EFSA-GMO-DE (MON863

Transcription

1 Comments from National Competent Authority under Directive 2001/18/EC AT Ministry of Health and Women General comments 1. General Comment: The dossier can be regarded as extremely voluminous. Nevertheless some parts of the dossier can not be considered as relevant for a state-of-the-art risk assessment: e.g. acute oral toxicity studies in mice (Kimberly et. al (1991) or Naylor (1992)). Therefore it is suggested to limit the information given to Outside the remit of the GMO Panel. AT AT Ministry of Health and Women Ministry of Health and Women General comments D. Information relating to the GM plant actual studies that guarantee a comprehensive risk assessment. 7. Detection method: Under part VI Additional Information of GMOs (page4) it is quoted that because MON863xMON810 contains two DNA inserts, there is no single PCR detection method available for MON863xMON810 which permits differentiation of this product from its single-trait parents. [.] However, as for all plants in which inserts are combined by traditional breeding, the unambiguous detection of MON863xMON810 in mixed consignments of grain will require single grains to be subjected to detection methods for both MON863 and MON810, and to test positive for both. This will cause legal problems with these products containing stacked-genes. If MON810, MON863 and MON863xMON810 will get an approval for putting on the market, also a labelling threshold of 0,9% will apply according to Regulation 1829/2003. Therefore it remains unclear how analytical results of quantitative analysis of products containing all three commercialised maize-lines - have to be interpreted. 2. Information concerning maize MON810: is only partly given in this notification and further information concerning state-of-the-art data of an allergological and toxicological risk assessment is regarded as necessary. Moreover Austria had raised several reasoned objections against the placing on the market of this GMO in the past, resulting in a national import-ban [Ordinance of the Federal Minister of Womens Affairs and Consumer Protection concerning the prohibition of placing on the market of genetically modified maize (Zea Mays L.) line MON 810 in Austria (BGBl. II, 10. June 1999).] against MON 810. These scientific based arguments are still to be maintained. Outside the remit of the GMO Panel. The GMO Panel assessed several applications on hybrids containing MON810. Supported by these, the GMO Panel affirms its conclusions with respect to the potential impact of Cry1Ab toxin, as invoked in the safeguard clauses by Austria. In this context, the GMO Panel has assessed the molecular characterisation, compositional analysis, the toxicology and allergenicity of the respective hybrids. Further, the GMO Panel has assessed available data from the literature concerning the environmental impacts of Cry1Ab. No reports of adverse environmental impacts from comparable Cry1Ab expressing maize have been observed. Reports and reviews of studies of the effects of Cry1Ab on biodiversity, including the abundance of non-target and biocontrol species, indicate that significant adverse environmental effects due to Bt maize EFSA-GMO-DE Page 1 of 26

2 cultivation are unlikely. The GMO Panel therefore affirms its conclusions that, on the basis of current scientific knowledge, MON810 maize is unlikely to have adverse effects on human and animal health or to the environment in the context of its proposed uses. See EFSA scientific opinion related to genetically modified crops (Bt176 maize, MON810 maize, T25 maize, Topas 19/2 oilseed rape and Ms1xRf1 oilseed rape) subject to safeguard clauses invoked according to Article 16 of Directive 90/220/EEC (EFSA, 2006c). MON810 has been evaluated by the ACNFP and SCP (ACNFP, 1996; SCP, 1998b). The GMO-Panel has evaluated MON810 in several opinions and has addressed the scientific arguments put forward by Austria in BGBl. II, 10. June See opinions referenced in EFSA scientific opinion (EFSA, 2006c). AT Ministry of Health and Women D, 03 Information on the expression of the insert 3. Sampling procedures: 1) Comparing the given data concerning the concentration of cry3bb1 from field trials in the U.S.A. and Argentina, significant differences are evident with respect to MON863. The same remarkable differences are obvious concerning field trials in Argentina comparing the cry3bb1-concentration of MON863 and MON863xMON810. 2) Data from the USA are not available. Similar arguments are valid for the cry1ab-concentration of MON810 in comparison to the concentration of cry1ab in MON863xMON810: For example the mean value of cry1ab in MON863xMON810 in grain is higher than the given range for this protein-concentration in MON810. 3) It can be stated, that for an adequate risk assessment further field trials are regarded as necessary to gain enough plant material in order to carry out an adequate statistical analysis. The available information has been regarded as sufficient to carry out an adequate safety assessment. The Panel has not found any evidence to question the conclusions of the previous evaluations by the ACNFP and SCP which formed the basis for the authorisation of MON810 maize. Points 1) and 2) see section of the scientific opinion: Expression levels of Cry3Bb1, Cry1Ab, and NptII proteins were measured in samples of various maize tissues including kernels from maize hybrids cultivated during field trials in one season (Argentina, ). Cultivated maize lines included MON 863 x MON 810, MON 863, and MON 810. Cry3Bb1 and Cry1Ab levels in kernels of MON 863 x MON 810 were on average higher than their levels in the comparator lines MON 863 and MON 810. However, the ranges were broad and there were overlaps between the levels of Cry proteins expressed in the MON 863 and MON 810 parents and in MON 863 x MON 810 maize. This reflects variability in gene expression, which may have been influenced, for example, by environmental factors. In addition it is conceivable that different genetic backgrounds and heterosis had an influence on expression levels. EFSA-GMO-DE Page 2 of 26

