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New Draft Guidance on Allergy Assessment of Genetically Modified Plants from EFSA

The European Food Safety Authority (EFSA) has issued a new draft guidance on allergenicity testing of genetically modified (GM) plants and is permitting interested parties to comment on the guidance until September 25, 2016[1].  The draft guidance reflects new methodologies that could be applied in allergenicity assessment and recent EU legislation on GM food which mandates inclusion of analyses for certain allergens in the compositional analysis of genetically modified crops. Topics addressed in the guidance are non-IgE-mediated immune adverse reactions to foods, in vitro protein digestibility testing to predict potential allergenicity of a newly expressed protein, and quantification of existing allergens in the recipient plant.

According to EFSA, non-IgE-mediated adverse immune reactions to food antigens include food-protein induced enterocolitis (FPIES), eosinophilic diseases of the gut (e.g. eosinophilic esophagitis and proctocolitis) and celiac disease. Because knowledge about the immune mechanisms for FPIES and eosinophilic diseases is lacking and because celiac disease is well characterized, EFSA stated that the allergenicity assessment of newly expressed proteins with regard to non-IgE mediated immune reactions should focus on celiac disease. Celiac disease, which is caused by an immune response to gluten, affects approximately 0.5 to 1% of the population in industrialized countries. EFSA recognizes that at least three factors contribute to the immunogenicity of gluten: resistance to proteolytic degradation, specific recognition by transglutaminase 2, and peptide binding properties of the class II major histocompatibility complex molecules HLA-DQ2.5 and HLA-DQ8. To adequately assess the potential for a food to cause celiac disease, EFSA recommends the following Phase 1 tests: 1) database searches with known celiac disease peptide sequences and motif searches to identify potential hazardous sequences and 2) in silico peptide modelling. When potentially hazardous sequences which cannot be disregarded by in silico testing are identified, more laborious tests such as in vitro digestibility, HLA-DQ-peptide binding assays and/or testing with T cell clones derived from patients with celiac disease need to be performed to determine the safety profile of the protein/peptide under assessment. Details on how these tests should be performed and criteria for positive responses are provided in the guidance document.

Regarding in vitro testing to assess the potential allergenicity of a newly expressed protein, EFSA points out several limitations of the pepsin resistance test, including use of an extremely acidic pH and excess pepsin compared to the protein substrate, lack of correlation of allergenicity with stability to pepsin digestion, and inability of the pepsin test to reflect changes in the digestive process that occur over the lifespan or in people with gastrointestinal disease. Based on these limitations, EFSA has proposed several changes to the allergy assessment of a new protein including use of digestion conditions based on the range of conditions found in vivo, which encompass the needs of special groups and those receiving medication, such as antacids.

EFSA does not specify a protocol for the in vitro test but proposes use of a minimum of two gastrointestinal test conditions which encompass the most extensive (including the current low pH/high pepsin classical pepsin resistance test conditions) and the least extensive digestion conditions, reflective of those found in children or individuals taking antacid medication (e.g. high pH/low pepsin). Both pepsin tests should be followed by a simulated intestinal digestion test with trypsin and chymotrypsin. If pancreatin is used instead of trypsin and chymostrypsin, proteolytic, lipolytic and amylolytic activity of the extract should be determined and the amount of pancreatin added should be based on trypsin activity.  Samples should be taken at various time points during the gastric and intestinal digestion steps to monitor peptide fragment evolution. Sampling time points should permit identification of transient and persistent peptides based on kinetic parameters.  A standardized method of detection should be used, suitable for profiling of both large resistant fragments and lower molecular weight peptides of ~1,000 Da (the average mass of a 9 residue peptide fragment). Because no single methodology can readily characterize the digestion of both proteins and peptides effectively, a combination of the best available methodologies should be used, such as SDS-PAGE and mass spectrometry.

When the plant receiving the new gene(s) is known to be allergenic, EFSA is recommending an assessment of endogenous allergenicity to determine whether there is a change in the overall allergenicity of the genetically modified plant compared to its non-genetically modified counterpart. The purpose of the assessment for innate allergens is to ensure that no unintended effects of the genetic modification alter the levels of endogenous allergens in a manner that would adversely impact human or animal health. The new guidance focuses on relevant allergens to be quantified in soybeans, quantification methods and interpretation of data. EFSA recommends using the OECD allergen list[2], the WNO/IUIS database[3], the FARRP allergen database[4] and Allergome[5]  to identify potential allergens, as well as published clinical studies for allergy to the food or specific allergens. Recommended methods for quantification include enzyme-linked immunosorbent assay (ELISA) or mass spectrometry. Individual allergens quantified by ELISA should be measured using purified monoclonal or polyclonal antibodies raised against each purified allergen molecule together with calibrated standards. If the levels of a specific allergen in a GM plant differ significantly from the levels observed in the appropriate comparator(s) and it falls outside the estimated range of natural variation, the biological relevance in relation to human and animal health needs to be assessed.

It is clear from this new guidance that EFSA shows a lack of confidence in the currently mandated battery of studies to assess the potential for genetically modified plants to trigger an allergic reaction(s). It is unclear whether or not the FDA will adopt similar requirements for the allergy assessment of genetically modified plants; however, in recent years, it is clear that the FDA is adopting a more precautionary, European-type stance regarding the safety of food ingredients.

[1] ESFA Panel of Genetically Modified Organisms (GMO) (2016). Draft guidance on allergenicity assessment of genetically modified plants. doi:10.2903/j.efsa.20YY.NNNN, available at

[2] OECD (2012). Revised consensus document on compositional considerations for new 452 varieties of soybean [Glycine max (L.) Merr.]: key food and feed nutrients, antinutrients, toxicants and allergens. Series on the Safety of Novel Foods and 454 Feeds No. 25. available at

[3] WHO/IUIS Allergen Nomenclature Sub-Committee (2016). Allergen nomenclature, available at

[4] The University of Nebraska-Lincoln (2010). FARRP allergen protein database, available at

[5] Allergome (2015), available at

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