Chronic Toxicity of Genetically Modified Maize with Cry1Ab-ma Gene and Its Effect on Serum Metabolites in Rats.

Genetic engineering has inserted the crystallin (Cry) gene of Bacillus thuringiensis into the genes of maize to cultivate a variety of transgenic insect-resistant maizes. At present, genetically modified maize with Cry1Ab-ma gene (maize CM8101) was in the stage of safety verification. In this study, a 1-year chronic toxicity test was carried out to evaluate the safety of maize CM8101. Wistar rats were selected for the experiment. Rats were randomly divided into three groups and fed the corresponding diets: genetically modified maize group (CM8101 group), parental maize group (Zheng58 group), and AIN group. Rat serum and urine were collected at the third, sixth, and twelfth months of the experiment, and viscera were collected at the end of the experiment for detection. Metabolomics was used to analyze the metabolites in the serum of rats at the 12th month. While the CM8101 group rats' diets were supplemented with 60% maize CM8101, no obvious poisoning symptoms were found in rats, and no poisoning death occurred. There were no negative effects on body weight, food intake, blood and urine indices, or organ histopathological examination results. Furthermore, metabolomics results revealed that, when compared to group differences, the gender of rats had a more obvious effect on metabolites. The CM8101 group primarily changed linoleic acid metabolism in female rats, while glyceropholipid metabolism was altered in male rats. In rats, consumption of maize CM8101 did not result in significant metabolic dysfunction.

Subacute toxicity studies of meals prepared from genetically modified potato overexpressing the StDREB1 or the VvWRKY2 transcription factor in rats.

BACKGROUND: Potato tubers from genetically modified plants overexpressing the StDREB1 or the VvWRKY2 transcription factors that exhibited improved tolerance to salt and resistance to Fusarium solani infection were characterized and evaluated for safety in a 30 day rat feeding study. Male Wistar rats were split into four groups and provided with a diet composed of 33% (w/w) of either one of the two genetically modified potatoes (GMPs), 33% of the commercial Spunta variety (Sp), or a control group fed with the basal rats' diet. The influence of the GMPs on rat behavior and overall health parameters was evaluated and compared with that of commercial potato (i.e. the Sp group) and control diet. RESULTS: Small differences were noticed in the chemical composition of the different tubers, but all the diets were adjusted to an identical caloric level. Results showed no sign of toxic or detrimental effects on the rats' overall health as a result of these diets. The rats fed with the GMPs meal showed hematological and biochemical compositions of the plasma comparable to the control groups. No histopathological damage nor any structural disorganization, severe congestion, or acute inflammation were noticed in the rats' tissues. CONCLUSION: Under these study conditions, the GMP diets did not induce any apparent or significant adverse effects on rats after 30 days of dietary administration in comparison with rats fed diets with the corresponding non-transgenic diet and the standard diet group. These two GMPs were therefore considered to be as safe as their commercial comparator. © 2022 Society of Chemical Industry.

Effects of transgenic Bacillus Thuringiensis maize (2A-7) on the growth and development in rats.

This study aimed to determine the effects of genetically modified insect-resistant maize (2A-7) on the growth and development in developing rats. Rats were fed a diet formulated with 2A-7 maize and were compared with rats fed a diet formulated with non-transgenic maize (CK group) and rats fed AIN-93G diet (BC group). 2A-7 maize was formulated into diets at ratios of 82.4% (H group) and 20.6% (L group); non-transgenic maize was formulated into diets at a ratio of 82.4%. From the first day of pregnancy, adult rats were divided into four groups and fed with the above four diets, respectively. Weaning on postnatal day 21, the diets of offspring were consistent with their parents. The results showed that body weight, hematology, serum biochemistry, organ weight, organ coefficients and allergenicity of offspring fed with 2A-7 maize were comparable with those in the CK and BC groups. In physiological and behavioral development experiments, there was no statistically significant difference among groups. Although mCry1Ab proteins were detected in organs and serum, no histopathological changes were observed among groups. In conclusion, A-7 maize cause no treatment-related adverse effects on offspring, indicating that 2A-7 maize is safe for developing rats.

Two generation reproduction toxicity study of GmDREB3 gene modified wheat in Wistar rats.

To study reproductive toxicity of gene modified wheat generated by introducing DREB3 (drought response element binding protein 3) gene, Wistar rats of were allocated into 3 groups and fed with DREB3 gene modified wheat mixture diet (GM group), non-gene modified wheat mixture diet (Non-GM group) and AIN-93 diet (Control group) from parental generation (F0) to the second offspring (F2). GM wheat and Non-GM wheat, Jimai22, were both formulated into diets at a ratio of 69.55% according to AIN93 diet for rodent animals. Compared with non-GM group, no biologically related differences were observed in GM group rats with respect to reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, reproductive organ pathology and developmental parameters such as body weight, body length, food consumption, neuropathy, behavior, immunotoxicity, hematology and serum chemistry. In conclusion, no adverse effect were found relevant to GM wheat in the two generation reproduction toxicity study, indicating the GM wheat is a safe alternative for its counterpart wheat regarding to reproduction toxicity.