3 4. Agronomic equivalence: In the dossier it is quoted that Given the toxicological profiles of the Cry proteins the Panel Regarding MON863xMON810 hybrid, we do not anticipate concludes that these data do not raise safety concerns. synergistic effects as a result of the genetic modification which Although raw data from the US field trial were not provided, could alter the agronomic characteristics. Due to the above the Panel considers the data from the Argentinean trials as mentioned facts as an example, further investigations are regarded sufficient to complete the risk assessment as necessary and it should not be anticipated that e.g. secondary effects (or else) could not happen. Point 3) see section of the scientific opinion: In this case, where both parental lines have been assessed in detail by the GMO Panel or are authorised in the EU, the Panel accepts that data for comparative assessment are obtained from one growing season of MON 863 x MON 810 maize. A draft guidance Risk Assessment of Plants Containing Genetic Modification Events Combined by Crossing has been under public consultation. ( o_consultations/gmo_hybrids_publcons.par.0001.file.tmp/g MO_hybrids_publconsul.pdf) See section 2.b of the draft guidance: Where the substantial equivalence of parental material containing genetically modified events has been fully tested in replicated field trials over at least 2 seasons, one years field trialing of events combined by crossing is acceptable where geographical localities are representative of the climatic conditions to which such crops will be exposed. Point 4) see section of the scientific opinion: The Panel does not anticipate interactions (i.e. synergistic or antagonistic) as a result of the genetic modification which could alter the agronomic characteristics. Furthermore, field trials performed with MON863 x MON810 maize did not show any agronomic differences. The Panel accepts the absence of further agronomic data. In addition the panel considers the hybrid to be compositionally equivalent to the comparators and the single events. Therefore the Panel concludes that the likelihood of occurrence of unintended effects is negligible. EFSA-GMO-DE Page 3 of 26

4 AT Ministry of Health and Women D, Allergenicity 5. Allergological risk assessment: The assessment of any potential allergenic effects of MON 810 x MON 863 is reduced to assessment of the allergenicity of the isolated proteins produced from the newly inserted genes [Goodman, Monsanto Company, App. V]. This is done primarily through literature review and research into databases for comparison of the introduced sequence with known allergens. 1) No experimental tests with the GMO itself have been conducted. The risk of an unintended enhancement of allergenic potential is therefore not thoroughly foreseeable. 2) Secondary effects resulting from the event of insertion, which might lead to new / unexpected allergenic qualities should be studied in depth. In the dossier it is quoted concerning the bioinformatic evaluation of DNA: These data demonstrate the lack of both structurally and immunologically relevant similarities toward allergens for all of the putative peptides analysed. These data also demonstrate the lack of structurally relevant similarities towards toxins or other pharmacologically active proteins for all of the putative peptides analysed. With regard to the answer of Monsanto concerning allergenicity it can be stated that the information given by the company is not sufficient to exclude with certainty a potential allergenic risk of the product in quest: Neither allergenicity nor lack of allergenicity of a protein can be unambiguously proven by comparing its sequence with known allergen sequences for at least two reasons: 3) a) The DNA and/or amino acid sequences of many allergens have been elucidated but the list of allergen sequences is still incomplete and continuously growing. On the other hand it has been shown that isoforms of allergens without allergenic activity exist, which differ from the allergen in only few amino acids. The latter would be wrongly classified as allergens by sequence comparisons. 4) b) Even if a given protein per se does not represent an allergen, its expression in another host organism may indirectly upregulate the expression of potential allergens. It is therefore recommended to compare the engineered plant/plant product with that of the parent/wildtype plant/plant product regarding IgE reactivity to establish whether the transgenic organism represents a more potent allergen source than the parent/wildtype organism for already Points 1) and 2) See section 7.9 of the GMO Panel Guidance Document (2006a): The specific allergy risk of GMOs is associated with i) exposure to newly expressed protein(s) that can be present in edible parts of the plants or in the pollen and ii) with alterations to the allergenicity of the whole plant and derived products e.g. due to over-expression of natural endogenous allergens as an unintended effect of the genetic modification. i) Given this lack of complete predictability of allergenicity of a newly expressed protein, it is necessary to obtain, from several steps in the risk assessment process, a cumulative body of evidence which minimises any uncertainty with regard to the protein(s) in question. This approach is internationally accepted and has been conducted as described in section of the scientific opinion. ii) The issue is to demonstrate that the GM crop will not be more allergenic than the non GM comparator because of an unintended effect of the insertion of the transgene. If the host of the introduced gene is known to be allergenic, any potential change in the allergenicity of the whole GM food should be tested by comparison of the allergen repertoire with that of the conventional non-gm variety. It should be pointed out that these approaches should be applied on a case-by-case basis depending on the available information on the allergenic potential of the host. Furthermore see section of the scientific opinion: This issue does not appear relevant to the Panel, however, since maize is not considered a common allergenic food. Food allergies to maize are of low frequency and mainly occur in populations of specific geographic areas. Rare cases of occupational allergy to corn dust have been reported. There is no reason to expect that the use of GM maize will significantly increase the intake and exposure to maize. Therefore a possible overexpression of any endogenous EFSA-GMO-DE Page 4 of 26

