Extended one generation reproductive toxicity study and effect on gut flora of genetically modified rice rich in ß-carotene in wistar rats.

To evaluate the reproductive toxicity of gene modified rice generated by introducing phytoene synthase (Psy) and bacterial phytoene desaturase (CrtI) from maize and Erwinia uredovora, Wistar rats were allocated into 3 groups and fed with Psy and CrtI gene modified rice mixture diet (GM group), non-gene modified rice mixture diet (non-GM group), and AIN-93 diet (Blank control group) from parental generation (F0) to the offsprings (F1). GM rice, Heijinmi (HJM) and Non-GM rice, Heishuai (HS), were both formulated into diets at ratios of 73.5% and 75.5% according to the AIN93 diet for rodent animals, respectively. Relative to the non-GM group, no biologically relevant differences were observed in GM group rats concerning reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, and reproductive organ pathology. The developmental parameters results were not significantly different from the non-GM group such as body weight, food consumption, developmental neurotoxicity, behavior, hematology, and serum chemistry. In terms of immunotoxicity, the IgG indicators of offspring from the GM group improved in contrast with the non-GM group. Additional gut flora analysis of F0 generation rats resulted as that the treatment elicited an increased gut microflora diversity of F0 rats. And no horizontal gene transfer of Psy and CrtI genes in rats fed a GM rice HJM diet. In conclusion, we found no adverse effects related to GM rice in the extended one-generation reproductive toxicity study, indicating that GM rice is a safe alternative for its counterpart rice regarding reproductive toxicity.

A 90-day rodent feeding study with grain for genetically modified maize L4 conferring insect resistance and glyphosate tolerance.

A 90-day rat feeding study was performed to conduct a safety assessment on L4, a multi-gene genetically modified maize, conferring "Bt" insect resistance and glyphosate tolerance. A total of 140 Wistar rats were assigned to seven groups, 10 animals/group/sex, which comprised three genetically modified groups fed diets containing different concentrations of L4, three corresponding non-genetically modified groups fed diets containing different concentrations of zheng58 (parent plants), and a basal diet group fed the standard basal diet for 13 weeks. The fed diets contained L4 and Zheng58 at w/w% percentages of 12.5%, 25.0%, and 50% of the total. Animals were evaluated on some research parameters, including general behaviour, body weight/gain, feed consumption/efficiency, ophthalmology, clinical pathology, organ weights, and histopathology. Throughout the feeding trial, all animals were in good condition. No mortality and no biologically relevant effects or toxicologically significant alterations were observed in the total research parameters of the rats in the genetically modified groups compared with those in the basal diet group or their corresponding non-genetically modified groups. No adverse effects were observed in any of the animals. The results indicated that L4 is as safe and wholesome as conventional, non-genetically modified control maize.

No Adverse Effects of Stacked Bacillus thuringiensis Maize on the Midge Chironomus riparius.

Material from genetically engineered maize producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) may enter aquatic ecosystems and expose nontarget organisms. We investigated the effects on life table parameters of the midge Chironomus riparius (Diptera: Chironomidae) of SmartStax maize leaves, which contain six different Cry proteins targeting Lepidoptera and Coleoptera pests, in two plant backgrounds. For midge development and emergence, 95% confidence intervals for the means of six conventional maize lines (Rheintaler, Tasty Sweet, ES-Eurojet, Planoxx, EXP 258, and EXP 262), were used to capture the natural range of variation. For reproduction, lowest and highest means were used. The natural range of variation allows one to judge whether observed effects between Bt maize and the closest non-Bt comparator are likely to be of biological relevance. No adverse effects on C. riparius were observed with any Bt maize line compared with the respective non-Bt counterpart. Development time was shorter when females were fed Bt maize than when they were fed non-Bt maize, but this effect was not considered adverse. Development time, emergence ratio, sex ratio, and larvae/egg rope measured for Bt maize were within the natural range of variation. Fecundity for the Bt lines was equal to or higher than that for the conventional lines. Future risk assessment studies may consider plant background effects and the natural range of variation to judge the relevance of observed differences between particular genetically engineered and non-genetically engineered plants. Environ Toxicol Chem 2022;41:1078-1088. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Edible Plant Sprouts: Health Benefits, Trends, and Opportunities for Novel Exploration.

