EVENT REPORT. European Food Safety Authority 3. European Food Safety Authority (EFSA), Parma, Italy

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1 Supporting Publications 2012:EN-317 EVENT REPORT Bilateral technical meeting between members of the EFSA Panels on Genetically Modified Organisms and Biological Hazards and the Austrian delegation on the national safeguard clause on GM potato EH , 2 ABSTRACT European Food Safety Authority 3 European Food Safety Authority (EFSA), Parma, Italy A bilateral technical meeting was held on 6 February 2012 between members of the EFSA Panels on Genetically Modified Organisms and Biological Hazards and Austrian experts to discuss issues related to the safeguard clause invoked by Austria for genetically modified (GM) potato EH The aim of the meeting was to listen to the arguments put forward by the Austrian delegation and to obtain clarification on scientific issues addressed in the Austrian document supporting its national safeguard clause on potato EH An observer from the European Commission (EC) presented the legal background and mandate by the EC. Scientific issues discussed related to the antibiotic resistance marker gene (nptii), the molecular characterisation and the postmarket environmental monitoring of GM potato EH European Food Safety Authority, 2012 KEY WORDS GMOs, potato, Solanum tuberosum, EH , Amflora, Austria, safeguard clause, human and animal health, environment, Directive 2001/18/EC 1 Question No EFSA-Q Disclaimer: The views or positions expressed in this publication do not necessarily represent in legal terms the official position of the European Food Safety Authority. The European Food Safety Authority assumes no responsibility or liability for any errors or inaccuracies that may appear. 3 Acknowledgement: EFSA wishes to thank the following members of the Joint GMO BIOHAZ Working Group on the Austrian safeguard clause for GM potato EH , Lieve Herman, Sirpa Kärenlampi and John Threlfall, and the following member of the GMO Working Group on Environment, Antoine Messéan, for the preparatory work on this scientific output and EFSA staff, Jaime Aguilera, Ana Gomes, Ernesto Liebana, Sylvie Mestdagh and Irina Olaru for the support provided to this scientific output. Any enquiries related to this output should be addressed to gmo@efsa.europa.eu Suggested citation: European Food Safety Authority; Bilateral technical meeting between members of the EFSA Panels on Genetically Modified Organisms and Biological Hazards and the Austrian delegation on the national safeguard clause on GM potato EH Supporting Publications 2012:EN-317. [9 pp.]. Available online: European Food Safety Authority, 2012

2 SUMMARY A bilateral technical meeting was held on 6 February 2012 between members of the EFSA Panels on Genetically Modified Organisms and Biological Hazards and Austrian experts to discuss issues related to the safeguard clause invoked by Austria for genetically modified (GM) potato EH The aim of the meeting was to listen to arguments put forward by the Austrian delegation and to obtain clarification on scientific issues addressed in the Austrian document supporting its national safeguard clause on potato EH An observer from the European Commission (EC) presented the legal background and mandate by the EC. Scientific issues discussed related to the antibiotic resistance marker gene (nptii), the molecular characterisation and the post-market environmental monitoring of GM potato EH Supporting publications 2012:EN-317 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Commission... 4 Terms of reference as provided by the European Commission... 4 Consideration Introduction Presentation of legal background and mandate by the European Commission Introduction to the safeguard clause by the National Competent Authority and technical discussion Antibiotic resistance marker gene (nptii) Molecular characterisation Post-market environmental monitoring... 8 Conclusions... 8 Documentation provided to EFSA... 9 References... 9 Supporting publications 2012:EN-317 3

