Statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of genetically modified plants
2021; Wiley; Volume: 19; Issue: 1 Linguagem: Inglês
10.2903/j.efsa.2021.6350
ISSN1831-4732
AutoresHanspeter Naegeli, Jean‐Louis Bresson, Tamás Dalmay, Ian Crawford Dewhurst, M. Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejátko, F. Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, G. Savoini, Eve Veromann, Fabio Veronesi, Antonio Fernández Dumont,
Tópico(s)Occupational exposure and asthma
ResumoEFSA JournalVolume 19, Issue 1 e06350 StatementOpen Access Statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of genetically modified plants EFSA Panel on Genetically Modified Organisms (GMO), Corresponding Author EFSA Panel on Genetically Modified Organisms (GMO) GMO_secretariat_applications@efsa.europa.eu Correspondence:GMO_secretariat_applications@efsa.europa.euSearch for more papers by this authorHanspeter Naegeli, Hanspeter NaegeliSearch for more papers by this authorJean-Louis Bresson, Jean-Louis BressonSearch for more papers by this authorTamas Dalmay, Tamas DalmaySearch for more papers by this authorIan Crawford Dewhurst, Ian Crawford DewhurstSearch for more papers by this authorMichelle M Epstein, Michelle M EpsteinSearch for more papers by this authorLeslie George Firbank, Leslie George FirbankSearch for more papers by this authorPhilippe Guerche, Philippe GuercheSearch for more papers by this authorJan Hejatko, Jan HejatkoSearch for more papers by this authorFrancisco Javier Moreno, Francisco Javier MorenoSearch for more papers by this authorEwen Mullins, Ewen MullinsSearch for more papers by this authorFabien Nogué, Fabien NoguéSearch for more papers by this authorNils Rostoks, Nils RostoksSearch for more papers by this authorJose Juan Sánchez Serrano, Jose Juan Sánchez SerranoSearch for more papers by this authorGiovanni Savoini, Giovanni SavoiniSearch for more papers by this authorEve Veromann, Eve VeromannSearch for more papers by this authorFabio Veronesi, Fabio VeronesiSearch for more papers by this authorAntonio Fernandez Dumont, Antonio Fernandez DumontSearch for more papers by this author EFSA Panel on Genetically Modified Organisms (GMO), Corresponding Author EFSA Panel on Genetically Modified Organisms (GMO) GMO_secretariat_applications@efsa.europa.eu Correspondence:GMO_secretariat_applications@efsa.europa.euSearch for more papers by this authorHanspeter Naegeli, Hanspeter NaegeliSearch for more papers by this authorJean-Louis Bresson, Jean-Louis BressonSearch for more papers by this authorTamas Dalmay, Tamas DalmaySearch for more papers by this authorIan Crawford Dewhurst, Ian Crawford DewhurstSearch for more papers by this authorMichelle M Epstein, Michelle M EpsteinSearch for more papers by this authorLeslie George Firbank, Leslie George FirbankSearch for more papers by this authorPhilippe Guerche, Philippe GuercheSearch for more papers by this authorJan Hejatko, Jan HejatkoSearch for more papers by this authorFrancisco Javier Moreno, Francisco Javier MorenoSearch for more papers by this authorEwen Mullins, Ewen MullinsSearch for more papers by this authorFabien Nogué, Fabien NoguéSearch for more papers by this authorNils Rostoks, Nils RostoksSearch for more papers by this authorJose Juan Sánchez Serrano, Jose Juan Sánchez SerranoSearch for more papers by this authorGiovanni Savoini, Giovanni SavoiniSearch for more papers by this authorEve Veromann, Eve VeromannSearch for more papers by this authorFabio Veronesi, Fabio VeronesiSearch for more papers by this authorAntonio Fernandez Dumont, Antonio Fernandez DumontSearch for more papers by this author First published: 12 January 2021 https://doi.org/10.2903/j.efsa.2021.6350 Requestor: EFSA Question number: EFSA-Q-2020-00314 Panel members: Hanspeter Naegeli, Jean-Louis Bresson, Tamas Dalmay, Ian Crawford Dewhurst, Michelle M Epstein, Leslie George Firbank, Philippe Guerche, Jan Hejatko, Francisco Javier Moreno, Ewen Mullins, Fabien Nogué, Nils Rostoks, Jose Juan Sánchez Serrano, Giovanni Savoini, Eve Veromann and Fabio Veronesi. Acknowledgements: The Panel wishes to thank the members of the Working Group on Allergenicity, the members of the Stakeholder Consultative group 'Focus Group', the hearing experts Clare Mills and Henk van Loveren, and the EFSA staff Anna Lanzoni and Elisabeth Waigmann for the support provided to this scientific output. Adopted: 26 November 2020 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract This statement supplements and updates the GMO Panel guidance document on allergenicity of genetically modified (GM) plants published in 2017. In that guidance document, the GMO Panel considered that additional investigations on in vitro protein digestibility were needed before providing any additional recommendations in the form of guidance to applicants. Thus, an interim phase was proposed to assess the utility of an enhanced in vitro digestion test, as compared to the classical pepsin