Allergenicity assessment of new or modified protein-containing food sources and ingredients.

The growing world population, changing dietary habits, and increasing pressure on agricultural resources are drivers for the development of novel foods (including new protein sources as well as existing protein sources that are produced or used in an alternative way or in a different concentration). These changes, coupled with consumer inclination to adopt new dietary trends, may heighten the intake of unfamiliar proteins, or escalate consumption of specific ones, potentially amplifying the prevalence of known and undiscovered food allergies. Assessing the allergenicity of novel or modified protein-based foods encounters several challenges, including uncertainty surrounding acceptable risks and assessment criteria for determining safety. Moreover, the available methodological tools for gathering supportive data exhibit significant gaps. This paper synthesises these challenges, addressing the varied interpretations of "safe" across jurisdictions and societal attitudes towards allergenic risk. It proposes a comprehensive two-part framework for allergenicity assessment: the first part emphasises systematic consideration of knowledge and data requirements, while the second part proposes the application of a generic assessment approach, integrating a Threshold of Allergological Concern. This combined framework highlights areas that require attention to bridge knowledge and data gaps, and it delineates research priorities for its development and implementation.

How does dose impact on the severity of food-induced allergic reactions, and can this improve risk assessment for allergenic foods?: Report from an ILSI Europe Food Allergy Task Force Expert Group and Workshop.

Quantitative risk assessment (QRA) for food allergens has made considerable progress in recent years, yet acceptability of its outcomes remains stymied because of the limited extent to which it has been possible to incorporate severity as a variable. Reaction severity, particularly following accidental exposure, depends on multiple factors, related to the allergen, the host and any treatments, which might be administered. Some of these factors are plausibly still unknown. Quantitative risk assessment shows that limiting exposure through control of dose reduces the rates of reactions in allergic populations, but its impact on the relative frequency of severe reactions at different doses is unclear. Food challenge studies suggest that the relationship between dose of allergenic food and reaction severity is complex even under relatively controlled conditions. Because of these complexities, epidemiological studies provide very limited insight into this aspect of the dose-response relationship. Emerging data from single-dose challenges suggest that graded food challenges may overestimate the rate of severe reactions. It may be necessary to generate new data (such as those from single-dose challenges) to reliably identify the effect of dose on severity for use in QRA. Success will reduce uncertainty in the susceptible population and improve consumer choice.

Approaches to assess IgE mediated allergy risks (sensitization and cross-reactivity) from new or modified dietary proteins.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks.

Can we identify patients at risk of life-threatening allergic reactions to food?

Anaphylaxis has been defined as a 'severe, life-threatening generalized or systemic hypersensitivity reaction'. However, data indicate that the vast majority of food-triggered anaphylactic reactions are not life-threatening. Nonetheless, severe life-threatening reactions do occur and are unpredictable. We discuss the concepts surrounding perceptions of severe, life-threatening allergic reactions to food by different stakeholders, with particular reference to the inclusion of clinical severity as a factor in allergy and allergen risk management. We review the evidence regarding factors that might be used to identify those at most risk of severe allergic reactions to food, and the consequences of misinformation in this regard. For example, a significant proportion of food-allergic children also have asthma, yet almost none will experience a fatal food-allergic reaction; asthma is not, in itself, a strong predictor for fatal anaphylaxis. The relationship between dose of allergen exposure and symptom severity is unclear. While dose appears to be a risk factor in at least a subgroup of patients, studies report that individuals with prior anaphylaxis do not have a lower eliciting dose than those reporting previous mild reactions. It is therefore important to consider severity and sensitivity as separate factors, as a highly sensitive individual will not necessarily experience severe symptoms during an allergic reaction. We identify the knowledge gaps that need to be addressed to improve our ability to better identify those most at risk of severe food-induced allergic reactions.

Application of scientific criteria to food allergens of public health importance.