5 sensitized patients. The potentially increased ability of the transgenic organism versus the parent/wildtype organism to induce de novo IgE responses (i.e., allergic sensitization) needs to be compared by immunization experiments [ Prof. Rudolf Valenta, Head of Department of Pathophysiology, University of Vienna, personal communication to the CA.]. 5) Concerning the results of the above mentioned risk assessment of the company, it must be stated again, that comprehensive risk protein, which is not known to be allergenic, would be unlikely to alter the overall allergenicity of the whole plant or the allergy risk for consumers. In section of the GMO Panel Guidance Document (2006a): In line with the recommendations of the Codex ad hoc Intergovernmental Task Force on Foods Derived from assessment as described in Spök et.al.[spök A, Hofer H, Valenta R, Biotechnology an integrated, stepwise, case-by-case Kienzl-Plochberger K, Lehner P, Gaugitsch H: Toxikologie und approach should be used in the assessment of possible Allergologie von GVO Produkten, Monographie M-109, allergenicity of newly expressed proteins. Umweltbundesamt, Wien, 2002] should be carried out. The recommendations given for a standardized and harmonized approach to the generation, presentation and interpretation of data concerning allergenicity of GM products are based on in depth scientific studies, performed by experienced scientists in the field. The proposed tests should be performed by the notifier and the Points 3) and 4) See section of the scientific opinion: a possible overexpression of any endogenous protein, which is not known to be allergenic, would be unlikely to alter the overall allergenicity of the whole plant or the allergy risk for consumers. resulting data provided in order to guarantee a high level of safety and public confidence in the in the approach taken. Point 5): regarding the remark on using a comprehensive risk assessment strategy the Panel emphasises that it assesses the safety and potential allergenicity of the GM Products on a case-by-case basis following the criteria laid down in EFSA s Guidance Document on GM Plants (EFSA, 2006a). The EFSA guidance document is in line with the Recommendation of the Codex ad hoc Intergovernmental Task Force on Foods AT Ministry of Health and Women D, 12 Environment al Monitoring Plan 6. Monitoring plan and General Surveillance: Although the company is of the opinion that the genetically modified maize poses no or negligible risk for potential adverse effects on human health and the receiving environment resulting from the import and use of this GMO in the EU and therefore the overall environmental risk posed by this genetically modified higher plant is negligible and no specific strategies for risk management are required, the Competent Authority of Austria is nevertheless of the opinion that a monitoring plan according to the provisions of annex VII of directive 2001/18/EC that takes into account the accidental release has to be added to the dossier. The general surveillance plan at hand lays down only general principles. It is regarded as necessary that it should be elaborated in more detail, i.e. specification of Derived from Biotechnology (CAC, 2003). In relation to accidental release, see section of the scientific opinion:...the likelihood of unintended environmental effects due to the establishment and spread of this maize will be no different to that of MON863 or MON810 maize and traditionally bred maize. The GMO Panel evaluated routes of exposure of MON863 x MON810 maize to the environment e.g. through animal faeces. See section of the scientific opinion. The GMO Panel comments on the scientific quality of the monitoring plan. EFSA has published guidance and opinion on PMEM (EFSA, 2006a,b) following a broad consultation with stakeholders, including national competent authorities. EFSA-GMO-DE Page 5 of 26

