Genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities and the speed of altering genetic material in organisms. So-called genome editing is increasingly being used to achieve agriculturally relevant novel traits and/or genetic combinations in both plants and animals, although predominantly as proof of concept studies, with commercial growing or rearing so far limited to the U.S. and Canada. However, there are numerous reports of unintended effects such as off-target effects, unintended on-target effects and other unintended consequences arising from genome editing, summarised under the term genomic irregularities. Despite this, the searching for genomic irregularities is far from routine in these studies and protocols vary widely, particularly for off-target effects, leading to differences in the efficacy of detection of off-target effects. Here, we describe the range of specific unintended effects associated with genome editing. We examine the considerable possibilities to change the genome of plants and animals with SDN-1 and SDN-2 genome editing (i.e. without the insertion of genes conferring the novel trait) and show that genome editing techniques are able to produce a broad spectrum of novel traits that, thus far, were not possible to be obtained using conventional breeding techniques. We consider that the current EU risk assessment guidance for GMOs requires revision and broadening to capture all potential genomic irregularities arising from genome editing and suggest additional tools to assist the risk assessment of genome-edited plants and animals for the environment and food/animal feed in the EU.

New challenges arise in risk assessment when genetically engineered (GE) plants can persist and propagate in the environment as well as produce viable offspring. Next generation effects can be influenced by heterogeneous genetic backgrounds and unexpected effects can be triggered in interaction with environmental conditions. Consequently, the biological characteristics of the original events cannot be regarded as sufficient to conclude on hazards that may emerge in following generations. Potential hazards identified by the European Food Safety Authority (EFSA) include exacerbating weed problems, displacement and even extinction of native plant species. However, there are reasons for concern that might escape the environmental risk assessment (ERA) because EFSA only takes into account the characteristics of the original events, leaving aside unintended or unexpected next generation effects emerging from spontaneous propagation and gene flow. From our review of the publications available and the analysis of risk assessment as performed, we conclude that the risk assessment of GE organisms able to persist and spontaneously propagate in the environment actually suffers from a high degree of spatio-temporal complexity causing many uncertainties. To deal with this problem, we recommend establishing 'cut-off criteria' in risk assessment that include factual limits of knowledge. It is proposed that these criteria are applied in a specific step within risk assessment, i.e. 'spatio-temporal controllability' that uses well-defined biological characteristics to delineate some of the boundaries between known and unknowns. This additional step in risk assessment will foster robustness in the process and can substantially benefit the reliability and overall conclusiveness of risk assessment and decision-making on potential releases.

The study of combined effects of pesticides represents a challenge for toxicology. In the case of the new growing generation of genetically modified (GM) plants with stacked traits, glyphosate-based herbicides (like Roundup) residues are present in the Roundup-tolerant edible plants (especially corns) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated in this work. Here we have tested for the very first time Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human embryonic kidney cell line 293, as well as their combined actions with Roundup, within 24 h, on three biomarkers of cell death: measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7 inductions. Cry1Ab caused cell death from 100 ppm. For Cry1Ac, under such conditions, no effects were detected. The Roundup tested alone from 1 to 20 000 ppm is necrotic and apoptotic from 50 ppm, far below agricultural dilutions (50% lethal concentration 57.5 ppm). The only measured significant combined effect was that Cry1Ab and Cry1Ac reduced caspases 3/7 activations induced by Roundup; this could delay the activation of apoptosis. There was the same tendency for the other markers. In these results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined side-effects with other residues of pesticides specific to GM plants.

