History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk.

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

Evidence-based regulations for bioinformatic prediction of allergen cross-reactivity are needed.

The bioinformatic criteria adopted by regulatory agencies to predict the potential cross reactivity between newly expressed proteins in genetically engineered crops and known allergens involves amino acid identity thresholds and was formulated nearly two decades ago based on the opinion of allergy experts. Over the subsequent years, empirical evidence has been developed indicating that better bioinformatic tools based on amino acid similarity are available to detect real allergen cross-reactive risk while substantially reducing false-positive detections. Although the formulation of safety regulations, in the absence of empirical evidence, may require reliance on expert opinion, such expert opinion should not trump empirical evidence once it becomes available. The failure of regulation to maintain consistency with the best available scientific evidence diminishes its value and creates arbitrary barriers to the use of beneficial technologies by society.

Evidence runs contrary to digestive stability predicting protein allergenicity.

A dogma has persisted for over two decades that food allergens are more stable to digestion compared with non-allergenic proteins. This belief has become enshrined in regulations designed to assess the allergenic risk of novel food proteins. While the empirical evidence accumulated over the last 20+ years has largely failed to confirm a correlation between digestive stability and the allergenic status of proteins, even those who accept this finding often assert that this shortfall is the result of faulty assay design rather than lack of causality. Here, we outline why digestive stability may not in fact correlate with allergenic potential.

Safety evaluation of E12, W8, X17, and Y9 potatoes: Nutritional evaluation and 90-day subchronic feeding study in rats.

The nutritional and health effects of four biotech potato events, E12, W8, X17, and Y9, were evaluated in a subchronic rodent feeding study. E12 contains pSIM1278 insert DNA derived from potato and designed to down regulate potato genes through RNAi. These changes result in reduced black spot and reduced acrylamide. W8, X17, and Y9 contain the DNA inserts from pSIM1278 and pSIM1678 to further reduce acrylamide and express a gene from wild potato that protects against late blight. Rats were fed diets containing 20% cooked, dried potatoes from these four events and three conventional potato varieties. Compositional analyses of the processed potatoes and the rodent diets demonstrated comparability between the four events and their respective conventional varieties. Rats consumed the diets for 90 days and were evaluated for body weight, dietary intake, clinical signs, ophthalmology, neurobehavioral parameters, clinical pathology, organ weights, gross pathology, and histopathology. No adverse effects were observed as a result of test diet consumption. These results support the conclusion that foods containing E12, W8, X17, or Y9 potatoes are as safe, wholesome and nutritious as foods from conventional potato varieties.

DP-2Ø2216-6 maize does not adversely affect rats in a 90-day feeding study.

Maize plants containing event DP-2Ø2216-6 (DP202216), which confers herbicide tolerance through expression of phosphinothricin acetyltransferase and enhanced grain yield potential via temporal modulation of the native ZMM28 protein, were developed for commercialization. To address current regulatory expectations, a mandatory 90-day rodent feeding study was conducted to support the safety assessment. Diets containing 50% by weight of ground maize grain from DP202216, non-transgenic control, and 3 non-transgenic reference varieties, were fully characterized, along with the grain, and diets were fed to Crl:CD®(SD) rats for at least 90 days. As anticipated, no biologically-relevant effects or toxicologically-significant differences were observed on survival, body weight/gain, food consumption/efficiency, clinical and neurobehavioral evaluations, ophthalmology, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), organ weights, or gross and microscopic pathology parameters in rats fed a diet containing up to 50% DP202216 maize grain when compared with rats fed diets containing control or reference maize grains. The results of this study support the conclusion that maize grain from plants containing event DP-2Ø2216-6 is as safe and nutritious as maize grain not containing the event and add to the significant existing database of rodent subchronic studies demonstrating the absence of hazards from consumption of edible fractions of genetically modified plants.

Evaluation of an in vitro experimental platform of human polarized intestinal epithelial monolayers for the hazard assessment of insecticidal proteins.

Previous work demonstrated the utility of using human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on Transwell® filters to differentiate between hazardous and non-hazardous proteins. The current study seeks to further resolve appropriate concentrations for evaluating proteins of unknown hazard potential using the IEC experimental platform and leverages these parameters for evaluating the potential toxicity of insecticidal proteins characteristic of those expressed in genetically modified (GM) agricultural biotechnology crops. To establish optimal test protein concentrations, effects of several known hazardous (C. perfringens epsilon toxin, Listeriolysin O, Phaseolus vulgaris erythroagglutinin, E. coli Shiga toxin 1, C. difficile Toxin B and wheat germ agglutinin) and non-hazardous (Ara-h2, ß-lactoglobulin, fibronectin and Rubisco) proteins on IEC barrier integrity and cell viability were evaluated at concentration ranges. Two insecticidal proteins (AfIP-1A and AfIP-1B) were evaluated for effects in the IEC assay, a seven-day insecticidal bioassay, and assessed in a high-dose 14-day acute oral toxicity study in mice. The results obtained from the human in vitro IEC assay were consistent with results obtained from an in vivo acute oral toxicity study, both demonstrating that the combination of AfIP-1A and AfIP-1B do not exhibit any identifiable harmful impacts on mammalian cells.

