed on a case-by-case basis Risk assessment of GM plants containing stacked events Risk assessment establishing that the combination of events is stable Risk assessment establishing that no interactions between the stacked events occurs that may raise safety concerns Risk assessment of GM plants containing stacked events focussing on a) stability of inserts b) expression of the introduced genes and their products c) potential synergistic effects resulting from the combination of the events Risk assessment addressing all sub-combinations that occur by natural segregation from the GM plant. Risk assessment of sub-combinations taking into account the different exposure levels covered by the scope of the application Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) Requirements for the Molecular Characterisation and Food and Feed risk assessment as outlined in the EFSA guidance for risk assessment of food and feed from genetically modified plants 2011 Reference to relevant chapters of the EFSA guidance for risk assessment of food and feed from genetically modified plants 2011 A. Hazard identification and characterisation Study overview table - Please refer to the example in Table 2 of Appendix C of the EFSA Submission Guidance 3.1

83 Part II - Sci info (stack) 1. Information relating to the recipient or (where appropriate) parental plants a) Complete name: (a) family name (b) genus (c) species (d) subspecies (e) cultivar/breeding line or strain (f) common name b) Geographical distribution and cultivation of the plant, including the distribution within the Union c) Information on the recipient or parental plants relevant to their safety, including any known toxicity and/or allergenicity of constituents and the plant d) Data on the past and present use of the recipient organism. This information should include: i) the history of safe use for consumption as food and/or feed ii) information on how the plant is typically cultivated, transported and stored iii) whether special processing is required to make the plant safe to eat iv) the description of the normal role of the plant in the diet (e.g. which part of the plant is used as a food source, whether its consumption is important in particular subgroups of the population, what important macro- or micro-nutrients it contributes to the diet) Comments (up to 500 characters) Please insert your comments here Molecular Characterisation for GM plants containing stacked events 2.1 Information relating to the genetic modification Description of the methods used for the genetic modification Summary of the method used to obtain single events Reference where information can be found for the single events Source of nucleic acid used for transformation: Summary of the source of nucleic acid used for transformation to obtain single events

84 Part II - Sci info (stack) Reference where information can be found for the single events Nature and source of vector(s) used including nucleotide sequences intended for insertion Summary of the nature and source of vectors used to obtain single events Reference where information can be found for the single events Comments (up to 500 characters) Please insert your comments here 2.2 Information relating to the GM plant General description of the trait(s) and characteristics which have been introduced or modified - description of the trait(s), of the resulting changes on phenotype and metabolism of the plant, and of its intended use - description of the mode of action Information on the sequences actually inserted or deleted including the following elements: a) size and copy number of all detectable inserts, both complete and partial. This is typically determined by Southern analysis. Southern analyses Summary of the organisation of the inserted genetic material at each insertion site in the single events or in the case of deletion(s), size and function of the deleted region(s), including sub-cellular location(s) of insert(s) Figure and table of the single events structure and elements present within the inserts to assist the comprehension of the Southern analyses used to show integrity of inserts Reference where information can be found for the single events Bioinformatic analyses Bioinformatic overview table of information provided for single events (Appendix C Figure 3) Summary of findings of bioinformatic analyses for the single events (flanking & ORF)

85 Part II - Sci info (stack) In the case of GM plants containing stacked events, applicants should assess the safety of potential interactions between any unintended modifications at each insertion site Information on the expression of the inserted/modified sequence Overview table - Field trial for protein expression analyses (Appendix C Table 4) The following elements should be provided: - data from plants grown under representative conditions including a rationale for the selection of sites - information on specific treatments linked to the trait (e.g. use of herbicides) - rationale for selection of analysed parts - rationale for selection of samples during development - data from minimum three growing sites or one site over three seasons a) methods used - information on specificity of methods used - raw data of protein expression analysis (including reference in main text) b) the range and mean values for the levels of the newly produced protein(s) (or levels of the relevant RNA(s), protein(s) or metabolite(s)) For GM plants containing stacked events for food, feed, import and processing purposes - levels should be compared, in the same field trials, with the levels in any set of GM plants that have all been risk assessed and that include between them all of the events stacked in the GM plant under assessment, and no others For GM plants containing stacked events, which include cultivation in the scope - Depending on the trait, information for the assessment of impacts on target and non-target organisms - data for plants grown under conditions representative of typical cultivation practices in Europe Genetic stability of the inserted/modified sequence and phenotypic stability of the GM plant Summary of stability analyses conducted for single events including evaluation of the safety implications of loss of function of specific genes from multi-gene expression cassettes - comparison of the structure of the inserts to the structure of the inserts in the corresponding single events - analysis carried out on plant materials representative of those aimed for commercial production

86 Part II - Sci info (stack) Comments (up to 500 characters) Please insert your comments here 2.3 Conclusion - information on expression of the insert - information on stability -information on possible interactions between the events - conclusion if genetic modification(s) raise(s) any issues regarding the potential for producing new toxins or allergens conclusion if unintended changes were identified Comments (up to 500 characters) Please insert your comments here 3. Comparative assessment 3.1 Criteria for the selection of comparator(s) Information on the breeding scheme (pedigree) in relation to the GM plant, the conventional counterpart and/or other comparator(s) used in the risk assessment (Appendix C) Details on the nature of the conventional counterpart and/or other comparator(s) Justification for the selection of the conventional counterpart and/or other comparator(s) (NB it is recommended that this selection is done in accordance to chapter of the EFSA guidance document) Justification for the selection of non-gm reference varieties used to estimate the variability required to set equivalence limits (as explained in chapter of the FF guidance)

87 Part II - Sci info (stack) Comments (up to 500 characters) Please insert your comments here 3.2 Field trials: experimental design and statistical analysis Experimental design Test materials included in field trials - GM plant - conventional counterpart and/or other comparator(s) - non-gm reference varieties - For herbicide tolerant GM plants: the GM plant exposed to the intended herbicide(s), the comparator treated with conventional herbicide management regimes and the GM plant treated with the same conventional herbicide management regimes (please refer the EFSA comparator guidance) Description of field trials including information on - geographical locations (including indication on a map) - crop rotation(s)/crops grown in previous seasons in the same plots - management of the field before sowing - date of sowing - soil type - agronomic practices during cultivation including, but not limited to, the following examples: --- herbicide use and other weed management practices --- pest management practices --- fertilizer regime --- irrigation - climatic and other cultivation/environmental conditions during growth - time of harvest - conditions during harvest and storage of the harvested material If the field trial includes one GM plant only, information on the following items should be provided: - Description of the field trial design, including, but not limited to, the following elements:

88 -- Blocking (e.g. completely randomised or randomised block experimental design) -- Rationale underlying choice of locations (NB the different sites selected for the trials should be representative of the range of receiving environments where the crop will be grown) Part II - Sci info (stack) -- Choice of reference lines in each location (NB at each site, there should be at least three appropriate non-gm reference varieties of the crop that have a known history of safe use, which must also be identical between all replicates) -- Identity of the GM plant, conventional counterpart, other comparator(s) and references within the different replicates in each location (NB within each site the GM plant, the conventional counterpart (and additional comparators, where appropriate) should be identical for all replicates) The field trial design respects the following requirements: - At each site: at least three appropriate non-gm reference varieties of the crop that have a known history of safe use - At least 4 replicates for each test material in the particular locations - If less than three non-gm references have been included at a particular site, at least six replicates per site - Each field trial should be replicated at a minimum of eight sites - At least six different reference varieties are used over the entire set of trials - all test materials are randomised to plots within a single field at each site If the field trial includes more than one GM plant of the same species, the following two criteria should be met: i. Occurrence of each of the comparator(s) in the same block as the particular GM plant ii. Randomization of the test materials within each block If the number of plots per block required for such a trial were to exceed 16, a description of the following items: - Description of whether a partially balanced incomplete block design has been used. If so, the following two criteria have been met: i. Each of the comparator(s) always occurs together with its respective GM plant in the same block. ii. All of the non-gm reference varieties appear in each of the incomplete blocks and are fully randomised with the GM plants and their comparator(s). Statistical analysis Raw data in an editable form

89 Part II - Sci info (stack) The programming code used for the statistical analysis in an editable form For each endpoint, the following two tests are performed: - difference test - equivalence test Statistical models used to derive confidence and equivalence limits: - Model used for the calculation of confidence limits for both tests (a linear mixed model is recommended) - Model used for the calculation of equivalence limits for the equivalence test (a mixed model, slightly different from the previous model, is recommended) For each endpoint analysed, the following details should be given: (a) the assumptions underlying the analysis (b) full specification of the mixed models chosen, including fixed and random effects (c) results of any test of interaction between the test materials and sites (d) fixed effects, together with the appropriate estimated residual variation with which it is compared, and variance components for the random factors (e) estimated degrees of freedom (f) any other relevant statistics Discussion of the likely impact of other growing conditions not tested in the trial Graphical display for all endpoints Discussion of all significant differences observed between the GM plant, its comparator and, where applicable, any other test material, focussing on their biological relevance Comments (up to 500 characters) Please insert your comments here 3.3 Compositional analysis Description of the quality standards applied for sampling, analysis and preparation of the material tested Description of the samples analysed, including but not limited to the following: - raw agricultural commodity/ies

90 Part II - Sci info (stack) - processed products (food and feed, food ingredients, feed materials, food and feed additives or food flavourings) Description of the compounds analysed The compounds selection has been in line with OECD consensus documents Description of the categories of parameters that have been analyzed, including but not limited to key macro- and micro-nutrients and other parameters falling in the following categories: - proximates (including moisture and total ash) - anti-nutritional compounds - natural toxins - other secondary plant metabolites characteristic for specific plant species - vitamins and minerals - for oil-rich plants and/or plants used for oil production: fatty acid profile - for plants used as an important protein source: amino acid profile For compounds not listed by OECD, justification for their selection, including the following - metabolites and additional compounds likely to be affected/formed as a result of the particular genetic modification but not mentioned by OECD, to be selected on a case-by-case basis For GM plants containing stacked events the primary concern for risk assessment is to establish that the combination of events is stable and that no interactions between the stacked events, that may raise safety concerns compared to the single events, occur (please refer to the EFSA MC/FF and the comparator guidance documents) A table comprising compositional data (combined site results) including means and ranges for all materials analysed for all single events stacked in the GM plant, the GM plant containing stacked events (which is subject to the application) and all controls Footnotes to this table indicating how these values for the single events were obtained (e.g. number of field sites, season(s), location(s), number of replicates, references to previous applications)

91 Part II - Sci info (stack) Comments (up to 500 characters) Please insert your comments here 3.4 Agronomic and phenotypic characteristics Protocols for these field trials and a description of whether they follow the specifications made in chapter of the EFSA MC/FF guidance document Raw data on agronomic and phenotypic characteristics Description of whether these field trials are different from those for the compositional analysis Comments (up to 500 characters) Please insert your comments here Effects of processing Detailed description of the different processing technologies Assessment whether the processing and/or preserving technologies applied are likely to modify the characteristics of the GM end-products compared with their non-gm counterparts Justification for not performing toxicological tests with the processed products For GM plants with modified metabolism: Scientific rationale for the selected approach to assess processed products For GM plants containing new compounds: - evaluation of effects of processing on these new compounds - evaluation of need for specific processing measures - evaluation whether the processing results in formation of new compounds

92 Part II - Sci info (stack) Comments (up to 500 characters) Please insert your comments here 3.6 Conclusions The conclusions should contain the following statements a) Concluding statement on the agronomic and phenotypic characteristics of the GM plant being, except for the introduced trait(s), different or not to those of its comparator and/or equivalent to the non-gm reference varieties, taking into account natural variation b) Concluding statement on compositional characteristics of the GM plant and derived food and feed being, except for the introduced trait(s), different or not to those of its comparator and/or equivalent to the non-gm reference varieties, taking into account natural variation c) Concluding statement on whether further investigation is needed for those characteristics showing differences between the GM plants and derived food and feed and the comparator, or showing lack of equivalence with respect to non-gm reference varieties, taking into account natural variation d) Concluding statement on whether there are indications of interactions between the combined events in the case of GM plants containing stacked events Comments (up to 500 characters) Please insert your comments here 4. Toxicological assessment Justification for performing toxicological tests Justification for not performing toxicological tests The toxicological assessment considers a) presence and levels of newly expressed proteins 3.1.4

