Repeated dose toxicity study to assess the safety and reproductive/developmental safety of 7-methyl-2H-1,5-Benzodioxepin-3(4H)-One (Calone®) and the read across approach to its biodegradation metabolite 7-methyl-2H-1,5-benzodioxepin-3-ol (Calol).

7-Methyl-2H-1,5-benzodioxepin-3(4H)-one (Calone®) is used in fragrances to impart a marine note. It is produced industrially at volumes requiring repeated dose and developmental/reproductive toxicology data (OECD TG 422) under European chemicals legislation (i.e., REACH). Additionally, Japanese chemicals legislation requires evaluation of Calone® biodegradability and identification of metabolites in an environmental biodegradation test. 7-Methyl-2H-1,5-benzodioxepin-3-ol (Calol) was the sole metabolite identified following biodegradation and a 28-day repeated dose toxicity study (OECD TG 407) would normally be required to support registration in Japan. The current paper presents results showing no adverse effects in the parental, reproductive, or developmental phases of an OECD TG 422 study following dietary administration of Calone® to rats at targeted doses of up to 1000 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was the highest administered dose of 791 and 922 mg/kg/day for males and females, respectively. An in vitro metabolism study conducted with rat and human liver microsomes demonstrated that greater than 90% of Calone® was metabolically reduced into Calol, the same metabolite observed in the environmental biodegradation test. Accordingly, the results from the OECD TG 422 study with Calone® are directly applicable to Calol and it would be expected to have the same NOAEL.

Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

Evaluation of the History of Safe Use of the Maize ZMM28 Protein.

The ZMM28 protein encoded by the zmm28 gene is endogenous to maize. DP202216 maize was genetically modified to increase and extend expression of the zmm28 gene relative to native zmm28 gene expression, resulting in plants with enhanced grain yield potential. Evaluation of the history of safe use (HOSU) is one component of the safety assessment framework for a newly expressed protein in a GM crop. The deduced amino acid sequence of the introduced ZMM28 protein in DP202216 maize is identical to the ZMM28 protein in nonmodified conventional maize. The ZMM28 protein has also been found in selected varieties of sweet corn kernels, and closely related proteins are found in other commonly consumed food crops. Concentrations of the ZMM28 protein in event DP202216 maize, conventional maize, and sweet corn are reported. This information supports, in part, the evaluation of HOSU, which can be leveraged in the safety assessment of the ZMM28 protein. Additional studies will be considered in the food and feed safety assessment of the DP202216 maize event.

Safety assessment of coleopteran active IPD072Aa protein from Psuedomonas chlororaphis.

The ipd072Aa gene from Pseudomonas chlororaphis encodes the IPD072Aa protein which confers protection against certain coleopteran pests when expressed in genetically modified (GM) plants. A weight of evidence approach was used to assess the safety of the IPD072Aa protein. This approach considered the history of safe use of the source organism and bioinformatic comparison of the protein sequence with known allergenic and toxic proteins. The IPD072Aa protein was assessed for resistance to degradation in the presence of simulated gastric fluid containing pepsin as well as heat stability. There was no hazard identified with the IPD072Aa protein. Furthermore, an acute oral toxicity study found no evidence of adverse effects. Collectively, these studies support the human health safety assessment of the IPD072Aa protein.

Risk-Only Assessment of Genetically Engineered Crops Is Risky.

The risks of not considering benefits in risk assessment are often overlooked. Risks are also often evaluated without consideration of the broader context. We discuss these two concepts in relation to genetically engineered (GE) crops. The health, environmental, and economic risks and benefits of GE crops are exemplified and presented in the context of modern agriculture. Misattribution of unique risks to GE crops are discussed. It is concluded that the scale of modern agriculture is its distinguishing characteristic and that the greater knowledge around GE crops allows for a more thorough characterization of risk. By considering the benefits and risks in the context of modern agriculture, society will be better served and benefits will be less likely to be forgone.

