Use of species sensitivity distributions to characterize hazard for insecticidal traits.

Species sensitivity distributions (SSDs) have a long history of use within environmental risk assessment to help make informed decisions about the potential risks associated with a variety of environmental stressors. Current risk assessments for transgenic crops conveying insect protection incorporate bioassays conducted on both pest and non-pest insects. As technology developers commercialize new insecticidal traits, SSDs combine the results of these individual studies to construct what may be a better picture of potential environmental risks. While SSDs have been used by technology developers and regulatory agencies to support transgenic crop risk assessments, they are not yet routine in the industry.

An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins.

Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([(3)H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk ß-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles.

Experiences in Engaging the Public on Biotechnology Advances and Regulation.

Public input is often sought as part of the biosafety decision-making process. Information and communication about the advances in biotechnology are part of the first step to engagement. This step often relies on the developers and introducers of the particular innovation, for example, an industry-funded website has hosted various authorities to respond to questions from the public. Alternative approaches to providing information have evolved, as demonstrated in sub-Saharan Africa where non-governmental organizations and associations play this role in some countries and subregions. Often times, those in the public who choose to participate in engagement opportunities have opinions about the overall biosafety decision process. Case-by-case decisions are made within defined regulatory frameworks, however, and in general, regulatory consultation does not provide the opportunity for input to the overall decision-making process. The various objectives on both sides of engagement can make the experience challenging; there are no clear metrics for success. The situation is challenging because public input occurs within the context of the local legislative framework, regulatory requirements, and the peculiarities of the fairly recent biosafety frameworks, as well as of public opinion and individual values. Public engagement may be conducted voluntarily, or may be driven by legislation. What can be taken into account by the decision makers, and therefore what will be gathered and the timing of consultation, also may be legally defined. Several practical experiences suggest practices for effective engagement within the confines of regulatory mandates: (1) utilizing a range of resources to facilitate public education and opportunities for understanding complex technologies; (2) defining in advance the goal of seeking input; (3) identifying and communicating with the critical public groups from which input is needed; (4) using a clearly defined approach to gathering and assessing what will be used in making the biosafety decision; and (5) communicating using clear and simple language. These practices create a foundation for systematic methods to gather, acknowledge, respond to, and even incorporate public input. Applying such best practices will increase transparency and optimize the value of input from the public.

Dynamic role and importance of surrogate species for assessing potential adverse environmental impacts of genetically engineered insect-resistant plants on non-target organisms.

Surrogate species have a long history of use in research and regulatory settings to understand the potentially harmful effects of toxic substances including pesticides. More recently, surrogate species have been used to evaluate the potential effects of proteins contained in genetically engineered insect resistant (GEIR) crops. Species commonly used in GEIR crop testing include beneficial organisms such as honeybees, arthropod predators, and parasitoids. The choice of appropriate surrogates is influenced by scientific factors such as the knowledge of the mode of action and the spectrum of activity as well as societal factors such as protection goals that assign value to certain ecosystem services such as pollination or pest control. The primary reasons for using surrogates include the inability to test all possible organisms, the restrictions on using certain organisms in testing (e.g., rare, threatened, or endangered species), and the ability to achieve greater sensitivity and statistical power by using laboratory testing of certain species. The acceptance of surrogate species data can allow results from one region to be applied or "transported" for use in another region. On the basis of over a decade of using surrogate species to evaluate potential effects of GEIR crops, it appears that the current surrogates have worked well to predict effects of GEIR crops that have been developed (Carstens et al. GM Crops Food 5:1-5, 2014), and it is expected that they should work well to predict effects of future GEIR crops based on similar technologies.

Emerging Agricultural Biotechnologies for Sustainable Agriculture and Food Security.