93 Part II - Sci info (stack) b) potential presence of other new constituents c) possible changes in the levels of endogenous constituents beyond normal variation d) impact of other changes in composition due to the genetic modification 4.1 Standardised Guidelines for Toxicity Tests For each toxicity test: Description of the use of test methods (NB internationally agreed test methods described by the OECD or by the European Commission should be used for toxicity testing) For each toxicity test: Justification for the selection of a specific test protocol For each toxicity test: Explanation and justification of any adaptation of these protocols, or use of any methods that differ from these protocols For each toxicity test: If performed in EU, the test follows principles of good laboratory practice as laid down by Directive 2004/10/EC For each toxicity test: If performed outside EU, the test follows the OECD Principles of Good Laboratory Practice (GLP) For each feeding study: - description of experimental design - description of statistical approach - endpoints measurements including feed intake, body weight, animal performance and, if applicable, bioavailability of nutrients For studies other than toxicological studies: A description of the standards and quality systems under which they have been conducted (NB they should be performed under ISO or GLP standards or other appropriate quality assurance systems) Comments (up to 500 characters) Please insert your comments here 4.2 Assessment of newly expressed proteins All newly expressed proteins are assessed

94 Documentation of a proper use and safe consumption as food and feed of both the plant and the newly expressed proteins Part II - Sci info (stack) If a protein produced by microorganisms is used, the following information should be provided "de minimis" so as to be able to sufficiently demonstrate equivalence of the tested protein to the newly expressed protein as expressed in the GM plant: - comparisons of the molecular weight, - comparison of amino acid sequence, - comparison of post-translational modification, - comparison of immunological reactivity - comparison of functional characteristics and biochemical activity Evaluation of significance of differences between the GM plant expressed protein and its microbial substitute with respect to their potential safety impact Demonstration of safety of newly expressed proteins including a) molecular and biochemical characterisation of the newly expressed proteins including - the molecular weight - the amino acid sequence - post-translational modifications - a description of the function - functional characteristics and biochemical activity. In the case of newly expressed enzymes, information on the enzyme activities including the temperature and ph range for optimum activity, substrate specificity and possible reaction products Evaluation of potential interactions between the newly expressed proteins and other plant constituents with respect to safety impact b) up-to-date bioinformatic search for homology - to toxins - to proteins exerting a normal metabolic or structural function - bioinformatic overview table (Appendix D Table 3) and reference to it in the main text c) information on the stability of the protein under the relevant processing and storage conditions for the food and feed derived from the GM plant - examination of influences of temperature and ph changes - characterisation of potential modification(s) of the proteins (e.g. denaturation) and/or production of stable protein fragments generated through processing and storage d) data concerning the resistance of the newly expressed protein to proteolytic enzymes - e.g. in vitro investigations using appropriate and standardised tests

95 Part II - Sci info (stack) - characterisation and evaluation of stable breakdown products with regard to the potential risks associated with their biological activity e) repeated dose toxicity studies using laboratory animals Repeated dose toxicity study OR Information demonstrating the safety of the newly expressed protein (including its mode of action) AND Information demonstrating that the protein is not structurally and functionally related to proteins adversely affecting human or animal health Repeated dose 28-day oral toxicity study in rodents with the newly expressed protein in line with the following requirements: - carried out according to principles specified in OECD guideline sufficient number of animals per group e.g. 10/sex (EFSA recommendation) in order to obtain an adequate statistical power Discussion of the outcome of the 28-day toxicity study, and justify whether further targeted investigations are needed When two or more proteins are newly expressed in the GM plant - assessment of possible synergistic or antagonistic interactions between two or more newly expressed proteins and their relevance to human or animal health Comments (up to 500 characters) Please insert your comments here 4.3. Assessment of new constituents other than proteins Safety assessment of identified new constituents including an evaluation of their toxic potency 4.4 Assessment of altered levels of food and feed constituents Risk assessment (if the genetic modification altered the content of the food and feed constituents beyond the levels expected for the non-gm counterpart), based on- and including a description of - the following items: - physiological function and/or toxic properties of these constituents as well as the anticipated changes in intake levels

96 Part II - Sci info (stack) Justification for performing tests to assess altered levels of food and feed constituents Justification for not performing tests to assess altered levels of food and feed constituents Comments (up to 500 characters) Please insert your comments here 4.5 Assessment of the whole food and/or feed derived from GM plants Justification for performing whole food and/or feed testing Justification for not performing whole food and/or feed testing 90-day feeding study in rodents (if the composition of the food and/or feed derived from GM plant is substantially modified or there are any indications for the potential occurrence of unintended effects based on the preceding molecular, compositional or phenotypic analyses) In the case of GM plants containing stacked events 90-day feeding study in rodents (if there are indications of possible interactions between the events that may cause adverse effects that cannot be tested otherwise) A description of the design and performance of 90-day feeding study in rodents, in particular a description if and how the following principles have been respected: - performance according to the principles of OECD guideline 408 following an adapted protocol - description and justification of adaptations of guideline test groups (NB normally a minimum of two test doses and a negative control are used) - statement explaining as to whether the highest dose is the maximum achievable without causing nutritional imbalance - statement explaining as to whether the lowest dose contains the tested food and/or feed in an amount at least equivalent to the one consumed by humans or animals - information on how the natural variation of test parameters is derived, e.g. from historical background data and/or from the inclusion of reference varieties in the experiments?

97 Part II - Sci info (stack) - statistical analysis (NB this should focus on the detection of possible differences between the test material and its control) Discussion of the outcome of the 90-day feeding study including a justification if further tests are needed Description of other studies than a 90-feeding study that have been provided If so, a justification for this/these study/studies Interpretation of animal studies Evaluation of changes in test parameters, in particular with respect to the following issues: (i) relationship with the applied doses, (ii) possible correlations with changes in other biologically related parameters, (iii) incidental occurrence, (iv) gender specificity and (v) normal biological variation When a difference is noted at the highest dose applied, consideration of other factors to determine whether there is a relationship with treatment. Comments (up to 500 characters) Please insert your comments here 4.6 Conclusions The conclusions of the toxicological assessment should include - but need not be limited to - the following items: a) A conclusion on whether the available information on the newly expressed protein(s) and other new constituents resulting from the genetic modification gives indications, or not, of adverse effects in particular, whether and at which dose levels adverse effects were identified in specific studies b) A conclusion on whether the information on natural constituents, with levels different from those in the comparator, provides indications of adverse effects, in particular, whether and at which dose levels adverse effects were identified in specific studies

98 Part II - Sci info (stack) c) A conclusion on whether toxicologically relevant effects have been identified from the animal studies made on the whole food and feed derived from the GM plant, compared to its comparators Evaluation of the results of the toxicological assessment in the light of anticipated intake of the food and feed derived from GM plants Comments (up to 500 characters) Please insert your comments here 5. Allergenicity assessment 5.1 Assessment of allergenicity of the newly expressed protein Indication whether the source of the transgene is allergenic Assessment of the newly expressed proteins for a possible role in the elicitation of gluten-sensitive enteropathy or other enteropathies which are not IgE-mediated (if the introduced genetic material is obtained from wheat, rye, barley, oats or related cereal grains) Weight of evidence approach for the assessment of possible allergenicity of the newly expressed protein(s) considering the following: a) Amino acid sequence homology comparison between the newly expressed protein and known allergens using a comprehensive database - amino acid sequence homology - structural similarities - bioinformatic overview table (Appendix C Figure 3) and reference to it in the main text Description of the algorithms applied Alignment uses the following criterion: 35% sequence identity to a known allergen over a window of at least 80 amino acids (minimum requirement) - Sequence alignment parameters used in the analysis [all parameters, including calculation of percent identity (PID), are provided] b) Specific serum screening Justification for not performing specific serum screening Justification for performing specific serum screening Specific serum screening should be performed in the following cases:

99 Part II - Sci info (stack) 1) the source of the introduced gene is considered allergenic (select "yes" if applicable); 2) there is any indication of relationship between the newly expressed protein and a known allergen, based on sequence homology or structure similarity (select "yes" if applicable) Specific serum screening study report c) Pepsin resistance and in-vitro digestibility tests if these data have not yet been provided as part of the assessment of potential toxicity of the newly expressed proteins, data on pepsin resistance and in-vitro digestibility should address the following issues: - Pepsin resistance test performed under standardized conditions (e.g. simulated gastric fluid, low ph, fixed pepsin:protein ratio) - Additional in-vitro digestibility tests using different conditions (e.g. pepsin incubations with varying ph values) so as to assess the digestibility of the newly expressed protein in individuals with impaired digestive function - Additional in-vitro digestibility tests in the presence of matrix components, if applicable, that can influence the newly expressed protein's digestibility - Additional in-vitro digestibility tests with the newly expressed protein following conditions representative to food and feed processing, if applicable, that can influence the newly expressed protein's digestibility d) based on outcome of a to c, additional tests for - and data on - potential allergenicity of the newly expressed proteins (optional, e.g. in-vitro and in-vivo data) other tests Comments (up to 500 characters) Please insert your comments here 5.2 Assessment of the allergenicity of the whole GM plant Description of whether the recipient plant species is a major allergenic organism in its own right Justification for not performing allergenicity tests with the whole GM plant Justification for performing allergenicity tests with the whole GM plant

100 Part II - Sci info (stack) Test of any potential change in the allergenicity of the GM plant by comparison of the allergen repertoire with that of its appropriate comparator (e.g. 2-dimensional immunoblots using individual patients' sera) (if the recipient of the introduced gene is known to be allergenic) Comments (up to 500 characters) Please insert your comments here 5.3 Adjuvanticity Consideration of possible role of the newly expressed proteins as adjuvants (if known functional aspects of the newly expressed protein or structural similarity to known strong adjuvants may indicate possible adjuvant activity) Comments (up to 500 characters) Please insert your comments here Conclusions Conclusions indicating whether - the novel protein(s) is (are) likely to be allergenic - the food derived from GM plant is likely to be more allergenic than the comparator Further characterisation of the food derived from the GM plant in the light of anticipated intake and appropriate conditions for placing on the market (if there is a likelihood of allergenicity in one of the above mentioned cases) Labelling proposal (if there is a likelihood of allergenicity in one of the above mentioned cases)

101 Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 6. Nutritional assessment Nutritional evaluation to demonstrate that the introduction of food and feed derived from a GM plant into the market is not nutritionally disadvantageous to humans and animals, respectively. This evaluation should include an assessment of: (i) the nutritional relevance of newly expressed proteins and other new constituents; (ii) the changes in the levels of endogenous constituents in the GM plant and derived food and feed; (iii) the potential alterations in the total diet for the consumers/animals. 6.1 Nutritional assessment of food derived from GM plants Justification for not performing nutritional tests Justification for performing nutritional tests based on a nutritional assessment of food derived from GM plants considering a) the composition of the food with regard to the levels of nutrients and anti-nutrients b) the bioavailability and biological efficacy of nutrients in the food, also taking into account the potential influences of transport, storage and expected treatment of the foods c) the anticipated dietary intake of the food and resulting nutritional impact Comments (up to 500 characters) Please insert your comments here Nutritional assessment of feed derived from GM plants Justification for not performing nutritional tests

102 Justification for performing nutritional tests based on a nutritional assessment of feed derived from GM plants considering - Nutritional assessment using target animal species (if compositional characteristics of the feed derived from a GM plant are different and/or not equivalent to those of its comparator) For nutritionally modified GM plants: Livestock study using an appropriate control diet with similar nutrient profile formulated with a non-gm control supplemented with the specific nutrient as present in the GM plant (if the GM plant contains introduced traits, such as altered nutrient density or levels, affecting animal performance) Target animal feeding studies should span: (i) from the growing and/or the finishing period to slaughter for chickens, pigs, and cattle; (ii) the major part of the lactation cycle for dairy cows; and (iii) the laying cycle for laying hens or quails For each feeding study: - description of experimental design - description of statistical approach - endpoints measurements always including feed intake, body weight, animal performance and bioavailability of nutrients ti t Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 6.3 Conclusions Conclusions of the nutritional assessment indicating: - if the food and feed is nutritionally equivalent to its comparator, taking into account natural variation - in case of lack of equivalence, if the identified changes have an impact on the anticipated intake of the food and feed if the unintended effects of the genetic modification, either identified during hazard identification or assumed based on the preceding molecular, compositional and phenotypic analyses, have affected the nutritional value of the food and feed

