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.

Composition of forage and grain from genetically modified DP202216 maize is equivalent to non-modified conventional maize (Zea mays L.).

DP202216 maize was genetically modified to increase and extend the expression of the zmm28 gene relative to native zmm28 gene expression, resulting in plants with enhanced grain yield potential. Standard nutritional and compositional parameters for maize grain and forage (e.g., proximates, fiber, minerals, amino acids, fatty acids, vitamins, anti-nutrients, secondary metabolites) from DP202216 maize were compared to grain and forage from non-modified near-isoline maize (control). Three amino acids (glycine, methionine, and serine) and two vitamins (vitamin B1 and vitamin B3) were statistically different between DP202216 and control maize grain but were not statistically different when adjusted using the false discovery rate method. These analyte values also fell within the ranges of natural variation of non-modified commercial maize varieties supporting that statistical differences were not biologically relevant. The composition of grain and forage from DP202216 maize is comparable to grain and forage from non-modified maize with a history of safe use.

Expression of endogenous proteins in maize hybrids in a multi-location field trial in India.

Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2-26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12-64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.

Safety Assessment of Food and Feed from GM Crops in Europe: Evaluating EFSA's Alternative Framework for the Rat 90-day Feeding Study.

Regulatory-compliant rodent subchronic feeding studies are compulsory regardless of a hypothesis to test, according to recent EU legislation for the safety assessment of whole food/feed produced from genetically modified (GM) crops containing a single genetic transformation event (European Union Commission Implementing Regulation No. 503/2013). The Implementing Regulation refers to guidelines set forth by the European Food Safety Authority (EFSA) for the design, conduct, and analysis of rodent subchronic feeding studies. The set of EFSA recommendations was rigorously applied to a 90-day feeding study in Sprague-Dawley rats. After study completion, the appropriateness and applicability of these recommendations were assessed using a battery of statistical analysis approaches including both retrospective and prospective statistical power analyses as well as variance-covariance decomposition. In the interest of animal welfare considerations, alternative experimental designs were investigated and evaluated in the context of informing the health risk assessment of food/feed from GM crops.

Survival of Corn Earworm (Lepidoptera: Noctuidae) on Bt Maize and Cross-Pollinated Refuge Ears From Seed Blends.

Refuge is mandated in the United States where genetically modified maize (Zea mays L.) expressing insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) are cultivated. Currently, refuge is deployed in different ways including blocks, field strips, or seed blends containing Bt and non-Bt maize. Seed blends provide practical advantages for refuge implementation. However, concerns related to the movement of insect larvae, potential differential survival of heterozygous resistant larvae, reduction in insect production, and cross-pollination of ears resulting in sublethal selection, have delayed seed blend use for Lepidoptera in the southern United States, where maize plantings are used as refuge for Helicoverpa zea (Boddie). In this study, we evaluated the relative survival of H. zea in Bt events and in seed blends compared with pure stand refuge and the relative survival of H. zea on the individual components of the pyramid 1507xMON810xMIR162. The results showed variation on the production of H. zea in refuge plants from seed blends compared with pure stand refuge plants. The relative survival of H. zea on the events 1507, MON810, MIR162, and 1507xMON810xMIR162 ranked similarly across the three locations tested. These results can be used in computer simulation modeling efforts to evaluate the feasibility of seed blends as a refuge deployment strategy with the pyramid 1507xMON810xMIR162. Because the reduction on survival of H. zea due to blending was variable, a sensitivity analysis that includes all possible scenarios of reduction in survival should be considered.

Model-based tolerance intervals derived from cumulative historical composition data: application for substantial equivalence assessment of a genetically modified crop.

Compositional analysis is a requisite component of the substantial equivalence framework utilized to assess genetically modified (GM) crop safety. Statistical differences in composition data between GM and non-GM crops require a context in which to determine biological relevance. This context is provided by surveying the natural variation of key nutrient and antinutrient levels within the crop population with a history of safe use. Data accumulated from various genotypes with a history of safe use cultivated in relevant commercial crop-growing environments over multiple seasons are discussed as the appropriate data representative of this natural variation. A model-based parametric tolerance interval approach, which accounts for the correlated and unbalanced data structure of cumulative historical data collected from multisite field studies conducted over multiple seasons, is presented. This paper promotes the application of this tolerance interval approach to generate reference ranges for evaluation of the biological relevance of statistical differences identified during substantial equivalence assessment of a GM crop.

Environmental effects on allergen levels in commercially grown non-genetically modified soybeans: assessing variation across north america.