4 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION In February 2006, the European Food Safety Authority (EFSA) adopted an opinion related to the placing on the market of GM potato EH for cultivation and industrial starch production, following a notification submitted by BASF Plant Science to the Swedish Authorities. On 2 March 2010, the European Commission (EC) adopted a decision authorising the placing on the market of GM potato EH for cultivation and industrial starch production. In June 2010, Austria notified to the European Commission its Ordinance BGBI. II No 125/2010 of 27/04/2010 implementing a national safeguard measure prohibiting the placing on the market of GM potato EH for cultivation purposes in Austria and provided scientific elements in support of its decision. In order for the EC to follow-up on this safeguard clause in accordance with Article 23 of Directive 2001/18/EC, it was deemed appropriate by EC that EFSA would assess the scientific elements provided by Austria. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION EFSA was requested in accordance with Article 29 of Regulation (EC) No 178/2002 to assess the scientific information submitted by the Austrian Authorities justifying their national safeguard measure concerning GM potato EH and to identify whether these new scientific elements might lead the GMO Panel to reconsider its opinion on GM potato EH from Supporting publications 2012:EN-317 4

5 CONSIDERATION MINUTES OF THE MEETING The report below reflects the common understanding of EFSA and the Austrian delegation of the meeting held on 6 February This report is not, and cannot be regarded as, representative of the position, the views or the policy of the European Food Safety Authority or of any national or European Union institution, agency or body. Participants Austrian delegation Andreas Heissenberger Environment Agency Austria, Department of Land Use and Biosafety Sanda Pasc Alexandra Ribarits Dietmar Vybiral Federal Ministry of Health, Department of Genetic Engineering Austrian Agency for Health and Food Safety, Institute for Seed Head of the Delegation Federal Ministry of Health, Department of Genetic Engineering EFSA GMO Panel EFSA BIOHAZ Panel EFSA GMO Unit EFSA BIOHAZ Unit European Commission Markus Wögerbauer Austrian Agency for Health and Food Safety, Data Statistic Risk Assessment, GMO Risk Assessment Lieve Herman, Sirpa Kärenlampi, Antoine Messéan John Threlfall Ana Gomes, Sylvie Mestdagh, Irina Olaru, Elisabeth Waigmann (Chair) Ernesto Liebana Martine Delanoy, Sabine Pelsser (DG SANCO) In the course of a bilateral technical meeting (venue: video/telemeeting; duration: 2 hours) between experts from the EFSA Panels on Genetically Modified Organisms (GMO Panel) and Biological Hazards (BIOHAZ Panel) and Austrian experts, issues related to a safeguard clause invoked by Austria for potato EH were discussed. 1. Introduction Welcome The Acting Head of the EFSA GMO Unit chaired the meeting and welcomed the Austrian delegation, members of the EFSA GMO and BIOHAZ Panels and observers from the European Commission. The Chair clarified the aim of the bilateral meeting, which was to listen to the arguments put forward by the Austrian delegation and to obtain clarification on scientific issues addressed in the Austrian document supporting its national safeguard clause on potato EH The agenda of the meeting had been agreed upon by EFSA and the Austrian delegation during communication prior to the meeting. The Chair briefly went through the agreed agenda of the meeting by pointing out how the meeting would be structured and how the Austrian delegation would be given the opportunity to present its arguments. Supporting publications 2012:EN-317 5

6 Tour de table Participants introduced themselves during a tour de table. 2. Presentation of legal background and mandate by the European Commission A representative from the European Commission (EC, Directorate-General on Health and Consumers (DG SANCO)) recalled the historical and legal background of the Austrian safeguard clause under discussion. She explained that the Austrian national measure was subject to a request from the EC, directed to EFSA, to provide a scientific opinion based on the documents submitted by Austria. 3. Introduction to the safeguard clause by the National Competent Authority and technical discussion The Austrian delegation clarified its position, and arguments related to the use and safety of potato EH Concerning the antibiotic resistance marker (ARM) gene (nptii), the Austrian delegation confirmed that the main line of argument related to an ongoing research project the results of which cannot be discussed before the evaluation phase but which is expected to be completed at the end of This was again emphasised in response to a direct question from the BIOHAZ Panel member about the status of the current experimentation. In light of the lack of any new experimental evidence relating to transfer of the ntpii gene, the Austrian delegation proposed discussing the impacts and relevance of a low horizontal gene transfer frequency and the