103 Part II - Sci info (stack) - in case of GM plants containing stacked events, if there are changes in nutritional value due to additive, synergistic or antagonistic effects of the gene products. This may be particularly relevant where the combined expression of the newly introduced genes affects biochemical pathways. This assessment will clearly require a case-by-case approach Comments (up to 500 characters) Please insert your comments here B. Exposure assessment - Anticipated intake/extent of use Information on - intended function - dietary role - expected level of use of the food and feed derived from GM plants Determination of the concentrations of the newly expressed proteins, other new constituents and endogenous constituents with levels altered as a result of the genetic modification (e.g. due to changes in metabolic pathways) in those parts of the GM plant intended for food or feed use. Estimation of intake taking into account influences of processing storage, and expected treatment of the food and feed Assessment of the anticipated change in total intake of a constituent realistic as well as worst intake scenarios (if the genetic modification has resulted in an altered level of this constituent, or if a new constituent occurs naturally in other food and feed products) Estimation of the anticipated average and maximum intake levels of the food and feed based on representative recent consumption data for products derived from the respective conventional plants. Identification of particular groups of the population with an expected high exposure and should consider them within the risk assessment. Description of any assumption made on the exposure assessment Data on anticipated import and production quantities 3.2

104 Part II - Sci info (stack) Information on known or anticipated human/animal intake considering all possible routes of exposure Report of any adverse effect(s) of exposure due to individuals professional activities (e.g. farming, seed processing) and studies to further characterise these adverse effects (for this point refer to section of the guidance document) Comments (up to 500 characters) Please insert your comments here C. Risk characterisation Comprehensive risk characterisation considering all available evidence from analyses including - molecular characterisation - phenotypic and agronomic analyses - compositional analyses - toxicity assessment - allergenicity assessment - exposure assessment Description and quantification of uncertainties Demonstration that a) the consumption of food and feed derived from GM plants is as safe as the respective comparators b) the food derived from a GM plant is not nutritionally disadvantageous for the consumer compared to the food which is intended to replace c) the feed derived from a GM plant does not nutritionally disadvantageous for animals compared to the feed which is intended to replace d) the feed derived from a GM plant does not harm or mislead the consumer by impairing distinctive features of the animal products compared to conventionally produced feed Indication of assumptions during risk assessment Information justifying the inclusion or non inclusion of a proposal for labelling in accordance with Articles 5(3)(f) and 17(3)(f) of Reg. (EC) No 1829/

105 Part II - Sci info (stack) Comments (up to 500 characters) Please insert your comments here D. Post Market Monitoring (PMM) of food and feed derived from GM plants Justification for performing or not a post market monitoring Monitoring plan, if applicable Comments (up to 500 characters) Please insert your comments here 3.4 E. ERA Requirements for the ERA as outlined in the EFSA guidance on the ERA of genetically modified plants 2010 Reference to relevant section of the EFSA guidance on the environmental risk assessment (ERA) of genetically modified plants Introduction The issues outlined in chapters 2.3.1, 2.3.2, and of the ERA guidance document should be considered throughout the ERA. EFSA does not expect a dedicated section on these chapters in the submitted application

106 Part II - Sci info (stack) 2. General approach of the ERA The ERA considers the principles of the EFSA guidance on ERA of GM plants (2010) and - is science-based, transparent and performed on a caseby-case basis - follows a systematic approach (6 steps, 7 areas of risks) - follows a comparative approach - addresses uncertainties The ERA considers - immediate and/delayed, direct and indirect effects - intended effects - unintended effects (event-specific ) taking into account the data collected/generated from i) the molecular characterisation ii) the compositional analysis iii) the agronomic and phenotypic characterisation iv) the GM plant-environment interactions taking into account in planta data The ERA considers the scope of the application and the different levels and routes of exposure to the GM plant 2.1 Choice of comparators Description of comparator(s) For GM plants containing stacked events The conventional counterpart, if available, should be used as the comparator If the conventional counterpart not available, non GM line derived from the breeding scheme used to develop the GM plant non GM line with agronomic properties as similar as possible to the GM plant containing the stacked events The following information is provided - Breeding scheme of the GM plant - Breeding scheme of all chosen comparator(s) - Justification for the selection of the comparator(s) - Details and justification of treatments and management regimes 2.2 Receiving environments The relevant receiving environment(s) is/are described including the following: - characteristics of the receiving environments - representative management systems - range of relevant biotic and abiotic interactions 2.1 and

107 Justification of representativeness of the receiving environments Justification of representativeness of the selected management systems Consideration of a worst-case scenario Consideration of the presence of other GM plants in the same receiving environments Justification that the generated data are relevant for other receiving environments and risk conclusions are valid for other receiving environments 2.3 General statistical principles An overview of statistical design and analysis for each study presented in the ERA part of the application For each ERA related study, Appendix G -ERA-statistical design and analysis is compiled Consideration of uncertainties Discussion of the level of uncertainty in the ERA in comparison with the current uncertainties displayed in the scientific literature Description of the types of uncertainties encountered and considered during the different risk assessment steps (steps 1 to 5) Description of the relative importance of these types of uncertainties and their influence on the assessment outcome Highlight and quantification as far as possible of uncertainties inherent in the different steps of the ERA Definition as precisely as possible of the terms for the expression of risks and associated uncertainties 2.4 Long-term effects Potential long-term effect(s) are identified and described by a desk study in the 7 areas of risk (chapters of the ERA guidance document) and classified according to - category 1 of long-term effects: result of chronic exposure - category 2 of long-term effects: result of increase in spatial and temporal complexity The long-term effects are addressed in each specific area of risk including - methods, approaches and data sets used to reach conclusions - the basis of and justification for the conclusions - cross-link to parts of the post-market environmental monitoring (PMEM) plan designed to observe possible longterm effects 2.5 For GM plants containing stacked events Part II - Sci info (stack)

108 For GM plant containing stacked events / for Import and Processing Part II - Sci info (stack) ERA of GM plant containing stacked events shall address all sub-combinations as occurring by natural segregation Each sub-combination (from natural segregation) has been evaluated, discussed and conclusions for the ERA provided For GM plant containing stacked events / for cultivation of the higher stacked events only Consideration of the full range of environmental issues of concern including change in management of the higher stacked events compared to lower stacked events or single events already risk assessed Consideration of all other sub-combinations of these events that may occur by natural segregation (e.g. volunteers) Each sub-combination (from natural segregation) has been evaluated, discussed and conclusions for the ERA provided For GM plant containing stacked events / for cultivation of the higher stacked events and specified sub-combinations Consideration of the full range of environmental issues of concern of the higher stacked events and the specified subcombinations for cultivation Description of the management of each of the cultivated subcombinations individually and assessment of their environmental impact Consideration of all other sub-combinations bi of these events that may occur by natural segregation (e.g. volunteers) Each sub-combination (from natural segregation) has been evaluated, discussed and conclusions for the ERA provided Comments (up to 500 characters) Please insert your comments here 3. Specific areas of risks 3.1. Persistence and invasiveness including plant-to-plant gene flow Step 1: Problem formulation A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) 3 3.1

109 Part II - Sci info (stack) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) information on the conditions of the production systems and relevant semi-natural and natural habitats Step 2: Hazard characterisation Species-specific background information Description of the parental species including information on - the reproductive biology - the characteristics associated with weediness and invasiveness - the factors limiting persistence and invasiveness - the hybridisation and introgression potential with any sympatric compatible relatives Stage 1: Event-specific information on - the seed germination characteristics (see Appendix D ERA agronomic characteristics) - the phenotype under agronomic conditions For each field trial the following Appendices are compiled: Appendix D ERA agronomic characteristics Appendix G ERA statistical design and analysis - the reproductive biology of the GM plant - the potential for seed persistence leading to volunteer occurrence Conclusions of stage 1 assessment Potential unintended effects, resulting from the transformation process, have been shown not to alter the fitness of the GM plant compared to the conventional counterpart in stage 1? if YES, then GM trait specific information can be used in the subsequent stages For plants that can either reproduce or overwinter in the EU consideration of stage 2 For plants that can either reproduce or overwinter: Stage 2: Trait-specific information The applicant has addressed the following questions (see Figure 4 of the ERA guidance document 2010) a) Will the GM plant be more persistent than conventional counterpart under agricultural conditions? b) Will the GM trait increase the fitness of the GM plant or compatible relative under agricultural conditions? c) Can the GM plant form feral populations under EU conditions?

110 Part II - Sci info (stack) d) Can the GM plant hybridise with sympatric compatible relatives outside production systems? Conclusions of stage 2 assessment If feral populations are likely and/or if hybridisation is plausible: Stage 3: Trait-specific information The applicant has addressed the following questions (see Figure 4 of the ERA guidance document 2010) a) Will the GM trait alter the fitness of feral plants or compatible relatives in semi-natural habitats? b) Will the GM trait alter the range of feral plants or populations of compatible relatives? Conclusions of stage 3 assessment If altered fitness or the ability to occupy new niches are demonstrated: Stage 4: Trait-specific information The applicant has addressed the following question (see Figure 4 of the ERA guidance document 2010) a) Will the GM trait caused populations of feral plants or compatible relatives to change in size? Conclusions of stage 4 assessment Step 3: Exposure characterisation Exposure characterisation for each hazard identified in step and Identification and description of pathway(s) of exposure : Step 4: Risk characterisation ti Risk characterisation is provided for all identified risks Information on the acceptability of the characterised risk(s) (within the range defined as acceptable during the problem formulation) 3.1.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies 3.1.6: Step 6: Conclusions Conclusions on - the impact of the GM plant and/or hybridising relatives in the production systems - the impact of the GM plant and/or hybridising relatives in semi-natural and natural habitats

111 - the acceptability of the anticipated harm - the risk management strategies needed to mitigate any harm Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 3.2. Plant to micro-organisms gene transfer Step 1: Problem formulation A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) The problem formulation should focus on - the molecular characterization of the DNA sequence inserted, including promoters is given - the presence of antibiotic marker gene (ARM) - the homologies between inserted plant DNA sequences and DNA sequences from relevant microbial recipients - the presence of recipient micro-organisms for transgenic DNA in the receiving environment(s) - Selective conditions enhancing the probability of dissemination and maintenance of the genetic material from the GM plant in natural microbial communities - the persistence of the GM plant material after harvest the potential for long-term establishment of the genetic material from the GM plants in natural microbial communities Hazard characterisation Characterisation of each hazard identified in step Assessment of prevalence and distribution of genes Step 3: Exposure characterisation Exposure characterisation for each hazard identified in step and 3.2.2

112 Exposure characterisation is taking into account - the sub-cellular location and copy number of the recombinant DNA - the environmental routes of exposure of the GM plants and the recombinant DNA - the stability of the DNA in the relevant environment(s) The exposure characterisation is considering the different routes of exposure in the receiving environment(s) - the plant production system - the food and feed chain - the gastro-intestinal system 3.2.4: Step 4: Risk characterisation Risk characterisation is provided for each identified risk, e.g. by estimating - the estimated probability of occurrence Part II - Sci info (stack) - any positive selection pressure in receiving environment - the magnitude of the consequences of the adverse effect(s) 3.2.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies 3.2.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s) The potential impacts are also evaluated for indirect effects on biogeochemical cycles Comments (up to 500 characters) Please insert your comments here 3.3 Interactions between the GM plant and target organisms Step 1: Problem formulation 3.3

113 Part II - Sci info (stack) Description of the target organisms A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) Step 2: Hazard characterisation Evaluation of the potential hazards identified in step 1 e.g. for the target organisms to develop resistance Background information on - the biology, life cycle, ecology and/or behaviour of the target organisms - the resistance mechanisms - the heritability and linkages to virulence, fitness and selective advantage - the distribution of the target organism and its resistant populations in European environments - the host range of the target organism - the population genetics and epidemiology of susceptible and resistant target organisms - the frequency of resistant individuals or resistance allele(s) - the mode of action of the transgenic products towards the target organisms - the baseline susceptibility of the target organisms to the transgenic products Various scenarios are considered, including a worst case scenario Step 3: Exposure characterisation Data characterising the exposure of target organisms to the GM plants should include - expression level of the transgenic products in plant tissues consumed by TO - estimation of the levels of intake of the transgenic product(s) at various development stages of the target organisms - influence of the expression level and its variability on the interaction between the GM plant and the target organism - proportion of the population of the target organisms exposed to the GM plant in the receiving environment(s)

