A Weight-of-the-Evidence Approach for Evaluating, in Lieu of Animal Studies, the Potential of a Novel Polysaccharide Polymer to Produce Lung Overload.

The United States Environmental Protection Agency (EPA) is concerned about the respiratory effects caused by respirable particles of water-insoluble high molecular weight polymers. The EPA has proposed a tiered approach to evaluate polymer lung overload, a kinetic event. Kinetic polymer lung overload in itself is not necessarily adverse, however, inhalation of respirable particulate matter can have adverse effects (i.e., inflammation, fibrosis, etc.). If Tier I testing demonstrates that particles may reach the distal lung (i.e., a non-negligible amount of respirable particles/droplets ≤10 µm in diameter and lack of biosolubility), then animal inhalation testing in Tiers II-IV would be requested. In silico, in chemico, and in vitro alternatives should be considered versus in vivo testing for animal welfare purposes. An in chemico measure of biosolubility was used to demonstrate that a novel α-1,3-glucan polysaccharide, made by enzymatic polymerization of glucose from sucrose, is biosoluble and fits a simple exponential decay model with a half-life on the order of 66 days. The multiple-path particle dosimetry (MPPD) in silico model was used to predict lung burden for the novel α-1,3-glucan polysaccharide. MPPD was validated with measurements in rats exposed to a toner particulate and showed good agreement with lung burden measurements. A simulated 24 month rat exposure yielded 10-20 times less lung burden for the polysaccharide compared to the toner at equivalent exposure concentrations. The MPPD model was refined to include biosolubility data for the polysaccharide polymer. Data for amorphous silica were used to validate the clearance model, and the model incorporating dissolution predicted the amorphous silica lung burden within 20% of measured values. Human equivalent concentrations (HECs) were calculated for each toner rat exposure concentration. HECs were also determined for the polysaccharide at exposure concentrations yielding the same predicted internal doses as the toner. The in vitro, in chemico and in silico studies described here for the novel polysaccharide provide a useful weight of evidence approach in the absence of animal studies for the evaluation of polymer substances where polymer lung overload may be a concern.

Allergenicity prediction of novel and modified proteins: Not a mission impossible! Development of a Random Forest allergenicity prediction model.

Alternative and sustainable protein sources (e.g., algae, duckweed, insects) are required to produce (future) foods. However, introduction of new food sources to the market requires a thorough risk assessment of nutritional, microbial and toxicological risks and potential allergic responses. Yet, the risk assessment of allergenic potential of novel proteins is challenging. Currently, guidance for genetically modified proteins relies on a weight-of-evidence approach. Current Codex (2009) and EFSA (2010; 2017) guidance indicates that sequence identity to known allergens is acceptable for predicting the cross-reactive potential of novel proteins and resistance to pepsin digestion and glycosylation status is used for evaluating de novo allergenicity potential. Other physicochemical and biochemical protein properties, however, are not used in the current weight-of-evidence approach. In this study, we have used the Random Forest algorithm for developing an in silico model that yields a prediction of the allergenic potential of a protein based on its physicochemical and biochemical properties. The final model contains twenty-nine variables, which were all calculated using the protein sequence by means of the ProtParam software and the PSIPred Protein Sequence Analysis program. Proteins were assigned as allergenic when present in the COMPARE database. Results show a robust model performance with a sensitivity, specificity and accuracy each greater than ≥85%. As the model only requires the protein sequence for calculations, it can be easily incorporated into the existing risk assessment approach. In conclusion, the model developed in this study improves the predictability of the allergenicity of new or modified food proteins, as demonstrated for insect proteins.

Industrial microbial enzyme safety: What does the weight-of-evidence indicate?

