Correction to: Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In the original publication, the starting point in time for the three feeding trials.

Enhanced foreign protein accumulation in Nicotiana benthamiana leaves co-infiltrated with a TMV vector and plant cell cycle regulator genes.

In this short communication, we report that the cell cycle checkpoint genes At-CycD2 and At-CDC27a from Arabidopsis thaliana enhance the transient heterologous protein expression in Nicotiana benthamiana. We selected a well-studied and widely used virus expression vector based on TMV for the delivery of recombinant proteins into the host plant. Co-infiltration of TMV-gfp and binary expression vectors carrying the At-CycD2 and At-CDC27a genes, respectively, resulted in enhanced GFP fluorescence in agroinoculated leaves. These findings corresponded with the observation of (1) higher mRNA levels for TMV and gfp and (2) increased GFP protein accumulation. Furthermore, by co-delivery of the TMV-scFv-TM43-E10 and At-CycD2/At-CDC27a expressing constructs we observed an enhanced amount of the scFv-TM43-E10 antibody fragment compared to the delivery of the TMV-scFv-TM43-E10 alone. We anticipate that this finding might be adapted for enhancing foreign protein production in N. benthamiana as the host plant.

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.

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.

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.

Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts.

Novel plant genome editing techniques call for an updated legislation regulating the use of plants produced by genetic engineering or genome editing, especially in the European Union. Established more than 25 years ago and based on a clear distinction between transgenic and conventionally bred plants, the current EU Directives fail to accommodate the new continuum between genetic engineering and conventional breeding. Despite the fact that the Directive 2001/18/EC contains both process- and product-related terms, it is commonly interpreted as a strictly process-based legislation. In view of several new emerging techniques which are closer to the conventional breeding than common genetic engineering, we argue that it should be actually interpreted more in relation to the resulting product. A legal guidance on how to define plants produced by exploring novel genome editing techniques in relation to the decade-old legislation is urgently needed, as private companies and public researchers are waiting impatiently with products and projects in the pipeline. We here outline the process in the EU to develop a legislation that properly matches the scientific progress. As the process is facing several hurdles, we also compare with existing frameworks in other countries and discuss ideas for an alternative regulatory system.

Enhancing the interpretation of statistical P values in toxicology studies: implementation of linear mixed models (LMMs) and standardized effect sizes (SESs).

In this paper, we compare the traditional ANOVA approach to analysing data from 90-day toxicity studies with a more modern LMM approach, and we investigate the use of standardized effect sizes. The LMM approach is used to analyse weight or feed consumption data. When compared to the week-by-week ANOVA with multiple test results per week, this approach results in only one statement on differences in weight development between groups. Standardized effect sizes are calculated for the endpoints: weight, relative organ weights, haematology and clinical biochemistry. The endpoints are standardized, allowing different endpoints of the same study to be compared and providing an overall picture of group differences at a glance. Furthermore, in terms of standardized effect sizes, statistical significance and biological relevance are displayed simultaneously in a graph.

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.

Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU.

Several new plant breeding techniques (NPBTs) have been developed during the last decade, and make it possible to precisely perform genome modifications in plants. The major problem, other than technical aspects, is the vagueness of regulation concerning these new techniques. Since the definition of eight NPBTs by a European expert group in 2007, there has been an ongoing debate on whether the resulting plants and their products are covered by GMO legislation. Obviously, cover by GMO legislation would severely hamper the use of NPBT, because genetically modified plants must pass a costly and time-consuming GMO approval procedure in the EU. In this review, we compare some of the NPBTs defined by the EU expert group with classical breeding techniques and conventional transgenic plants. The list of NPBTs may be shortened (or extended) during the international discussion process initiated by the Organization for Economic Co-operation and Development. From the scientific point of view, it may be argued that plants developed by NPBTs are often indistinguishable from classically bred plants and are not expected to possess higher risks for health and the environment. In light of the debate on the future regulation of NPBTs and the accumulated evidence on the biosafety of genetically modified plants that have been commercialized and risk-assessed worldwide, it may be suggested that plants modified by crop genetic improvement technologies, including genetic modification, NPBTs or other future techniques, should be evaluated according to the new trait and the resulting end product rather than the technique used to create the new plant variety.

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu ) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is 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 two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties 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 408. 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 subchronic exposure, independently of the two different genetic backgrounds of the event.

Utilization of PVX-Cre expression vector in potato.

Trait genes are usually introduced into the plant genome together with a marker gene. The last one becomes unnecessary after transgene selection and characterization. One of the strategies to produce transgenic plants free from the selectable marker is based on site-specific recombination. The present study employed the transient Cre-lox system to remove the nptII marker gene from potato. Transient marker gene excision involves introduction of Cre protein in lox-target plants by PVX virus vector followed by plant regeneration. Using optimized experimental conditions, such as particle bombardment infection method and application of P19 silencing suppressor protein, 20-27% of regenerated plants were identified by PCR analysis as marker-free. Based on our comparison of the recombination frequencies observed in this study to the efficiency of other methods to avoid or eliminate marker genes in potato, we suggest that PVX-Cre mediated site-specific excisional recombination is a useful tool to generate potato plants without superfluous transgenic sequences.

Microscopic analysis of severe structural rearrangements of the plant endoplasmic reticulum and Golgi caused by overexpression of Poa semilatent virus movement protein.

Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the "triple gene block" (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.

Problem formulation in the environmental risk assessment for genetically modified plants.

Problem formulation is the first step in environmental risk assessment (ERA) where policy goals, scope, assessment endpoints, and methodology are distilled to an explicitly stated problem and approach for analysis. The consistency and utility of ERAs for genetically modified (GM) plants can be improved through rigorous problem formulation (PF), producing an analysis plan that describes relevant exposure scenarios and the potential consequences of these scenarios. A properly executed PF assures the relevance of ERA outcomes for decision-making. Adopting a harmonized approach to problem formulation should bring about greater uniformity in the ERA process for GM plants among regulatory regimes globally. This paper is the product of an international expert group convened by the International Life Sciences Institute (ILSI) Research Foundation.

Options to Reform the European Union Legislation on GMOs: Post-authorization and Beyond.

We discuss options to reform the EU genetically modified organism (GMO) regulatory framework, make risk assessment and decision-making more consistent with scientific principles, and lay the groundwork for international coherence. In this third of three articles, we focus on labeling and coexistence as well as discuss the political reality and potential ways forward.

Options to Reform the European Union Legislation on GMOs: Risk Governance.

Here, we discuss options to reform the EU genetically modified organism (GMO) regulatory framework, to make risk assessment and decision-making more consistent with scientific principles, and to lay the groundwork for international coherence. We discussed the scope and definitions in a previous article and, thus, here we focus on the procedures for risk assessment and risk management.

Options to Reform the European Union Legislation on GMOs: Scope and Definitions.

We discuss options to reform the EU genetically modified organisms (GMO) regulatory framework, make risk assessment and decision-making more consistent with scientific principles, and lay the groundwork for international coherence. The first in a three-part series, this article focuses on reform options related to the scope of the legislation and the GMO definition.

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism.

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.