114 - baseline frequency of resistant individuals or resistance/virulence alleles - deployment of other GM plants expressing similar traits in the receiving environment 3.3.4: Step 4: Risk characterisation Risk characterisation is provided for each identified risk identified, e.g. - evolving resistance - developing undesired changes in the interaction between the target plant pathogens and the GM plants in the receiving environment(s) 3.3.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies An IRM plan is presented Annex II-ERA-IRM is compiled Cross link with PMEM taking into account risk management strategies 3.3.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s) Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 3.4 Interactions of the GM plant with non-target organisms (NTOs) Step 1: Problem formulation The following elements are considered - the plant and the objective of the inserted trait(s) are clearly described - the receiving environments are clearly described - the selected NTO focal species are clearly described - the selected NTO focal species are commonly present in European environments 3.4

115 - if the NTOs are NOT commonly present in European environments, are justifications provided? A problem formulation is given including identification of potential hazards identification of pathways of exposure of NTOs to plant/plant products) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) Was the stepwise approach (Figure 5 of the ERA GD 2010) followed to select focal NTO species to be tested? According to this stepwise approach, did you address the following questions step 1: identification of NT functional groups likely to be exposed to the GM plant step 2:categorisation of NT species from identified functional groups did you also consider endangered NT species or species of economic/cultural value? step 3: ranking species based on the ecological criteria step 4: final selection of focal species Step 2: Hazard characterisation Was a tiered approach followed to assess effects on NTO? Did you test at least one focal NTO species per functional group identified? Did you provide tier 1a studies? Did you provide tier 1b (in planta) studies? Did you provide tier 2 studies? If not, justification provided Did you provide tier 3 studies? If not, justification provided For each tier study provided, please indicate the selected assessment and measurements endpoints, the experimental details of the study and the trigger values to move between tiers (see Appendix F) Appendix F - ERA NTO Appendix G - ERA statistical design and analysis Did you also assess unintended effects based on a weight-ofevidence approach? Did you consider the following data? (1) molecular data Part II - Sci info (stack)

116 Part II - Sci info (stack) (2) compositional data (3) data from agronomic & phenotypic field trials (4) GM plant-environment interactions taking into account in planta data Did you provide field-generated data from outside EU? Step 3: Exposure characterisation The exposure of NTO to the newly inserted product(s)/gm plant is evaluated, considering the (0) scope of the application, (1) characteristics of the NTO (e.g. spatial distribution, trophic levels, feeding habits) (2) characteristics of the GM plant, its transgene(s) and the products thereof (e.g. spatial distribution, pollen dispersal & deposition, time/location of pollen, shed, product concentration in the various parts of the plant over the growing season) (3) characteristics of the host plant(s) (e.g. range and spatial distribution of host plants) (4) and other external factors (e.g. rainfall, agricultural management practices) 3.4.4: Step 4: Risk characterisation Risk characterisation is provided for each identified risk. Specific characterization and quantification of the identified risk(s) for each selected endpoint (1) in the production site of the GM plant? (2) outside the production site in different habitats where relevant exposure of sensitive NTO may occur? 3.4.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies Were the management strategies designed for worstcase scenario of high exposure? Do they comply with common principles of good agricultural practices like crop rotations, integrated pest management? 3.4.6: Step 6: Conclusions

117 The conclusions are provided, taking into account any proposed risk management strategie(s) (1) in the production site of the GM plant (2) outside the production site in different habitats where relevant exposure of sensitive NTO may occur? Conclusion on intended effects on NTOs Conclusion on unintended effects on NTOs Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 3.5 Impacts of the specific cultivation, management and harvesting techniques Step 1: Problem formulation A problem formulation is given including identification of potential hazards identification of pathways of exposure (plant / environment) identification of aspects of the environment to be protected (protection goals) risk hypothesis to be tested definition of assessment & measurement endpoints definition of acceptable effect size (limits of concern) 3.5 Identification of the various representative management and production systems in which the GM plant might be introduced Identification of potential changes of receiving environment(s) and management and production systems which are foreseeable in the near future Description how the introduction of the GM plant might alter the existing management and production systems, taking into consideration direct and indirect effects Identification of relevant assessment endpoints representing the aspects of the environment(s) that need to be protected from adverse effects due to changes in cultivation, management and harvesting techniques. Identification of the potential adverse effects that may result from the changes in management and production systems in a range of different environments, taking account of anticipated future changes in agriculture associated with other drivers

118 Part II - Sci info (stack) Step 2: Hazard characterisation For each representative management and production system: Identification of the possible environmental adverse effects due to the change in management practices and cultivation practices, including the cultivation of other plants - Consideration of the potential impact of the GM plant on the cultivation of other plants and of its consequences. - Consequences of risk management measures identified in other chapter sections are being considered ; Information on the potential long-term and indirect environmental impacts of the management and production systems in countries where the GM plant is/has been grown (even outside EU) Models are used to support the risk assessment Step 3: Exposure characterisation 3 scenarios for exposure characterisation are considered - a "field level" or "substitution" scenario is described - a "landscape scenario" or "typical" scenario is described - a "worst-case" scenario is described A "fourth" scenario is described considering the potential adoption of other GM plants in the receiving environment Models are used to support the scenario analysis 3.5.4: Step 4: Risk characterisation Risk characterisation is provided for each scenario analysis, e.g. assessment as to whether the specific GM management practices cause greater, similar or lower adverse environmental effects than the current management and production systems they are likely to replace Models are used to complement applicant's statement and clarify uncertainties 3.5.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If needed, proposal and definition of the management strategies Assessment of efficacy and reliability of each management strategy Information on the expected reduction in risk associated with the management strategies Cross link with PMEM taking into account risk management strategies Information on whether

119 - the proposed management and production systems are consistent with the environmental protection goals and - the strategies proposed do not pose more harm than non- GM management strategies Models are used to complement applicant's statement and clarify uncertainties 3.5.6: Step 6: Conclusions Conclusions taking into account any proposed risk management strategie(s) The conclusions are taking into account effects of further potential changes in the receiving environment(s) and farming systems Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack) 3.6 Effects on biogeochemical processes Step 1: Problem formulation A problem formulation is given including identify potential hazards identify pathways of exposure (plant / environment) identify aspect of the environment to be protected (protection goals) formulate risk hypothesis to be tested define assessment & measurement endpoints define acceptable effect size (limits of concern) Identify if GM plants and their associated management have potential adverse effects on biogeochemical processes compared to the effects of a range of current production systems (link to 3.5) - at production site - in the wider environment Step 2: Hazard characterisation An assessment is provided whether the hazard identified in step 1 would have additional adverse effects relative to current production practice Step 3: Exposure characterisation The exposure of the hazard characterised in step 2 are discussed 3.6

120 The assessment of the GM plant and its management affecting biogeochemical processes in the production site is provided The assessment of the GM plant and its management affecting biogeochemical processes in the wider environment is provided The assess the potential exposure to GM plant products through manure or organic plant matter, (imported as fertilizer or soil amendment derived from faeces animal fed GMO) or derived from other bioproducts of industrial processes is provided 3.6.4: Step 4: Risk characterisation Risk characterisation is provided for each risk identified and is carried out both at the production site and in the wider environment The risk characterisation demonstrates that the GM plant and its management do not have more adverse effects on biogeochemical cycles than any present system 3.6.5: Step 5: Risk management strategies Information on whether any risk management strategies are needed If yes, the management strategies are proposed and defined The efficacy and reliability of each management strategy are discussed The final level of risk, after applying the management strategies, is provided Cross link with PMEM taking into account risk management strategies Step 6: Conclusions The conclusions are provided, taking into account any risk management strategies The conclusions consider in both production site and the wider environment The conclusions consider long-term effects of adverse changes in biogeochemical processes and address indirect effects on biogeochemical processes as a consequences of altered production practices related to GM plant Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack)

121 Part II - Sci info (stack) 3.7. Effects on human and animal health The issue is considered in the application Reference is given to the MC-FF GM plant guidance document and to the allergenicity guidance document If the applicant is for non-food or non-feed purposes, reference is given to the EFSA GMO Panel guidance document (EFSA, 2009) 3.8. Overall risk evaluation and conclusions The overall evaluation of the risk of the GM plant in the receiving environment(s) is provided The overall evaluation is taking into account - the risk characterisation - any risk management strategies proposed - assumptions made during the ERA - nature and magnitude of the uncertainties associated Cross link with PMEM taking into account risk management strategies 4. PMEM Plan for General Surveillance (GS) Consideration of the scope of the application and the level of exposure The GS plan relies on the following tools: - GMO-focused systems like farmer questionnaires - existing monitoring networks - literature review Identification of risk(s) or critical uncertainty during the ERA A Case-Specific Monitoring (CSM) plan is provided considering the risk(s) identified during the ERA including any uncertainty on risk management measures An Insect-Resistant Management (IRM) plan is provided If yes, - Appendix E - ERA IRM is compiled - A strategy for managing resistance (e.g. High dose/refuge) is provided - A proposal to train and educate the farmers (e.g. to implement refuges) is provided - A proposal to monitor the implementation of resistance management measures is provided - A proposal to monitor the change in susceptibility of target pests is provided Information on data quality, management and statistical analyses Reporting the results of monitoring on an annual basis

122 Review and adaptation proposed Comments (up to 500 characters) Please insert your comments here Part II - Sci info (stack)

123 Part III Cartagena Protocol Part III - Cartagena Protocol Cartagena Protocol with minimum information as specified in Annexes II and III of Regulation 1946/2003 on a) Name and contact details of the applicant Yes, provided No, not provided (justification required) Not applicable (justification required) For EFSA use only EFSA comments/quest EFSA agrees ions to applicants b) Name and contact details of the authority responsible for the decision c) Name and identity of the living modified organism d) Description of the gene modification, the technique used, and the resulting characteristics of the living modified organism e) Any unique identification of the living modified organism f) Taxonomic status, common name, point of collection or acquisition, and characteristics of recipient organism or parental organisms related to biosafety g) Centres of origin and centres of genetic diversity of the recipient organism and/or the parental organisms and a description of the habitats where the organisms may persist or proliferate. h) Taxonomic status, common name, point of collection or acquisition, and characteristics of the donor organism or organisms related to biosafety i) Approved uses of the living modified organism j) A risk assessment report consistent with Annex III of the Cartagena protocol i. Characteristics of the living modified organism that may have adverse effects on biological diversity or on human health

124 Part III Cartagena Protocol ii. Likelihood of (these) adverse effects iii. Evaluation of the consequences of these adverse effects iv. Estimation of the overall risk (likelihood and consequences of the adverse effects) v. Recommendation if the risks are acceptable or manageable (managing strategies) vi. If the level of risk is uncertain: provision of information on the specific issues of concern, implementation of risk management strategies, monitoring of the living modified organism k) Suggested methods for the safe handling, storage, transport and use, including packaging, labelling, documentation, disposal and contingency procedures, where appropriate.

125 Part IV Labelling Part IV - Labelling Yes, provided No, not provided (justificat ion required) Not applicable (justification required) For EFSA use only EFSA agrees EFSA comments/questions to applicants The application shall include Proposal for labelling in accordance with Articles and Articles of Regulation (EC) No 1829/2003

126 Part V Methods of detection Part V - Methods of detection, sampling and reference materials Yes, provided No, not provided (justificat ion required) Not applicabl e (justificat ion required) For EFSA use only EFSA agrees EFSA comments/questions to applicants Methods of detection, sampling Reference to the place where the reference material can be accessed shall be provided in the application.

127 Part VI Add info Part VI - Additional information to be provided for GM plants and/or food/feed containing or consisting of GM plants Yes, provided No, not provided (justificat ion required) Not applicabl e (justificat ion required) For EFSA use only EFSA agrees EFSA comments/questio ns to applicants The information required by Annex III to Directive 2001/18/EC shall be provided where it is not covered by the requirements of other parts of the application. Proposed commercial names of the products and names of GMOs contained therein, and any name or code used to identify the GMO. Name and address of the person responsible for the placing on the market. Name and address of the supplier(s) of control samples. Description of how the product and the GMO as or in product are intended to be used. Description of the geographical area(s) and environments where the product is intended to be used. Intended categories of users of the product. Information on the genetic modification, which can be used for the detection and identification of particular GMO products to facilitate post-marketing control. Proposed labelling on a label or in an accompanying document. Measures to take in case of unintended release or misuse. Specific instructions for storage and handling. Specific instructions for carrying out monitoring and reporting to the notifier and, if required, to the competent authority (in line with Dir. 2001/18/EC, Annex VII part C) Proposed restrictions in the approved use of the GMO.

128 Part VI Add info Proposed packaging Estimated production in and/or imports to the Community Proposed additional labelling.