As global populations continue to increase, agricultural productivity will be challenged to keep pace without overtaxing important environmental resources. A dynamic and integrated approach will be required to solve global food insecurity and position agriculture on a trajectory toward sustainability. Genetically modified (GM) crops enhanced through modern biotechnology represent an important set of tools that can promote sustainable agriculture and improve food security. Several emerging biotechnology approaches were discussed in a recent symposium organized at the 13th IUPAC International Congress of Pesticide Chemistry meeting in San Francisco, CA, USA. This paper summarizes the innovative research and several of the new and emerging technologies within the field of agricultural biotechnology that were presented during the symposium. This discussion highlights how agricultural biotechnology fits within the context of sustainable agriculture and improved food security and can be used in support of further development and adoption of beneficial GM crops.

Building Better Environmental Risk Assessments.

Risk assessment is a reasoned, structured approach to address uncertainty based on scientific and technical evidence. It forms the foundation for regulatory decision-making, which is bound by legislative and policy requirements, as well as the need for making timely decisions using available resources. In order to be most useful, environmental risk assessments (ERAs) for genetically modified (GM) crops should provide consistent, reliable, and transparent results across all types of GM crops, traits, and environments. The assessments must also separate essential information from scientific or agronomic data of marginal relevance or value for evaluating risk and complete the assessment in a timely fashion. Challenges in conducting ERAs differ across regulatory systems - examples are presented from Canada, Malaysia, and Argentina. One challenge faced across the globe is the conduct of risk assessments with limited resources. This challenge can be overcome by clarifying risk concepts, placing greater emphasis on data critical to assess environmental risk (for example, phenotypic and plant performance data rather than molecular data), and adapting advances in risk analysis from other relevant disciplines.

Transgenic maize event TC1507: Global status of food, feed, and environmental safety.

Maize (Zea mays) is a widely cultivated cereal that has been safely consumed by humans and animals for centuries. Transgenic or genetically engineered insect-resistant and herbicide-tolerant maize, are commercially grown on a broad scale. Event TC1507 (OECD unique identifier: DAS-Ø15Ø7-1) or the Herculex®(#) I trait, an insect-resistant and herbicide-tolerant maize expressing Cry1F and PAT proteins, has been registered for commercial cultivation in the US since 2001. A science-based safety assessment was conducted on TC1507 prior to commercialization. The safety assessment addressed allergenicity; acute oral toxicity; subchronic toxicity; substantial equivalence with conventional comparators, as well as environmental impact. Results from biochemical, physicochemical, and in silico investigations supported the conclusion that Cry1F and PAT proteins are unlikely to be either allergenic or toxic to humans. Also, findings from toxicological and animal feeding studies supported that maize with TC1507 is as safe and nutritious as conventional maize. Maize with TC1507 is not expected to behave differently than conventional maize in terms of its potential for invasiveness, gene flow to wild and weedy relatives, or impact on non-target organisms. These safety conclusions regarding TC1507 were acknowledged by over 20 regulatory agencies including United States Environment Protection Agency (US EPA), US Department of Agriculture (USDA), Canadian Food Inspection Agency (CFIA), and European Food Safety Authority (EFSA) before authorizing cultivation and/or food and feed uses. A comprehensive review of the safety studies on TC1507, as well as some benefits, are presented here to serve as a reference for regulatory agencies and decision makers in other countries where authorization of TC1507 is or will be pursued.

Bringing policy relevance and scientific discipline to environmental risk assessment for genetically modified crops.

Although public opinion is important in deciding what is valued by society, governments have determined that scientific expertise is required to evaluate potential environmental effects of genetically modified (GM) crops. We suggest how to evaluate rigorously the environmental effects of GM crops in the context of a scientific investigation. Following a disciplined scientific approach to environmental risk assessment (ERA) for GM crops should help resolve controversy in identifying and addressing risk.

Assessing the ecological risks from the persistence and spread of feral populations of insect-resistant transgenic maize.

