Scientific Strategy for GMO R isk Assessment within EU Scientific Strategy for GMO R isk Assessment within EU Yi Liu Scientific officer Taiex workshop on biosafety AGR Kiev, April 2012 Committed since 2002 to ensuring that Europe’s food is safe

2 Committed since 2002 to ensuring that Europe’s food is safe EFSA Guidance Documents on GMO Plants PLANTS Guidance for risk assessment of food and feed from GM plants (2011) Environmental Risk Assessment (ERA) of GM Plants (2010) Supporting documents Application submission guidance (2012) Selection of comparators for the risk assessment of GM plants (2011) Statistical considerations (2009) Use of animal feeding trials for safety assessment of whole GM food/feed (2008) Scientific Committee 90-day study on whole food/feed in rodents (2011) Allergenicity assessment of GM plants and microorganisms (2010) Post market environmental monitoring – PMEM (2011) Potential impacts on non-target organisms (2010) Guidance for non-food/non-feed use of GM plants (2009)

3 Committed since 2002 to ensuring that Europe’s food is safe EFSA Guidance Documents  Documents for:  applicants: to follow in their risk assessment  evaluators: to assess the risk assessment  Living documents: e.g GD being updated with FF GD (2011) and ERA GD (2010), more prescriptive, but the principles remain  Detailed list of all the information necessary to assess the safety of GM plants and derived food/feed products

4 Committed since 2002 to ensuring that Europe’s food is safe GMO RA methodology and principles Science-based Comparative approachWeight-of-evidence Case-by-case principle Step-by-step principle (tiered approach) Scopes of GMO applications Food (human consumption)  GMO for food use  Food containing or consisting of GMOs  Food produced from or containing ingredients produced from GMO Feed (animal consumption)  GMO for feed use  Feed containing or consisting of GMOs  Feed produced from GMOs Release into the environment  Import and processing  Seeds and plant propagation material for cultivation

5 Committed since 2002 to ensuring that Europe’s food is safe RA principle logic Comparative approach Compare the GMO and derived products to their non-GM counterparts Assessment of the identified differences regarding environmental, food/feed safety and nutritional impact Intended effects = those occurring because of the genetic modification Unintended effects = additional effects which were NOT the objective of the genetic modification

6 Committed since 2002 to ensuring that Europe’s food is safe Case-by-case approach Within the frame of the RA approach described in the GD, the GMO Panel requires a CASE-by-CASE approach In the context of the intended uses of the GM product In light of the evaluation of all available scientific information (application + MS comments + scientific literatures) In case the Panel needs more specific information: the applicant is asked for additional information.

7 Committed since 2002 to ensuring that Europe’s food is safe Guidance documents for Food Feed RA Per section introductory paragraphs to explain why information is required Per section summary of conclusion Requirements for molecular characterisation Assessment of protein expression Requirements for the RA of GM plants containing stacked events Criteria for selection of appropriate comparator(s) under different scenarios (comparator GD 2011) Design of field trials for compositional / agronomic / phenotypic traits (stats opinion 2010) Statistical analysis of field trial data: difference / equivalence tests (stats opinion 2010) Toxicological assessment: reference to internationally agreed protocols Allergenicity assessment: newly expressed proteins & whole GM food/feed (allergenicity opinion 2010) Animal feeding studies: when considered necessary (EFSA 2008, 2011)

8 Committed since 2002 to ensuring that Europe’s food is safe Molecular Characterisation (1) Description of methods used for the genetic modification Method for the transformation & relevant bibliographic references Source and characterisation of nucleic acid used for transformation The complete sequence of nucleic acid intended to be inserted, including information on deliberate alteration(s) to the corresponding donor sequence(s). Nature and source of vector(s) used Physical map with the indication of the position of e.g. relevant restriction sites, primers used in PCR, probes for Southern analysis.

9 Committed since 2002 to ensuring that Europe’s food is safe Molecular Characterisation (2) General description of traits introduced or modified Description including their mode of action (e.g. novel proteins, gene silencing traits). Information on the sequences actually inserted/deleted Sequence information from the 5’ and 3’ flanking regions with the aim of identifying interruptions of known genes. Bioinformatic analyses performed using up-to-date databases and searching for intra- and inter-species similarity. ORFs present within the insert and spanning the junction sites, analysed for similarity to known toxins and allergens.

10 Committed since 2002 to ensuring that Europe’s food is safe Molecular Characterisation (3a) Information on the expression of the inserted/modified sequences Other than protein expression data (RNA, metabolites) are now foreseen for non-protein based traits (e.g. RNA interference). Expression data derived from plants grown under conditions representative of typical cultivation practices (i.e. from field trials). The minimum requirement is defined as: 1 site/3 seasons, 3 sites/one season or any permutation of these. The specificity of the protein analysis method should be demonstrated.