Slow alignment of GMO allergenicity regulations with science on protein digestibility.

The current science on food allergy supports the dual allergen exposure hypothesis where sensitization to allergenic proteins is favored by dermal and inhalation exposure, and tolerization against allergy is favored by exposure in the gut. This hypothesis is bolstered by the epidemiological evidence showing that regions where children are exposed early in life to allergenic foods have lower rates of allergy. This led medical experts to replace the previous recommendation to exclude commonly allergenic foods from the diets of young children with the current recommendation that such foods be introduced to children early in life. Past beliefs that lowering gut exposure would reduce the likelihood that a protein would be allergenic led regulators and risk assessors to consider digestively stable proteins to be of greater allergenic risk. This resulted in international guidance and government regulations for newly expressed proteins in genetically engineered crops that aligned with this belief. Despite empirical results showing that allergens are no more digestively stable than non-allergens, and that gut exposure favors tolerization over sensitization, regulations have not come into alignment with the current science prompting developers to continue to engineer proteins for increased digestibility. In some rare cases, this could potentially increase sensitization risk.

Safety assessment of the insecticidal protein IPD079Ea from the fern, Ophioglossum pendulum.

As agricultural biotechnology continues to develop solutions for addressing crop pests through newly expressed proteins from novel source organisms, with different modes or sites of action and/or different spectra of activity, the safety of these proteins will be assessed. The results of hazard-identification and characterization studies for the insecticidal protein IPD079Ea, which is derived from a fern (Ophioglossum pendulum) and active against the maize pest western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae) are provided. Collectively these results indicate that IPD079Ea is unlikely to present a hazard to human or animal health and support the safety of genetically modified maize expressing IPD079Ea.

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

Erroneous Belief that Digestive Stability Predicts Allergenicity May Lead to Greater Risk for Novel Food Proteins.

There continues to be an erroneous belief that allergens (especially food allergens) are more resistant to gastrointestinal digestion than non-allergens. Government regulations based on this erroneous belief may result in technology developers altering the amino acid sequences of digestively stable native proteins to create digestively unstable modified versions for expression in genetically engineered crops. However, an investigation where a known stable allergen was modified to make it more digestible eliminated the protein's ability to tolerize against allergy in a mouse model, which is consistent with the dual allergen exposure hypothesis. Thus, the false belief that digestive stability increases the allergenic risk of novel food proteins (e.g., such as expressed in genetically engineered crops) could, in some cases, lead to introduction of digestively unstable modified protein versions with greater sensitization risk. However, it is noteworthy that developers have historically been very effective at preventing allergens from being introduced into crops based on the other components of the weight-of-evidence assessment of allergenic risk such that no newly expressed protein in any commercialized genetically engineered crop has ever been documented to cause allergy in anyone.

Evaluation of the safety and nutritional equivalency of maize grain with genetically modified event DP-Ø23211-2.

Feeding studies were conducted with rats and broiler chickens to assess the safety and nutrition of maize grain containing event DP-Ø23211-2 (DP23211), a newly developed trait-pyramid product for corn rootworm management. Diets containing 50% ground maize grain from DP23211, non-transgenic control, or non-transgenic reference hybrids (P0928, P0993, and P1105) were fed to Crl:CD®(SD) rats for 90 days. Ross 708 broilers were fed phase diets containing up to 67% maize grain from each source for 42 days. Body weight, gain, and feed conversion were determined for comparisons between animals fed DP23211 and control diets in each study. Additional measures included clinical and neurobehavioral evaluations, ophthalmology, clinical pathology, organ weights, and gross and microscopic pathology for rats, and carcass parts and select organ yields for broilers. Reference groups were included to determine if any observed significant differences between DP23211 and control groups were likely due to natural variation. No diet-related effects on mortality or evaluation measures were observed between animal fed diets produced with DP23211 maize grain and animal fed diets produced with control maize grain. These studies show that maize grain containing event DP-Ø23211-2 is as safe and nutritious as non-transgenic maize grains when fed in nutritionally adequate diets. The results are consistent with previously published studies, providing further demonstration of the absence of hazards from edible-fraction consumption of genetically modified plants.

Nutrient composition and safety evaluation of simulated isobutanol distillers dried grains with solubles and associated fermentation metabolites when fed to male Ross 708 broiler chickens (Gallus domesticus).