6 implementation procedure and steps: Information provided concerning the general surveillance raises several questions, e.g.: Who is responsible of providing traders and processors with the mentioned relevant information? Who is responsible of informing the European feed industry? According to Annex VII, C.5 of The information supplied by the applicant is in line with this guidance. See section 5.3 of the scientific opinion: The scope of the monitoring plan provided by the applicant is directive 2001/18/EG it is necessary that the design of the in line with the intended uses for the GMO since this does not monitoring plan [identifies] who (notifier, users) will carry out the various tasks [ ] and who is responsible for ensuring that the monitoring plan is set into place and carried out appropriate [ ] How are traders or processors of maize grains supposed to assess any potential adverse effects on the environment or human health? cover cultivation. The Panel advises that appropriate management systems should be in place to restrict seeds of GM maize entering cultivation, as the latter requires specific approval under Directive 2001/18/EC or Regulation (EC) No 1829/2003. In general this part of the dossier should be in accordance with Annex VII of directive 2001/18/EG (which is also relevant in this See section 5.2 of the PMEM opinion (EFSA, 2006b): context) and the relating guidance notes complementing this Details of the specific plans and methods of monitoring in Annex. E.g. the monitoring plan should contain information on how often the data have to be checked and analysed; this is amongst other details not the case in concrete monitoring plan. Import of grains of MON 863 x MON 810 does not necessarily mean that there is no environmental impact. Experience as well as results of surveys and inspections in Austria [Heissenberger et al., 2003, Research Report 4/03, Federal Ministry of Social Security and Generations (now Federal Ministry of Health and Women), each country should not be included in the original application. The GMO Panel advises that the application should describe the general approaches and methods that the applicant would apply in different commercialisation sites, including the type of dialogue that would be established with risk managers in each Member State. ( ) Thus detailed local arrangements will be developed by the applicant after the application has been accepted ( ). Vienna] have shown unintended or technically unavoidable contamination (e.g. through transport and processing respectively) leading to the unintended release of seed or grains. Although maize See section of the GMO Panel Guidance Document (EFSA, 2006a): has a weak capability of surviving outside cultivation and of Knowing the limitations of existing monitoring systems, it is overwintering, consequences concerning implementation of any future potential co-existence of different agricultural systems (with or without GMO) are possible. The notifier should act in a more proactive way and approach end-users directly to ask for any observed effects. In this regard appropriate training and education important for the applicant to describe the processes and criteria that will be used for selecting and evaluating existing monitoring systems for supplying data related to the unanticipated adverse effects of GM plants in the general surveillance. on how to assess any potential adverse effect is regarded necessary. According to article 20 of directive 2001/18/EC the competent authority shall be informed immediately about any new information with regard to the risks of the GMO which becomes available after written consent. Therefore the suggestion of the notifier to only inform in case of confirmed adverse effects can not be accepted. DK Danish Forest and Nature General comments Denmark finds that validation of quantitative PCR-methods done by ENGL should be in place before placing on the market. Outside the remit of the GMO Panel. EFSA-GMO-DE Page 6 of 26

7 Agency DE Federal Office of Consumer Protection and (BVL) General comments 1) The application is following Annex III of the EFSA GMO Panel Guidance Document (2006a) and is considered a valid application. DE Federal Office of Consumer Protection and (BVL) D, 02 Information on the sequences actually inserted or deleted Comments of the German Federal Agency for Nature Conservation (BfN): 1) The Federal Agency for Nature Conservation considers, that the application has a number of deficits. A major problem are formal deficits of the technical dossier, which lacks references to the appendices and for this reason cannot be considered as comprehensive. The contents of the numerous appendices are not presented in a structured way. Moreover, the overview of the content of the application offers no help to identify the contents of the appendices. With regard to the limited time span available for the annotation of the application we ask EFSA to demand a revised technical dossier from the applicant and 2) to extend the time for annotation by MS. Comments of the German Federal Agency for Nature Conservation (BfN): 1) Southern blot technique can be considered to detect only largescale features of the insertion site (Wilson et al. 2004). More reliable and state-of-the-art techniques such as PCR and DNA sequencing should be employed to check the assumption that transgenes in Mon863xMon810 do not differ from the parent lines Mon863 and Mon810 respectively. 2) In addition the southern blot pictures give the impression of changed concentrations of the respective sequence segments. Wilson, A., Latham, J., and Steinbrecher, R., (2004), Genome scrambling - myth or reality? Transformation-induced mutations in transgenic crop plants. EcoNexus Technical Report, pp.1-35; Report.pdf 2) The time frame for the Member States s consultation is defined in Articles 6.4 and 18.4 of the Regulation (EC) 1829/2003 (related to Competent Authorities under Directive 2001/18/EC only and extended by EFSA to all members of the GMO EFSAnet). 1) As traditional breeding methods were used in the production of MON863 x MON810 maize, no genetic modification was involved and thus the molecular structures of the DNA inserts in MON863 and MON810 were expected to remain unchanged in MON863 X MON810. Introduction of plasmid DNA, deletions of part of the introduced sequence, and other local DNA modifications often associated to the genetic modification process are not expected in traditional breeding. Thus, a Southern blot analysis is considered sufficient to confirm the conservation of the transgenes. The Southerns provided by the applicant, performed using different enzymes covering the entire insert and flanking regions, confirmed the conservation of the transgenes. The DNA sequences of the inserts in maize MON863 and MON810 are available in section D.2(e) of the technical dossier (Part I). 2) Southerns do not present any new band that could be indicative of a new copy of a fragment or a complete transgene. Moreover, Southern blots are not necessarily quantitative and differences in signal intensity do not indicate any safety issues. EFSA-GMO-DE Page 7 of 26