The consumption of plant sprouts as part of human day-to-day diets is gradually increasing, and their health benefit is attracting interest across multiple disciplines. The purpose of this review was to (a) critically evaluate the phytochemicals in selected sprouts (alfalfa, buckwheat, broccoli, and red cabbage), (b) describe the health benefits of sprouts, (c) assess the recent advances in sprout production, (d) rigorously evaluate their safety, and (e) suggest directions that merit special consideration for further novel research on sprouts. Young shoots are characterized by high levels of health-benefitting phytochemicals. Their utility as functional ingredients have been extensively described. Tremendous advances in the production and safety of sprouts have been made over the recent past and numerous reports have appeared in mainstream scientific journals describing their nutritional and medicinal properties. However, subjects such as application of sprouted seed flours in processed products, utilizing sprouts as leads in the synthesis of nanoparticles, and assessing the dynamics of a relationship between sprouts and gut health require special attention for future clinical exploration. Sprouting is an effective strategy allowing manipulation of phytochemicals in seeds to improve their health benefits.

Equivalence tests for safety assessment of genetically modified crops using plant composition data.

The evaluation of compositional characteristics of plants harvested from field trials is an important step in the safety assessment of a genetically modified crop and its derived products for food and feed. The European Food Safety Authority (EFSA) evaluates safety by testing for equivalence between the GM genotype and other genotypes, typically with a history of safe use. Here, a new equivalence test is proposed, which addresses issues with the EFSA test. The method is motivated by a recently proposed equivalence test for analysis of data from animal feeding trials. In order to be suitable for practical safety assessment, the new method has a statistical power set to a desired value, e.g. 95%, by construction. In addition, we assess distributions rather than average values. This way, equivalence limits can also be established when there is limited genotypic variation. The original EFSA equivalence test breaks down in this case. The method is illustrated by its application to data from a field study on maize grain. Simulation studies indicate that the proposed test has appropriate performance characteristics and is competitive with respect to recently proposed alternatives, including the EFSA/EU equivalence test.

Mass spectrometric analysis of digesta does not improve the allergenicity assessment of GM crops.

An investigation of the potential allergenicity of newly expressed proteins in genetically modified (GM) crops comprises part of the assessment of GM crop safety. However, allergenicity is not completely predictable from a definitive assay result or set of protein characteristics, and scientific opinions regarding the data that should be used to assess allergenicity are continuously evolving. Early studies supported a correlation between the stability of a protein exposed to digestive enzymes such as pepsin and the protein's status as a potential allergen, but over time the conclusions of these earlier studies were not confirmed. Nonetheless, many regulatory authorities, including the European Food Safety Authority (EFSA), continue to require digestibility analyses as a component of GM crop risk assessments. Moreover, EFSA has recently investigated the use of mass spectrometry (MS), to make digestion assays more predictive of allergy risk, because it can detect and identify small undigested peptides. However, the utility of MS is questionable in this context, since known allergenic peptides are unlikely to exist in protein candidates intended for commercial development. These protein candidates are pre-screened by the same bioinformatics processes that are normally used to identify MS targets. Therefore, MS is not a standalone allergen identification method and also cannot be used to predict previously unknown allergenic epitopes. Thus, the suggested application of MS for analysis of digesta does not improve the poor predictive power of digestion assays in identifying allergenic risk.

Genetically Modified Plants: Nutritious, Sustainable, yet Underrated.

