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.

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.

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.

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.

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.

Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.

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.

The Sheep Erythrocyte T-Dependent Antibody Response (TDAR).

The sheep erythrocyte T-dependent antibody response (TDAR) evaluates the ability of animals sensitized in vivo to produce primary IgM antibodies to sheep erythrocytes (sRBC). The assay enumerates the number of antigen-specific IgM antibody-producing cells in the spleen. When exposure to the test material takes place in vivo, as does sensitization, the actual quantification of the number of antibody-producing cells occurs ex vivo. Following the animal being euthanized, a single-cell suspension of spleen cells is prepared. These spleen cells containing the IgM-secreting plasma cells are incubated in a semisolid matrix of agar, sheep erythrocytes, and guinea pig serum as a single-cell layer between a Petri dish and glass cover slip. After a 3-h incubation period, lysis of sRBCs around each of the IgM-secreting antigen-specific plasma cells results in the formation of a clear plaque, which can easily be counted. The TDAR has been found to be the most sensitive functional assay for evaluating effects on the immune system, particularly the humoral immune component in young adult rodents. Data suggest, however, that it may not be possible to measure the TDAR in preweaning rodent pups due to the immature status of their immune cells. Nevertheless, the TDAR to sheep erythrocytes still remains the gold standard for evaluating the potential adverse effects of xenobiotics on the immune system.

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.

Food and Feed Safety of Genetically Engineered Food Crops.

The first genetically engineered (GE) food crop (tomato) was introduced in 1995, followed by the successful development and commercial release of maize, soybeans, cotton, canola, potatoes, papaya, alfalfa, squash, and sugar beets with specific new genetic traits. Even though the safety of every new GE crop has been evaluated by various regulatory authorities throughout the world prior to its commercial release, the ongoing public debate about the safety of food and feed derived from GE plants has not abated. Such debates often overshadow an important fact that all crops used as human food or animal feed include varieties that have been developed through conventional breeding and selection over hundreds or thousands of years, or through intentional but random mutagenesis. Developing food crops through such breeding practices result in large-scale genomic changes in the resulting crops, and these genomic changes do not undergo molecular characterization. In contrast, new GE crops are developed using well-characterized DNA fragments and the resulting crops are tested and evaluated with much greater scrutiny. This document reviews the safety data and information of GE crops and foods obtained from them.

Functional classification of protein toxins as a basis for bioinformatic screening.

Proteins are fundamental to life and exhibit a wide diversity of activities, some of which are toxic. Therefore, assessing whether a specific protein is safe for consumption in foods and feeds is critical. Simple BLAST searches may reveal homology to a known toxin, when in fact the protein may pose no real danger. Another challenge to answer this question is the lack of curated databases with a representative set of experimentally validated toxins. Here we have systematically analyzed over 10,000 manually curated toxin sequences using sequence clustering, network analysis, and protein domain classification. We also developed a functional sequence signature method to distinguish toxic from non-toxic proteins. The current database, combined with motif analysis, can be used by researchers and regulators in a hazard screening capacity to assess the potential of a protein to be toxic at early stages of development. Identifying key signatures of toxicity can also aid in redesigning proteins, so as to maintain their desirable functions while reducing the risk of potential health hazards.

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.

Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins.

BACKGROUND: The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). MAIN BODY: The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. CONCLUSION: The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

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.

An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins.

Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([(3)H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk ß-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles.

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.

Transgenic maize event TC1507: Global status of food, feed, and environmental safety.

Maize (Zea mays) is a widely cultivated cereal that has been safely consumed by humans and animals for centuries. Transgenic or genetically engineered insect-resistant and herbicide-tolerant maize, are commercially grown on a broad scale. Event TC1507 (OECD unique identifier: DAS-Ø15Ø7-1) or the Herculex®(#) I trait, an insect-resistant and herbicide-tolerant maize expressing Cry1F and PAT proteins, has been registered for commercial cultivation in the US since 2001. A science-based safety assessment was conducted on TC1507 prior to commercialization. The safety assessment addressed allergenicity; acute oral toxicity; subchronic toxicity; substantial equivalence with conventional comparators, as well as environmental impact. Results from biochemical, physicochemical, and in silico investigations supported the conclusion that Cry1F and PAT proteins are unlikely to be either allergenic or toxic to humans. Also, findings from toxicological and animal feeding studies supported that maize with TC1507 is as safe and nutritious as conventional maize. Maize with TC1507 is not expected to behave differently than conventional maize in terms of its potential for invasiveness, gene flow to wild and weedy relatives, or impact on non-target organisms. These safety conclusions regarding TC1507 were acknowledged by over 20 regulatory agencies including United States Environment Protection Agency (US EPA), US Department of Agriculture (USDA), Canadian Food Inspection Agency (CFIA), and European Food Safety Authority (EFSA) before authorizing cultivation and/or food and feed uses. A comprehensive review of the safety studies on TC1507, as well as some benefits, are presented here to serve as a reference for regulatory agencies and decision makers in other countries where authorization of TC1507 is or will be pursued.

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.