resistance test. Historically, resistance to degradation by pepsin using the classical pepsin resistance test has been considered as additional information, in a weight-of-evidence approach, for the assessment of allergenicity and toxicity of newly expressed proteins in GM plants. However, more recent evidence does not support this test as a good predictor of allergenic potential for hazard. Furthermore, there is a need for more reliable systems to predict the fate of the proteins in the gastrointestinal tract and how they interact with the relevant human cells. Nevertheless, the classical pepsin resistance test can still provide some information on the physicochemical properties of novel proteins relating to their stability under acidic conditions. But other methods can be used to obtain data on protein's structural and/or functional integrity. It is acknowledged that the classical pepsin resistance test is embedded into international guidelines, e.g. Codex Alimentarius and Regulation (EU) No 503/2013. For future development, a deeper understanding of protein digestion in the gastrointestinal tract could enable the framing of more robust strategies for the safety assessment of proteins. Given the high complexity of the digestion and absorption process of dietary proteins, it is needed to clarify and identify the aspects that could be relevant to assess potential risks of allergenicity and toxicity of proteins. To this end, a series of research questions to be addressed are also formulated in this statement. Summary This statement addresses in vitro protein digestibility tests in allergenicity and protein safety assessment of genetically modified (GM) plants. The main focus was to supplement and update the GMO Panel guidance document on the allergenicity of GM plants (EFSA GMO Panel, 2017) by reviewing the recent scientific literature on the usefulness of in vitro protein digestion in allergenicity risk assessment and protein safety, and data from the EFSA external report published by Mackie et al. (2019). Data from the classical pepsin resistance test measuring resistance to degradation by pepsin has been used in a weight-of-evidence approach to assess the potential allergenicity and toxicity of newly expressed proteins in GM plants. However, there is debate on its predictive value for protein allergenicity, although the classical pepsin resistance test provides information on certain physicochemical properties of novel proteins with implications for their stability, structure and function. Nevertheless, other methods can be used to obtain data on protein's structural and/or functional integrity. The evidence supporting the resistance to degradation by pepsin as a direct predictor of allergy is weak with several publications describing the pepsin test as a poor predictor of allergenicity. It is also acknowledged that the classical pepsin resistance test is embedded into international guidelines, such as Codex Alimentarius and Regulation (EU) No 503/2013. There is a need for more reliable systems to predict the fate of the proteins in the gastrointestinal (GI) tract and how they interact with the relevant cells in the human body. Resistance to protein digestion using GI models might contribute to the protein safety assessment because most dietary proteins are digested into small peptides and amino acids. Thus, a better understanding of protein–GI tract interactions might build a more robust assessment strategy. It is generally accepted that a protein which is completely digested in the GI tract would not be absorbed as an intact and functionally active molecule and is unlikely allergenic. Proteins that are resistant to digestive proteolysis might be more effective at stimulating the immune system and might be absorbed by small intestine epithelial cells in an active form that could be potentially toxic. However, a protein is not necessarily allergenic or toxic if it undergoes slow or limited protein digestibility. It is necessary to fully understand the complexity of digestion and absorption of dietary proteins to develop GI models to assess the usefulness of corresponding in vitro tests for protein allergenicity and toxicity. For example, it is necessary to understand what the usefulness of in vitro digestion is in the overall protein safety assessment; what the most suitable in vitro digestion models are; what constitutes an adequate test item (e.g. purified proteins vs whole food and/or representative food extracts); what criteria should be used to identify digestion fragments as relevant for the risk assessment (size, abundance, persistence, etc.); what follow-up actions would be required to assess the relevant proteins/fragments identified in previous steps; and, finally, how this information can be integrated into a weight-of-evidence approach. Addressing these and other issues are essential for considering in vitro digestion into the overall safety assessment of proteins and will be the basis of a subsequent EFSA scientific opinion which will be published in 2021. 