With the exception for the potential skin and eye irritating effects of some proteases, and the well-documented potential for respiratory sensitization in case of work place exposure, enzymes in general don't produce acute toxicity, dermal sensitization; genotoxicity, or repeated dose oral toxicity. Acute inhalation, reproduction, chronic toxicity, and carcinogenicity are not relevant for enzymes. Several hundred mutagenicity studies have been conducted on bacterial and mammalian cells using a variety of enzymes. No positive findings were observed. > 225 90-day studies have been performed and submitted to EFSA with no adverse findings, including in the bone marrow. The data showing no adverse effects for enzyme preparations also confirms that microbial metabolites and fermentation materials lack toxicity as well. Exposure to enzyme products is also minimal as recommended use levels are low, generally <0.1% (wt/wt). The weight-of-evidence indicates that there are no concerns for oral toxicity of enzymes in general, nor genotoxicity. Therefore, the continued routine practice of performing genotoxicity and 90-day studies on enzyme preparations as part of approval requirements is questionable, and establishing general health-based guidance values for enzymes may be considered. A criterion for our assertion that general health-based guidance values be established is to select and use suitable non-toxigenic microbial production strains, per decision tree guidelines.

Allergenic sensitization versus elicitation risk criteria for novel food proteins.

The value of criteria used in the weight-of-evidence assessment of allergenic risk of genetically modified (GM) crops has been debated. This debate may originate, in part, from not specifying if the criteria are intended to contribute to the assessment of sensitization risk or elicitation risk. Here, this distinction is explicitly discussed in the context of exposure and hazard. GM crops with structural relationships with known allergens or sourced from an organism known to cause allergy (hazard) are screened for IgE-antibody reactivity using serum from sensitized individuals. If IgE reactivity is observed, the GM crop is not developed. While digestive and heat stability impact exposure and thus the elicitation risk to sensitized individuals, these attributes are not interpretable relative to sensitization risk. For novel food proteins with no identified hazard, heat stability cannot be validly assessed because relevant IgE antibodies are not available. Likewise, the uncertain and sometime non-monotonic dose relationship between oral exposure to allergens and sensitization makes digestive stability a poor predictor of sensitization risk. It is hoped that by explicitly distinguishing between sensitization risk and elicitation risk, some of the debate surrounding the weight-of evidence criteria for predicting the allergenic risk of GM crops can be resolved.

Allergenic potential of novel proteins - What can we learn from animal production?

Currently, risk assessment of the allergenic potential of novel proteins relies heavily on evaluating protein digestibility under normal conditions based on the theory that allergens are more resistant to gastrointestinal digestion than non-allergens. There is also proposed guidance for expanded in vitro digestibility assay conditions to include vulnerable sub-populations. One of the underlying rationales for the expanded guidance is that current in vitro assays do not accurately replicate the range of physiological conditions. Animal scientists have long sought to predict protein and amino acid digestibility for precision nutrition. Monogastric production animals, especially swine, have gastrointestinal systems similar to humans, and evaluating potential allergen digestibility in this context may be beneficial. Currently, there is no compelling evidence that the mechanisms sometimes postulated to be associated with allergenic sensitization, e.g. antacid modification of stomach pH, are valid among production animals. Furthermore, examples are provided where non-biologically representative assays are better at predicting protein and amino acid digestibility compared with those designed to mimic in vivo conditions. Greater emphasis should be made to align in vitro assessments with in vivo data.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Genetic basis and detection of unintended effects in genetically modified crop plants.

In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled "Genetic Basis of Unintended Effects in Modified Plants" was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging "omics" technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that "unintended" does not necessarily mean "harmful". This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics.

Retrospective evaluation of the impact of functional immunotoxicity testing on pesticide hazard identification and risk assessment.

Conduct of a T-cell-dependent antibody response (TDAR) assay in rodents according to Environmental Protection Agency (EPA) Test Guideline OPPTS 870.7800 is now required for chemical pesticide active ingredients registered in the United States. To assess potential regulatory impact, a retrospective analysis was developed using TDAR tests conducted on 78 pesticide chemicals from 46 separate chemical classes. The objective of the retrospective analysis was to examine the frequency of positive responses and determine the potential for the TDAR to yield lower endpoints than those utilized to calculate reference doses (RfDs). A reduction in the TDAR response was observed at only the high-dose level in five studies, while it was unaltered in the remaining studies. Importantly, for all 78 pesticide chemicals, the TDAR no-observed-adverse-effect levels (TDAR NOAELs) were greater than the NOAELS currently in use as risk assessment endpoints. The TDAR NOAELs were higher than the current EPA-selected endpoints for the chronic RfD, short-term, intermediate and long-term exposure scenarios by 3-27,000, 3-1,688, 3-1,688 and 4.9-1,688 times, respectively. Based on this analysis, conduct of the TDAR assay had minimal impact on hazard identification and did not impact human health risk assessments for the pesticides included in this evaluation. These data strongly support employment of alternative approaches including initial weight-of-evidence analysis for immunotoxic potential prior to conducting functional immunotoxicity testing for pesticide active ingredients.