One source of potential harm from the cultivation of transgenic crops is their dispersal, persistence and spread in non-agricultural land. Ecological damage may result from such spread if the abundance of valued species is reduced. The ability of a plant to spread in non-agricultural habitats is called its invasiveness potential. The risks posed by the invasiveness potential of transgenic crops are assessed by comparing in agronomic field trials the phenotypes of the crops with the phenotypes of genetically similar non-transgenic crops known to have low invasiveness potential. If the transgenic and non-transgenic crops are similar in traits believed to control invasiveness potential, it may be concluded that the transgenic crop has low invasiveness potential and poses negligible ecological risk via persistence and spread in non-agricultural habitats. If the phenotype of the transgenic crop is outside the range of the non-transgenic comparators for the traits controlling invasiveness potential, or if the comparative approach is regarded as inadequate for reasons of risk perception or risk communication, experiments that simulate the dispersal of the crop into non-agricultural habitats may be necessary. We describe such an experiment for several commercial insect-resistant transgenic maize events in conditions similar to those found in maize-growing regions of Mexico. As expected from comparative risk assessments, the transgenic maize was found to behave similarly to non-transgenic maize and to be non-invasive. The value of this experiment in assessing and communicating the negligible ecological risk posed by the low invasiveness potential of insect-resistant transgenic maize in Mexico is discussed.

Three-year field monitoring of Cry1F, event DAS-01507-1, maize hybrids for nontarget arthropod effects.

Field studies were conducted over a 3-yr period to investigate the potential effects of cultivating transgenic maize hybrids containing a Cry1F insect-resistant protein on nontarget arthropod abundance. The narrow spectrum of activity of Cry1F against a subset of lepidopteran pest species would not suggest broad-spectrum effects on nontarget arthropods. However, because of the insecticidal nature of Bt proteins, an alternate hypothesis is that some nontargets may be affected by exposure to the protein. To examine this hypothesis at the field level, monitoring for nontarget organism abundance was initiated at four locations across the U.S. Corn Belt from 2004 through 2006. At each location, paired fields (approximately 0.8 ha each) of commercial Cry1F maize hybrids and isogenic nontransgenic control hybrids were planted. Sampling methods used to monitor nontarget organisms included visual surveillance, sticky cards, pitfall traps, and litterbags. Data were analyzed using multivariate analyses to look for a general community level response to the treatments. Analysis of variance was conducted on individual taxa to detect differences distinct from the primary community response. Community level analyses of the nontarget arthropod abundance showed no significant impact on community abundance when comparing Bt with non-Bt maize fields. Analyses of the individual taxa also showed no significant differences in abundance between Bt and non-Bt fields. Results of these studies confirm earlier laboratory testing and support the hypothesis that Cry1F maize does not produce adverse effects on nontarget arthropods occurring in maize fields.

Assessment of risk of insect-resistant transgenic crops to nontarget arthropods.

An international initiative is developing a scientifically rigorous approach to evaluate the potential risks to nontarget arthropods (NTAs) posed by insect-resistant, genetically modified (IRGM) crops. It adapts the tiered approach to risk assessment that is used internationally within regulatory toxicology and environmental sciences. The approach focuses on the formulation and testing of clearly stated risk hypotheses, making maximum use of available data and using formal decision guidelines to progress between testing stages (or tiers). It is intended to provide guidance to regulatory agencies that are currently developing their own NTA risk assessment guidelines for IRGM crops and to help harmonize regulatory requirements between different countries and different regions of the world.

Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats.