1 Introduction 1.1 Background In 2017, the scientific Panel on Genetically Modified Organisms of the European Food Safety Authority (hereafter referred to as the 'GMO Panel') published a supplementary guidance document on allergenicity risk assessment of genetically modified (GM) plants addressing non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity of plant constituents (EFSA GMO Panel, 2017). In relation to in vitro protein digestibility, the GMO Panel considered that additional investigations were needed before providing any additional recommendations in the form of guidance to applicants. Thus, an interim phase was proposed to assess the utility of an enhanced in vitro digestion test, with a review undertaken in the final year. As a consequence, in July 2017, EFSA launched a procurement call entitled: 'In vitro protein digestibility'. The aim of the project was to outsource the protocol development and the production of experimental data using a refined in vitro protein degradation test, following the principles described in the EFSA GMO Panel supplementary guidance document (Annex B of the EFSA GMO Panel, 2017). The outcome of the EFSA procurement has recently been published in the EFSA website11 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/sp.efsa.2019.EN-1765 (Mackie et al., 2019). In accordance with the procedure described in the EFSA GMO Panel supplementary guidance document (EFSA GMO Panel, 2017), an ad hoc Allergenicity working group of the GMO Panel has been established to address to what extent the in vitro digestion test adds value to the allergenicity risk assessment of GM plants and to the protein safety assessment in general, and what further actions are needed to move forward this field of risk assessment. Furthermore, the Allergenicity working group will also focus on current gaps in knowledge and on future development needs for the overall allergenicity and protein safety assessment. In this context, the first action of this working group is the publication of this current statement addressing the usefulness of in vitro protein digestion in allergenicity and protein safety assessment. Subsequently, a scientific opinion on recommendations for future developments in the field of allergenicity assessment (research needs), and protein safety, in general, will be delivered by the GMO Panel in 2021. 1.2 Terms of Reference The European Food Safety Authority (EFSA) asked the Panel on Genetically Modified Organisms (GMO Panel) to develop a GMO Panel statement on in vitro protein digestibility tests in allergenicity and protein safety assessment of GM plants. This statement supplements and updates the GMO Panel guidance document on allergenicity of GM plants (EFSA GMO Panel, 2017), considering the information from the EFSA external report published last year (Mackie et al., 2019) and the state-of-the-art in science on the topic. 2 Data and methodologies 2.1 Data In delivering this statement, the EFSA GMO Panel took into account the considerations on in vitro protein digestion described in its latest guidance document on allergenicity (EFSA GMO Panel, 2017) and information from the EFSA external report on in vitro protein digestion recently published (Mackie et al., 2019), including related scientific publications retrieved from the public domain. The GMO Panel also considered comments raised by a Stakeholder Consultative Group following the activities of the GMO Panel Allergenicity working group.22 https://www.efsa.europa.eu/en/news/allergenicity-assessment-gm-plants-stakeholders-support-working-group 2.2 Methodologies The GMO Panel considered the principles described in its guidance documents and scientific opinions on allergenicity (EFSA GMO Panel, 2010, 2011, 2017), Regulation (EU) No 503/201333 Commission Implementing Regulation (EU) No 503/2013 of 3 April 2013 on applications for authorisation of genetically modified food and feed in accordance with Regulation (EC) No 1829/2003 of the European Parliament and of the Council and amending Commission Regulations (EC) No 641/2004 and (EC) No 1981/2006 Text with EEA relevance OJ L 157, 8.6.2013, p. 1–48. and other relevant international guidelines (Codex Alimentarius 2003, 2009). 