Measurement of endogenous allergens in genetically modified soybeans--short communication.

The measurement of endogenous allergens is required by the European Commission (EC) as part of the compositional analysis for GM products from host plants that are common causes of food allergy, such as soybean (EC Implementing Regulation No. 503/2013). In each case, the EC Implementing Regulation indicates that analysis be conducted on identified allergens as specified in the Organization of Economic Cooperation and Development (OECD) consensus documents on compositional considerations for new plant varieties. This communication discusses the methods available to measure endogenous allergens as well as the endogenous soybean allergens that should be analyzed. It is suggested herein that in conjunction with the 2012 OECD consensus document on soybean, any list of soybean allergens should be based on clinically relevant data among publicly available allergen databases and peer-reviewed scientific publications, and the ability to measure the identified allergen. Based on a detailed analysis of the scientific literature, the following key points are recommended: (1) the acceptance of serum-free, quantitative analytical method data as an alternative to traditional IgE reactivity qualitative or semi-quantitative data for evaluation of endogenous soybean allergen content; (2) eight of the 15 potential allergens listed in the OECD soybean consensus document (Gly m 3, Gly m 4, Gly m Bd28K, Gly m Bd30K, Gly m 5, Gly m 6, Gly m 8, and Kunitz trypsin inhibitor) have both appropriate supporting clinical data and sufficient sequence information to be evaluated in comparative endogenous soybean allergen studies; and (3) the remaining seven proteins (Gly m 1, Gly m 2, unknown 50kDa protein, unknown 39kDa protein, P-22-25, lipoxygenase and lectin) lack sufficient data for clear classification as confirmed allergens and/or available sequence information and should not be currently included in the measurement of endogenous soybean allergens in the compositional analysis for the EU.

Determination of OTNE-induced thyroid effects within an adverse outcome pathway (AOP) network.

Octahydro-tetramethyl-naphthalenyl-ethanone (OTNE) is a synthetic fragrance ingredient. OTNE was evaluated in repeated-dose toxicological studies. Target organs via oral and dermal routes were the liver and skin/liver, respectively. Effects were observed on the thyroid and thyroid hormones, suggesting hypothalamic-pituitary-thyroid axis perturbation. We investigated the molecular initiating event(s) (MIEs), key events (KEs), and adverse outcomes of OTNE-induced thyroid perturbation within an adverse outcome pathway (AOP). Data were generated using new approach methodologies (NAMs) on human, mouse, and/or rat receptors exploring MIEs using in vitro receptor ligand-binding assays for androstane receptor variant 3 (CAR), farnesoid X receptor (FXR), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptors alpha, delta, and gamma (PPARα, δ, and γ), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AhR). These data inform an AOP network where CAR, FXR, and PXR activation serve as MIEs with thyroid perturbation occurring as secondary effects. These data represent a robust evaluation using NAMs for mapping OTNE-induced thyroid effects and identifying activation of receptor-ligand binding as MIEs in lieu of additional in vivo experimentation. These data indicate the observed thyroid effects are secondary to liver effects and the thyroid effects, therefore, should not be the basis for assessing potential OTNE-induced human health hazards.

Cashmeran as a potential endocrine disrupting chemical: What does the weight-of-the-evidence indicate?

Cashmeran is a fragrance ingredient. Risk assessments are available but have not focused on its endocrine disruptor potential. The objective was to evaluate Cashmeran as a potential endocrine-disrupting chemical (EDC). The assessment was based on data from US EPA's CompTox Chemicals Dashboard, the Danish (Q)SAR Database, in vitro assays, and in vivo studies. ToxCast assays related to estrogen, androgen, thyroid, and steroidogenesis modalities were Inactive at non-cytotoxic concentrations. In vitro assays demonstrated no estrogenic activity in a human cervical epithelioid carcinoma HeLa cell line and indicated only weak agonist estrogenic activity in Chinese Hamster Ovary (CHO)-K1 cells. In the same test, no agonist or antagonist activity was detected for human androgen receptor (hAR) and thyroid hormone receptor ß (hTHRß) binding. The Danish QSAR database didn't indicate any ED potential. There were no adverse endocrine related effects in either a 90-day repeated gavage dosing study or a reproductive and developmental screening study. Regarding ED potential for environment, the data from two limited environmental ED related studies on Cashmeran did not raise any concern. Data from in vitro and in vivo studies were considered for environmental ED concern. Based on the weight-of-the-evidence, Cashmeran is not expected to cause endocrine effects.