Combating malnutrition is one of the greatest global health challenges. Plant-based foods offer an assortment of nutrients that are essential for adequate nutrition and can promote good health. Unfortunately, the majority of widely consumed crops are deficient in some of these nutrients. Biofortification is the umbrella term for the process by which the nutritional quality of food crops is enhanced. Traditional agricultural breeding approaches for biofortification are time consuming but can enhance the nutritional value of some foods; however, advances in molecular biology are rapidly being exploited to biofortify various crops. Globally, genetically modified organisms are a controversial topic for consumers and governmental agencies, with a vast majority of people apprehensive about the technology. Golden Rice has been genetically modified to contain elevated ß-carotene concentrations and is the bellwether for both the promise and angst of agricultural biotechnology. Although there are numerous other nutritional targets of genetically biofortified crops, here I briefly summarize the work to elevate iron and folate concentrations. In addition, the possibility of using modified foods to affect the gut microbiota is examined. For several decades, plant biotechnology has measured changes in nutrient concentrations; however, the bioavailability of nutrients from many biofortified crops has not been demonstrated.

Plant breeding involving genetic engineering does not result in unacceptable unintended effects in rice relative to conventional cross-breeding.

Advancements in -omics techniques provide powerful tools to assess the potential effects in composition of a plant at the RNA, protein and metabolite levels. These technologies can thus be deployed to assess whether genetic engineering (GE) causes changes in plants that go beyond the changes introduced by conventional plant breeding. Here, we compare the extent of transcriptome and metabolome modification occurring in leaves of four GE rice lines expressing Bacillus thuringiensis genes developed by GE and seven rice lines developed by conventional cross-breeding. The results showed that both types of crop breeding methods can bring changes at transcriptomic and metabolic levels, but the differences were comparable between the two methods, and were less than those between conventional non-GE lines were. Metabolome profiling analysis found several new metabolites in GE rice lines when compared with the closest non-GE parental lines, but these compounds were also found in several of the conventionally bred rice lines. Functional analyses suggest that the differentially expressed genes and metabolites caused by both GE and conventional cross-breeding do not involve detrimental metabolic pathways. The study successfully employed RNA-sequencing and high-performance liquid chromatography mass spectrometry technology to assess the unintended changes in new rice varieties, and the results suggest that GE does not cause unintended effects that go beyond conventional cross-breeding in rice.

Safety assessment of transgenic Cry2Aa rice to a generalist predator, Paederus fuscipes Curtis (Coleoptera: Staphylinidae).

The insecticidal crystal proteins of Cry2A family from Bacillus thuringiensis (Bt) are important candidate proteins expressed in gene pyramiding Bt crops. A transgenic rice line (T2A-1) harboring a synthetic Cry2A* (Cry2Aa) gene showed effective resistance to some lepidopteran rice pests. As a generalist predator in rice ecosystems, the rove beetle (Paederus fuscipes) can prey on many rice insect pests such as planthoppers. Considering the possible exposure of Cry2Aa to P. fuscipes through tritrophic food chain, it is necessary to assess the potential risks of T2A-1 rice to this predator. In this study, a tritrophic experiment was conducted to assess the prey-mediated effects of Cry2Aa on P. fuscipes through the T2A-1 rice-Nilaparvata lugens-P. fuscipes food chain. After preying on N. lugens nymphs reared on T2A-1, no accumulated Cry2Aa could be detected in P. fuscipes adults, despite Cry2Aa being detected in N. lugens. In addition, no harmful effects were detected on the life table parameters of P. fuscipes in this tritrophic chain. Additionally, direct exposure to a high dose of purified Cry2Aa protein, representing the worst case scenario, showed no significant adverse effects on the development of P. fuscipes. These results showed that transgenic Cry2Aa rice had no harmful effects on P. fuscipes.

Comparing agronomic and phenotypic plant characteristics between single and stacked events in soybean, maize, and cotton.