59122 is a transgenic maize line containing event DAS-59122-7 that expresses the corn rootworm (CRW) specific pesticidal Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 and the phosphinothricin-N-acetyltransferase (PAT) protein from Streptomyces viridochromogenes for tolerance to the herbicidal ingredient glufosinate-ammonium. For the current study, 59122 maize grain, non-transgenic near-isogenic maize grain (091), and a commercially available non-transgenic reference maize grain (33R77) were grown under conditions simulating commercial farming practices. Adult Sprague-Dawley rats (12/sex/group) were fed diets formulated with 35% maize grain from either 59122, 091, or 33R77, or one of two separate lots of commercially available rodent chow prepared with commercially available corn (35%) in accordance with the standards of Purina Mills Labdiet 5002 for approximately 90 days. All diets possessed similar nutritional and contaminant profiles. The transgenic proteins were detected only in diets prepared with 59122 maize grain and were stable over the course of the study. Compared to control groups, no adverse diet-related differences were observed in rats fed diets formulated with 59122 maize grain with respect to body weight/gain, food consumption/efficiency, clinical signs of toxicity, mortality, ophthalmology, neurobehavioral (FOB and motor activity) assessments, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), and pathology (organ weights and gross and microscopic pathology). Results from this study indicate that 59122 maize grain is nutritionally equivalent to and as safe as conventional maize grain.

Thirteen week feeding study with transgenic maize grain containing event DAS-Ø15Ø7-1 in Sprague-Dawley rats.

Maize line 1507, containing event DAS-Ø15Ø7-1 (1507), is a genetically modified (GM) maize plant that expresses the cry1F gene from Bacillus thuringiensis (Bt) sbsp. aizawai and the phosphinothricin-N-acetyltransferase (pat) gene from Streptomyces viridochromogenes throughout the plant including in the grain expression of the Cry1F protein confers in planta resistance to the European corn borer (ECB; Ostrinia nubilalis Hübner: Crambidae) and other lepidopteran pests. Expression of the PAT protein confers tolerance to the herbicidal active ingredient glufosinate-ammonium. The current study evaluated the nutritional performance of rats fed diets containing 1507 maize grain in a subchronic rodent feeding study. The grains in this study, 1507, its near-isogenic control (33P66), and a non-GM commercial hybrid (33J56) contained similar amounts of proximates, amino acids, minerals, anti-nutrients, and secondary metabolites. The subchronic feeding study compared standard toxicology response variables in rats fed diets containing 1507 maize grain with those in rats fed diets containing non-GM maize grains. All diets were prepared according to the specifications of PMI Nutrition International, LLC Certified Rodent LabDiet 5002 (PMI) 5002). Diets were fed ad libitum to Sprague-Dawley rats for approximately 90 days. In-life response variables included indicators of dietary performance and weekly evaluations for clinical signs of toxicity. No toxicologically significant differences were observed in the nutritional performance variables, clinical and neurobehavioral signs, ophthalmology, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), organ weights, and gross and microscopic pathology between any pair of treatment groups. These results demonstrate that 1507 maize grain is as safe and as nutritious as non-GM maize grain.

A tiered system for assessing the risk of genetically modified plants to non-target organisms.

Representatives of the developers of modern agricultural biotechnology are proposing a tiered approach for conducting non-target organism risk assessment for genetically modified (GM) plants in Europe. The approach was developed by the Technical Advisory Group of the EuropaBio Plant Biotechnology Unit (http://www.europabio.org/TAG.htm) and complements other international activities to harmonize risk assessment. In the European Union (EU), the principles and methods to be followed in an environmental risk assessment for the placing on the market of GM plants are laid out in Annex II of Directive 2001/18/EC on the deliberate release into the environment of GMOs, Commission Decision 2002/623/EC and Regulation (EC) No. 1829/2003. Additional information is provided in the European Food Safety Authority guidance document of 2004. However, risk assessment for effects to non-target organisms could benefit from further clarification and remains the subject of much discussion in Europe. The industry-wide approach developed by EuropaBio is based on the fundamental steps of risk evaluation, namely hazard and exposure assessment. It follows a structured scheme including assessment planning, product characterization and assessment of hazard/exposure (Tier 0), single high dose and dose response testing (Tier 1), refined hazard characterization and exposure assessment (Tier 2) and further refined risk assessment experiments (Tier 3). An additional tier (Tier 4) was included to reflect the fact that post-market activities such as monitoring are required under Directive 2001/18/EC. The approach is compatible with conditions of commercial release in the EU and around the world.