11 Committed since 2002 to ensuring that Europe’s food is safe Molecular Characterisation (3b) Information on the expression of the inserted/modified sequence List of comparator(s) that are needed for single and stacked events. Where appropriate, e.g. HR crops, the effect of specific treatment(s) linked to the trait should be assessed for single events. For stacked events, expression data for specific treatment(s) linked to the trait(s) are only required if data obtained from the single events indicate a potential safety concern. For cultivation scope, depending on the trait, information may be required to assess the impact on target and non-target organisms. In such cases, data from various parts of the plant are required from plants grown under conditions representative of typical European cultivation practices.

12 Committed since 2002 to ensuring that Europe’s food is safe Molecular Characterisation (4) Genetic and phenotypic stability genetic stability of the insert(s), phenotypic stability, and inheritance pattern(s) of the introduced trait(s). For single events, data should usually be provided from five generations or vegetative cycles. For stacked events, the structure of the insert should be compared to their respective single events, using plant materials representative for commercial production. Safety implications of the loss-of-function of gene(s) in a multi- gene cassette should be considered.

13 Committed since 2002 to ensuring that Europe’s food is safe RA of Stacked events RA of singles is a pre-requisite RA of higher stack should address all sub-combinations from natural segregation whenever relevant, sub-combinations from targeted breeding approaches Focus on: Stability of inserts Expression of the introduced genes and their products Potential interactions (synergistic or antagonistic effects) Choice of Comparator: Conventional counterpart or Negative-segregant or Any set of GM plants already RA (experimental data) non-GM reference varieties in the field for the equivalence component of the comparative assessment

14 Committed since 2002 to ensuring that Europe’s food is safe RA of stacks: RA of singles is a pre-requisite RA of plant containing stacked events: requires the RA of plant containing single events Allows a comprehensive evaluation of stacks on the basis of knowledge acquired during the evaluation of all singles involved. Allows the evaluation of potential interactions: stacked events in comparison to single events. Allows flexibility on the choice of comparator (when conventional counterpart is not available). If RA of all singles involved is not completed, EFSA will stop the clock for the stack. EFSA will re-start the clock for the stack once the RA of all singles is completed and no outstanding issues remain. EFSA may require an update of the stack application to include cross reference to the singles dossiers.

15 Committed since 2002 to ensuring that Europe’s food is safe RA of stacks: sub-combinations Sub-combinations of events Natural segregation Targeted breeding programs Sub-combinations of events for crops with segregating progeny (e.g. maize): All sub-combinations arising by natural segregation should be addressed (EFSA GD, 2011). Import & processing applications should cover (scope) all sub-combinations independently of their origin (either arising by natural segregation or by targeted breeding programs; EC request, 2010 ).

16 Committed since 2002 to ensuring that Europe’s food is safe RA of stacks: sub-combinations (cont) Sub-combinations of events for crops without segregating progeny (e.g. cotton): Sub-combinations should not be included in the scope (Panel may require demonstration of lack of segregation). For each sub-combination intended for marketing, EFSA requires the presentation of a separate application.

17 Committed since 2002 to ensuring that Europe’s food is safe Comparator – single events FF risk assessment The comparator should be the conventional counterpart, Additional comparators may be included if deemed useful (e.g. negative segregant ). Source: available at Terminology Conventional counterpart:  non-GM isogenic variety (vegetatively propagated)  genotype as close as possible (sexually propagated) Comparator: all other cases Non-GM reference varieties: those in the field to establish ranges of natural variation (equivalence test)

18 Committed since 2002 to ensuring that Europe’s food is safe Comparator – stacked events by conventional breeding Pre-requisite: the risk assessment of the GM plants containing the events independently (i.e. GM plants containing single events) is available (opinions adopted). The comparator of choice is the conventional counterpart When the conventional counterpart is not available negative segregant(s) derived from crosses between GM plants containing events which have been risk assessed and which are all stacked in the GM plant under assessment or any set of GM plants that have all been risk assessed on the basis of experimental data and that include between them all of the events stacked in the GM plant under assessment, and no others.