Q-X1-P-X2 motif search for potential celiac disease risk has poor selectivity.

The European Food Safety Authority (EFSA) recently published guidelines for assessment of potential celiac disease risk for newly expressed proteins in genetically modified (GM) crops. This novel step-wise approach prescribes, in part, how to conduct sequence identity searches between a newly expressed protein and known celiac disease peptides including a Q/E-X1-P-X2 amino acid motif. To evaluate the specificity of the recommended sequence identity searches in the context of risk assessment, protein sequences from celiac disease causing crops, as well as from crops not associated with celiac disease, were compared with known HLA-DQ restricted epitopes and searched for the presence of motifs followed by peptide analysis. Searches for the presence of the Q/E-X1-P-X2-motif were found to generate a high proportion of false-positive hits irrelevant to celiac disease risk. Identification of a 9mer exact match between a newly expressed protein and the known celiac disease peptides (recommended by the guideline) along with a supplementary sequence comparisons (suggested by FARRP/AllergenOnline) is considered better suited to more specifically capture the potential risk of a newly expressed protein for celiac disease.

The food safety of DP-356Ø43 soybeans on SD rats reflected by physiological variables and fecal microbiota during a 90-day feeding study.

Soybean is an important food resource for the eastern countries and herbicide-tolerant genetically modified soybeans (GMS) were widely developed to deal with weeds problems. Unprocessed soybean flour instead of dehulled and defatted soybean meal was used to reflect the safety of soybean food in whole. Rats were given formulated diets containing DP-356Ø43 or non-GM soybean JACK at an incorporation rate of 7.5%, 15%, or 30% (w/w), respectively for 90 days. Targeted traditional toxicological response variables were measured to reflect the holistic health of animals. No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. The results demonstrate that the soybean DP-356Ø43 is as safe for consumption as conventional soybean JACK. In the current study, the effect of a herbicide-tolerant GMS DP-356043 on identified intestinal microbiota was evaluated in a rodent feeding study compared with its conventional control JACK. Feces samples from rats consuming different diets were collected before the start of the experiment (time 0) and at monthly intervals (at the end of the 1st, 2nd and 3rd months) over the course of 90 days. Six types of bacterias shared by humans and rats were detected with Q-PCR. The results of QPCR indicated that the GMS 356Ø43 had a comparable effect on the abundance of Bifidobacterium group, Clostridium perfringens subgroup, Escherichia coli, and Bacteroides-Prevotella group as the non-GMS JACK.

Isoline use in crop composition studies with genetically modified crops under EFSA guidance - Short communication.

The European Food Safety Authority (EFSA) oversees the safety evaluation of genetically modified (GM) crops in the European Union. EFSA requires inclusion of commercial non-GM reference lines and a non-GM isoline in crop composition studies with GM crops. Reference lines are used to construct equivalence limits for each compositional analyte. Results for the GM line are compared with these equivalence limits to assess compositional equivalence between the GM crop and the non-GM crop. If compositional equivalence cannot be concluded from this comparison, then results for the non-GM isoline can be used to determine if this finding is likely the result of the background non-GM genetics of the GM crop. If this latter comparison is not sufficient to assess the compositional safety of the GM crop, then a biological-relevance assessment for the analytes in question can be completed taking into account the greater body of knowledge of composition for the crop and diets. Thus, the isoline is a useful comparator but not required to assess the compositional safety of the GM crop, and therefore, unavoidable genotype differences between the isoline and GM line should not be grounds for rejection of compositional studies where the biological relevance of potential non-equivalence is addressed.

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.

In vitro studies with human intestinal epithelial cell line monolayers for protein hazard characterization.

