Criteria to evaluate the reliability of interaction studies with insecticidal proteins.

This paper recommends five criteria to evaluate the reliability of interaction studies with insecticidal proteins. However, these criteria are broadly applicable to an interaction analysis with any type of substance. The recommended criteria reflect the consensus of the literature on interaction analysis from decades of research in fields such as pharmacology and toxicology. The criteria can be used to interrogate the experimental design, assay methodology, data analysis, and interpretation of the results. These criteria will be useful to researchers to help identify the strengths and potential weaknesses of interaction studies and to help define the limits of interpretation of the data. The criteria will also be useful to risk assessors evaluating the reliability of interaction data as part of an environmental risk assessment, and to inform a weight of evidence analysis when there are contradictory results. In addition, these criteria can be used prospectively by researchers to help avoid common pitfalls that are apparent in some interaction studies. Five examples have been provided, with studies from the literature, that demonstrate how these criteria can be objectively and consistently applied to score the reliability of interaction studies with insecticidal proteins that differ in design and methodology.

Sequence-Activity Relationships for the Snf7 Insecticidal dsRNA in Chrysomelidae.

The responsiveness of insects to oral delivery of insecticidal dsRNA has been shown to be dependent on dsRNA length and sequence match. Previous work with the western corn rootworm (WCR, Diabrotica virgifera virgifera; Coleoptera: Chrysomelidae) demonstrated that at least one ≥21 nt match must be present in the DvSnf7 dsRNA of approximately ≥60 base-pairs (bp) for activity. Further data is needed on the activity of <21 nt matches along with characterization of relationship between activity and the number of ≥21 nt matches. To characterize the sequence-activity relationship for insecticidal dsRNA further, the activity of orthologous Snf7 dsRNAs with 19, 20, and 21 nt contiguous matches against WCR was compared. Neither 19 nor 20 nt sequence matches were active, supporting that a ≥21 nt sequence match is required for activity. The relationship between the number of 21 nt matches with activity of Snf7 dsRNA orthologs from several Chrysomelid species was characterized using WCR and Colorado potato beetle (CPB, Leptinotarsa decemlineata; Coleoptera Chrysomelidae). For WCR, there was a strong relationship between an increasing number of 21 nt matches and increased activity (i.e., lower LC50 values). A similar relationship was observed for CPB with an exception for a single ortholog, which may be related to the exceptionally high rate of polymorphisms in CPB. Overall, these results demonstrate a general relationship between the number of 21 nt matches and activity, and this relationship could be used to inform a testing and assessment plan for an ecological risk assessment for an insecticidal dsRNA.

Review and analysis of the potential for glyphosate to interact with the estrogen, androgen and thyroid pathways.

Glyphosate was recently evaluated for its potential to interact with the estrogen, androgen and thyroid (EAT) hormone pathways, including steroidogenesis, under the United States Environmental Protection Agency's (USEPA) Endocrine Disruptor Screening Program (EDSP), then by Germany, the rapporteur Member State who led the European Annex 1 renewal for glyphosate, and then by the European Food Protection Agency (EFSA) also as part of the Annex 1 renewal for glyphosate. Under the EDSP, 11 Tier 1 assays were run following the USEPA's validated 890-series test guidelines and included five in vitro and six in vivo assays to evaluate the EAT pathways. Steroidogenesis was evaluated as part of the estrogen and androgen pathways. An up-to-date critical review has been conducted that considered results from the EDSP Tier 1 battery, guideline regulatory studies and an in-depth analysis of the literature studies that informed an endocrine assessment. A strength of this evaluation was that it included data across multiple levels of biological organization, and mammalian and nonmammalian test systems. There was strong agreement across the in vitro and in vivo Tier 1 battery, guideline studies and relevant literature studies, demonstrating that glyphosate does not interact with EAT pathways including steroidogenesis. Based on an analysis of the comprehensive toxicology database for glyphosate and the literature, this review has concluded that glyphosate does not have endocrine-disrupting properties through estrogen, androgen, thyroid and steroidogenic modes of action. © 2020 Society of Chemical Industry.

Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Assessing the Potential for Interaction in Insecticidal Activity Between MON 87751 × MON 87701 Produced by Conventional Breeding.