19 Committed since 2002 to ensuring that Europe’s food is safe The guidance takes into account : - re-transformation - co-transformation - transformation with mutiple gene cassettes The issue of possible segregation of the new event(s) is discussed and, depending on the outcomes, the comparator requested are based on the requirements for the GM plants containing single and stacked events. Comparator – events stacked by other methods

20 Committed since 2002 to ensuring that Europe’s food is safe Comparator - Cases where the comparative approach is not applicable GM plants with major modifications in metabolic pathways, possibly leading to extensive compositional alterations (e.g. nutritionally enhanced foods) GM plants with traits facilitating adaptation to environmental stresses (e.g. drought/salinity).  Selection of appropriate comparators may be difficult. FF risk assessment should focus on specific characteristics, should be based on a comprehensive safety and nutritional assessment of the GM plant and derived food and feed per se.

21 Committed since 2002 to ensuring that Europe’s food is safe Comparator for ERA When comparative approach is applicable, ERA: Case-by-case basis depending on the plant’s characteristics and on the purpose of the study for which the comparator is used (details in EFSA, 2010). When comparative approach is NOT applicalbe ERA: focus on the environmental impacts/management of the GM plant compared to what is currently grown and/or against environmental protection goals. Comparators should be chosen on a case-by-case basis: Non-GM line derived from the breeding scheme used to develop the GM plant Non-GM plant with agronomic properties as similar as possible to the GM plant Non-GM line having other characteristics as close as possible to those of the GM plant.

22 Committed since 2002 to ensuring that Europe’s food is safe GM C CV 1 CV 2 CV 3 CV 4 GM C CV 1 CV 2 CV 3 GM C CV 1 CV 2 CV 4 GM C CV 1 CV 5 CV 6 GM C CV 5 CV 6 CV 7 CV 8 GM C CV 5 CV 6 CV 7 GM C CV 1 CV 7 CV 8 GM C CV 1 CV 5 CV 7 CV 8 CV 9 must be the same GM, non-GM comparator at each site must be at least 8 sites, over one or more years may be different commercial varieties at each site must be at least 6 commercial varieties over all the sites GM, non-GM comparator & commercial varieties are all randomised and replicated at each site Design for field trial

23 Committed since 2002 to ensuring that Europe’s food is safe Design for field trials - herbicide tolerant crops RA of herbicide-tolerant GM plants, containing single or stacked events, the experimental design should include a comparison of: GM plants exposed to the intended herbicide Comparator treated with conventional herbicide management regimes GM plants treated with the same conventional herbicide management regimes

24 Committed since 2002 to ensuring that Europe’s food is safe Statistical analysis Test of Difference: to verify whether the GMO is different from the non-GM comparator (identification of possible hazard) Test of Equivalence: to verify whether the GMO is equivalent to appropriate reference varieties (natural variation) YES biologically relevant NO biologically NOT relevant YES within natural range of variation NOoutside natural range of variation Difference Equivalence Source: available at

25 Committed since 2002 to ensuring that Europe’s food is safe Toxicological assessment Focused on Presence and levels of newly expressed proteins Potential presence of other constituents Possible changes in the levels of endogenous constituents (beyond normal variation) Impact of other changes linked to the modification No. OECD Title 402Acute Dermal Toxicity 406Skin Sensitisation 407Repeated Dose 28-day Oral Toxicity Study in Rodents 408Repeated Dose 90-Day Oral Toxicity Study in Rodents 410Repeated Dose Dermal Toxicity:21/28-Day 415One-Generation Reproduction Toxicity 416Two-Generation Reproduction Toxicity Study 417Toxicokinetics 421Reproduction/Developmental Toxicity Screening Test 471 Bacterial reverse mutation test 473 In vitro mammalian chromosome aberration test 474 Mammalian erythrocyte micronucleus test 475 Mammalian bone marrow chromosome aberration test 476 In vitro mammalian cell gene mutation test 479 In vitro sister chromatid exchange (SCE) assay in mammalian cells 482 DNA damage and repair, unscheduled DNA synthesis in mammalian cells in vitro 487 Draft guideline on: In vitro mammalian cell micronucleus test Non-exhaustive lists of OECD guidelines selectively applicable for (geno)toxicological testing for GMO RA GLP recommended

26 Committed since 2002 to ensuring that Europe’s food is safe Animal feeding studies (not mandatory) Safety on newly-expressed proteins: Repeated dose 28-day oral tox-study (unless safety – including mode of action – is documented). Safety & nutrition of whole food/feed: When: if composition is substantially modified, or if molecular and comparative analysis indicate possible unintended effects When (stacks): if indication for interactions What: 90-day toxicity study in rodents When being not equivalent, supplementary comparative nutritional studies on rapidly growing animals. Source: available at

27 Committed since 2002 to ensuring that Europe’s food is safe Allergenicity assessment Allergenicity is not an intrinsic fully predictable property of a protein. Genetic diversity and variability in atopic humans as well as geographic and environmental factors are important. A cumulative body of evidence is necessary for the risk assessment. Source: available at  weight-of-evidence approach to assess  likelihood of the novel protein to be allergenic  likelihood of the GM plant to be more allergenic than the comparator ( endogenous allergens).