8 DE Federal Office of Consumer Protection and (BVL) D, 03 Information on the expression of the insert Point 1) see section of the scientific opinion: In this case, where both parental lines have been assessed in detail by the GMO Panel or are authorised in the EU, the Panel accepts that data for comparative assessment are obtained from one growing season of MON 863 x MON 810 maize. The expression levels of the Bt-toxins are generally lower when Btprotein levels are compared from corn harvested from Argentinian field trials to those from US-trials. The notifier is asked to give reasons and to make a statement about expression stability of the transgenes in the hybrid. He is also asked to provide the respective data from the US trial. Additional comments of the German Federal Agency for Nature Conservation (BfN): 1) Overall the expression of Bt-proteins in Mon863 and Mon863xMon810 must be considered high in comparison to other Bt-corn. Nevertheless the expression is not well characterized. The analysis of expression levels in Mon863xMon810 maize is limited to four field sites during the growing season in Argentina (MSL-17266). Since expression can be affected by climatic conditions, soil fertility, agricultural practice or unknown gene-environment interactions, the data presented in MSL gives only a crude estimate of the expression. 2) Further data as well as an analysis of the between-site variability of all available data should be provided before a market release of Mon863xMon810 maize. 3) In this context data from MSL and MSL show up to 300% difference between expression levels of Cry3Bb1 in Mon863 between the Argentinean and US trials. This leads to the conclusion, that gene-environment interactions of the GMO need further consideration and were not sufficiently addressed in the first place. MSL shows increased expression levels of both Cry3Bb1 and Cry1Ab in Mon863xMon810 compared with Mon863 and Mon810 respectively. 4) We recommend to analyze these differences statistically with an extended data set. Differences in expression between the hybrid and the single trait GMO indicate that the effect of the stacked traits is not simply additive. In the context of risk assessment these findings call for rigorous testing of other non expected effects of the combined toxins on human and animal health. A draft guidance Risk Assessment of Plants Containing Genetic Modification Events Combined by Crossing has been under public consultation. ( o_consultations/gmo_hybrids_publcons.par.0001.file.tmp/g MO_hybrids_publconsul.pdf) See section 2.b in this draft guidance: Where the substantial equivalence of parental material containing genetically modified events has been fully tested in replicated field trials over at least 2 seasons, one years field trialling of events combined by crossing is acceptable where geographical localities are representative of the climatic conditions to which such crops will be exposed. Point 2) See section 3.3 of the scientific opinion: Comparison of MON 863 x MON 810 maize with controls, both single-trait parental lines and various commercial reference hybrids showed statistically significant differences in several compounds. The Panel considers that the levels of these compounds are within normal ranges of variation and there is no need for further assessment in MON 863 x MON 810 maize. Furthermore, based upon these results in addition to those obtained for single-trait parental GM lines MON 863 and MON 810 during multiple seasons, the Panel considers the likelihood of occurrence of unintended effects as negligible. Point 3) See section of the scientific opinion: This reflects variability in gene expression, which may have been influenced, for example, by environmental factors. In addition it is conceivable that different genetic backgrounds EFSA-GMO-DE Page 8 of 26

9 and heterosis had an influence on expression levels. The Panel concludes that these data do not raise safety concerns. Given the toxicological profiles of the Cry proteins the Panel concludes that these data do not raise safety concerns. DE Federal Office of Consumer Protection and (BVL) D, 04 Information on how the GM plant differs from the recipient plant in: Comments of the German Federal Agency for Nature Conservation (BfN): With regard to a final assessment further information is required. Information including parameters, methods and proper statistical tests should be given to establish the phenotypic and ecologic equivalence of Mon863xMon810 with conventional maize (isolines) and maize with the two single traits. The applicant assumes that the single traits or the combination of the traits by conventional breeding will not alter the plant properties in any unexpected way simply because this was not intended. This is clearly not acceptable. Because unexpected adverse effects should be given special attention in GMO, results from field trials before the market release play an important role in the risk assessment. However, studies for Mon863xMon810 containing relevant information were not provided. The data provided to show the agronomic equivalence of Mon863 (Pilcher et al. 2001; Monsanto Petition No E) do not use the full data from field releases but are restricted to data from one year and four locations in the US. Moreover, variability between the experimental sites were not analyzed and parameters observed allow no assessment of any potential change in ecological characteristics. 4) The Panel agrees that difference in expression may occur, however given the toxicological profile of the Cry proteins, and compositional equivalence between the hybrid, comparators and single events the panel concludes that this does not raise safety concerns. Phenotypic stability can not be demonstrated in plants since their capacity to change phenotype in different environments, also known as phenotypic plasticity is a well known phenomenon. See section of the scientific opinion: The Panel does not anticipate interactions (i.e. synergistic or antagonistic) as a result of the genetic modification which could alter the agronomic characteristics. Furthermore, field trials performed with MON863 x MON810 maize did not show any agronomic differences. The Panel accepts the absence of further agronomic data. See section 3.3 of the scientific opinion: Comparison of MON 863 x MON 810 maize with controls, both single-trait parental lines and various commercial reference hybrids showed statistically significant differences in several compounds. The Panel considers that the levels of these compounds are within normal ranges of variation and there is no need for further assessment in MON 863 x MON 810 maize. Furthermore, based upon these results in addition to those obtained for single-trait parental GM lines MON 863 and MON 810 during multiple seasons, the Panel considers the likelihood of occurrence of unintended effects as negligible. EFSA-GMO-DE Page 9 of 26