Scientific criteria for identifying allergenic foods of public health importance (Björkstén, B., Crevel, R., Hischenhuber, C., Løvik, M., Samuels, F., Strobel, S., Taylor, S.L., Wal, J.-M., Ward, R., 2008. Criteria for identifying allergenic foods of public health importance. Regulatory Toxicology and Pharmacology 51(1), 42-52) have been further refined to incorporate an assessment of the strength of available scientific evidence (van Bilsen, J.H., Ronsmans, S., Crevel, R.W., Rona, R.J., Przyrembel, H., Penninks, A.H., Contor, L., Houben, G.F., 2011. Evaluation of scientific criteria for identifying allergenic food of public health importance. Regulatory Toxicology and Pharmacology 60, 281-289). A multi-disciplinary group was invited to critically test the refined approach. They independently evaluated selected publications on coconut, soy and/or peanut allergy, scored them using the newly developed level of evidence criteria, and debated proposed approaches for combining and utilising the scores to measure the overall impact of an allergen in public health impact assessments. The evaluation of selected publications using the modified criteria produced a relatively consistent result across the experts. These refined criteria were judged to be a way forward for the identification of allergenic foods of public health importance, and for prioritisation of allergen risk management and future data gathering. The debate to combine available evidence when assessing whether an allergenic food is of sufficient public health importance to warrant active management led to proposals on how to weight and combine evidence on allergen severity, potency and prevalence. The refined criteria facilitate a debate to find a meaningful sequence of steps to summarise the available information in relation to a food allergen.

Evaluation of scientific criteria for identifying allergenic foods of public health importance.

Identification of allergenic foods of public health importance should be based on well-defined criteria. Björkstén et al. (2008) proposed that the criteria should assess the evidence for an IgE mechanism, the reaction, the potency and the severity of the effect of the food and its prevalence. This study evaluated the application of the proposed criteria based on published reports. Publications were selected from two databases to test whether the descriptions for ranking the level of evidence for each criterion were unambiguous and covered the full range of levels of evidence regarding seven foods, five known to be allergenic and two negative controls. The options available to rank the quality of evidence were appropriate but needed refinement to improve clarity and conceptual value. The criteria were helpful to assess known IgE-dependent allergens, and to exclude the non-allergenic substances. The criteria framework discriminated between papers with high, moderate and low quality of evidence. The advantage of using the proposed criteria is to make the decision-making process and rationale explicit. The framework helps to identify gaps in knowledge and to uncover the level of heterogeneity of the evidence thus guiding research and providing a basis for sound risk management decisions.

Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.

Approaches to risk assessment in food allergy: report from a workshop ''developing a framework for assessing the risk from allergenic foods".

A workshop was organised to investigate whether risk assessment strategies and methodologies used in classical/conventional toxicology may be used for risk assessment of allergenic foods, to discuss the advantages and limitations of different approaches and to determine the research needed to move the area forward. Three possible approaches to safety assessment and risk assessment for allergenic foods were presented and discussed: safety assessment using NOAEL/LOAEL and uncertainty factors, safety assessment using Benchmark Dose and Margin of Exposure (MoE), and risk assessment using probabilistic models. The workshop concluded that all the three approaches to safety and risk assessment of allergenic foods should continue to be considered. A particular strength of the MoE and probabilistic approaches is that they do not rely on low-dose extrapolations with its inherent issues. Probabilistic modelling is considered to be the most promising approach for use in population risk assessment (which is a particular focus for risk managers). For all approaches, further improvement of input data is desirable, particularly data on consumption patterns/food choices in food allergic consumers, data on minimum eliciting doses and data that can be used to evaluate whether the whole population at risk has been modelled accurately. Specific research topics were identified.

Thresholds for food allergens and their value to different stakeholders.

Thresholds constitute a critical piece of information in assessing the risk from allergenic foods at both the individual and population levels. Knowledge of the minimum dose that can elicit a reaction is of great interest to all food allergy stakeholders. For allergic individuals and health professionals, individual threshold data can inform allergy management. Population thresholds can help both the food industry and regulatory authorities assess the public health risk and design appropriate food safety objectives to guide risk management. Considerable experience has been gained with the double-blind placebo-controlled food challenge (DBPCFC), but only recently has the technique been adapted to provide data on thresholds. Available data thus vary greatly in quality, with relatively few studies providing the best quality individual data, using the low-dose DBPCFC. Such high quality individual data also form the foundation for population thresholds, but these also require, in addition to an adequate sample size, a good characterization of the tested population in relation to the whole allergic population. Determination of thresholds at both an individual level and at a population level is influenced by many factors. This review describes a low-dose challenge protocol developed as part of the European Community-funded Integrated Project Europrevall, and strongly recommends its wider use so that data are generated that can readily increase the power of existing studies.