Genetically modified (GM) crops are one of the most valuable tools of modern biotechnology that secure yield potential needed to sustain the global agricultural demands for food, feed, fiber, and energy. Crossing single GM events through conventional breeding has proven to be an effective way to pyramid GM traits from individual events and increase yield protection in the resulting combined products. Even though years of research and commercialization of GM crops show that these organisms are safe and raise no additional biosafety concerns, some regulatory agencies still require risk assessments for these products. We sought out to investigate whether stacking single GM events would have a significant impact on agronomic and phenotypic plant characteristics in soybean, maize, and cotton. Several replicated field trials designed as randomized complete blocks were conducted by Monsanto Regulatory Department from 2008 to 2017 in field sites representative of cultivation regions in Brazil. In total, twenty-one single and stacked GM materials currently approved for in-country commercial use were grown with the corresponding conventional counterparts and commercially available GM/non-GM references. The generated data were presented to the Brazilian regulatory agency CTNBio (National Biosafety Technical Committee) over the years to request regulatory approvals for the single and stacked products, in compliance with the existing normatives. Data was submitted to analysis of variance and differences between GM and control materials were assessed using t-test with a 5% significance level. Data indicated the predominance of similarities and neglectable differences between single and stacked GM crops when compared to conventional counterpart. Our results support the conclusion that combining GM events through conventional breeding does not alter agronomic or phenotypic plant characteristics in these stacked crops. This is compatible with a growing weight of evidence that indicates this long-adopted strategy does not increase the risks associated with GM materials. It also provides evidence to support the review and modernization of the existing regulatory normatives to no longer require additional risk assessments of GM stacks comprised of previously approved single events for biotechnology-derived crops. The data analyzed confirms that the risk assessment of the individual events is sufficient to demonstrate the safety of the stacked products, which deliver significant benefits to growers and to the environment.

Results from ten years of post-market environmental monitoring of genetically modified MON 810 maize in the European Union.

In European regulations for the deliberate release into the environment of genetically modified organisms (GMO), the objective of General Surveillance in Post-Market Environmental Monitoring is defined as the identification of the occurrence of adverse effects of the GMO or its use which were not anticipated in the environmental risk assessment (ERA). Accompanying the commercial cultivation in the EU of maize event MON 810, General Surveillance was implemented by Monsanto, the authorization holder, on a voluntary basis. We carried out a statistical analysis on the pooled results of ten years of farmer questionnaires, which were a part of this General Surveillance, amounting to 2,627 farmer fields in eight European countries in the period 2006-2015. This analysis did not reveal any unexpected adverse effects associated with the cultivation of MON 810. Results from farmer questionnaires confirmed that the cultivation of MON 810 resulted in a significant reduction in the use of pesticides, efficient protection against the target pests, and healthier, higher yielding crops compared to conventional maize. MON 810 also had reduced susceptibility to disease and pests when compared to conventional maize. Monitoring characteristics related to environment and wildlife revealed no significant differences between MON 810 and conventional maize. Literature searches, that were also conducted as part of General Surveillance, identified a comprehensive set of publications addressing environmental safety as well as food and feed safety aspects of MON 810. None of the publications indicated any adverse effect of MON 810 that was not anticipated in the initial ERA, nor did they lead to a change in the conclusions of the initial risk assessment that demonstrated the safety of MON 810. The development of resistance by the target pests (Ostrinia nubilalis, ECB and Sesamia nonagrioides, MCB) was the only potential adverse effect identified in the ERA of MON 810 cultivation in the EU. The extensive safety data package for MON 810, the robust weight of evidence demonstrating both its safety and benefits, and the history of safe use of MON 810 for 15 years in the EU, indicates that focussing the General Surveillance of MON 810 on literature searches and farmer complaint systems would be appropriately protective. This will allow the identification of potential adverse effect not anticipated in the initial ERA without the intensive effort and organizational challenges of farmer questionnaires.

Consideration of familiarity accumulated in the confined field trials for environmental risk assessment of genetically modified soybean (Glycine max) in Japan.