10 A draft guidance Risk Assessment of Plants Containing Genetic Modification Events Combined by Crossing has been under public consultation. ( o_consultations/gmo_hybrids_publcons.par.0001.file.tmp/g MO_hybrids_publconsul.pdf) DE Federal Office of Consumer Protection and (BVL) D, 05 Genetic stability of the insert and phenotypic stability of the GM plant Comments of the German Federal Agency for Nature Conservation (BfN): We do not share the opinion of the applicant that the information on the stability of the single trait lines is appropriate to conclude that Mon863xMon810 is genetically and phenotypically stable. See also comments on D.4. and D.7.4. See section 2.b of the draft guidance: Where the substantial equivalence of parental material containing genetically modified events has been fully tested in replicated field trials over at least 2 seasons, one years field trialling of events combined by crossing is acceptable where geographical localities are representative of the climatic conditions to which such crops will be exposed. See section 2.2 of the scientific opinion: The EFSA GMO Panel guidance document (.) states that when events have been combined by the interbreeding of existing approved GM lines the need for further molecular analysis will depend, on a case-by-case basis, on the nature of the genetic modifications involved. However, there is no a priori or biological reason to assume that traditional interbreeding of independent approved GM lines will pose any additional risk through a compromised stability of copy number and insert structure. Phenotypic stability can not be demonstrated in plants since their capacity to change phenotype in different environments, also known as phenotypic plasticity is a well known phenomenon. See section of the scientific opinion: A bioinformatic analysis of DNA sequences spanning the 5 and 3 junctions of the insert was undertaken. Identified open reading frames were analyzed to test for the creation of a potential peptide with homology to known allergens, toxins or proteins that display adverse health effects and these were not found. The genetic stability of the inserted DNA in event MON 863 was demonstrated by Southern blot analysis of genomic DNA from nine plant generations and segregation data for EFSA-GMO-DE Page 10 of 26

11 the Cry3Bb1 trait was studied using Chi square analysis of Mendelian inheritance data over five generations ( ). DE Federal Office of Consumer Protection and (BVL) D, Comparative assessment Comments of the German Federal Agency for Nature Conservation (BfN): As in the analysis of Bt-expression levels, the compositional analysis relies solely on the 1999/2000 Argentinean field trials. The compositional analysis shows a substantial number of statistical differences between Mon863xMon810 and the control lines (Total comparisons 290; 71 significant differences with Mon846; 59 significant differences with Mon863; 122 significant differences with Mon810; 142 significant differences with commercial lines). The large number of statistical differences (up to 49%) in combination with the higher expression levels in Mon863xMon810 compared to the single trait lines, raise profound doubts that Mon863xMon810 maize can be regarded as substantial equivalent to conventional maize varieties. In addition to the above it should also be emphasized that the Panel uses a weight of evidence approach in risk assessment and does not necessarily rely on data from one approach. See section 3.3 of the scientific opinion: Comparison of MON 863 x MON 810 maize with controls, both single-trait parental lines and various commercial reference hybrids showed statistically significant differences in several compounds. The Panel considers that the levels of these compounds are within normal ranges of variation and there is no need for further assessment in MON 863 x MON 810 maize. Furthermore, based upon these results in addition to those obtained for single-trait parental GM lines MON 863 and MON 810 during multiple seasons, the Panel considers the likelihood of occurrence of unintended effects as negligible. A draft guidance Risk Assessment of Plants Containing Genetic Modification Events Combined by Crossing has been under public consultation. ( o_consultations/gmo_hybrids_publcons.par.0001.file.tmp/g MO_hybrids_publconsul.pdf) DE Federal Office of Consumer Protection and (BVL) D, Agronomic traits Comments of the German Federal Agency for Nature Conservation (BfN): With regard to a final assessment further information is required. Information including parameters, methods and proper statistical tests should be given to establish the phenotypic and ecologic equivalence of Mon863xMon810 with conventional maize and maize with the two single traits. The assumption of the See section 2.b in the draft guidance: Where the substantial equivalence of parental material containing genetically modified events has been fully tested in replicated field trials over at least 2 seasons, one years field trialling of events combined by crossing is acceptable where geographical localities are representative of the climatic conditions to which such crops will be exposed. A draft guidance Risk Assessment of Plants Containing Genetic Modification Events Combined by Crossing has been under public consultation. ( o_consultations/gmo_hybrids_publcons.par.0001.file.tmp/g MO_hybrids_publconsul.pdf) EFSA-GMO-DE Page 11 of 26