Background, aim, and scopeThis review deals with publications concerning the mode of action of Bt proteins and their potential synergism with extrinsic factors. The aim was to assess the impact of those factors especially regarding selectivity and efficacy of Bt toxins and to discuss possible gaps in current risk assessment of genetically engineered plants expressing Bt toxins.Main featuresThe review shows that several extrinsic factors are able to influence the selectivity and efficacy of Bt toxins. The findings are seen as being relevant for risk assessment in Bt plants. This conclusion is derived by discussing current state of knowledge about the mode of action of Bt proteins, unexpected effects on non-target organism, and the way how modified Bt toxins are expressed in genetically engineered plants.ResultsSeveral publications have been identified that show that certain factors and synergism can impact efficacy and selectivity of Bt toxins. These extrinsic factors are various and include other Bt toxins or parts from the spore of Bacillus thuringiensis as well as certain enzymes, environmental stress, non-pathogenic microorganisms, and infectious diseases.DiscussionResearch on the underlying mechanism of observed synergism might help to explain some of the effects found in non-target organisms. In general, possible synergism of Bt toxins with extrinsic factors can be relevant for risk assessment of genetically engineered Bt plants since they expose a modified Bt toxin to the environment under various conditions and over a long period of time.ConclusionsRisk assessment of genetically engineered plants should put into question the general assumption of a high selectivity and a linear dose–response relationship in the toxicity of Bt proteins. Both selectivity and efficacy can be influenced by synergism, which can provoke unexpected and undesired effects in non-target organisms.PerspectivesIt is suggested that systematic research be promoted on synergism between Bt toxins and potential extrinsic factors that could impact the spectrum of susceptible organisms. This research should become a prerequisite for risk assessment of Bt plants.

About 20 years after the market introduction of the first GM plants, we review whether or not uncontrolled spread occurred. We summarise cases documented in the scientific literature and derive conclusions for the regulation of the authorisation of new events. Several cases documented in North and Central America and Japan show that transgenes have spread beyond cultivation areas. Important examples are bentgrass (Agrostis stolonifera), oilseed rape (Brassica napus) and cotton (Gossypium hirsutum). Several factors can be identified as relevant for transgene dispersal in the environment. Grasses (Poaceae), in particular, show a high potential for persistence and invasiveness, and wild relatives that can cross with the crop plants are a major factor in the unintended spread of the transgenes. There are significant uncertainties in predicting which transgenes will escape and how they will interact with the environment. For example, climate change is likely to have a major impact on the invasive potential of some plant species. The uncontrolled spread of transgenes is therefore a remaining challenge for regulators. We discuss some of these issues in the context of EU regulations since these regulations explicitly refer to the precautionary principle in the assessment of uncertainties. We found the that the precautionary principle as established in EU Directive 2001/18 can only be applied where efficient measures are available to remove genetically engineered organisms from the environment should this become necessary. If a removal from the environment would not be practically feasible, undesirable developments could not be mitigated.

The introduction of herbicide-tolerant (HT) genetically engineered (GE) soybeans has raised new challenges for the European risk assessment of imported food and feed. Food and feed products derived from these plants may show specific patterns of chemical residues and altered nutritional composition. Furthermore, there has been a substantial increase in the usage of herbicides in soybean production due to the emergence of resistant weeds. This concerns particular glyphosate-based herbicides and also other herbicides. In this review, we give an overview of available data regarding glyphosate application on HT GE soybeans in North and South America. We have further compared this data with herbicide applications in experimental field trials conducted by the industry. We conclude that field trials carried out for risk assessment purposes do not generally represent the real agronomic conditions in commercial HT GE plant cultivation. In most cases, neither the applied dose nor the number of applications match real conditions. This finding is especially relevant for risk assessment since a review of relevant publications shows that the amount and timing of spraying glyphosate as a complementary herbicide onto HT GE plants can impact their composition; this is relevant to EFSA comparative risk assessment of GMOs. Further, closely related issues were identified that overlap with EU GMO and pesticide regulation, but are not currently considered. These issues concern indirect, cumulative and combinatorial effects as well as the assessment of mixed toxicity. Consequently, current risk assessment practice for HT GE plants cannot be considered to fulfil EU regulatory standards which require the safety of food and feed to be demonstrated. It is much more likely that concerns about the health risks of HT GE plant material used for food and feed have been underestimated. We therefore conclude that the EU risk assessment of food and feed derived from HT GE plants needs substantial improvement.