Saccharomyces cerevisiae genetically engineered to enhance butanol production will be used in a manufacturing process similar to that of fuel ethanol production, including co-production of distillers products for animal feed. A poultry feeding trial was conducted with simulated isobutanol-derived dried distillers grains with solubles (bDDGS), comprising non-fermentable corn solids and heat-inactivated Butamax modified yeast (BMY), to determine potential health effects. Simulated dried distillers grains were produced in 2 variants: bDDGS containing 10% (B10) or 50% (B50) BMY. The BMY concentrations were selected based on a conservative estimate from ethanol-derived distillers grains (eDDGS) approximating 2.5 and 12-fold margins of exposure. The B10 and B50 DDGS were evaluated in a 42-day feeding trial using male Ross 708 broiler chickens fed diets containing eDDGS, B50 DDGS, or B10 DDGS without or with isobutanol, 2,3-butanediol, and isobutyric acid metabolites each at target concentrations of 2 (B10-2), 5 (B10-5), or 10 (B10-10) times the anticipated specification limit in the commercial product. Diets were fed (n = 50 broilers/treatment) in 3 phases: starter phase with 8% DDGS and grower and finisher phases each with 15% DDGS. No statistically significant differences or diet-related effects on mortality, clinical pathology, or organ weights, and no microscopic observations associated with consumption of diets containing B10, B50, or B10 supplemented with metabolites at any targeted exposure level were observed. A lower (P < 0.05) mean absolute bursa of Fabricius weight in the B10-10 group compared to the B10 group was considered to be within the range of biological variability. A non-significant trend toward lower weight, gains, and feed intake, and higher feed:gain ratio was observed in the B10-10 group, and was considered a non-adverse palatability effect of consuming high concentrations of metabolites. These results demonstrate that consumption of phase diets containing simulated DDGS from a novel isobutanol production process was well-tolerated.

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.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics.

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.

Safety evaluation of AB-LIFE(®) (Lactobacillus plantarum CECT 7527, 7528 and 7529): Antibiotic resistance and 90-day repeated-dose study in rats.

AB-LIFE(®) is a probiotic product consisting of equal parts of three strains of Lactobacillus plantarum (CECT 7527, 7528, and 7529) blended with inert excipients. Whole genome sequencing was performed on each of the three strains. Antibiotic resistance was evaluated by genomic mining for resistance genes, and assessment for transferability. No risk of transfer potential was identified for any antibiotic resistance genes in the three strains. AB-LIFE(®) was evaluated for potential subchronic oral toxicity in rats, with dosages of 300 and 1000 mg/kg BW/day (equivalent to 5.55 × 10(10) and 1.85 × 10(11) CFU/kg BW/day). Survival of the three test strains through the gastrointestinal tract was supported by fecal analysis. No adverse effects were identified with respect to in-life parameters, clinical or anatomic pathology, translocation, or fecal chemical analyses. The no-observed-adverse-effect level (NOAEL) for AB-LIFE(®) in male and female rats was 1000 mg/kg BW/day (1.85 × 10(11) CFU of AB-LIFE(®)/kg BW/day), the highest dose level evaluated. These results, in conjunction with a previous acute toxicity study in rats, support the conclusion that AB-LIFE(®) is safe for human consumption.

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.

Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development.

Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair. In adult mice, type II cell proliferation was low during room air and hyperoxia exposure but increased during recovery in room air and then declined to control levels by day 7. Eight weeks later, type II cell number and alveolar compliance were indistinguishable from those in room air controls. In newborn mice, type II cell proliferation markedly increased between birth and postnatal day 7 before declining by postnatal day 14. Exposure to hyperoxia between postnatal days 1 and 4 inhibited type II cell proliferation, which resumed during recovery and was aberrantly elevated on postnatal day 14. Eight weeks later, recovered mice had 70% fewer type II cells and 30% increased lung compliance compared with control animals. Recovered mice also had higher levels of T1alpha, a protein expressed by type I cells, with minimal changes detected in genes expressed by vascular cells. These data suggest that perinatal hyperoxia adversely affects alveolar development by disrupting the proper timing of type II cell proliferation and differentiation into type I cells.

Hyperoxic ventilated premature baboons have increased p53, oxidant DNA damage and decreased VEGF expression.

Hyperoxia is implicated in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. High levels of supplemental oxygen can result in microvascular endothelial cell death and may disrupt lung development. In postnatal animals, hyperoxia inhibits expression of vascular endothelial growth factor (VEGF), which is required for normal vascular development. A potential mechanism of oxygen effects on VEGF is induction of p53, a transcription factor that represses VEGF gene transcription. Oxidant DNA damage can increase p53. We used a moderately premature baboon model of hyperoxia to examine p53, oxidant DNA damage, and VEGF expression. Fetal baboons delivered at 140 d of gestation (75% of term) were ventilated with 100% oxygen or oxygen as needed for 6 or 10 d. Lungs from the 10-d 100% oxygen animals had increased nuclear p53, compared with the oxygen as needed animals. The mechanism of increased p53 was probably related to oxidant DNA damage, which was documented by increased oxidized guanine. Dual fluorescent confocal microscopy found increased oxidized guanine in mitochondrial DNA of distal lung epithelial cells. Distal epithelial cell VEGF expression was decreased and p21, another downstream target of p53, was increased in the distal epithelium of the hyperoxic animals. These data show that p53 is induced in hyperoxic fetal lung epithelium and are consistent with p53 repression of VEGF expression in these cells. The findings suggest that oxidant DNA damage may be a mechanism of increased p53 in hyperoxic fetal lung.

Loss of Gadd45a does not modify the pulmonary response to oxidative stress.

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.