12 applicant that no such differences exist is not documented. Because unexpected adverse effects should be given special attention in GMO, results from field trials before the market release play an See section 2.b in the draft guidance: Where the substantial equivalence of parental material containing genetically modified events has been fully tested in important role in the risk assessment. However, studies for replicated field trials over at least 2 seasons, one years field Mon863xMon810 containing relevant information were not provided. The data provided to show the agronomic equivalence of Mon863 (Pilcher et al. 2001; Monsanto Petition No E) do trialling of events combined by crossing is acceptable where geographical localities are representative of the climatic conditions to which such crops will be exposed. not use the full data from field releases but are restricted to data from one year and four locations in the US. Moreover, variability between the experimental sites were not analyzed. See section of the scientific opinion: The Panel does not anticipate interactions (i.e. synergistic or antagonistic) as a result of the genetic modification which could alter the agronomic characteristics. Furthermore, field trials performed with MON863 x MON810 maize did not show any agronomic differences. The Panel accepts the absence of further agronomic data. DE DE Federal Office of Consumer Protection and (BVL) Federal Office of Consumer Protection and D, Effect of the production and processing D, Toxicology Comments of the German Federal Agency for Nature Conservation (BfN): The applicant states that no effects of processing are expected when comparing Mon863xMon810 maize with traditional maize. However, no data or studies are presented to show that this assumption is valid. The statement that any alteration in Mon863xMon810 maize was not intended or only of agronomic nature cannot be sufficient to address any unintended effects. Comments of the German Federal Agency for Nature Conservation (BfN): With regard to the large differences observed in the compositional analysis and the expression studies, testing of the whole GM food/feed is crucial to obtain the necessary information See section 3.3 of the scientific opinion: Comparison of MON 863 x MON 810 maize with controls, both single-trait parental lines and various commercial reference hybrids showed statistically significant differences in several compounds. The Panel considers that the levels of these compounds are within normal ranges of variation and there is no need for further assessment in MON 863 x MON 810 maize. Furthermore, based upon these results in addition to those obtained for single-trait parental GM lines MON 863 and MON 810 during multiple seasons, the Panel considers the likelihood of occurrence of unintended effects as negligible. See section of the scientific opinion: Based on the data of the compositional analysis of the raw agricultural commodities of MON 863 x MON 810 maize and the non-gm maize, the Panel is of the opinion that there are no reasons to assume that the stability of the processed products derived from this maize would be different from the non-gm processed products. See section of the scientific opinion: However, an additional 90-day rat study with MON 863 x MON 810 maize was requested by EFSA in order for the Panel to finalise its opinion. EFSA-GMO-DE Page 12 of 26

13 (BVL) about any adverse unintended effects of Mon863xMon810 maize on human or animal health. Only one test with the GMO (broiler study MSL-18163), however, has been carried out. The broiler feeding study is a pure yield study with nearly no physiological data relating to the health status of the animals. As a consequence the safety of Mon863xMon810 maize for human or animal health cannot be assessed from this study. To complete the risk assessment EFSA demanded a 90-day-chronic rat study from the applicant. This study has not been submitted yet. A final risk assessment is therefore not possible. The rat study, in our opinion, should be supplemented by other chronic studies with ruminants and swine which will be exposed to Mon863xMon810 feed. The applicant states that Cry3Bb1 and Cry1Ab have different modes of See section 4.3 of the scientific opinion: The results of 90-day sub-chronic rodent studies do not indicate adverse effects from consumption of maize lines MON 863 x MON 810 and the Panel concludes that there are no resultant concerns over its safety. The Panel considers that the data from the 90-day rat feeding study with grain from MON 863 x MON 810 maize are sufficient to conclude that there is no reason to assume that the MON 863 x MON 810 is different from the conventional maize. action and the proteins functions under very different physiological conditions (see p. 57 of the technical dossier). The assumption seems not to be justified since both proteins are comparatively similar (gut toxins, receptor bound molecules induce pore formation and desintegration of the gut tissue). Additive or synergistic effects therefore can be rather expected and need to be addressed before a market release. DE Federal Office of Consumer Protection and (BVL) D, Allergenicity Comments of the German Federal Agency for Nature Conservation (BfN): To assess the allergenicity potential the weight of evidence approach is recommended by the applicant. However the given reasoning neglects important evidence for allergic potential of the Cry proteins. Homology of sequences 6 to 8 amino acids in length are considered potentially significant because allergenic epitopes can be this small (Metcalfe et al, 1996; FAO-WHO, 2001). Gendel found that Cry3A and -lactoglobulin, a milk allergen, shared sequences 7-10 amino acids in length. He also identified sequences of 9-12 amino acids shared by Cry1Ab and vitellogenin, an egg yolk allergen. Gendel concluded that: the similarity between Cry1A(b) and vitellogenin might be sufficient to warrant additional evaluation (Gendel, 1998). In addition Noteborn also found that Cry1Ab possessed relatively significant thermostability comparable to that of the Lys mutant Cry9C protein found in StarLink maize (Noteborn, 1998 in Schubert et Freese 2004). These results should trigger further studies in the context of the assessment of allergenicity. We recommend to demand such studies according the state of the art (see also Spök et al. 2002, See section 7.9 of the GMO Panel Guidance Document (2006a): The specific allergy risk of GMOs is associated with i) exposure to newly expressed protein(s) that can be present in edible parts of the plants or in the pollen and ii) with alterations to the allergenicity of the whole plant and derived products e.g. due to over-expression of natural endogenous allergens as an unintended effect of the genetic modification. i) Given this lack of complete predictability of allergenicity of a newly expressed protein, it is necessary to obtain, from several steps in the risk assessment process, a cumulative body of evidence which minimises any uncertainty with regard to the protein(s) in question. This approach is internationally accepted and has been conducted as described in section of the scientific opinion. EFSA-GMO-DE Page 13 of 26