Pyramiding (combining) of plant incorporated protectants (PIPs) with insecticidal activity in genetically engineered crops is a strategy used to improve efficacy as well as delay potential resistance for a specific group of targets. In some countries, a regulatory risk assessment is required for breeding "stacks" expressing multiple PIPs and these countries may require an assessment of potential interaction among the PIPs. This study evaluated whether combining soybean events MON 87551 and MON 87701 results in a toxicological interaction that effects a species that is controlled by each event. MON 87751 coexpresses the Cry1A.105 and Cry2Ab2 proteins and MON 87701 expresses the Cry1Ac protein. EC50 values for MON 87751 and MON 87701 were comparable in diet-incorporation bioassays using corn earworm (Lepidoptera: Noctuidae, Helicoverpa zea) and the observed combined activity of the stack was consistent with predictions of additivity (i.e., no interaction). Under the concentration and response addition models, predicted and observed median effect levels differed by <10%. These results demonstrate independent action at the median effect level between the insecticidal activity of MON 87751 and MON 87701. Taken together, no interaction between these PIPs and acceptable margins of safety for the individual proteins to nontarget organisms, it is appropriate to bridge back to the risk assessments for the individual products that demonstrated environmental safety of stack products containing both MON 87751 and MON 87701.

Overcoming Challenges of Incorporating Higher Tier Data in Ecological Risk Assessments and Risk Management of Pesticides in the United States: Findings and Recommendations from the 2017 Workshop on Regulation and Innovation in Agriculture.

Pesticide regulation requires regulatory authorities to assess the potential ecological risk of pesticides submitted for registration, and most risk assessment schemes use a tiered testing and assessment approach. Standardized ecotoxicity tests, environmental fate studies, and exposure models are used at lower tiers and follow well-defined methods for assessing risk. If a lower tier assessment indicates that the pesticide may pose an ecological risk, higher tier studies using more environmentally realistic conditions or assumptions can be performed to refine the risk assessment and inform risk management options. However, there is limited guidance in the United States on options to refine an assessment and how the data will be incorporated into the risk assessment and risk management processes. To overcome challenges to incorporation of higher tier data into ecological risk assessments and risk management of pesticides, a workshop was held in Raleigh, North Carolina. Attendees included representatives from the United States Environmental Protection Agency, United States Department of Agriculture, National Oceanic and Atmospheric Administration, universities, commodity groups, consultants, nonprofit organizations, and the crop protection industry. Key recommendations emphasized the need for 1) more effective, timely, open communication among registrants, risk assessors, and risk managers earlier in the registration process to identify specific protection goals, address areas of potential concern where higher tier studies or assessments may be required, and if a higher tier study is necessary that there is agreement on study design; 2) minimizing the complexity of study designs while retaining high value to the risk assessment and risk management process; 3) greater transparency regarding critical factors utilized in risk management decisions with clearly defined protection goals that are operational; and 4) retrospective analyses of success-failure learnings on the acceptability of higher tier studies to help inform registrants on how to improve the application of such studies to risk assessments and the risk management process. Integr Environ Assess Manag 2019;15:714-725. © 2019 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Distinguishing between endocrine disruption and non-specific effects on endocrine systems.

The endocrine system is responsible for growth, development, maintaining homeostasis and for the control of many physiological processes. Due to the integral nature of its signaling pathways, it can be difficult to distinguish endocrine-mediated adverse effects from transient fluctuations, adaptive/compensatory responses, or adverse effects on the endocrine system that are caused by mechanisms outside the endocrine system. This is particularly true in toxicological studies that require generation of effects through the use of Maximum Tolerated Doses (or Concentrations). Endocrine-mediated adverse effects are those that occur as a consequence of the interaction of a chemical with a specific molecular component of the endocrine system, for example, a hormone receptor. Non-endocrine-mediated adverse effects on the endocrine system are those that occur by other mechanisms. For example, systemic toxicity, which perturbs homeostasis and affects the general well-being of an organism, can affect endocrine signaling. Some organs/tissues can be affected by both endocrine and non-endocrine signals, which must be distinguished. This paper examines in vitro and in vivo endocrine endpoints that can be altered by non-endocrine processes. It recommends an evaluation of these issues in the assessment of effects for the determination of endocrine disrupting properties of chemicals. This underscores the importance of using a formal weight of evidence (WoE) process to evaluate potential endocrine activity.