BackgroundMON89788 was the first genetically engineered soybean worldwide to express a Bt toxin. Under the brand name Intacta, Monsanto subsequently engineered a stacked trait soybean using MON89788 and MON87701—this stacked soybean expresses an insecticidal toxin and is, in addition, tolerant to glyphosate. After undergoing risk assessment by the European Food Safety Authority (EFSA), the stacked event was authorised for import into the EU in June 2012, including for use in food and feed. This review discusses the health risks associated with Bt toxins present in these genetically engineered plants and the residues left from spraying with the complementary herbicide.ResultsWe have compared the opinion published by EFSA [1] with findings from other publications in the scientific literature. It is evident that there are several issues that EFSA did not consider in detail and which will need further assessment: (1) There are potential combinatorial effects between plant components and other impact factors that might enhance toxicity. (2) It is known that Bt toxins have immunogenic properties; since soybeans naturally contain many allergens, these immunogenic properties raise specific questions. (3) Fully evaluated and reliable protocols for measuring the Bt concentration in the plants are needed, in addition to a comprehensive set of data on gene expression under varying environmental conditions. (4) Specific attention should be paid to the herbicide residues and their interaction with Bt toxins.ConclusionsThe case of the Intacta soybeans highlights several regulatory problems with Bt soybean plants in the EU. Moreover, many of the issues raised also concern other genetically engineered plants that express insecticidal proteins, or are engineered to be resistant to herbicides, or have those two types of traits combined in stacked events. It remains a matter of debate whether the standards currently applied by the risk assessor, EFSA, and the risk manager, the EU Commission, meet the standards for risk analysis defined in EU regulations such as 1829/2003 and Directive 2001/18. While this publication cannot provide a final conclusion, it allows the development of some robust hypotheses that should be investigated further before such plants can be considered to be safe for health and the environment. In general, the concept of comparative risk assessment needs some major revision. Priority should be given to developing more targeted approaches. As shown in the case of Intacta, these approaches should include: (i) systematic investigation of interactions between the plant genome and environmental stressors as well as their impact on gene expression and plant composition; (ii) detailed investigations of the toxicity of Bt toxins; (iii) assessment of combinatorial effects taking into account long-term effects and the residues from spraying with complementary herbicides; (iv) investigation into the impact on the immune and hormonal systems and (v) investigation of the impact on the intestinal microbiome after consumption....

Gene drive organisms are a recent development created by using methods of genetic engineering; they inherit genetic constructs that are passed on to future generations with a higher probability than with Mendelian inheritance. There are some specific challenges inherent to the environmental risk assessment (ERA) of genetically engineered (GE) gene drive organisms because subsequent generations of these GE organisms might show effects that were not observed or intended in the former generations. Unintended effects can emerge from interaction of the gene drive construct with the heterogeneous genetic background of natural populations and/or be triggered by changing environmental conditions. This is especially relevant in the case of gene drives with invasive characteristics and typically takes dozens of generations to render the desired effect. Under these circumstances, “next generation effects” can substantially increase the spatial and temporal complexity associated with a high level of uncertainty in ERA. To deal with these problems, we suggest the introduction of a new additional step in the ERA of GE gene drive organisms that takes 3 criteria into account: the biology of the target organisms, their naturally occurring interactions with the environment (biotic and abiotic), and their intended biological characteristics introduced by genetic engineering. These 3 criteria are merged to form an additional step in ERA, combining specific “knowns” and integrating areas of “known unknowns” and uncertainties, with the aim of assessing the spatiotemporal controllability of GE gene drive organisms. The establishment of assessing spatiotemporal controllability can be used to define so‐called “cut‐off criteria” in the risk analysis of GE gene drive organisms: If it is likely that GE gene drive organisms escape spatiotemporal controllability, the risk assessment cannot be sufficiently reliable because it is not conclusive. Under such circumstances, the environmental release of the GE gene drive organisms would not be compatible with the precautionary principle (PP). Integr Environ Assess Manag 2020;16:555–568. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)