Comparative assessment of multiple criteria for the in silico prediction of cross-reactivity of proteins to known allergens.

Genetically modified crops are becoming important components of a sustainable food supply and must be brought to market efficiently while also safeguarding the public from cross-reactivity of novel proteins to known allergens. Bioinformatic assessments can help to identify proteins warranting further experimental checks for cross-reactivity. This study is a large-scale in silico evaluation of assessment criteria, including searches for: alignments between a query and an allergen having ≥ 35% identity over a length ≥ 80; any sequence (of some minimum length) found in both a query and an allergen; any alignment between a query and an allergen with an E-value below some threshold. The criteria and an allergen database (AllergenOnline) are used to assess 27,243 Viridiplantae proteins for potential allergenicity. (A protein is classed as a "real allergen" if it exceeds a test-specific level of identity to an AllergenOnline entry; assessment of real allergens in the query set is against a reduced database from which the identifying allergen has been removed.) Each criterion's ability to minimize false positives without increasing false negative levels of current methods is determined. At best, the data show a reduction in false positives to ∼6% (from ∼10% under current methods) without any increase in false negatives.

Workshop proceedings: challenges and opportunities in evaluating protein allergenicity across biotechnology industries.

A workshop entitled "Challenges and Opportunities in Evaluating Protein Allergenicity across Biotechnology Industries" was held at the 51st Annual Meeting of the Society of Toxicology (SOT) in San Francisco, California. The workshop was sponsored by the Biotechnology Specialty Section of SOT and was designed to present the science-based approaches used in biotechnology industries to evaluate and regulate protein allergenicity. A panel of experts from industry and government highlighted the allergenicity testing requirements and research in the agricultural, pharmaceutical/biopharma, and vaccine biotechnology industries and addressed challenges and opportunities for advancing the science of protein allergenicity. The main learning from the workshop was that immunoglobulin E-mediated allergenicity of biotechnology-derived products is difficult to assess without human data. The approaches currently being used to evaluate potential for allergenicity across biotechnology industries are very different and range from bioinformatics, in vitro serology, in vivo animal testing, in vitro and in vivo functional assays, and "biosimilar" assessments (ie, biotherapeutic equivalents to innovator products). The challenge remains with regard to the different or lack of regulatory requirements for allergenicity testing across industries, but the novel approaches being used with bioinformatics and biosimilars may lead to opportunities in the future to collaborate across biotechnology industries.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 µg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

The COMPARE Database: A Public Resource for Allergen Identification, Adapted for Continuous Improvement.

Motivation: The availability of databases identifying allergenic proteins via a transparent and consensus-based scientific approach is of prime importance to support the safety review of genetically-modified foods and feeds, and public safety in general. Over recent years, screening for potential new allergens sequences has become more complex due to the exponential increase of genomic sequence information. To address these challenges, an international collaborative scientific group coordinated by the Health and Environmental Sciences Institute (HESI), was tasked to develop a contemporary, adaptable, high-throughput process to build the COMprehensive Protein Allergen REsource (COMPARE) database, a publicly accessible allergen sequence data resource along with bioinformatics analytical tools following guidelines of FAO/WHO and CODEX Alimentarius Commission. Results: The COMPARE process is novel in that it involves the identification of candidate sequences via automated keyword-based sorting algorithm and manual curation of the annotated sequence entries retrieved from public protein sequence databases on a yearly basis; its process is meant for continuous improvement, with updates being transparently documented with each version; as a complementary approach, a yearly key-word based search of literature databases is added to identify new allergen sequences that were not (yet) submitted to protein databases; in addition, comments from the independent peer-review panel are posted on the website to increase transparency of decision making; finally, sequence comparison capabilities associated with the COMPARE database was developed to evaluate the potential allergenicity of proteins, based on internationally recognized guidelines, FAO/WHO and CODEX Alimentarius Commission.