28 Committed since 2002 to ensuring that Europe’s food is safe Allergenicity assessment Assessment of the newly expressed protein (1) Amino acid sequence homology comparison: i) aligment-based criterion: involving 35% sequence identity over a window of at least 80 amino acids is a minimal requirement. ii) for short peptidic fragments (e.g. ORFs) a search for sequences of continuous identical amino acids. Specific serum screening: if there is indication of sequence homology and/or the source of the gene is allergenic: i) individual (not pooled) sera from well-characterised allergic individuals. ii) IgE-binding assays

29 Committed since 2002 to ensuring that Europe’s food is safe Allergenicity assessment Assessment of the newly expressed protein (2) Pepsin resistance and other in vitro digestibility tests: i) more physiological conditions as well as specific sub-population (e.g. infants) to be considered. ii) interactions between the protein and matrix, and effect of processing to be taken into account. Other tests might provide additional information: i) in vitro cell based assays ii) in vivo tests on animal models When evidence shows potential adjuvant activity, the possible role of these proteins as adjuvants should be considered.

30 Committed since 2002 to ensuring that Europe’s food is safe Allergenicity assessment Assessment of the whole GM plant If recipient is known to be allergenic, allergen repertoire should be compared between GM plant and its comparator. -Performed by analytical methodologies, e.g. proteomics, together with allergic human sera as probes. -To minimise the use of human sera, preliminary information from the inclusion of allergens in the compositional analysis and using sera from animals experimentally sensitised. -The integrated process applies to edible components and the pollen of GM plants (i.e. covers both food and respiratory allergy risk). -Available information on the prevalence of occupational allergy in workers or farmers should be provided.

31 Committed since 2002 to ensuring that Europe’s food is safe ERA Guidance Document Systematic approach 7 area of risks 6 steps as described in Directive 2001/18/EC

32 Committed since 2002 to ensuring that Europe’s food is safe ERA Guidance Document Source: available at Strategies for ERA of GM plants Cross-cutting issuesSpecific areas of risk 1.Choice of comparators 2.Statistical considerations 3.Long-term effects 4.Receiving environments 5.Stacked events 1.Persistence and invasiveness 2.Horizontal gene transfer 3.Target organisms (TO) 4.Non-target organisms (NTO) 5.Farming practices 6.Biogeochemical processes 7.Human and animal health

33 Conclusions ERA & PMEM Cross-cutting considerations Comparators Receiving environments Statistics Long-term effects Stacks Step 1 Step 2 Step 6 Step 3 Step 5 Step Persistence & invasiveness Horizontal Gene Transfer Target Organisms Non- Target Organisms Impact of cultivation practices Impact on biogeochemical processes 2345 Human and animal health ERA Guidance Document

34 Committed since 2002 to ensuring that Europe’s food is safe ERA as starting point  Overall conclusions of ERA, including identified risk, if any, and critical uncertainty (e.g., knowledge gap)  CSM to confirm assumptions of the ERA (case-by-case) & GS to monitor unanticipated adverse effects (Mandatory!) Interplay RA-RM-PMEM Source: available at

35 Committed since 2002 to ensuring that Europe’s food is safe Methodology for general surveillance GS is not hypothesis-driven (as targeting unanticipated adverse effects) but focused on protection goals. When setting up the GS plan, applicants should consider the complementary tools for GS. At the desk: comprehensive literature review At field scale: farmers survey for first-hand info At landscape level: existing networks  surveying the environment (e.g., fauna)  surveying production and land use (e.g., fertilisers, pesticides)

36 Committed since 2002 to ensuring that Europe’s food is safe GS reporting Annually & periodically (e.g., every 3 years) Request for raw data e.g., re-analysis of farmer questionnaires for MON810 Applicant remains sole responsible for PMEM but… GS goes beyond monitoring of GMOs  Monitoring of agro-ecosystems necessitating close cooperation between ≠ parties  Common platform for PMEM data collection & storage Close collaboration amongst applicants, RA and RM

37 Committed since 2002 to ensuring that Europe’s food is safe Risk assessment of GMOs at EFSA All guidance documents are available at Further questions on guidance documents to:

38 Yi Liu EFSA GMO Unit Thank you for your attention!