129 Part VII - Summary Part VII - Summary 1. General Information provided not provided not relevant/ not applicable For EFSA use only EFSA agrees EFSA comments/question s to applicants 1.1 Details of application a) Member state of application b) Application number c) Name of the product (commercial and other names) d) Date of acknowledgement of valid application 1.2. Applicant a) Name of applicant b) Address of applicant c) Name and address of the representative of the applicant established in the Union (if the applicant is not established in the Union) 1.3. Scope of the application 1.4 Is the product or the uses of the associated plant protection product(s) already authorised or subject to another authorisation If yes, specify 1.5. Has the GM plant been notified under Part B of Directive 2001/18/EC? If no, refer to risk analysis data on the basis of the elements of Part B of Directive 2001/18/EC 1.6. Has the GM plant or derived products been previously notified for marketing in the Community under Part C of Directive 2001/18/EC? If yes, specify

130 Part VII - Summary 1.7. Has the product been notified in a third country either previously or simultaneously? If yes, specify 1.8. General description of the product a) Name of the recipient or parental plant and the intended function of the genetic modification b) Types of products planned to be placed on the market according to the authorisation applied for and any specific form in which the product must not be placed on the market (seeds, cut-flowers, vegetative parts, etc.) as a proposed condition of the authorisation applied for c) Intended use of the product and types of users d) Any specific instructions and/or recommendations for use, storage and handling, including mandatory restrictions proposed as a condition of the authorisation applied for e) If applicable, geographical areas within the EU to which the product is intended to be confined under the terms of the authorisation applied for. f) Any type of environment to which the product is unsuited g) Any proposed packaging requirements h) Any proposed labelling requirements in addition to those required by law and when necessary a proposal for specific labelling in accordance with Articles 13(2), (3) and 25(2)(c), (d) and 25(3) of Regulation (EC) No 1829/2003. In the case of GMO plants, food and/or feed containing or consisting of GMO plants, a proposal for labelling has to be included complying with the requirements of Annex IV, A(8) of Directive 2001/18/EC. i) Estimated potential demand (i) In the Union (ii) In export markets for EU supplies j) Unique identifier in accordance with Regulation (EC) No 65/2004

131 Part VII - Summary 1.9. Measures suggested by the applicant to take in case of unintended release or misuse as well as measures for disposal and treatment 2. Information relating to (A) the recipient or (B) (where appropriate) parental plants 2.1. Complete name a) Family name b) Genus c) Species d) Subspecies e) Cultivar/breeding line f) Common name 2.2. Geographical distribution and cultivation of the plant, including the distribution within the Union 2.3. Information concerning reproduction (for environmental safety aspects) a) Mode(s) of reproduction b) Specific factors affecting reproduction c) Generation time 2.4. Sexual compatibility with other cultivated or wild plant species (for environmental safety aspects) 2.5. Survivability (for environmental safety aspects) a) Ability to form structures for survival or dormancy b) Specific factors affecting survivability 2.6. Dissemination (for environmental safety aspects)

132 Part VII - Summary a) Ways and extent of dissemination b) Specific factors affecting dissemination 2.7. Geographical distribution and cultivation of the plant, including the distribution in Europe of the compatible species 2.8. In the case of a plant species not grown in the Member State(s), description of the natural habitat of the plant, including information on natural predators, parasites, competitors and symbionts (for environmental safety aspects) 2.9. Other potential interactions, relevant to the GM plant, of the plant with organisms in the ecosystem where it is usually grown or used elsewhere, including information on toxic effects on humans, animals and other organisms (for environmental safety aspects) 3. Molecular Characterisation 3.1. Information relating to the genetic modification a) Description of the methods used for the genetic modification b) Nature and source of vector used c) Source of donor DNA, size and intended function of each constituent fragment of the region intended for insertion 3.2. Information relating to the GM plant Description of the trait(s) and characteristics which have been introduced or modified Information on the sequences actually inserted or deleted a) The copy number of all detectable inserts, both complete and partial b) In case of deletion(s), size and function of the deleted region(s) c) Sub-cellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or maintained in a non-integrated form), and methods for its determination d) The organisation of the inserted genetic material at the insertion site e) In case of modifications other than insertion or deletion, describe function of the modified genetic material before and after the modification

133 Part VII - Summary Information on the expression of the insert a) Information on developmental expression of the insert during the life cycle of the plant b) Parts of the plant where the insert is expressed Genetic stability of the insert and phenotypic stability of the GM plant Information on how the GM plant differs from the recipient plant in a) Mode(s) and/or rate of reproduction b) Dissemination c) Suvivability d) Other differences Any change to the ability of the GM plant to transfer genetic material to other organisms a) Plant to bacteria gene transfer b) Plant to plant gene transfer: 4. Comparative Analysis 4.1. Choice of the conventional counterpart and additional comparators 4.2. Experimental design and statistical analysis of data from field trials for comparative analysis Description of the experimental design (Number of locations, growing seasons, geographical spread, replicates and number of commercial varieties in each location) Selection of material and compounds for analysis 4.4. Comparative analysis of agronomic and phenotypic characteristics 4.5. Effect of processing

134 Part VII - Summary 5. Toxicology a) Toxicological testing of newly expressed proteins b) Testing of new constituents other than proteins c) Information on natural food and feed constituents d) Testing of the whole GM food/feed 6. Allergenicity a) Assessment of allergenicity of the newly expressed protein b) Assessment of allergenicity of the whole GM plant 7. Nutritional assessment a) Nutritional assessment of GM food b) Nutritional assessment of GM feed 8. Exposure assessment - Anticipated intake/extent of use 9. Risk characterisation for the safety assessment of GM food and feed 10. Post-market monitoring of GM food/feed 11. Environmental assessment Mechanism of interaction between the GM plant and target organisms Potential changes in the interactions of the GM plant with the biotic environment resulting from the genetic modification a) Persistence and invasiveness b) Selective advantage or disadvantage c) Potentioal for gene transfer

135 Part VII - Summary d) Interactions between the GM plant and target organisms e) Interactions of the GM plant with non-target organisms f) Effects on human health g) Effects on animal health h) Effects on biogeochemical processes i) Impacts of the specific cultivation, management and harvesting techniques Potential interactions with the abiotic environment 12. Environmental monitoring plan a) General (risk assessment, background information) b) Interplay between environmetnal risk assessment and monitoring c) Case-specific GM plant monitoring (approach, strategy, method and analysis) d) General surveillance of the impact of the GM plant (approach, strategy, method and analysis) e) Reporting the results of monitoring 13. Detection and event-specific identification techniques for the GM plant 14. Information relating to previous releases of the GM plant and/or derived products History of previous releases of the GM plant notified under Part B of the Directive 2001/18/EC and under Part B of Directive 90/220/EEC by the same notifier a) Notification number b) Conclusions of post-release monitoring c) Results of the release in respect to any risk to human health and the environment (submitted to the Competent Authority according to Article 10 of Directive 2001/18/EC)

136 Part VII - Summary History of previous releases of the GM plant carried out outside the Community by the same notifier a) Release country b) Authority overseeing the release c) Release site d) Aim of the relaese e) Duration of the release f) Aim of post-releases monitoring g) Duration of post-releases monitoring h) Conclusions of post-releases monitoring i) Results of the release in respect to any risk on human health and the environment

137 Appendix C APPENDIX C: EXAMPLES OF FIGURES AND TABLES FOR PART II This appendix contains examples of the types of figures and tables that may be included in an application. Figures and tables are useful to provide an overview of studies in the application and snap-shots of each study, to add clarity to parts of a study with illustrations, and to streamline the risk assessment process. These figures and tables should not be viewed as precise templates as the data in each application differs. They are non-binding, omission of certain details in the exemplar tables or figures does not mean these data are not necessary, for example, the fatty acid analysis in Table 2 contains only a limited number of fatty acids. Other formats of these figures and tables will be accepted, provided that the same aim is achieved. Table of contents 1. Example of an overview table indicating the title of the studies, the section they relate to and the type of information Example of study overview table.4 A. GM plant containing a single event....4 B. GM plant containing stacked events Example of overview table on bioinformatic analyses...15 A. GM plant containing a single event B. GM plant containing stacked events Example of a summary table related to a field trial for the protein expression analysis Example of a breeding tree Examples of Southern data representation..19 1

138 Appendix C Table 1: Example of a general overview table of data provided in the Part II of an application indicating the title of the studies, the section they relate to and the type of information they contain. This table should be provided as a separate appendix. Every time additional information is provided, this table should be updated and provided as a separate appendix. SECTION NAME TITLE RELATED SECTION IN PART II INFORMATION 1 Main text (no additional info needs to be added) e.g. Appendix X e.g. Molecular characterisation of insert e.g. A.2.1 New document 2 Non-CI Appendices From Apxx 3 -flanking Updated study 4 CI Appendices References 5 1 Some additional information to clarify the way information can be provided is given in footnotes 2 This analyses can be found in the current application 3 This study has also been provided in the frame of applicationxx 4 A study had been provided in the frame of a previous application but has been updated in the current application 5 No description is expected, except for key studies they should be added (maximum 10) 2

139 Appendix C Table 2: Example of application overview table focussing on study reports based on an application for herbicide tolerance GM maize. This table should be provided as a separate appendix. Every time additional information is provided, this table should be updated and provided as a separate appendix. A. GM plant containing single event APPLICATION IDENTIFICATION CODE (EVENT NAME) Single event Comparators event name conventional counterpart commercial varieties newly expressed proteins protein A protein B n.a. n.a. Traits n.a. n.a. Breeding tree (Appendix xx Pxx) (Appendix xx Pxx) Scope 1. Food 1.1 GM plants for food use 1.2 Food containing or consisting of GM plants 1.3 Food produced from GM plant s or containing ingredients produced from GM plants 2. Feed 2.1 GM plants for feed use 2.2 Feed containing or consisting of GM plants 2.3 Feed produced from GM plants 3. GM plants for environmental release 3.1 Import and processing 3.2 Seeds and plant propagating material for cultivation in Europe Overview of the anticipated uses and products (Appendix xx page xx) MOLECULAR CHARACTERISATION Information on the zygosity of the insert in the to be commercialised plant e.g. F1 hybrid hemizygous for the newly introduced genes Information on the biology of the crop (self- or cross-pollinator) Insert structure and backbone presence (sequence) (Appendix xx) Number of Inserts & copy number of the newly introduced gene A gene B No backbone sequence present or partial vector backbone present (including following elements x & y) name of the gene promoter driving expression of gene A and gene B Ref to sequence (Appendix xx) Bioinformatic analyses Ref to bioinformatic overview table (Appendix xx): Flanking sequence (Appendix xx): ORF analyses Stability/integrity (Segregation) (Appendix xx) Genetic stability stable insertion in nucleus confirmed by PCR and Southern on x generations (Fn, BCx). negative control used in Southern analyses:e.g. near-isogenic NGMx/NGMy or commercial hybrid xx negative control used in Southern and PCR n.a. n.a. 3

140 Appendix C (Appendix xx) Phenotypic stability APPLICATION IDENTIFICATION CODE (EVENT NAME) Single event event name consistent expression level of proteins A and B in x generations (Fn, BCx). segregation analysis of on x generations (Fn, BCx). Protein expression Ref to protein field trial overview table (Appendix xx) Field trial year (production plan ID) -country, nr sites, x hybrid (NGMx(BCxFx)/NGMy & NGMz(BCxFx)/NGMy) - zygosity of the insert in the analysed plants and a detailed description on the grain content if segregation occurs -leaves (developmental stage xx and xx), roots, pith, silk, pollen, whole plants at anthesis stage and kernels. -treated and untreated with targeted herbicide regime (Appendix xx) Field trial year (production plan ID) -country, nr sites, (NGMx(BCxFx)/NGMy) -leaves (developmental stage xx and xx), roots (developmental stage xx and xx), whole plants at four growth stages, kernels, piths, silk, pollen. -treated and untreated with targeted herbicide regime Other molecular studies (Appendix xx) e.g. RT-PCR on ORF3 Comparators conventional counterpart analyses e.g. near-isogenic NGMx/NGMy or commercial hybrid xx control (e.g. near-isogenic NGMx/NGMy) used to test specificity of antibody negative control e.g. nearisogenic NGMx/NGMy commercial varieties Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines e.g. Genetic stability was only shown in 4 generations due to the long generation time of the species (see main text page xx) n.a. n.a. Compositional analysis COMPARATIVE ANALYSIS (Appendix xx) Field trial year (country, nr sites, production plan ID) NGMx(BCxFx)/NGMy (BCxFx) Herbicide regime sprayed unsprayed not applicable nr parameter analyzed in forage o Proximate (ash, fat, moisture, protein, carbohydrates, ADF, NDF) o mineral (Ca, P) nr parameter analyzed in grain o o proximates (ash, fat, moisture, protein, carbohydrates, ADF, NDF) minerals (Ca, copper, Fe,Mg, manganese, P, potassium, selenium, Na, Zn) o amino acids composition (18) o fatty acides (16:0 Palmitic, 18:0 Stearic, 18:1 Oleic, 18:2 Linoleic, 18:3 Linolenic) o vitamins (A, B1, B2, B3, B6, B9, E) o secondary metabolites and anti nutrients (ferulic acid, p-coumaric acid, inositol, phytic acid, trypsin inhibitor, furfural, raffinose) near-isogenic NGMx/ NGMy nr. commercial hybrid + ranges of natural variation (ILSL, 2006) (OECD, 2002) (add info year-mon-date Appendix xx) Field trial year (country, nr sites, production plan ID) near-isogenic NGMz/ NGMy nr. commercial hybrid 4