3 Assessment Resistance to degradation by pepsin using the classical pepsin resistance test (Astwood et al., 1996) has been considered as an additional element, together with other relevant information, in a weight-of-evidence approach for the assessment of novel proteins introduced into GM plants (Metcalfe et al., 1996). The pepsin resistance test was intended to evaluate the susceptibility of the protein to digestion under fixed conditions in vitro, as a relevant parameter for the identification of potential allergens in risk assessment (FAO/WHO, 2001). Codex Alimentarius initially introduced this test in an international context in 2003 where stability to degradation by representative gastric/intestinal proteases and model systems was considered relevant not only for the allergenicity but also the toxicity assessment (Codex Alimentarius, 2003, 2009). As a consequence, this test has been embedded in other international guidance documents3,44 http://www.fao.org/food/food-safety-quality/gm-foods-platform/en (EFSA GMO Panel, 2011). Since 1996, adaptations to the original classical pepsin resistance test have been implemented to provide information on the susceptibility of a protein to digestion in a more reproducible manner (Thomas et al., 2004). However, the direct correlation between resistance to pepsin digestion and allergenicity per se is uncertain (Kenna and Evans, 2000; Fu, 2002; Fu et al., 2002; Herman et al., 2007; EFSA GMO Panel, 2017; Costa et al., 2021). Furthermore, the principles of the classical pepsin digestion protocol do not mimic the physiological conditions of the human gastric digestion (EFSA GMO Panel, 2011, 2017; Bøgh and Madsen, 2016). All these elements have raised questions on the overall utility of the classical pepsin resistance test in the risk assessment of proteins. At the time, Codex Alimentarius (2003, 2009) indicated that alternative protocols to the classical pepsin resistance test, aimed to assess susceptibility to digestion by proteases, could be used when adequate justification is provided. EFSA previously highlighted the limitations of the classical pepsin resistance test in protein safety assessment, and recommended that in vitro protein digestibility could be evaluated using other in vitro digestibility methods designed to more closely simulate the conditions of the human digestion process (EFSA GMO Panel, 2010, 2011, 2017). This has prompted the development of alternative protocols aimed at improving the knowledge of proteins behaviour during the digestive process (EFSA GMO Panel, 2017; Mackie et al., 2019; Torcello-Gómez et al., 2020a,b). In this statement, the GMO Panel considers the information on in vitro protein digestion described in its latest guidance document on allergenicity (EFSA GMO Panel, 2017) and information from an EFSA external report on in vitro protein digestion (Mackie et al., 2019). In addition, the GMO Panel also reviews recent scientific literature dealing with the usefulness of in vitro protein digestion in allergenicity risk assessment and the overall protein safety assessment, framing recommendations for future development of a field with great potential. 3.1 Protein digestibility and relevance to risk assessment The intestinal tract is the largest mucosal surface of the human body. It is lined by a single layer of columnar intestinal epithelial cells that forms a barrier between the intestinal lumen and host connective tissue and strongly participates in innate immunity (Artis, 2008). Virtually all nutrients from the diet are absorbed into the bloodstream across the epithelial cell layer forming the small intestinal mucosa (Kiela and Ghishan, 2016). Oral exposure to dietary proteins, as a general class of substances, is a necessary physiological process. Nevertheless, a very small number of food proteins have been identified to be capable of causing adverse effects, in terms of allergy or toxicity when consumed and these effects may be observed within the gut (Markell et al., 2017) and/or at extraintestinal sites, such as those food allergens responsible for the oral allergy syndrome (Carlson and Coop, 2019). Resistance to digestion by pepsin and pancreatic endoproteases is informative for protein safety assessment because the normal fate of most dietary proteins is digestion into small peptides and individual amino acids which pass into the systemic circulation via transcellular or paracellular routes (Caspary, 1992). This reinforces the fact that digestion affects exposure to intact protein. Codex Alimentarius (2003, 2009) refers to protein in vitro digestion tests as relevant studies for assessing protein safety, mainly allergenicity but also protein toxicity. On the one hand, the generally accepted position is that if a protein is completely digested in the GI tract, the potential for its absorption from the digestive tract as an intact and functionally active macromolecule would be in most cases negligible, minimising the likelihood that the protein could exert adverse effects such as allergenicity (Pali-Schöll et al., 2018) and/or toxicity (Hammond et al., 2013; Tafazoli et al., 2019). On the other hand, evidence of slow or limited protein digestibility does not indicate on its own that the protein is necessarily allergenic or toxic, but proteins that are at least partially resistant to digestive proteolysis might be more effective at stimulating the immune system because possible immunogenic structures are persisting longer in the gut (Shan et al., 2002; Fernandez et al., 2019; Pilolli et al., 2019) and might be absorbed by epithelial cells of the