Review and recommendations on criteria to evaluate the relevance of pesticide interaction data for ecological risk assessments.

Mixing pesticides with different modes of action can provide a wider spectrum of control with fewer applications compared to using single active ingredients and is essential for comprehensive management of pest resistance. Mixture studies with pesticides are performed to assess compatibility, combined efficacy, and potential for toxicological interactions that damage crops. The purpose of this paper is to review and recommend previously published scientific criteria for evaluating the quality, relevance and interpretability of data on toxicological interactions and to demonstrate a methodology for applying them objectively to mixtures studies used in ecological risk assessment. The recommended criteria reflect the consensus of the literature on interaction analysis from decades of research in pharmacology and toxicology and are broadly applicable to mixtures of drugs, pesticides, industrial chemicals and food additives. They are useful for researchers who design and analyze interaction studies, for risk assessors who use interaction data in risk assessments, and for those who make risk management decisions pertaining to pesticides. This paper describes our methodology for assessing data on the combined activity of pesticides and then discusses how to interpret such data in the context of an ecological risk assessment. Examples have been drawn primarily from studies with herbicides and nontarget plants, and several example analyses have been included that can inform whether mixture data are sufficiently reliable and relevant for use in regulatory decision making.

A critical review of the environmental occurrence and potential effects in aquatic vertebrates of the potent androgen receptor agonist 17ß-trenbolone.

Trenbolone acetate is widely used in some parts of the world for its desirable anabolic effects on livestock. Several metabolites of the acetate, including 17ß-trenbolone, have been detected at low nanograms per liter concentrations in surface waters associated with animal feedlots. The 17ß-trenbolone isomer can affect androgen receptor signaling pathways in various vertebrate species at comparatively low concentrations/doses. The present article provides a comprehensive review and synthesis of the existing literature concerning exposure to and biological effects of 17ß-trenbolone, with an emphasis on potential risks to aquatic animals. In vitro studies indicate that, although 17ß-trenbolone can activate several nuclear hormone receptors, its highest affinity is for the androgen receptor in all vertebrate taxa examined, including fish. Exposure of fish to nanograms per liter water concentrations of 17ß-trenbolone can cause changes in endocrine function in the short term, and adverse apical effects in longer exposures during development and reproduction. Impacts on endocrine function typically are indicative of inappropriate androgen receptor signaling, such as changes in sex steroid metabolism, impacts on gonadal stage, and masculinization of females. Exposure of fish to 17ß-trenbolone during sexual differentiation in early development can greatly skew sex ratios, whereas adult exposures can adversely impact fertility and fecundity. To fully assess ecosystem-level risks, additional research is warranted to address uncertainties as to the degree/breadth of environmental exposures and potential population-level effects of 17ß-trenbolone in sensitive species. Environ Toxicol Chem 2018;37:2064-2078. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances.

A SETAC Pellston Workshop® "Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)" was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS-not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17ß-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267-279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16.

The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some agricultural ecosystems (e.g. pollinators, decomposers, and natural enemies).

Aquatic fate of a double-stranded RNA in a sediment---water system following an over-water application.

Determining the rate of biodegradation of double-stranded RNA (dsRNA) in the environment is an essential element of a comprehensive risk assessment of an RNA-based agricultural product. This information is used during problem formulation to define relevant routes and durations of environmental exposure for in planta-expressed dsRNA. Although exposure to biotechnology-derived crops expressing dsRNA traits in the aquatic environment is predicted to be minimal, little is known regarding the fate of dsRNA in these environments. To assess exposure to aquatic environments, a study was conducted to measure the rate of biodegradation of DvSnf7 dsRNA in aerobic water-sediment systems. Aquatic systems containing natural water and sediments that varied in physical and chemical characteristics were treated with dsRNA by applying DvSnf7 dsRNA directly to the water column. In the present study, DvSnf7 dsRNA dissipated rapidly from the water phase and was undetectable within 7 d as measured by QuantiGene (Affymetrix) and a sensitive insect bioassay in these diverse systems. Degradation kinetics estimated a half-life (time to 50% dissipation [DT50]) of less than 3 d and a time to 90% dissipation of approximately 4 d. Further analysis indicated that DvSnf7 dsRNA had DT50 values of less than 6 d in both sediment-free systems containing natural water and systems with only sediment. Taken together, the results of the present study indicate that dsRNA-based agricultural products rapidly degrade and consequently are unlikely to persist in aquatic environments. Environ Toxicol Chem 2017;36:727-734. © 2016 SETAC.