Soybean (Glycinemax) is a hugely valuable soft commodity that generates tens of billions of dollars annually. This value is due in part to the balanced composition of the seed which is roughly 1:2:2 oil, starch, and protein by weight. In turn, the seeds have many uses with various derivatives appearing broadly in processed food products. As is true with many edible seeds, soybeans contain proteins that are anti-nutritional factors and allergens. Soybean, along with milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, and wheat, elicit a majority of food allergy reactions in the United States. Soybean seed composition can be affected by breeding, and environmental conditions (e.g., temperature, moisture, insect/pathogen load, and/or soil nutrient levels). The objective of this study was to evaluate the influence of genotype and environment on allergen and anti-nutritional proteins in soybean. To address genetic and environmental effects, four varieties of non-GM soybeans were grown in six geographically distinct regions of North America (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania). Absolute quantification of proteins by mass spectrometry can be achieved with a technique called multiple reaction monitoring (MRM), during which signals from an endogenous protein are compared to those from a synthetic heavy-labeled internal standard. Using MRM, eight allergens were absolutely quantified for each variety in each environment. Statistical analyses show that for most allergens, the effects of environment far outweigh the differences between varieties brought about by breeding.

Comments on the paper "A statistical assessment of differences and equivalences between genetically modified and reference plant varieties" by van der Voet et al. 2011.

van der Voet et al. (2011) describe statistical methodology that the European Food Safety Authority expects an applicant to adopt when making a GM crop regulatory submission. Key to their proposed methodology is the inclusion of reference varieties in the experimental design to provide a measure of natural variation amongst commercially grown crops. While taking proper account of natural variation amongst commercial varieties in the safety assessment of GM plants makes good sense, the methodology described by the authors is shown here to be fundamentally flawed and consequently cannot be considered fit for purpose in its current form.

Two-generation reproductive and developmental toxicity assessment of dietary N-acetyl-L-aspartic acid in rats.

N-acetyl-l-aspartic acid (NAA) is a component of the mammalian central nervous system (CNS) that has also been identified in a number of foods. This paper reports the outcome of a reproductive toxicology study conducted with NAA in Sprague-Dawley rats. NAA was added to diets at target doses of 100, 250 and 500 mg/kg of body weight/day and administered for two consecutive generations. A carrier control group was administered diet with no added NAA and a comparative control group was given aspartate (ASP), the constituent amino acid of NAA, at a target dose of 500 mg/kg of body weight/day. The study evaluated OECD 416 reproductive performance variables and additional segments to assess potential developmental effects, neurobehavioural and ophthalmologic function, and the concentrations of NAA or ASP in brain and plasma. No biologically significant differences were observed in any reproductive response variables, neurobehavioural tests, ophthalmologic examinations, body weights, feed consumption, or organ weights. Further, no test substance related mortalities or adverse clinical, neurohistopathologic or histopathologic findings were observed. Under the conditions of this study, the highest target dose of NAA, 500 mg/kg of body weight/day, represents the no-observed-adverse-effect-level (NOAEL) for reproductive and systemic toxicity, and neurotoxicity for Sprague-Dawley rats.

Nickel-cysteine binding supported by phosphine chelates.

The effect of chelating phosphines was tested on the structure and pH-dependent stability of nickel-cysteine binding. (1,2-Bis(diphenylphosphino)ethane (dppe) and 1,1,1-tris[(diphenylphosphino)methyl]ethane (triphos) were used with three different cysteine derivatives (L-cysteine, Cys; L-cysteine ethyl ester, CysEt; cystamine, CysAm) to prepare complexes of the form (dppe)NiCysR(n+) and (triphos)NiCysR(n+) (n = 0 for Cys; n = 1 for CysEt and CysAm). Similar 31P {1H} NMR spectra for all (dppe)NiCysRn+ confirmed their square-planar P2NiSN coordination spheres. The structure of [(dppe)NiCysAm]PF6 was also confirmed by single-crystal X-ray diffraction methods. The (triphos)NiCysAm+ and (triphos)NiCysEt+ complexes were fluxional at room temperature by 31P NMR. Upon cooling to -80 degrees C, all gave spectra consistent with a P2NiSN coordination sphere with the third phosphorus uncoordinated. Temperature-dependent 31P NMR spectra showed that a trans P-Ni-S pi interaction controlled the scrambling of the coordinated triphos. In aqueous media, (dppe)NiCys was protonated at pH approximately 4-5, leading to possible formation of a nickel-cysteinethiol and eventual cysteine loss at pH < 3. The importance of N-terminus cysteine in such complexes was demonstrated by preparing (dppe)NiCys-bead and trigonal-bipyramidal Tp*NiCys-bead complexes, where Cys-bead represents cysteine anchored to polystyrene synthesis beads and Tp*- = hydrotris(3,5-dimethylpyrazolyl)borate. Importantly, results with these heterogeneous systems demonstrated the selectivity of these nickel centers for cysteine over methionine and serine and most specifically for N-terminus cysteine. The role of Ni-S pi bonding in nickel-cysteine geometries will be discussed, including how these results suggest a mechanism for the movement of electron density from nickel onto the backbone of coordinated cysteine.