Probabilistic risk assessment model for allergens in food: sensitivity analysis of the minimum eliciting dose and food consumption.

Previously, TNO developed a probabilistic model to predict the likelihood of an allergic reaction, resulting in a quantitative assessment of the risk associated with unintended exposure to food allergens. The likelihood is estimated by including in the model the proportion of the population who is allergic, the proportion consuming the food and the amount consumed, the likelihood of the food containing an adventitious allergen and its concentration, and the minimum eliciting dose (MED) distribution for the allergen. In the present work a sensitivity analysis was performed to identify which parts of the model most influence the output. A shift in the distribution of the MED reflecting a more potent allergen, and an increase in the proportion of the population consuming a food, increased the number of estimated allergic reactions considerably. In contrast, the number of estimated allergic reactions hardly changed when the MEDs were based on a more severe response, or when the amount of food consumed was increased. Development of this work will help to generate a more accurate picture of the potential public health impact of allergens. It highlights areas where research is best focused, specifically the determination of minimum eliciting doses and understanding of the food choices of allergic individuals.

The prevalence, cost and basis of food allergy across Europe.

The development of effective management strategies to optimize the quality of life for allergic patients is currently hampered by a lack of good quality information. Estimates of how many individuals suffer from food allergy and the major foods involved vary widely and inadequacies of in vitro diagnostics make food challenges the only reliable means of diagnosis in many instances. The EuroPrevall project brings together a multidisciplinary partnership to address these issues. Cohorts spanning the main climatic regions of Europe are being developed in infants through a birth cohort, community surveys in school-age children and adults and an outpatient clinic study. Confirmatory double-blind placebo-controlled food challenge diagnosis is being undertaken using foods as they are eaten with titrated doses to allow no-effect and lowest-observable effect levels for allergenic foods to be determined. The cohorts will also facilitate validation of novel in vitro diagnostics through the development of the EuroPrevall Serum Bank. Complementary studies in Ghana, western Siberia, India and China will allow us to gain insights into how different dietary patterns and exposure to microorganisms affect food allergies. New instruments to assess the socioeconomic impact of food allergy are being developed in the project and their application in the clinical cohorts will allow, for the first time, an assessment to be made of the burden this disease places on allergy sufferers and their communities.

Hazard characterisation in food allergen risk assessment: the application of statistical approaches and the use of clinical data.

A structured approach to assess the risk to allergic individuals from food allergens requires as a first step the experimental measurement of minimum eliciting doses in a population that is as representative as possible of the relevant allergic population, using a standardised protocol. These doses are established in controlled challenge studies, but logistical and statistical constraints mean that a proportion of the allergic population may still be at risk of reacting at doses below those which have been or could feasibly be tested. However, statistical modelling of the dose distribution resulting from such challenges permits inferences to be drawn about the proportion of allergic individuals that are likely to react to specified (low) amounts of residual allergen in food. However, different statistical models, which all provide good fits to the experimental data yield different values outside the experimental range. Consequently, the outputs from these models require a form of validation, which demonstrates how close the predictions are to reality. In addition to characterisation of the hazard, for each allergenic food this validation requires information about exposure to undeclared allergen, the actual number of reactions taking place in the wider allergic population, and the prevalence of allergy to that food.

Lack of immunogenicity of ice structuring protein type III HPLC12 preparation administered by the oral route to human volunteers.

Before a novel protein can be used in foods, its potential allergenicity must be assessed. In this study, healthy volunteers consumed ice structuring protein (ISP) Type III preparation or a control material 5 days a week for a total of 8 weeks. General measures of health were recorded during the study, and the immunogenicity of the protein was assessed by monitoring the levels of IgG and IgE antibodies specific for ISP Type III. The participants remained in good health throughout the study and during the 4 week follow-up period. No IgG or IgE antibodies specific for ISP Type III were detected in the blood of the participants. Investigations of immunogenicity in man have not been previously applied in the context of safety evaluation and they do not form part of the regimens proposed for the evaluation of protein allergenicity. Consequently no standardised protocols exist for such studies, nor any background against which to interpret the results. Nevertheless, the absence of an immune response using a protocol which could have been expected to result in a response with a strongly immunogenic protein, confirms the conclusions of earlier published work, and attests to the lack of allergenicity of ISP Type III preparation.