The GMO90+ Project: Absence of Evidence for Biologically Meaningful Effects of Genetically Modified Maize-based Diets on Wistar Rats After 6-Months Feeding Comparative Trial.

The GMO90+ project was designed to identify biomarkers of exposure or health effects in Wistar Han RCC rats exposed in their diet to 2 genetically modified plants (GMP) and assess additional information with the use of metabolomic and transcriptomic techniques. Rats were fed for 6-months with 8 maize-based diets at 33% that comprised either MON810 (11% and 33%) or NK603 grains (11% and 33% with or without glyphosate treatment) or their corresponding near-isogenic controls. Extensive chemical and targeted analyses undertaken to assess each diet demonstrated that they could be used for the feeding trial. Rats were necropsied after 3 and 6 months. Based on the Organization for Economic Cooperation and Development test guideline 408, the parameters tested showed a limited number of significant differences in pairwise comparisons, very few concerning GMP versus non-GMP. In such cases, no biological relevance could be established owing to the absence of difference in biologically linked variables, dose-response effects, or clinical disorders. No alteration of the reproduction function and kidney physiology was found. Metabolomics analyses on fluids (blood, urine) were performed after 3, 4.5, and 6 months. Transcriptomics analyses on organs (liver, kidney) were performed after 3 and 6 months. Again, among the significant differences in pairwise comparisons, no GMP effect was observed in contrast to that of maize variety and culture site. Indeed, based on transcriptomic and metabolomic data, we could differentiate MON- to NK-based diets. In conclusion, using this experimental design, no biomarkers of adverse health effect could be attributed to the consumption of GMP diets in comparison with the consumption of their near-isogenic non-GMP controls.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

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.

Safety assessment of genetically modified milk containing human beta-defensin-3 on rats by a 90-day feeding study.

In recent years, transgenic technology has been widely applied in many fields. There is concern about the safety of genetically modified (GM) products with the increased prevalence of GM products. In order to prevent mastitis in dairy cows, our group produced transgenic cattle expressing human beta-defensin-3 (HBD3) in their mammary glands, which confers resistance to the bacteria that cause mastitis. The milk derived from these transgenic cattle thus contained HBD3. The objective of the present study was to analyze the nutritional composition of HBD3 milk and conduct a 90-day feeding study on rats. Rats were divided into 5 groups which consumed either an AIN93G diet (growth purified diet for rodents recommended by the American Institute of Nutrition) with the addition of 10% or 30% HBD3 milk, an AIN93G diet with the addition of 10% or 30% conventional milk, or an AIN93G diet alone. The results showed that there was no difference in the nutritional composition of HBD3 and conventional milk. Furthermore, body weight, food consumption, blood biochemistry, relative organ weight, and histopathology were normal in those rats that consumed diets containing HBD3. No adverse effects were observed between groups that could be attributed to varying diets or gender.

Variability of control data and relevance of observed group differences in five oral toxicity studies with genetically modified maize MON810 in rats.

The data of four 90-day feeding trials and a 1-year feeding trial with the genetically modified (GM) maize MON810 in Wistar Han RCC rats performed in the frame of EU-funded project GRACE were analysed. Firstly, the data obtained from the groups having been fed the non-GM maize diets were combined to establish a historical control data set for Wistar Han RCC rats at the animal housing facility (Slovak Medical University, Bratislava, Slovakia). The variability of all parameters is described, and the reference values and ranges have been derived. Secondly, the consistency of statistically significant differences found in the five studies was analysed. In order to do so, the body weight development, organ weight, haematology and clinical biochemistry data were compared between the studies. Based on the historical control data, equivalence ranges for these parameters were defined, and the values measured in the GM maize-fed groups were compared with these equivalence ranges. Thirdly, the (statistical) power of these feeding studies with whole food/feed was assessed and detectable toxicologically relevant group differences were derived. Linear mixed models (LMM) were applied, and standardized effect sizes (SES) were calculated in order to compare different parameters as well as to provide an overall picture of group and study differences at a glance. The comparison of the five feeding trials showed a clear study effect in the control data. It also showed inconsistency both in the frequency of statistically significant differences and in the difference values between control and test groups.

Effect of genetically modified corn on the jejunal mucosa of adult male albino rat.