To date, there have been 160 regulatory approvals for environmental safety in Japan for the major genetically modified (GM) crops, including corn, soybean, canola and cotton. Confined field trials (CFTs) have been conducted in Japan for all single events, which contain various traits. The accumulated information from these previously conducted CFTs, as well as the agronomic field study data from other countries, provides a rich source of information to establish "familiarity" with the crops. This familiarity can be defined as the knowledge gained through experience over time, and used to inform the environmental risk assessments (ERA) of new GM crops in Japan. In this paper, we compiled agronomic data from the CFTs performed in Japan for 11 GM soybean events which obtained food, feed and environmental safety approvals from regulatory agencies in Japan. These CFTs were conducted by multiple developers according to Japan regulations to support the ERA of these GM soybean, covering standard measurement endpoints evaluated across developers in Japan. With this dataset, we demonstrate how familiarity gained from the CFTs of GM soybeans in Japan can be used to inform on the ERA of new GM soybean events. By leveraging this concept of familiarity, we discuss potential enhancements to the ERA process for GM soybean events in Japan.

Subchronic feeding toxicity studies of drought-tolerant transgenic wheat MGX11-10 in Wistar Han RCC rats.

A subchronic toxicity study were conducted in Wistar Han RCC rats to evaluate the potential health effects of genetically modified (GM), drought-tolerant wheat MGX11-10. Rats were fed a rodent diet formulated with MGX11-10 and were compared with rats fed a diet formulated with its corresponding non-transgenic control Jimai22 and rats fed a basal diet. MGX11-10 and Jimai22 were ground into flour and formulated into diets at concentrations of 16.25, 32.5, or 65%, w/w% and fed to rats (10/sex/group) for 13 weeks. Compared with rats fed Jimai22 and the basal-diet group, no biologically relevant differences were observed in rats fed the GM diet with respect to body weight/gain, food consumption/efficiency, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, clinical chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, the MGX11-10 diets did not cause any treatment-related effects in rats following at least 90 days of dietary administration as compared with rats fed diets with the corresponding non-transgenic control diet and the basal-diet group. The MGX11-10 diets are considered equivalent to the diets prepared from conventional comparators. The results demonstrated that MGX11-10 wheat is as safe and wholesome as the corresponding non-transgenic control wheat.

No impact of transgenic cry1Ie maize on the diversity, abundance and composition of soil fauna in a 2-year field trial.

Soil fauna play an essential role in the soil ecosystem, but they may be influenced by insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) maize. In this study, a 2-year field trial was conducted to study the effects of transgenic cry1Ie maize, a type of Bt maize (Event IE09S034), on soil fauna, with the near-isogenic line non-Bt maize (Zong 31) as a control. The soil animals were collected with Macfadyen heat extractor and hand-sorting methods, respectively, and their diversity, abundance and community composition were calculated. Then, the effects of maize type, year, sampling time and soil environmental factors on the soil fauna were evaluated by repeated-measures ANOVA, redundancy analysis (RDA) and nonmetric multidimensional scaling (nMDS). Repeated-measures ANOVA showed that the diversity and abundance of the soil fauna were not affected by maize type, while they were significantly influenced by year and sampling time. Furthermore, for both the Macfadyen and hand-sorting methods, RDA indicated that soil fauna community composition was not correlated with maize type (Bt and non-Bt maize) but was significantly correlated with year, sampling time and root biomass. In addition, it was significantly related to soil pH according to the hand-sorting method. nMDS indicated that soil fauna community composition was significantly correlated with year and sampling time; however, it was not associated with maize type. In this study, we collected soil faunal samples according to the Macfadyen and hand-sorting methods and processed the obtained data with ANOVA, RDA, and nMDS in three ways, and our data indicate that transgenic cry1Ie maize (Event IE09S034) had no substantial influence on the diversity, abundance or community composition of the soil fauna.

Evolving Regulatory Landscape for Genome-Edited Plants.