potential formation of mosaic genes involving nptii. The molecular characterisation and post-market environmental monitoring were also discussed Antibiotic resistance marker gene (nptii) An Austrian delegate highlighted the fact that a low horizontal gene transfer frequency (also acknowledged in the EFSA opinion on ARM; EFSA, 2009) could allow a rare event to occur and have significant relevance. As an example, the Austrian delegate referred to the formation of mosaic genes in the penicillin-binding proteins (PBPs) of Streptococcus pneumoniae and its public health consequences and asked the GMO and BIOHAZ Panels why they had not taken into account the formation of mosaic genes during their ARM gene risk assessment. An EFSA GMO Panel member indicated that mosaic genes in the context of antibiotic resistance are known only for highly transformable bacteria and certain antibiotic resistance genes. Mosaic genes have not been found among aminoglycoside phosphotransferase genes. In addition, the rate of DNA transfer between bacteria is higher than that from plant DNA to bacteria. For a recombination event and formation of a mosaic gene to occur, nptii would need to be present in bacteria in the environment. If this gene were already present in the environment, then the recombination frequency among bacteria would be expected to be much higher than between plant DNA and bacteria. Consequently, the potential risk of the formation of mosaic genes would be related to the environmental presence of the nptii gene in the bacterial community and to the fact that the bacteria to bacteria transfer rate is several orders of magnitude greater than that between plant DNA and bacteria. The Austrian delegate agreed that both the transfer and the recombination frequencies are higher among bacteria than those for the transfer and recombination of DNA from GM plants into bacteria. Nevertheless, a rare event (i.e. transfer of a mutated plant-derived nptii gene; involvement of plantderived copies of nptii in the formation of gene mosaics with resident nptii or other aminoglycoside phosphotransferase genes both potentially leading to alterations in the substrate specificity of the involved antibiotic resistance determinants) cannot be excluded, thereby resulting in possible clinical relevance (Heinemann and Traavik, 2004). The Austrian delegate also agreed that there is no published literature about the formation of mosaic genes among the seven classes of aminoglycoside phosphotransferases. But he was unaware of any scientific efforts targeted at looking systematically for the presence or absence of mosaic sequence patterns in aminoglycoside phosphotransferase genes. Thus, the absence of evidence could not be regarded a priori as evidence for the absence of that Supporting publications 2012:EN-317 6

7 phenomenon. According to the Austrian delegate the formation of mosaic genes is a common theme in bacterial evolution. As such, mosaic gene formation is not restricted to pbp genes or to highly transformable bacteria (van Hoek et al., 2008; Didelot and Maiden, 2010). Such mosaic gene formation should therefore be experimentally evaluated before drawing final conclusions on the potential risk of the nptii gene. As quantitative data on the prevalence of nptii on bacterial populations are also not available, conclusions on the prevalence of nptii cannot be drawn. An EFSA GMO Panel member asked the Austrian experts why a mosaic nptii gene would make a significant contribution to substrate specificity. The Austrian delegate explained his concern regarding the inactivation of amikacin, should the spectrum of substrate specificity be altered for nptii. He also referred to the example of S. pneumoniae, a case in which formation of mosaic genes occurred in spite of low frequency rates for the recombination of small DNA fragments Molecular characterisation An Austrian delegate made a general statement that Austria had found several shortcomings in the data contained in the applications for potato EH , as submitted to EFSA. These flaws increased the degree of uncertainty and did not allow Austria to draw reliable conclusions from the addressed data packages. The Chair reminded the participants that the safeguard clause was bound to address information made available after the date of consent for potato EH The Austrian delegates agreed that there had been no new data on the molecular characterisation of potato EH made available after the date of consent. The GMO Panel members agreed that the quality of data presented in the original potato EH dossiers was