Genetically modified (GM) plants expressing insecticidal traits offer a new strategy for crop protection. GM-corn contains Bacillus thuringiensis (Bt) genes producing delta endotoxins in the whole plant. Diet can influence the characteristics of the gastrointestinal tract altering its function and structure. The aim of this study was to evaluate the effect of GM-corn on the histological structure of jejunal mucosa of adult male albino rat using different histological, immunohistochemical and morphometrical methods. Twenty adult male albino rats were divided into two equal groups; control and GM-corn fed group administered with 30% GM-corn for 90days. Specimens from the jejunum were processed for light and electron microscopy. Immunohistochemical study was carried out using antibody against proliferating cell nuclear antigen (PCNA). Different morphometrical parameters were assessed. Specimens from GM-corn fed group showed different forms of structural changes. Focal destruction and loss of the villi leaving denuded mucosal surface alternating with stratified areas were observed, while some crypts appeared totally disrupted. Congested blood capillaries and focal infiltration with mononuclear cells were detected. Significant upregulation of PCNA expression, increase in number of goblet cells and a significant increase in both villous height and crypt depth were detected. Marked ultrastructural changes of some enterocytes with focal loss of the microvillous border were observed. Some enterocytes had vacuolated cytoplasm, swollen mitochondria with disrupted cristae and dilated rough endoplasmic reticulum (rER). Some cells had dark irregular nuclei with abnormally clumped chromatin. It could be concluded that consumption of GM-corn profoundly alters the jejunal histological structure.

Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota.

In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM Bacillus thuringiensis (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded in vitro with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Ecological risk assessment for DvSnf7 RNA: A plant-incorporated protectant with targeted activity against western corn rootworm.

MON 87411 maize, which expresses DvSnf7 RNA, was developed to provide an additional mode of action to confer protection against corn rootworm (Diabrotica spp.). A critical step in the registration of a genetically engineered crop with an insecticidal trait is performing an ecological risk assessment to evaluate the potential for adverse ecological effects. For MON 87411, an assessment plan was developed that met specific protection goals by characterizing the routes and levels of exposure, and testing representative functional taxa that would be directly or indirectly exposed in the environment. The potential for toxicity of DvSnf7 RNA was evaluated with a harmonized battery of non-target organisms (NTOs) that included invertebrate predators, parasitoids, pollinators, soil biota as well as aquatic and terrestrial vertebrate species. Laboratory tests evaluated ecologically relevant endpoints such as survival, growth, development, and reproduction and were of sufficient duration to assess the potential for adverse effects. No adverse effects were observed with any species tested at, or above, the maximum expected environmental concentration (MEEC). All margins of exposure for NTOs were >10-fold the MEEC. Therefore, it is reasonable to conclude that exposure to DvSnf7 RNA, both directly and indirectly, is safe for NTOs at the expected field exposure levels.

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.

Corn rootworm-active RNA DvSnf7: Repeat dose oral toxicology assessment in support of human and mammalian safety.

Genetically modified (GM) crops have been developed and commercialized that utilize double stranded RNAs (dsRNA) to suppress a target gene(s), producing virus resistance, nutritional and quality traits. MON 87411 is a GM maize variety that leverages dsRNAs to selectively control corn rootworm through production of a 240 base pair (bp) dsRNA fragment targeting for suppression the western corn rootworm (Diabrotica virgifera virgifera) Snf7 gene (DvSnf7). A bioinformatics assessment found that endogenous corn small RNAs matched ∼450 to 2300 unique RNA transcripts that likely code for proteins in rat, mouse, and human, demonstrating safe dsRNA consumption by mammals. Mice were administered DvSnf7 RNA (968 nucleotides, including the 240 bp DvSnf7 dsRNA) at 1, 10, or 100 mg/kg by oral gavage in a 28-day repeat dose toxicity study. No treatment-related effects were observed in body weights, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. Therefore, the No Observed Adverse Effect Level (NOAEL) for DvSnf7 RNA was 100 mg/kg, the highest dose tested. These results demonstrate that dsRNA for insect control does not produce adverse health effects in mammals at oral doses millions to billions of times higher than anticipated human exposures and therefore poses negligible risk to mammals.

Adaptation of the ToxRTool to Assess the Reliability of Toxicology Studies Conducted with Genetically Modified Crops and Implications for Future Safety Testing.

To determine the reliability of food safety studies carried out in rodents with genetically modified (GM) crops, a Food Safety Study Reliability Tool (FSSRTool) was adapted from the European Centre for the Validation of Alternative Methods' (ECVAM) ToxRTool. Reliability was defined as the inherent quality of the study with regard to use of standardized testing methodology, full documentation of experimental procedures and results, and the plausibility of the findings. Codex guidelines for GM crop safety evaluations indicate toxicology studies are not needed when comparability of the GM crop to its conventional counterpart has been demonstrated. This guidance notwithstanding, animal feeding studies have routinely been conducted with GM crops, but their conclusions on safety are not always consistent. To accurately evaluate potential risks from GM crops, risk assessors need clearly interpretable results from reliable studies. The development of the FSSRTool, which provides the user with a means of assessing the reliability of a toxicology study to inform risk assessment, is discussed. Its application to the body of literature on GM crop food safety studies demonstrates that reliable studies report no toxicologically relevant differences between rodents fed GM crops or their non-GM comparators.

A 90-day toxicology study of meat from genetically modified sheep overexpressing TLR4 in Sprague-Dawley rats.

Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.