141 Appendix C APPLICATION IDENTIFICATION CODE (EVENT NAME) Single event event name NGMx/NGMy(BCxFx) treated or untreated with target herbicide nr parameter analyzed in forage (as 200x) nr parameter analyzed in grain (more than 200x) o proximate (+ starch) o minerals (as 200x) o amino acids composition (as 200x) o fatty acids (+ 20:0 arachidic, 20:1 eicosenoic, 22:0 behenic) o vitamins (as 200x) o secondary metabolites and anti nutrients (as 200x) conventional counterpart Comparators commercial varieties + ranges of natural variation (ILSL, 2006) (OECD, 2002) Agronomic traits & phenotypic stability (Appendix xx) Field trial year (country, nr sites, production plan ID) NGMx/NGMy(BCxFx) nr agronomic traits nr disease trait were evaluated. Not all traits were recorded at all locations. (add info year-mon-date Appendix xx) statistical analysis TOXICITY Bioinformatics of newly expressed proteins to Toxin databases Ref to bioinformatic overview table (Appendix xx) BLASTP to Genbank non-redundant xx 201x Equivalence between microbial recombinant protein vs. plant protein (Appendix xx) Amino acid comparison alignment indicated on pxx (Appendix xx) comparison protein A produced by E.coli to the leaf extract bacterial strain used for producing recombinant protein plant tissue from which the native protein was extracted list type of analysis (e.g., concentration, purity, immunoreactivity, molecular weight, glycosylation and N-terminal aa and insecticidal activity was determined by SDS-PAGE, western, peptide mass mapping analysis, N-terminal sequence, glycosylation analysis, insect bioassay, etc). (Appendix xx) protein A produced by E.coli vs the plant extract (Appendix xx) protein B produced by E.coli vs the plant extract bacterial strain used for producing recombinant protein plant tissue from which the native protein was extracted list type of analysis (e.g., concentration, purity, immunoreactivity, molecular weight, glycosylation and N-terminal aa and insecticidal activity was determined by SDS-PAGE, western, peptide mass mapping analysis, N-terminal sequence, glycosylation analysis, insect bioassay, etc) near-isogenic NGMy/ NGMx leaf extract from e.g., a negative segregant nr. commercial hybrid n.a. Acute oral toxicity test (Appendix xx) protein A protein source: e.g., E.coli duration: e.g., 14 days dosage: e.g., 0 and 1250 mg protein / kg body weight animals (species, number): e.g., inbred mice (nr. Female + nr. male) negative control: e.g., corn oil 5

142 Appendix C (Appendix xx) protein B as above Repeated-dose oral toxicity test (Appendix xx) protein A APPLICATION IDENTIFICATION CODE (EVENT NAME) Single event event name protein source: e.g., E.coli duration: e.g., 14 days dosage: e.g., 0, 200, 1000 and 5000 mg protein / kg body weight animals (species, number): e.g., inbred mice (nr. Female + nr. male) negative control: e.g., corn oil (Appendix xx) protein B as above 90-day animal feeding study (Appendix xx) F2 grain NGMy/ NGMx(BCxFx) dosage: e.g., 10 or 41.5% of grain animals (species, number): e.g., inbred mice (Nr F + Nr M) diet component analysis statistical analysis: gm impact, gender impact Other toxicity studies (Appendix xx) conventional counterpart near-isogenic NGMy/ NGMx Comparators commercial varieties nr. commercial hybrid Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines e.g. why certain toxicity tests are not necessary (see main text page xx) ALLERGENICITY Bioinformatics of newly expressed proteins to Allergen databases Reference to bioinformatic overview table (Appendix xx) e.g., FARRP 201x Proteolytic degradation (Appendix xx) in vitro SGF digestibility assay (ph xx) on protein A (Appendix xx) in vitro SGF digestibility assay (ph xx) on protein B (Appendix xx) in vitro SIF digestibility assay on protein A (Appendix xx) in vitro SIF digestibility assay on protein B specify the host e.g. bacterial strain used for producing recombinant protein In vitro IgE binding assay (Appendix xx) Other immunological studies (Appendix xx) Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines e.g. why certain immunological tests are not necessary (see main text page xx) Exposure NUTRITIONAL ASSESSMENT 6

143 Appendix C APPLICATION IDENTIFICATION CODE (EVENT NAME) Single event event name (Technical dossier pxx) anticipated intake of proteins A and B from consuming crop xx in EU (Appendix xx pxx) previous exposure of human and animals to proteins A and B Nutritional assessment by animal study (Appendix xx) e.g. broiler study F2 grain NGMx/ NGMy(BCxFx) dosage animals (species, number): e.g., each genotype used Nr F + Nr M [nr birds/pen x nr pens], in total nr diet component analysis statistical analysis: gm impact, gender impact Other nutritional studies (Appendix xx) conventional counterpart isogenic NGMx/ NGMy Comparators commercial varieties nr. commercial variety Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines e.g. why certain nutritional tests are not necessary (see main text page xx) Note: ERA please fill in the Appendices D, E, F, G 1) for data generated in other relevant application, please indicate the EFSA application identification code. 2) please distinguish not applicable (n.a.) from not provided (n.p.), for the latter a justification shall be included. 3) a laboratory study shall be always cleared referred in the table, a reference includes (author name, year, study ID). 4)NGMx/NGMy is a example of genetic background of a GM maize hybrid. 5)BCxFx refers to the number of backcrosses and the number of selfing during plant breeding. 7

144 Appendix C B. GM plant containing stacked events Event name A x B x conventional counterpart APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties newly expressed proteins n.a. n.a. traits n.a. n.a. Breeding tree (Appendix xx page xx) (Appendix xx page xx) Scope 1. Food 2. Feed 1.1 GM plants for food use 1.2 Food containing or consisting of GM plants 1.3 Food produced from GM plant s or containing ingredients produced from GM plants 2.1 GM plants for feed use 2.2 Feed containing or consisting of GM plants 2.3 Feed produced from GM plants 3. GM plants for environmental release 3.1 Import and processing 3.2 Seeds and plant propagating material for cultivation in Europe Overview of the anticipated uses and products (Appendix xx Pxx) ISSUES CONSIDERED DURING THE SAFETY ASSESSMENT OF GM PLANTS CONTAINING STACKED EVENTS Assessment of interaction(s) (Appendix xx) list arguments in bullet points, indicate laboratory studies with clear reference Assessment of sub-combinations (Appendix xx) list arguments in bullet points, indicate laboratory studies with clear reference A B (add one column for each additional event) 8

145 Appendix C Event name A x B x conventional counterpart Insert structure insert structure/backbone sequence APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties MOLECULAR CHARACTERISATION Information on the zygosity of the insert in the to be commercialised plant e.g. F1 hybrid hemizygous for the newly introduced genes Information on the biology of the crop (self- or cross-pollinator) (Appendix xx) Integrity via method inserts: Control description n.a. Nr of inserts/nr of copies/backbone A B (add one column for each additional event) Nr of inserts/nr of copies/backbone Sequence Bioinformatic analyses Ref to bioinformatic overview table Flanking sequence Updated in this dossier/ up-to date in previous (see singles) E.g. See single n.a. n.a. Ref to current or previous dossier where the studies can be found n.a. n.a. Ref to current or previous dossier where the most up-to-date studies can be found Ref to current or previous dossier where the studies can be found Ref to current or previous dossier where the most up-to-date studies can be found ORF analysis Updated in this dossier/ up-to date in previous (see singles) n.a. n.a. Ref to current or previous dossier where the most up-to-date studies can be found Ref to current or previous dossier where the most up-to-date studies can be found Stability/integrity Genotypic (Appendix xx) Method & Number of generations control n.a. Method & Number of generations Method & Number of generations 9

146 Appendix C Event name A x B x conventional counterpart APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties A B (add one column for each additional event) Phenotypic (Appendix xx) Method & Number of generations n.a. Method & Number of generations Method & Number of generations Protein expression Ref to protein field trial overview table (Appendix xx) Year(s) (+ location and nr of sites) of studies in current application Year(s) (+ location and nr of sites) (Appendix xx) Control used to test specificity of antibody n.a. (Appendix xx) Year(s) (+ location and nr of sites) (Appendix xx) Year(s)(+ location and nr of sites) List tissues that were analyzed List tissues that were analyzed n.a. n.a. List tissues that were analyzed List tissues that were analyzed Other relevant info (zygosity of the insert in the analysed plant, indicate if inserts segregate in the analysed grain/seed, specific treatment) specific treatment n.a. n.a. specific treatment specific treatment Raw/data production plan ID Reference n.a. n.a. Reference Reference Data in related dossiers n.a. n.a. n.a. (APxx, Appendix xx) Year(s) (+ nr of sites) Other molecular studies (APxx, Appendix xx) Year(s) (+ nr of sites) Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines COMPARATIVE ANALYSIS Compositional analysis 10

147 Appendix C Event name A x B x conventional counterpart (Appendix xx) Field trial year (country, nr. site, production ID) (Appendix xx) compositional analysis (Appendix xx) statistical analysis nr parameter in forage NGMx/NGMy(BCxFx) list parameters nr parameter in grain APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Statistical analysis across location per site Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Statistical analysis across location per site Nr. varieties commercial Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Statistical analysis across location per site A B (add one column for each additional event) Herbicide regime Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Statistical analysis across location per site sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Statistical analysis across location per site B020x/B971x(BCxFx) list parameters Agronomic traits & phenotypic stability (Appendix xx) Field trial year (country, nr. site, production ID) (Appendix xx) agronomic study NGMx/NGMy(BCxFx) nr agronomic traits nr disease trait Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Nr. varieties commercial Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Herbicide regime sprayed unsprayed not applicable field trial (country, nr. of sites, production plan ID) Bioinformatics of newly expressed proteins to Toxin databases TOXICITY 11

148 Appendix C Event name A x B x conventional counterpart Reference to bioinformatic overview table (Appendix xx) BLASTP to e.g., Genbank nonredundant xx 201x APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties Database name & version Equivalence between microbial recombinant protein vs. plant protein (Appendix xx) based on data of single events Acute oral toxicity test (Appendix xx) assessment in light of data of single events Repeated-dose oral toxicity test (Appendix xx) assessment in light of data of single events protein source duration dosage animals (species, number) negative control protein source duration dosage animals (species, number) negative control n.a. n.a. Database name & version of the last update n.a. n.a. bacterial strain used for producing recombinant protein plant tissue from which the native protein was extracted n.a. n.a. protein source duration dosage animals (species, number) n.a. n.a. protein source duration dosage animals (species, number) A B (add one column for each additional event) Database name & version of the last update bacterial strain used for producing recombinant protein plant tissue from which the native protein was extracted protein source duration dosage animals (species, number) protein source duration dosage animals (species, number) 90-day animal feeding study (Appendix xx) diet component diet component diet component diet component diet component 12

149 Appendix C Event name A x B x conventional counterpart Other toxicity studies (Appendix xx) e.g., assessment of synergistic or antagonistic toxicity by combining newly expressed proteins APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties duration dosage animals (species, number) protein source duration dosage animals (species, number) duration dosage animals (species, number) duration dosage animals (species, number) negative control Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines ALLERGENICITY Bioinformatics of newly expressed proteins to Allergen databases Reference to bioinformatic overview table (Appendix xx) e.g., FARRP 201x Proteolytic degradation (Appendix xx) assessment in light of data of single events In vitro IgE binding assay (Appendix xx) Other immunological studies Database name & version duration dosage animals (species, number) A B (add one column for each additional event) duration dosage animals (species, number) n.a. n.a. n.a. n.a. n.a. n.a. Database name & version of the last update n.a. n.a. specify the host e.g. bacterial strain used for producing protein in vitro SGF in vitro SIF recombinant Database name & version of the last update specify the host e.g. bacterial strain used for producing protein in vitro SGF in vitro SIF recombinant 13