Evaluating the safety of newly expressed proteins in genetically modified (GM) crops is conducted prior to commercialization to determine whether they could present a hazard upon consumption. A multicomponent, weight of evidence approach has been applied to individual proteins that has often included acute oral toxicology studies. Based on resources required to produce and purify the proteins, the number of animals necessary for these studies and the fact that no evidence of hazard has been observed for any of the proteins tested to date, it is questionable whether acute toxicology studies should be conducted for all proteins. This article reviews the chronology of the acute toxicology study from its origins into application for hazard assessment and classification of individual substances including proteins expressed in GM crops. It further proposes that a physiologic approach using cultured intestinal epithelial cell (IEC) line monolayers as an in vitro model of the gastrointestinal system provides results relevant to the hazard characterization of proteins when necessary. Benefits of this approach would include reduced quantities of proteins for testing and minimization or elimination of animal studies while maintaining confidence in the safety assessment process.

Incorporation of in vitro digestive enzymes in an intestinal epithelial cell line model for protein hazard identification.

Relatively few proteins in nature produce adverse effects following oral exposure. Of those that do, effects are often observed in the gut, particularly on intestinal epithelial cells (IEC). Previous studies reported that addition of protein toxins to IEC lines disrupted monolayer integrity but innocuous dietary proteins did not. Studies presented here investigated the effects of innocuous (bovine serum albumin, ß-lactoglobulin, RuBisCO, fibronectin) or hazardous (phytohaemagglutinin-E, concanavalin A, wheat germ agglutinin, melittin) proteins that either were untreated or exposed to digestive enzymes prior to addition to Caco-2 human IEC line monolayers. At high concentrations intact fibronectin caused an increase in monolayer permeability but other innocuous proteins did not whether exposed to digestive enzymes or not. In contrast, all untreated hazardous proteins and those that were resistant to digestion (ex. wheat germ agglutinin) disrupted monolayer integrity. However, proteins sensitive to degradation by digestive enzymes (ex. melittin) did not adversely affect monolayers when exposed to these enzymes prior to addition to IEC line monolayers. These results indicate that in vitro exposure of proteins to digestive enzymes can assist in differentiating between innocuous and hazardous proteins as another component to consider in the overall weight of evidence approach in protein hazard assessment.

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.

Safety assessment of foods from genetically modified crops in countries with developing economies.

Population growth particularly in countries with developing economies will result in a need to increase food production by 70% by the year 2050. Biotechnology has been utilized to produce genetically modified (GM) crops for insect and weed control with benefits including increased crop yield and will also be used in emerging countries. A multicomponent safety assessment paradigm has been applied to individual GM crops to determine whether they as safe as foods from non-GM crops. This paper reviews methods to assess the safety of foods from GM crops for safe consumption from the first generation of GM crops. The methods can readily be applied to new products developed within country and this paper will emphasize the concept of data portability; that safety data produced in one geographic location is suitable for safety assessment regardless of where it is utilized.

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.

Characteristics and safety assessment of intractable proteins in genetically modified crops.

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

Thirteen week rodent feeding study with processed fractions from herbicide tolerant (DP-Ø73496-4) canola.

The potential health effects of meal and oil processed from seed of genetically modified (GM) canola plants (OECD unique identifier: DP-Ø73496-4; hereafter referred to as 73496 canola) containing an insert that expresses the GAT4621 protein conferring tolerance to nonselective herbicidal ingredient glyphosate were evaluated in a subchronic rodent feeding study. Sprague-Dawley rats (12/sex/group) were administered diets containing dehulled, defatted toasted canola meal (DH meal) and refined/bleached/deodorized canola oil (RBD oil) processed from seed of plants that were untreated (73496), sprayed in-field with glyphosate (73496GLY), the non-transgenic near-isogenic (091; control), or one of four commercially available non-GM reference canola varieties (45H72, 45H73, 46A65, 44A89). All diets were formulated as a modification of the standard laboratory chow PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (PMI® 5002). DH canola meal and RBD canola oil replaced all commodity soybean fractions typically incorporated in PMI® 5002. No toxicologically significant differences were observed between the test and control groups in this study. The results reported herein support the conclusion that DH meal and RBD oil processed from seed of 73496 canola are as safe and nutritious as DH meal and RBD oil processed from seed of non-GM canola.