Too clean, or not too clean: the hygiene hypothesis and home hygiene.

The 'hygiene hypothesis' as originally formulated by Strachan, proposes that a cause of the recent rapid rise in atopic disorders could be a lower incidence of infection in early childhood, transmitted by unhygienic contact with older siblings. Use of the term 'hygiene hypothesis' has led to several interpretations, some of which are not supported by a broader survey of the evidence. The increase in allergic disorders does not correlate with the decrease in infection with pathogenic organisms, nor can it be explained by changes in domestic hygiene. A consensus is beginning to develop round the view that more fundamental changes in lifestyle have led to decreased exposure to certain microbial or other species, such as helminths, that are important for the development of immunoregulatory mechanisms. Although this review concludes that the relationship of the hypothesis to hygiene practice is not proven, it lends strong support to initiatives seeking to improve hygiene practice. It would however be helpful if the hypothesis were renamed, e.g. as the 'microbial exposure' hypothesis, or 'microbial deprivation' hypothesis, as proposed for instance by Bjorksten. Avoiding the term 'hygiene' would help focus attention on determining the true impact of microbes on atopic diseases, while minimizing risks of discouraging good hygiene practice.

Information provision for allergic consumers--where are we going with food allergen labelling?

As the current treatment for food allergy involves dietary exclusion of the problem food, information for food-allergic consumers provided on food labels about the nature of allergenic ingredients is important to the management of their condition. The members of an EU-funded networking project, InformAll, focusing on developing strategies for the provision of credible, reliable sources of information for food allergy sufferers, regulators and the food industry, have been considering these matters with respect to food labelling. This paper presents an overview of the genesis of the new EU directive on food labelling, its relevance to food-allergic consumers and the problems that might arise if precautionary labelling becomes more widespread in response to concerns regarding inadvertent allergen contamination in foods. International efforts to define threshold levels of allergens able to trigger a reaction coupled with validated allergen detection methods are essential if the food industry is to implement effective hazard control procedures and address the problems of cross-contact allergens without devaluing the information provided to consumers on food labels.

Assessment of the safety of foods derived from genetically modified (GM) crops.

This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative.

Post-market surveillance of GM foods: applicability and limitations of schemes used with pharmaceuticals and some non-GM novel foods.

Post-market surveillance (PMS) is increasingly required by some regulatory authorities for the marketing approval of GM-Novel Foods. This requirement, in addition to a complete conventional safety assessment, aims to show that unexpected (adverse) effects do not occur after long-term everyday exposure. Large food manufacturers have systems to obtain feedback from consumers on their products. We show that such systems can be enhanced to collect information on possible health effects of specific products and relate these to intake in specific groups of consumers. The term post-launch monitoring (PLM) is proposed to distinguish the process from that used for pharmaceuticals. GM foods differ from branded products to which existing systems have been applied. The paper discusses whether and how such systems could be applied to GM foods and what additional elements would need to be incorporated in them. A PLM system should define and organize the flow of information between the different stakeholders. We conclude that because such data will be generated from a range of sources and will need to be collated, verified, and integrated, an independent agency will be essential to undertake this activity in order to balance the interests of all stakeholders and ensure public trust.

Assessment of the potential allergenicity of ice structuring protein type III HPLC 12 using the FAO/WHO 2001 decision tree for novel foods.