not perfect but reliable conclusions could be drawn by the GMO Panel on the basis of the total data package (EFSA, 2006a,b). The Austrian delegate pointed out the absence of data on the messenger RNA (mrna) expressed by the construct and that there could be different 3 ends, with a consequence for stability due to the loss of the nos terminator sequences. Moreover, he mentioned that the transgenic construct actually residing in EH was producing mrnas by convergent transcription starting in opposite directions from two flanking promoters leading to the formation of sirnas (small interfering RNAs), with potential pleiotropic silencing effects on other genes (Yan et al., 2006). The induction of RNA interference pathways was not intended by the initial application. Granule-bound starch synthase (GBSS) silencing was established on sodium dodecyl sulphate (SDS) polyacrylamide gel photos, which showed aberrant banding patterns besides the GBSS fragment between EH and the non-gm prevalent potato line. According to the Austrian delegate, both observations would require a thorough qualitative and quantitative analysis of the construct-derived GBSS mrna fragments and their effects on cellular metabolism. The applicant had failed to supply this information. Moreover, these convergent run-off mrna transcripts were the original effector molecules mediating the genetic modification, and thus should be dealt with and described in detail. A GMO Panel expert indicated that the lack of RNA analysis was not limiting, given that there were interpretable protein data demonstrating the loss of GBSS protein from the EH potato, which was the intended effect. The different banding pattern in the SDS-PAGE (polyacrylamide gel electrophoresis) analysis between the GM sample and the non-gm control was as might be expected. As in a non-gm potato starch 80 % of the extractable protein is GBSS, further evidence by Western blot analysis that the dominant correct-sized band was indeed GBSS was not considered necessary. It could also be expected that, in the absence of GBSS, other protein(s) dominate in a GM sample of similar total protein content. The (lower) band dominating in the GM sample was experimentally shown to be patatin, which is logical as patatins are a major protein family found in potato tubers. Stability has been addressed in the post-market environmental monitoring (PMEM) report, which has Supporting publications 2012:EN-317 7

8 just been received and evaluated by EFSA. The GMO Panel has concluded in its opinion on the PMEM report for potato EH that stability of the trait did not raise safety issues (EFSA, 2012) Post-market environmental monitoring An Austrian delegate indicated that the PMEM plan for potato EH , as submitted by the applicant, was very general and some exposure pathways (e.g. a possible increase in volunteers, the use of GM pulp for feed) had not been completely considered. The Austrian delegate also stressed that the changes in composition could lead to altered pest behaviour (e.g. making the GM potato more attractive to potato pests). The Austrian delegate also noted that the monitoring undertaken so far by the applicant and described in the monitoring report of 2010 did not fulfil the requirements of the authorisation decision, e.g. regarding studies to be carried out in the vicinity of the fields. A GMO Panel member noted that no new scientific knowledge had been provided by the Austrian Authorities indicating that changes in composition, such as those observed in potato EH , might significantly change its exposure to organisms feeding on potato or the persistence of potato EH over natural variation, or lead to adverse effects. The GMO Panel member also informed the meeting that the GMO Panel has recently assessed the 2010 PMEM report on potato EH , which included a specific field monitoring study on potato-feeding organisms in addition to general surveillance. On the basis of the available information, the outcomes of this 2010 report did not invalidate the conclusions of the previous risk assessment of potato EH by the EFSA GMO Panel. Although the EFSA GMO Panel had noted a number of weaknesses in the PMEM methodology and made several recommendations to improve the study design, there was no evidence that changes in composition in potato EH lead to adverse effects. As for exposure pathways, the GMO Panel member indicated that, in cases in which potato EH pulp is used as animal feed, the EFSA GMO Panel recommends that the Identity Preservation System, which already traces and records information on potato EH