small intestine in an active form in terms of potential toxicity (Delaney et al., 2008). Therefore, studies on resistance to digestion are considered as additional evidence together with other elements in a weight-of-evidence approach, mainly informing the hazard identification step, for the allergenicity and toxicological assessment of newly expressed proteins (Codex Alimentarius, 2003, 2009; EFSA GMO Panel, 2011, 2017; European Commission, 2013). Protease resistance of food proteins can also be a useful tool for assessing exposure (Akkerdaas et al., 2018). Digestion and absorption involve complex, highly integrated and regulated physiological processes. Key host factors include, for instance, the interaction of the digesta with the gut epithelial cells and the integrity of the intestinal epithelial barrier which determine the transport of nutrients. Likewise, a number of extrinsic factors, such as the presence of particular matrix components (e.g. fibre, lipids, polyphenols, anti-nutritive factors), food microstructure or food-processing conditions, may affect protein digestibility in the GI tract and these may be of importance when designing an in vitro model (Ekmay et al., 2017). Therefore, the simulation of such a complex system in its entirety using static unresponsive in vitro methods is very challenging (Butts et al., 2012), and the application of in vitro digestibility assays within a risk assessment frame should be tempered. Given the marked differences between the mechanisms underlying allergen- and toxin-induced adverse reactions, as well as between the inherent properties of potential allergens and toxins, the relevance of protein digestibility in the allergenicity and toxicity risk assessment processes will be separately discussed below. 3.1.1 Digestibility and the allergenicity risk assessment process Food allergy consists of two separate phases: i) sensitisation, a phase where the immune system develops hyperreactivity to the allergen, but no symptoms occur; and ii) elicitation, a phase where clinical manifestations are triggered (Fernandez et al., 2013; Selb et al., 2017). One of the main arguments questioning the relevance of protein stability and resistance to proteolytic digestion to the allergenicity risk assessment process, is that the evidence demonstrating that de novo sensitisation takes place through oral exposure via the gut immune system is weak or even missing (Herman and Ladics, 2018). Therefore, this could make digestive protein stability a poor predictor of sensitisation (Herman et al., 2020). Although it has been shown that impairing gastric digestion exerted by different means (e.g. suppression of gastric acid secretion, bariatric surgery) increases the risk for allergic sensitisation (Pali-Schöll et al., 2018), de novo allergies to plant and animal food allergens can be induced by inhalation (Ramirez and Bahna, 2009) or the dermal route (Kimber et al., 2014; Brough et al., 2020). Nevertheless, the distinction of IgE antibody-mediated responses to ingested food proteins compared to those responses induced by inhalation or dermal contact has been considered artificial. This is because, although there may be route-dependent differences in response, there appears to be a substantial overlap in allergens of significance across routes of exposure (Blackburn et al., 2015). This is in line with the fact that allergic sensitisation to food proteins can be achieved by any physiologic route of exposure in the presence of an appropriate environment (exogenous adjuvant) (Dunkin et al., 2011). Likewise, it has been shown that non-GI exposure to food proteins, i.e. via the cutaneous or respiratory route, may facilitate a subsequent sensitisation via the oral route without the use of adjuvants (Wavrin et al., 2014, 2015). On the one hand, survival in the GI tract of stable protein/peptide fragments would facilitate the maintenance of epitope expression and thereby facilitate the development of sensitisation (Pekar et al., 2018; Krutz et al., 2020); however, this attribute can be ascribed to some food allergens, but not to all of them (Bøgh and Madsen, 2016; EFSA GMO Panel, 2017; Fernandez et al., 2019; Verhoeckx et al., 2019). On the other hand, recent evidence has shown the capacity of the intestinal microbiota to metabolise food protein antigens or allergens, thereby, altering their immunogenicity (Caminero et al., 2019) or to produce metabolites that directly affect tolerogenic responses (Smith et al., 2013). Notwithstanding the role of route of exposure in sensitisation as described above, understanding how proteins are presented to the gut mucosal immune system may provide insight into how the balance between tolerance and sensitisation is regulated (Dearman et al., 2002; EFSA GMO Panel, 2017). When characteristics of known food allergens are examined, one of the most prominent traits attributed to food allergens is protein stability (Helm, 2001; Breiteneder