150 Appendix C Event name A x B x conventional counterpart (Appendix xx) APPLICATION IDENTIFICATION CODE (EVENT NAME) Stacked event Comparators for the stacked event Single events (obligatory) / Parent events (if available) commercial varieties Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines NUTRITIONAL ASSESSMENT Exposure (Technical dossier pxx) anticipated intake of proteins A, B from consuming crop xx in EU n.a. n.a. (Appendix xx pxx) previous exposure of human and animals to proteinx A, B A B (add one column for each additional event) Nutritional assessment by animal study (Appendix xx) e.g. broiler study diet component duration dosage Other nutritional studies (Appendix xx) animals (species, number) diet component duration dosage animals (species, number) diet component duration dosage animals (species, number) diet component duration dosage animals (species, number) diet component duration dosage animals (species, number) Rationale if certain studies were not deemed needed or not in line with EFSA GMO Panel guidelines ERA please fill in the Appendices D, E, F, G Note: 1) for data generated in other relevant application, please indicate the EFSA application identification code. 2) please distinguish not applicable (n.a.) from not provided (n.p.), for the latter a justification shall be included. 3) a laboratory study shall be always cleared referred in the table, a reference includes (author name, year, study ID). 4) NGMx/NGMy is a example of genetic background of a GM maize hybrid. 5) BCxFx refers to the number of backcrosses and the number of selfing during plant breeding. 14

151 Appendix C Table 3: Example of overview table on bioinformatic analyses. This table/these tables should be included or in the main text, or in the specific studies of the Part II of an application, or as a separate appendix. In case the bioinformatic analysis is updated these tables should be amended. Since the risk assessment performed by the EFSA GMO Panel may not start immediately after validity for applications for GM plants including the scope cultivation and applications for GM plants containing stacked events for which single event(s) have not been risk assessed, for these types of applications, the completeness check of the bioinformatic analyses will be limited to checking if the application includes: (1) a summary of the results, (2) an overview of the studies, related to the different aspects (flanking sequences, ORFs, newly expressed proteins, see below), and (3) a clear and correct reference where the studies (including the outputs) can be found. In the case of applications for GM plants containing stacked events, it will be accepted that bioinformatic studies are not included in the technical dossier in case they have been summarised and properly referred to in the main text. Please note that other formats of overview tables will be accepted as long as the information to be included in the example formats is summarised. A. GM plant containing single event Flanking sequences (both against DNA and protein databases) General Database 1 Date 2 Algorithms 3 in dossier 4 EST Database 1* Date 2 Algorithms 3 dossier 4 Ref. to place Ref. to place in Nucleotide 1 Protein 1 ORF analyses insert-plant (a) / insert-insert (b)* / whole insert (c) Allergen database 1 Date 2 Algorithms 3 in dossier 4 toxin*) database 1 Date 2 Algorithms 3 dossier 4 Ref. to place General (and Ref. to place in (a) Newly expressed proteins Protein 1 Protein 2 Allergen database 1 Date 2 Algorithms 3 in dossier 4 Ref. to place General or toxindatabase Ref. to place in 1 Date 2 Algorithms 3 dossier 4 1. e.g. Genbank non-redundant nucleotide, Genbank non-redundant protein, Genbank general/plant/species EST, FARRP vs. xx (including version and using official name) 2. release date of the version of the database used for the analysis 3. algorithm e.g. BLASTn, BLASTx, BLASTp, FASTA,... and indicate if default settings were used and if not which parameter was adjusted 4. application number, place in dossier (e.g. technical dossier, additional information with date); citation and internal reference number * include specifics in the table only when applicable and provided 15

152 Appendix C B. GM plant containing stacked events Flanking sequences (both against DNA and protein databases) General Ref. to place Ref. to place in Database 1 Date 2 Algorithms 3 in dossier 4 EST Database 1* Date 2 Algorithms 3 dossier 4 event 1 Nucleotide 1 Protein 1 event 2 Nucleotide 1 Protein 1 event 3 Nucleotide 1 Protein 1 event 1 (a) event 1 (b) event 2 event 3 ORF analyses insert-plant (a) / insert-insert (b)* / whole insert (c) Allergen Ref. to place General (and Ref. to place in database 1 Date 2 Algorithms 3 in dossier 4 toxin*) database 1 Date 2 Algorithms 3 dossier 4 Newly expressed proteins protein 1 protein 2 protein... Allergen Ref. to place in database 1 Date 2 Algorithms 3 dossier 4 General or toxindatabase Ref. to place in 1 Date 2 Algorithms 3 dossier 4 1. e.g. Genbank non-redundant nucleotide, Genbank non-redundant protein, Genbank general/plant/species EST, FARRP vs. xx (including version and using official name 2. release date of the version of the database used for the analysis 3. algorithm e.g. BLASTn, BLASTx, BLASTp, FASTA,... and indicate if default settings were used and if not which parameter was adjusted 4. application number, place in dossier (e.g. technical dossier, additional information with date); citation and internal reference number * include specifics in the table only when applicable and provided 16

153 Appendix C Table 4: Example of a summary table related to a field trial for the protein expression analyses. This table/these tables should be included in the specific study reports of the Part II of an application For each field trial (site) carried out to analyse the protein expression levels of the GM plant (including the controls such as GM plants containing single/related stacked events and/or non-gm comparator) a summary data sheet must be filled out. Therefore in one application multiple sheets may be required. Consider including tables for field trials described in previous or related applications submitted to EFSA. Field trial ID Protein(s) analysed A B Method of analysis (indicate if methods are identical between different field trials) Season Country/state/region (nr of sites) GM analysed with identification code, generation and genetic background Comparator(s) (non-gm; single events; parental lines-including genetic background) GM specific treatment(s)(such as specific herbicide) Tissues sampled/developmental stage (number of replicates) All tissues were analysed for each sites (if not please indicate) Report reference Production plan reference Raw data reference and kind of statistical analyses Reference where argumentation of choose of sites can be found Reference where argumentation of choose of tissues can be found 17

154 Appendix C Figure 1: Example of a breeding tree. This figure should be included or in the main text, or as a separate annex, if applicable. In case an additional generation was created and used in a study the figure should be amended. 18

155 Appendix C Examples of Southern data representation Similar figures and tables should be included or in the main text, or in the specific study report. Table 5: Genetic element A summary of genetic elements on the plasmid and in the insert Size Location Description, function and reference Figure 2: A schematic representation of the insert To support the Southern analysis EFSA requests that a schematic overview of the insert (final structure in the plant including any rearrangements/duplications/deletions) showing the position of the genetic elements, restriction sites, different probes/primers and the length of the different expected fragments is included. Table 6: A table with expected and observed fragments, including the information in which figure they can be found. Expected fragment Observed band Figure Please provide this for both samples and positive controls. Probe 1 Probe 2 Restriction Restriction enzyme(s) enzyme(s) combination A combination B Restriction enzyme(s) combination A Restriction enzyme(s) combination B 19

156 Appendix D1 EFSA identification code for the application (event name) Appendix D1 Schematic summary of data for field or greenhouse trial for agronomic and phenotypic characteristics within a season (single event) A schematic summary should be provided for each field trial conducted for the comparative analysis of agronomic and phenotypic characteristics. It should be stored in the folder Appendices, subfolder ERA_Appendices D to G. Study report of field trial (e.g. Appendix X or author et al. (year)): Field trial design: Field trial objective: Season (year) and dates: Location (country): Number of sites: Number of replicates: Type of plot design: Statistical power analysis: specify the name of the Appendix G ERA statistical design and analysis same as field trial for compositional analysis different 1. Information on the tested plant material Plant material Identification code in study report Replicates GM plant Comparator(s) 1.. Reference varieties Treatments Treatment Code 1. Treatment 1. n. Treatment n Genotype and name Specification of treatment (herbicide, insecticide, other) 1

157 Appendix D1 4. Information on agronomic and phenotypic characteristics (Field trials) Agronomic Evaluation characteristic time 1. Plant establishment and vigour Evaluation description Please specify how observations were evaluated and quantified (e.g. unit of measurement) Raw data provided Yes No 2. Time of flowering Yes and maturity No 3. Growth Yes No 4. Plant height Yes No 5. Dry matter Yes production No 6. Seed Yes No 7. Yield Yes characteristics No 8. Vernalisation Yes requirement No 9. Attractiveness to Yes pollinators No 10. Pollen shed & Yes viability 11. Pollen compatibility & morphology 12. Others Yes No No Yes No 5. Information on biotic and abiotic stressor(s) tested Biotic or abiotic stressors Characteristics analysed Raw data provided 1. Insect incidence Yes No 2. Diseases observation Yes No 3. Abiotic stressors Yes No 4. Others Yes No 2

158 Appendix D1 6. Dormancy and germination assessment and pollen morphology and viability assessment Reference to study Type of study Control Germination endpoint Replicates Summary of analyses Dormancy and germination Pollen morphology and viability Differences observed Biological relevance Conclusions For EFSA use 7. Summary of analysis from Tables 1 to 6 Agronomic and phenotypic characteristics Statistically significant differences Combined sites Individual sites Pleas e specif y Biological relevance Combined sites Individual sites Please specify Environmental observations Differences observed Combined sites Individual sites Please specify Biological relevance Please specify Conclusions For EFSA use 3

159 Appendix D2 EFSA identification code for the application (event name) Appendix D2 Schematic summary of data for field or greenhouse trial for agronomic and phenotypic characteristics within a season (GM plant containing stacked transformation events) A schematic summary should be provided for each field trial conducted for the comparative analysis of agronomic and phenotypic characteristics. It should be stored in the folder Appendices, subfolder ERA_Appendices D to G. Study report of field trial (e.g. Appendix X or author et al. (year)): Field trial design: Season (year) and dates: Location (country): Number of sites: Number of replicates: Type of plot design: Statistical power analysis: specify the name of the Appendix G ERA statistical design and analysis same as field trial for compositional analysis different Field trial objective: 1. Information on the tested plant material Plant material Identification code in study report Replicates GM plant containing stacked events ABC GM single event A GM single event B GM single event C Comparator(s) 1.. Reference varieties Treatments 1

160 Appendix D2 Treatment Code 1. Treatment 1. n. Treatment n Genotype and name Specification of treatment (herbicide, insecticide, other) 4. Information on agronomic and phenotypic characteristics Agronomic characteristic Evaluation time Raw data provided 1. Plant establishment and vigour 2. Time of flowering and maturity Evaluation description Please specify how observations were quantified (e.g. unit of measurement) Yes No Yes No 3. Growth Yes No 4. Plant height Yes No 5. Dry matter Yes production No 6. Seed Yes No 7. Yield Yes characteristics No 8. Vernalisation Yes requirement No 9. Yes Attractiveness No to pollinators 10. Pollen shed & viability 11. Pollen compatibility & morphology Yes No Yes No 12. Others Yes Comparison data single/stacked events Please specify if observations differed from data obtained on each single event (including assessment of biological relevance) 2

161 Appendix D2 No 5. Information on biotic and abiotic stressor(s) tested Biotic or abiotic stressors 1. Insect incidence 2. Diseases observation 3. Abiotic stressors Characteristics analysed Raw data provided Yes No Yes No Yes No Comparison data single/stacked events Please specify if observations differed from data obtained on each single event 4. Others Yes No 6. Dormancy and germination assessment and pollen morphology and viability assessment Reference to study Type of study Control Germination endpoint Replicates Summary of analyses Dormancy and germination Pollen morphology and viability Differences observed Biological difference Conclusions For EFSA use 3

162 Appendix D2 7. Summary of analysis from Tables 1 to 6 Agronomic and phenotypic characteristics (see 4.) Environmental observations (see 5. and 6.) Statistical differences Please specify Biological relevance Please specify Differences observed Please specify Biological relevance Combined Combined Combined sites sites sites Individual Individual Individual sites sites sites Please specify Conclusions For EFSA use 4