14 2003; Gaugitsch et al. 2004). FAO-WHO (2001) Evaluation of Allergenicity of Genetically Modified Foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology, Jan , DE Federal Office of Consumer Protection and (BVL) D, 08 Postmarket monitoring of GM food/feed Gaugitsch, Stirn, S. & Spök, A. (2003) Toxikologie und Allergologie von GVO-Produkten - Teil 2B. Untersuchung von Regelungen zur Sicherheitsbewertung von gentechnisch veränderten Lebensmitteln in der EU und den USA. Monographie 164B, Umweltbundesamt Wien. Gendel, S. (1998) The use of amino acid sequence alignments to assess potential allergenicity of proteins used in genetically modified foods. Advances in Food and Nutrition Research 42, Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L. and Fuchs, R.L. (1996) Assessment of the Allergenic Potential of Foods Derived from Genetically Engineered Crop Plants. Critical Reviews in Food Science and Nutrition 36(S), S Noteborn, H.P.J.M. (1998) Assessment of the Stability to Digestion and Bioavailability of the LYS Mutant Cry9C Protein from Bacillus thuringiensis serovar tolworthi. Unpublished study submitted to the EPA by AgrEvo, EPA MRID No Spök, A., Hofer, H., Valenta, R., Kienzl- Plochberger, K., Lehner, P. & Gaugitsch, H. (2002) Toxikologie und Allergologie von GVO-Produkten. Monographie 109, Umweltbundesamt Wien. Spök, A., Hofer, H., Gaugitsch & Stirn, S. (2003) Toxikologie und Allergologie von GVO-Produkten - Teil 2A: Untersuchung zur Praxis und Empfehlungen zur Standardisierung der Sicherheitsbewertung von gentechnisch veränderten Lebensmitteln. Monographie 164A, Umweltbundesamt Wien. The notifier should report on a yearly basis and not, as suggested, in a three years interval on the general surveillance. The notifier is also asked for giving measures how to prevent and/or to minimize the non-intended spread of maize kernels to the environment during transport and processing. Special care has to be taken to prevent unintended germination of viable seeds that are lost during transportation, as there is no allowance for the cultivation of Mon863xMon810. Additional comments of the German Federal Agency for Nature Conservation (BfN): According to Directive 2001/18/EC the application shall include a plan for monitoring in ii) The issue is to demonstrate that the GM crop will not be more allergenic than the non GM comparator because of an unintended effect of the insertion of the transgene. If the host of the introduced gene is known to be allergenic, any potential change in the allergenicity of the whole GM food should be tested by comparison of the allergen repertoire with that of the conventional non-gm variety. It should be pointed out that these approaches should be applied on a case-by-case basis depending on the available information on the allergenic potential of the host. Furthermore see section of the scientific opinion: This issue does not appear relevant to the Panel, however, since maize is not considered a common allergenic food. Food allergies to maize are of low frequency and mainly occur in populations of specific geographic areas. Rare cases of occupational allergy to corn dust have been reported. There is no reason to expect that the use of GM maize will significantly increase the intake and exposure to maize. Therefore a possible overexpression of any endogenous protein, which is not known to be allergenic, would be unlikely to alter the overall allergenicity of the whole plant or the allergy risk for consumers. See section of the scientific opinion: Also, the Panel is not aware of any new, validated tests that produce more relevant or accurate information on possible allergenicity of the protein and that would provide a higher guarantee of safety. Same comment as for section DE/D.12. environmental monitoring plan See answer in section DE/D.12 below EFSA-GMO-DE Page 14 of 26

15 order to identify effects of the GMO(s) and their use on human health or the environment. Since the applicant didn t provide a plan for a post market monitoring, a plan suitable to meet objectives defined in Annex VII of Directive 2001/18/EC and the supplementing guidance notes (2002/811/EC) is requested. Case specific monitoring: As stated by the notifier, the scope of the application is for all uses of Mon863xMon810 maize for food and feed and for industrial uses. In the application no information concerning the exposure of Mon863xMon810 maize and the corresponding Bt-proteins to the environment during or after the production process and during uses is given. In order to monitor possible exposure pathways of organic waste material or waste water (containing Mon863xMon810) to the environment, the detection of spread, persistence and accumulation of Bt-Toxin (Cry 3Bb1, Cry1Ab) in the receiving environments is requested. Further observations of possible impacts on organisms, food chains and habitats in the specific environment are required. Furthermore the environmental exposure of Mon863xMon810 and the corresponding Bt-toxins (Cry 3Bb1, Cry1Ab) during the use as food and feed should be part of the monitoring. General surveillance: According to Directive 2001/18/EC general surveillance is a compulsory part of the monitoring. The objective of general surveillance is to monitor potential cumulative long-term impacts on human health and the environment and to identify the occurrence of adverse effects of the GMO on human health and the environment which were not anticipated in the E.R.A. Therefore a monitoring plan for general surveillance is required. The general surveillance plan has to focus on possible pathways how Mon863xMon810 maize can get into the environment and how unforeseen adverse effects on human health and the environment can be linked to the dispersal of the GMO. Therefore, the applicant is requested to provide an appropriate monitoring plan to observe the spread, persistence and accumulation of the inserted genetic sequences and the corresponding proteins in organism and environmental media (soil, air, water, bodies). General requirements of the monitoring plan Both parts of the monitoring plan case-specific monitoring and general surveillance have to meet the following requirements: A fully specified list of EFSA-GMO-DE Page 15 of 26