and Mills, 2005; Costa et al., 2021). The term protein stability encompasses several properties, including not only proteolytic stability but also thermal stability, pH-dependent stability and physical stability (e.g. tendency to aggregate), among others (Li et al., 2019). Consequently, protein stability to digestion is one of several relevant parameters to consider in the weight-of-evidence approach (EFSA GMO Panel, 2010, 2011, 2017). The allergenicity of proteins is likely determined by several factors, such as the relationship between physicochemical and structural properties governing protein stability (James et al., 2018), abundance, size, point in time of exposure and duration of exposure to the immune system. Environmental conditions also need to be considered, including microbial exposure, immune-modulating components (adjuvant effect) of allergenic sources facilitating T helper 2 (Th2) immune responses, as well as the intrinsic effects of proteins on the innate and adaptive immune system (Scheurer et al., 2015). Food allergens with high stability to digestion and processing are able to reach the GI immune system in their IgE-reactive form and to elicit more severe reactions (Valenta et al., 2015). However, recent studies have demonstrated that some food allergens, when digested to small peptide fragments, may still retain both the sensitising and the IgE-reacting potential. This is because of the peptides' ability to hold together and to adopt three-dimensional structures similar to that of the native allergen under certain conditions (Prodic et al., 2018). For instance, post-translational modifications, such as disulphide bond formation (Apostolovic et al., 2016) or hydroxylation of proline residues (Bernard et al., 2015), as well as physicochemical properties that promote peptide aggregation (Bøgh et al., 2009, 2012; Radosavljević et al., 2020) have been identified as important determinants for preserving allergenic properties in digestion-resulting peptides derived from allergens found in plant- and animal-based food sources. Finally, protein digestibility is also central for the risk assessment of celiac disease where GI digestion is important in the delivery of immunologically active fragments to gut mucosal segments (Shan et al., 2002; Pilolli et al., 2019). 3.1.2 Digestibility and the toxicity risk assessment process Tools to investigate novel protein oral toxicity from GM plants include in silico,in vitro and in vivo studies, and information on the history of safe consumption of the proteins under assessment, combined in a weight-of-evidence approach. Noteworthy, the in vivo toxicity studies requested (i.e. 28-day toxicity studies, EFSA GMO Panel, 2011) are based on protocols developed for small molecules, but they can provide some information on the potential toxicity following ingestion of proteins. However, the studies normally will provide no information on the digestibility of the protein or whether any effects observed were due to the intact protein or particular domains or peptides derived from it. Therefore, information on the fate of the protein after ingestion is an important element to take into consideration in the toxicological (and allergenic assessment) of proteins in a weight-of-evidence approach. According to Codex Alimentarius (2003, 2009), the assessment of potential toxicity considers, among others stability of the protein to heat or food processing procedures and to degradation in appropriate representative gastric and intestinal model systems. The EFSA guidance document (EFSA GMO Panel, 2011) also refers to the need for data on resistance of the novel proteins to proteolytic enzymes using appropriate and standardised tests, as not only the intact protein but also stable fragments might pose concerns (Bethune and Khosla, 2008). In recent years, there has been increasing pressure to move away from animal models for the toxicological assessment because of ethical considerations (Krutz et al., 2020). In addition, the inherent properties of the so-called intractable proteins (e.g. proteins associated with lipid membranes) may prevent the performance of the traditional high-dose in vivo toxicology studies because of the technical limitations to produce a sufficient amount of high purity protein in a functionally active form as a part of overall safety assessment (Bushey et al., 2014; Eaton et al., 2017). This panorama could put the focus on the reinforcement of all weight-of-evidence elements used for the safety assessment, such as the concept of history of safe use, bioinformatic analysis, mode of action and specificity, in vitro digestibility, stability, expression levels and dietary intake evaluation. In relation to in vitro digestibility, while the use of a single enzyme (e.g. pepsin) in the context of risk assessment is clearly an oversimplification, it is also acknowledged that the implem
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