by-products, should be supplemented by the questionnaire outlined by the applicant. Regarding studies to be carried out in the vicinity of fields, the GMO Panel member mentioned that the above-mentioned assessment considered that outer rows cannot be the area that should be monitored as vicinity of the potato fields, acknowledged that the vicinity of potato fields might not be easy to define and requested the applicant to revise its definition of vicinity. Finally, the GMO Panel member acknowledged that there is always room to improve the methodology of the PMEM and that the PMEM strategy should not be seen as static but needs to be revised on a regular basis. CONCLUSIONS SUMMARISING REMARKS The Austrian delegation acknowledged the fruitful scientific discussion between the delegation and members of the EFSA GMO and BIOHAZ Panels and the opportunity to present its scientific arguments. In the absence of any new scientific data, the Austrian point of view was that these arguments justify the Austrian safeguard measure on potato EH The delegation agreed with the GMO Panel that, with regard to the PMEM, there is always room to improve the methodology. Furthermore, the PMEM strategy should not be seen as static but needs to be revised on a regular basis. The Austrian delegation appreciated that EFSA and its GMO and BIOHAZ Panels had dedicated this meeting to discussion of this Austrian safeguard clause. The EFSA GMO and BIOHAZ Panel experts were also thanked for the fruitful scientific discussion. Supporting publications 2012:EN-317 8

9 The Chair closed the meeting, thanking the Austrian delegation, the experts of the EFSA GMO and BIOHAZ Panels, and the observers from the European Commission. The Chair also informed the participants that a meeting report would be prepared by EFSA staff and sent for comments prior to publication. DOCUMENTATION PROVIDED TO EFSA 1. Letter, received 24 May 2011, with supporting document from Ladislav Miko, Deputy Director- General for the food chain EC, to Catherine Geslain-Lanéelle, Executive Director EFSA (ref SANCO/E1/SP/mb Ares(2011) ), requesting the assessment by EFSA of the scientific elements provided by Austria in support of its decision to implement a national safeguard measure under Article 23 of Directive 2001/18/EC for GM potato EH and comprising the following supporting document: REFERENCES Scientific arguments for an import ban of genetically modified potato (Solanum tuberosum L. line EH , notification C/SE/96/3501) in Austria. Didelot X and Maiden MC, Impact of recombination on bacterial evolution. Trends in Microbiology, 18(7), EFSA (European Food Safety Authority), 2006a. Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the notification (Reference C/SE/96/3501) for the placing on the market of genetically modified potato EH with altered starch composition, for cultivation and production of starch, under Part C of Directive 2001/18/EC from BASF Plant Science. The EFSA Journal, 323, EFSA (European Food Safety Authority), 2006b. Opinion of the Scientific Panel on Genetically Modified Organisms on an application (Reference EFSA-GMO-UK ) for the placing on the market of genetically modified potato EH with altered starch composition, for production of starch and food/feed uses, under Regulation (EC) No 1829/2003 from BASF Plant Science. The EFSA Journal, 324, EFSA (European Food Safety Authority), Statement of EFSA on the consolidated presentation of the joint Scientific Opinion of the GMO and BIOHAZ Panels on the Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants and the Scientific Opinion of the GMO Panel on Consequences of the Opinion on the Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants on Previous EFSA Assessments of Individual GM Plants. The EFSA Journal, 1108, 1 8. EFSA (European Food Safety Authority), Scientific Opinion on the annual Post-Market Environmental Monitoring (PMEM) report from BASF Plant Science Company GmbH on the cultivation of genetically modified potato EH in EFSA Journal, 10(2):2558. Heinemann JA and Traavik T, Problems in monitoring horizontal gene transfer in field trials of transgenic plants. Nature Biotechnology, 22(9), van Hoek AH, Mayrhofer S, Domig KJ, Flórez AB, Ammor MS, Mayo B and Aarts HJ, Mosaic tetracycline resistance genes and their flanking regions in Bifidobacterium thermophilum and Lactobacillus johnsonii. Antimicrobial Agents and Chemotherapy, 52(1), Yan H, Chretien R, Ye J and Rommens CM, New construct approaches for efficient gene silencing in plants. Plant Physiology, 141(4), Supporting publications 2012:EN-317 9