163 Appendix E EFSA identification code for the application (event name) Appendix E Schematic summary of information for Insect Resistance Management Appendix E is requested for applications of GM insect resistant plants with the scope seeds and plant propagating material for cultivation in the EU. The compiled appendix should be stored in the folder Appendices, subfolder ERA_Appendices D to G. 1. Information on the target specific spectrum List of target insect species 1. [name target organism] 2. n. 2. IRM plan and structure The IRM plan is High dose/refuge strategy Yes No Medium to low dose / refuge strategy Yes No Data on concentration of the insecticidal protein(s) in the GM plant are provided Yes No Data on proportion of target insects killed by the GM plant are provided Yes No Size of the refuge provided Yes No The IRM plan includes A monitoring for any potential evolution of resistance Yes No An educational programme Yes No A remedial action plan Yes No 3. Underlying assumptions Data on occurrence of resistance alleles in target insect population are provided Yes No Data on frequency of resistance alleles to the insecticidal proteins are provided Yes No If not provided, data are provided on o Efficacy of the GM plant in controlling target insects Yes No o Baseline susceptibility in the target insect Yes No Mating occur randomly between resistant and susceptible insects Yes No Data on mating and dispersal behaviour are provided Yes No 1

164 Appendix E Data on inheritance of resistance alleles (dominant, partially or fully recessive), Yes No including dominance value h, are provided Duration (i.e. number of generations) of susceptibility of target insects is considered Yes No Modelling prediction are used Yes No 2

165 Appendix F EFSA identification code for the application (event name) Appendix F Schematic summary of NTO studies (laboratory, greenhouse, field trials) For each functional group, a table should be compiled summarising the NTO studies submitted in support of the application. The compiled appendix should be included in the folder Appendices, subfolder ERA_Appendices D to G. 1. HERBIVORES Common name Reference Taxonomy (Order, Family, Genus, species) Appendix X or author et al., year Peer reviewed Yes/No Site of the experiment Country name Type of experiment Tier 1a, 1b, 2, 3 Stressor Standard toxin, purified product (from bacteria, from GM plants) GM plant, plant part (specify), etc. Event Comparator(s) Control (negative, positive) Number of replications Duration of the experiment Experimental design e.g. GM plant containing stacked events A x B (contains proteins A, B, ) Years and dates e.g. split-plot Taxonomy (Order, Family, Genus, species) Taxonomy (Order, Family, Genus, species) 1

166 Appendix F Development stage Source of specimen Test performed Feeding conditions Measurement Endpoint Larvae/adult In house colony, purchased from commercial suppliers, field collected Laboratory test Conditions e.g. diet (plant, plant part, artificial diet), [xx ug protein A/g of diet protein/ml xx ug protein B/g of diet Choice/no choice, ad libitum/fixed dose e.g. body mass, mortality, fecundity Agronomic and phenotypic field trials (for compositional analysis) Agronomic and ecological field trials (for compositional analysis) Environmental conditions Effects observed Reference to Appendix G ERA statistical design and analysis e.g. temperature, photoperiod, relative humidity Specify the name of the appendix For EFSA use 2

167 Appendix F 2. PREDATORS Common name Reference Taxonomy (Order, Family, Genus, species) Appendix X or author et al., year Peer reviewed Yes/No Site of the experiment Country name Type of experiment Tier 1a, 1b, 2, 3 Stressor Standard toxin, purified product (from bacteria, from GM plants) GM plant, plant part (specify), etc. Event Comparator(s) Control (negative, positive) Number of replications Duration of the experiment Experimental design Development stage Source of specimen Test performed Feeding conditions Measurement Endpoint e.g. GM plant containing stacked events A x B (contains proteins A, B, ) Years and dates e.g. split-plot Larvae/adult In house colony, purchased from commercial suppliers, field collected Laboratory test Conditions e.g. diet (plant, plant part, artificial diet), [xx ug protein A/g of diet protein/ml xx ug protein B/g of diet Choice/no choice, ad libitum/fixed dose e.g. body mass, mortality, fecundity Taxonomy (Order, Family, Genus, species) Agronomic and phenotypic field trials (for compositional analysis) Taxonomy (Order, Family, Genus, species) Agronomic and ecological field trials (for compositional analysis) 3

168 Appendix F Environmental conditions Effects observed Reference to Appendix G ERA statistical design and analysis e.g. temperature, photoperiod, relative humidity Specify the name of the appendix For EFSA use 4

169 Appendix F 3. POLLINATORS Common name Reference Taxonomy (Order, Family, Genus, species) Appendix X or author et al., year Peer reviewed Yes/No Site of the experiment Country name Type of experiment Tier 1a, 1b, 2, 3 Stressor Standard toxin, purified product (from bacteria, from GM plants) GM plant, plant part (specify), etc. Event Comparator(s) Control (negative, positive) Number of replications Duration of the experiment Experimental design Development stage Source of specimen Test performed Feeding conditions Measurement Endpoint e.g. GM plant containing stacked events A x B (contains proteins A, B, ) Years and dates e.g. split-plot Larvae/adult In house colony, purchased from commercial suppliers, field collected Laboratory test Conditions e.g. diet (plant, plant part, artificial diet), [xx ug protein A/g of diet protein/ml xx ug protein B/g of diet Choice/no choice, ad libitum/fixed dose e.g. body mass, mortality, fecundity Taxonomy (Order, Family, Genus, species) Agronomic and phenotypic field trials (for compositional analysis) Taxonomy (Order, Family, Genus, species) Agronomic and ecological field trials (for compositional analysis) 5

170 Appendix F Environmental conditions Effects observed Reference to Appendix G ERA statistical design and analysis e.g. Temperature, photoperiod, relative humidity Specify the name of the appendix For EFSA use 6

171 Appendix F 4. DECOMPOSERS Common name Reference Taxonomy (Order, Family, Genus, species) Appendix X or author et al., year Peer reviewed Yes/No Site of the experiment Country name Type of experiment Tier 1a, 1b, 2, 3 Stressor Standard toxin, purified product (from bacteria, from GM plants) GM plant, plant part (specify), etc. Event Comparator(s) Control (negative, positive) Number of replications Duration of the experiment Experimental design Development stage Source of specimen Test performed Feeding conditions Measurement Endpoint e.g. GM plant containing stacked events A x B (contains proteins A, B, ) Years and dates e.g. split-plot Larvae/adult In house colony, purchased from commercial suppliers, field collected Laboratory test Conditions e.g. diet (plant, plant part, artificial diet), [xx ug protein A/g of diet protein/ml xx ug protein B/g of diet Choice/no choice, ad libitum/fixed dose e.g. body mass, mortality, fecundity Taxonomy (Order, Family, Genus, species) Agronomic and phenotypic field trials (for compositional analysis) Taxonomy (Order, Family, Genus, species) Agronomic and ecological field trials (for compositional analysis) 7

172 Appendix F Environmental conditions Effects observed Reference to Appendix G ERA statistical design and analysis e.g. temperature, photoperiod, relative humidity Specify the name of the appendix For EFSA use 8

173 Appendix F 5. PARASITOIDS Common name Reference Taxonomy (Order, Family, Genus, species) Appendix X or author et al., year Peer reviewed Yes/No Site of the experiment Country name Type of experiment Tier 1a, 1b, 2, 3 Stressor Standard toxin, purified product (from bacteria, from GM plants) GM plant, plant part (specify), etc. Event Comparator(s) Control (negative, positive) Number of replications Duration of the experiment Experimental design Development stage Source of specimen Test performed Feeding conditions Measurement Endpoint e.g. GM plant containing stacked events A x B (contains proteins A, B, ) Years and dates e.g. split-plot Larvae/adult In house colony, purchased from commercial suppliers, field collected Laboratory test Conditions e.g. diet (plant, plant part, artificial diet), [xx ug protein A/g of diet protein/ml xx ug protein B/g of diet Choice/no choice, ad libitum/fixed dose e.g. body mass, mortality, fecundity Taxonomy (Order, Family, Genus, species) Agronomic and phenotypic field trials (for compositional analysis) Taxonomy (Order, Family, Genus, species) Agronomic and ecological field trials (for compositional analysis) 9

174 Appendix F Environmental conditions Effects observed Reference to Appendix G ERA statistical design and analysis e.g. temperature, photoperiod, relative humidity Specify the name of the appendix For EFSA use 10

175 Appendix G EFSA identification code for the application (event name) XXXX Appendix G Schematic summary of statistical design and analysis for each ERA study A schematic summary should be provided for each study conducted for the environmental risk assessment. All complied appendices should be included in the folder Appendices, subfolder ERA_Appendices D to G. Study report (e.g. [author] et al. (YYYY)): Field trial Semi-field trial Laboratory Tier study tier 1a tier 1b tier 2 tier 3 Equivalence test Difference test 1. Presentation of data Comments Prov ided Results are clearly presented, using standardized scientific units Raw data are provided Programming code used for the statistical analysis are present in an edible form Test materials are randomized to the experimental units The study is performed in accordance with international standards and protocols An experimental design protocol is provided An statistical analysis protocol is provided The mean, confidence limits and all equivalence limits are displayed on a graph Not prov ided Not relev ant 2. Requirement for General Statistical Principles List explicitly in words all the questions that the study was designed to address Re-stated each question in formal terms, including precise null hypothesis that was tested to answer the question Clear description and justification of each assumptions made A proof of difference is provided 1

176 Appendix G A proof of equivalence is provided For studies that use extra comparators, separate difference tests (between the GM plant and each of its different comparators) and separate equivalence tests (between the GM plant and each of its different comparators) are reported similarly 3. Requirement for each measurement endpoint Clear description of each measurement endpoint are provided Limits of concern for each measurement endpoints are described If limits of concern for lower-tier studies are less than for higher-tier studies, justification is provided Effect size desired to detect with the study is given and justification is provided Minimum effect size relevant on the receiving environment(s) given and justification provided Statement on how the chosen effect size relates to the limit of concern through the minimum relevant ecological effect that is deemed biological relevant is provided When many measurement endpoints have been included in a study (e.g. where the endpoints represent several NTO species), the results of all endpoints for which sufficient records have been obtained are reported, not just those deemed to be of particular biological or statistical interest. 4. Requirement for equivalence and difference test For the equivalence test, limit of concern are stated explicitly Statistical power if given The difference test has sufficient statistical power and justification are provided Power of each measurement endpoint of each difference test are provided at the planning stage of the study 5. Additional requirement for field trials Minimum levels of abundance of each taxa samples are described and justified (NTO field trials) 2

177 Appendix G The level of within-site replication is linked to the power analysis Justification of the selection of the different sites for the field trials is provided Each field trial is replicated over at least two years, each field trial over at least three sites. If not, justification is provided Field trials are performed in Europe Field trials are not performed in Europe and justification are provided 6. Reporting All significant differences observed are reported and discussed; focusing on their biological difference For simultaneous texts of difference and equivalence, each outcome from the graph is categorized and the respective appropriate conclusion drawn. Analysis addressed all field trials simultaneously and is based on the full dataset from all sites Each analysis has the potential to identify any interactions between sites and years and the test materials; for each measurement endpoint studied, explicit statement concerning the presence or absence of any such interactions is provided; if interactions are found, the possible reasons for their existence and the implications for the inferences drawn from the trials are discussed. A table or graph giving, for each site and year and for each (transformed) measurement endpoint, the means and standard errors of means of the GM plant and its conventional counterpart(s), and any other test material, where applicable is provided. 3

178 Appendix H DG JRC I3 Record for Quality System R19GP7/EURL Reception of Samples, Reagents and Methods Page 1 / 1 Date: 25/11/2011 Revision. 5 From Molecular Biology and Genomics Unit tel: European Commission - Joint Research Centre - IHCP fax: ISPRA (VA) Italy JRCI04/MBG/GVDE/ARES (2001) To : Applicant Applicant Adress of the applicant (contact) fax: Fax App. Contact App. Contact File No. EURL Dossier Ref. EFSA Dossier We have received the following goods, in relation with the file in reference: Samples on 01/01/2001 / Means of reception / Condition of reception List of samples received Reagents on 01/01/2001 / Means of reception / Condition of reception List of Reagents received Methods and documents on 01/01/2001 / Means of reception / Condition of reception List of documentation received Additive Remark (Optional) Name responsible Sample delivery 01/01/2001 This document is not a recognition of the quantity and/or quality of samples and reagents provided. EURL-GMFF will experimentally assess the quality and quantity of material and the performance of the method(s). The laboratory will use these products in accordance with the Regulation EU 1829/2003. EURL-GMFF will not sign and return any other acknowledgement of receipt. A copy of this document is sent to EFSA