Safety evaluation of a novel variant of consensus bacterial phytase.

A 90-day subchronic oral toxicity study was conducted to evaluate the safety of a consensus bacterial phytase variant 6-phytase (PhyG) for use as an animal feed additive. This phytase is produced by fermentation with a fungal (Trichoderma reesei) production strain expressing a biosynthetic variant of a consensus bacterial phytase gene assembled via ancestral reconstruction with sequence bias for the phytase from Buttiauxella sp. Rats were administered PhyG daily via oral gavage at dose-levels of 0 (distilled water), 250, 500 or 1000 mg total organic solids (TOS)/kg bodyweight (bw)/day (equivalent to 0, 112,500, 225,000 and 450,000 phytase units (FTU)/kg bw/day, respectively). No test article-related adverse effects were observed. A no-observed-adverse-effect level (NOAEL) for PhyG was established as 1000 mg TOS/kg bw/day, the highest test concentration. Based on this NOAEL and an estimate of broiler consumption determined from the proposed inclusion of the phytase in feed at the maximum recommended level (4000 FTU/kg), a margin of safety value of 1613 was calculated. Results of in vitro genotoxicity testing and in silico protein toxin evaluation further confirmed PhyG to be non-genotoxic and not likely to be a protein toxin upon consumption. These data support the safety of PhyG as an animal feed additive.

Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools.

A safety assessment process exists for genetically engineered crops that includes the evaluation of the expressed protein for allergenic potential. The objectives of this evaluation are twofold: (1) to protect allergic consumers from exposure to known allergenic or cross-reactive proteins, and (2) protect the general population from risks associated with the introduction of genes encoding proteins that are likely to become food allergens. The first systematic approach to address these concerns was formulated by Metcalfe et al. [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 from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36(5), 165-186.] and subsequently Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) [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. January 22-25, 2001. Rome, Italy]. More recently, Codex [Codex Alimentarius Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity. pp. 47-60], noting that no single factor is recognized as an identifier for protein allergenicity, suggested a weight of evidence approach be conducted that takes into account a variety of factors and approaches for an overall assessment of allergenic potential. These various recommendations are based on what is known about allergens, including the history of exposure and safety of the gene(s) source; amino acid sequence identity to human allergens; stability to pepsin digestion in vitro; protein abundance in the crop and processing effects; and when appropriate, specific IgE binding studies or skin-prick testing. Similarities and differences between these various suggested recommendations, as well as data gaps, are discussed. The US Environmental Protection Agency (EPA)'s Office of Research and Development (ORD) has initiated a targeted research effort to address data gaps and improve the various recommended methods/endpoints for assessing the allergenic risks associated with plant incorporated pesticides (PIPs) through both intramural and extramural (grant supported) research. The areas of primary focus for EPA include: (1) development and evaluation of animal models; (2) targeted or specific serological assays; and (3) structure-activity relationships. Details on the current as well as proposed EPA funded research are discussed. More recently US EPA has partnered with the National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health to support research in areas of mutual interest with respect to food allergy.

Assessment of the potential allergenicity of genetically-engineered food crops.

An extensive safety assessment process exists for genetically-engineered (GE) crops. The assessment includes an evaluation of the introduced protein as well as the crop containing the protein with the goal of demonstrating the GE crop is "as-safe-as" non-GE crops in the food supply. One of the evaluations for GE crops is to assess the expressed protein for allergenic potential. Currently, no single factor is recognized as a predictor for protein allergenicity. Therefore, a weight-of-the-evidence approach, which accounts for a variety of factors and approaches for an overall assessment of allergenic potential, is conducted. This assessment includes an evaluation of the history of exposure and safety of the gene(s) source; protein structure (e.g. amino acid sequence identity to human allergens); stability of the protein to pepsin digestion in vitro; heat stability of the protein; glycosylation status; and when appropriate, specific IgE binding studies with sera from relevant clinically allergic subjects. Since GE crops were first commercialized over 20 years ago, there is no proof that the introduced novel protein(s) in any commercialized GE food crop has caused food allergy.