New plant breeding innovations, including CRISPR-mediated genome editing, are enhancing the development of new plant varieties, many of which are on the cusp of entering market places internationally. The lack of clear and consistent regulatory policies globally for genome-edited plants, and their products, may limit the adoption and commercialization of these products; In contrast to the positive trends of regulatory agencies in Argentina, Brazil, Colombia, Chile, and the United States, which are at the forefront of a wave of countries that are implementing policies facilitating the development of genome-edited plants, the European Union has adopted a more restrictive approach that considers any new mutagenesis technique developed after 2001 as subject to regulation as genetically modified plants.

No Effect of Bt-transgenic Rice on the Tritrophic Interaction of the Stored Rice, the Maize Weevil Sitophilus Zeamais and the Parasitoid Wasp Theocolax elegans.

During Bt transgenic rice storage, Bt Cry1Ab/Cry1Ac fused protein is exposed to the maize weevil Sitophilus zeamais and the parasitoid wasp Theocolax elegans. We have carried out a long-term risk assessment for Bt rice to these non-target organisms in the storehouse. Effects of Bt rice on S. zeamais and T. elegans have been carefully detected in a laboratory experiment of over 5 years. The survival, development, fecundity, and longevity of the maize weevil were compared between Bt rice and non-Bt rice treatments for every 5 generations from generation 1 to 25. Moreover, the development, adult body size and sex ratio of T. elegans were compared between them parasitizing S. zeamais feeding on Bt rice or non-Bt rice. We found that although Bt Cry1Ab/Cry1Ac fused protein exists in the Bt rice grains and S. zeamais digestive tracts, Bt rice is not harmful to the maize weevil S. zeamais and its parasitoid T. elegans.

Equivalence limit scaled differences for untargeted safety assessments: Comparative analyses to guard against unintended effects on the environment or human health of genetically modified maize.

Safety assessments guard against unintended effects for human health and the environment. When new products are compared with accepted reference products by broad arrays of measurements, statistical analyses are usually summarised by significance tests or confidence intervals per endpoint. The traditional approach is to test for statistical significance of differences. However, absence or presence of significant differences is not a statement about safety. Equivalence limits are essential for safety assessment. We propose graphs to present the results of equivalence tests over the array of endpoints. It is argued that plots of the equivalence limit scaled difference (ELSD) are preferable over plots of the standardised effect size (SES) used previously for similar assessments. The ELSD method can be used either with externally specified equivalence limits or with equivalence limits estimated from (historical) data. The method is illustrated with two examples: first, environmental safety of MON810 Bt maize was assessed using field trial count data of arthropods; second, human safety of herbicide tolerant NK603 maize was assessed using haematological, biochemical and organ weight data from a 90-day rat feeding study. All assessed endpoints were classified in EFSA equivalence categories I or II, implying full equivalence or equivalence more likely than not.

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.

Subchronic toxicity study in rats evaluating genetically modified DAS-81419-2 soybean.

A 90-day feeding study in rats was conducted to evaluate the subchronic oral toxicity of genetically modified (GM) DAS-81419-2 soybean. Wistar rats were fed with diets containing toasted soybean meal produced from DAS-81419-2 soybean grain that expresses the Cry1F, Cry1Ac, and Pat proteins or containing conventional soybean at doses of 30.0%, 15.0%, 7.5%, or 0% (control group) for 90 consecutive days. The general behavior, body weight and food consumption were observed. At the middle and end of the experiment, blood, serum, and urine samples were collected for biochemical assays. At the conclusion of the study, the internal organs were weighed and histopathological examination was completed. The rats exhibited free movement and shiny coats without any abnormal symptoms or abnormal secretions in their noses, eyes, or mouths. There were no adverse effects on body weight in GM soybean groups and conventional soybean groups. No biological differences in hematological, biochemical, or urine indices were observed. No significant differences in relative organ weights were detected between the experimental groups and the control group. No histopathological changes were observed. Under the conditions of this study, DAS-81419-2 soybean did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.