The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein. Therefore, decision trees for evaluation of the risk have been developed, the latest being proposed by WHO/FAO early in 2001. Proteins developed using modern biotechnology and derived from fish are being considered for use in food and other applications, and since allergy to fish is well established, a potential risk from such proteins to susceptible human beings exists. The overall aim of the study was to investigate the potential allergenicity of an Ice Structuring Protein (ISP) originating from an arctic fish (the ocean pout, Macrozoarces americanus) using the newly developed decision tree proposed by FAO/WHO. The methods used were those proposed by FAO/WHO including amino acid sequence analysis for sequence similarity to known allergens, methods for assessing degradability under standardised conditions, assays for detection of specific IgE against the protein (Maxisorb RAST) and histamine release from human basophils. In the present paper we describe the serum screening phase of the study and discuss the overall application of the decision tree to the assessment of the potential allergenicity of ISP Type III. In an accompanying paper [Food Chem. Toxicol. 40 (2002) 965], we detail the specific methodology used for the sequence analysis and assessment of resistance to pepsin-catalysed proteolysis of this protein. The ISP showed no sequence similarity to known allergens nor was it stable to proteolytic degradation using standardised methods. Using sera from 20 patients with a well-documented clinical history of fish allergy, positive in skin prick tests to ocean pout, eel pout and eel were used, positive IgE-binding in vitro to extracts of the same fish was confirmed. The sera also elicited histamine release in vitro in the presence of the same extracts. The ISP was negative in all cases in the same experiments. Using the proposed decision tree, we demonstrated the safety of the ISP to patients already sensitised to fish, as well as to individuals potentially susceptible to producing IgE responses to proteins. Furthermore, the practicability of the new decision tree was confirmed.

Sequence analysis and resistance to pepsin hydrolysis as part of an assessment of the potential allergenicity of ice structuring protein type III HPLC 12.

The recently published WHO/FAO guidelines on the assessment of allergenicity of novel food proteins provide a strategy with which to approach the determination of the potential of novel proteins in foods to be allergens. Key to this strategy are the assessment of sequence similarity to known allergens and the assessment of the resistance to pepsin hydrolysis. Ice structuring proteins (also commonly referred to as anti-freeze or thermal hysteresis proteins) are a group of naturally occurring proteins that bind to ice and structure ice crystal formation. The amino acid sequence of the ice structuring protein (ISP) type III HPLC 12 (ISP type III) was compared in silico with the sequences of known allergens. Secondly, the resistance to pepsin hydrolysis of ISP type III and its glycoconjugates (produced in recombinant baker's yeast) was assessed. The results indicate that ISP type III has no sequence similarity with known allergenic proteins. Both ISP type III and ISP type III glycoconjugates contained within the fermentation product were hydrolysed readily by pepsin (50% loss in <10 min at pH 1.5) to give peptide fragments that were too small to be allergenic or to trigger cross-linking to IgE. In an accompanying study, we demonstrated that IgE from fish-allergic individuals did not bind ISP Type III. Therefore, in accordance with the WHO/FAO strategy, the assessment of ISP type III and ISP type III glycoconjugates by sequence analysis together with lack of resistance to pepsin hydrolysis and the absence of IgE binding supports the conclusion that both are unlikely to present a potential sensitisation hazard.

Antifreeze proteins: characteristics, occurrence and human exposure.

Antifreeze proteins (AFPs), also known as ice structuring proteins, bind to and influence the growth of ice crystals. Proteins with these characteristics have been identified in fish living in areas susceptible to ice formation and in numerous plants and insects. This review considers the occurrence of AFPs and relates it to the likely intake by human populations, with a view to forming a judgment about their safety in foods. Intake of AFPs in the diet is likely to be substantial in most northerly and temperate regions. Much of this intake is likely to be from edible plants, given their importance in the diet, but in some regions intake from fish will be significant. Inadequate data exist to estimate intakes from plants but estimates of intake of AFP from fish are presented for two countries with very different fish consumption, the USA and Iceland. Typical short-term exposure, for instance a portion of cod may contain up to 196 mg AFGP, while the AFP content of the same weight of ocean pout would be up to 420 mg. Average available fish AFP in the diet is calculated to be around 1-10 mg/day in the USA and 50-500 mg/day in Iceland, but these estimates are subject to considerable uncertainty. As far as can be ascertained, AFPs are consumed with no evidence of adverse health effects, either short- or long-term. Given the structural diversity of AFPs, one firm general conclusion that can be drawn from the history of consumption of AFPs is that their functional characteristics do not impart any toxicologically significant effect, in a way that, for instance, a property such as cholinesterase inhibition would. Furthermore, specifically in the case of fish AFPs where some consumption data are available, it is reasonable to infer a lack of allergenicity from the absence of reports of this effect.