Current limitations and recommendations to improve testing for the environmental assessment of endocrine active substances.

In the present study, existing regulatory frameworks and test systems for assessing potential endocrine active chemicals are described, and associated challenges are discussed, along with proposed approaches to address these challenges. Regulatory frameworks vary somewhat across geographies, but all basically evaluate whether a chemical possesses endocrine activity and whether this activity can result in adverse outcomes either to humans or to the environment. Current test systems include in silico, in vitro, and in vivo techniques focused on detecting potential endocrine activity, and in vivo tests that collect apical data to detect possible adverse effects. These test systems are currently designed to robustly assess endocrine activity and/or adverse effects in the estrogen, androgen, and thyroid hormone signaling pathways; however, there are some limitations of current test systems for evaluating endocrine hazard and risk. These limitations include a lack of certainty regarding: 1) adequately sensitive species and life stages; 2) mechanistic endpoints that are diagnostic for endocrine pathways of concern; and 3) the linkage between mechanistic responses and apical, adverse outcomes. Furthermore, some existing test methods are resource intensive with regard to time, cost, and use of animals. However, based on recent experiences, there are opportunities to improve approaches to and guidance for existing test methods and to reduce uncertainty. For example, in vitro high-throughput screening could be used to prioritize chemicals for testing and provide insights as to the most appropriate assays for characterizing hazard and risk. Other recommendations include adding endpoints for elucidating connections between mechanistic effects and adverse outcomes, identifying potentially sensitive taxa for which test methods currently do not exist, and addressing key endocrine pathways of possible concern in addition to those associated with estrogen, androgen, and thyroid signaling. Integr Environ Assess Manag 2017;13:302-316. © 2016 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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.

Characterizing a novel and sensitive method to measure dsRNA in soil.

Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics.

Assessing the potential for interaction between the insecticidal activity of two genetically engineered cotton events combined by conventional breeding: An example with COT102 × MON 15985.

Bollgard(®) III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex(®) cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity.

Glyphosate and aminomethylphosphonic acid chronic risk assessment for soil biota.

Glyphosate is a broad-spectrum herbicide used widely in agriculture, horticulture, private gardens, and public infrastructure, where it is applied to areas such as roadsides, railway tracks, and parks to control the growth of weeds. The exposure risk from glyphosate and the primary soil metabolite aminomethylphosphonic acid (AMPA) on representative species of earthworms, springtails, and predatory soil mites and the effects on nitrogen-transformation processes by soil microorganisms were assessed under laboratory conditions based on internationally recognized guidelines. For earthworms, the reproductive no-observed-effect concentration (NOEC) was 472.8 mg glyphosate acid equivalent (a.e.)/kg dry soil, which was the highest concentration tested, and 198.1 mg/kg dry soil for AMPA. For predatory mites, the reproductive NOEC was 472.8 mg a.e./kg dry soil for glyphosate and 320 mg/kg dry soil for AMPA, the highest concentrations tested. For springtails, the reproductive NOEC was 472.8 mg a.e./kg dry soil for glyphosate and 315 mg/kg dry soil for AMPA, the highest concentrations tested. Soil nitrogen-transformation processes were unaffected by glyphosate and AMPA at 33.1 mg a.e./kg soil and 160 mg/kg soil, respectively. Comparison of these endpoints with worst-case soil concentrations expected for glyphosate (6.62 mg a.e./kg dry soil) and AMPA (6.18 mg/kg dry soil) for annual applications at the highest annual rate of 4.32 kg a.e./ha indicate very low likelihood of adverse effects on soil biota. Environ Toxicol Chem 2016;35:2742-2752. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

No impact of DvSnf7 RNA on honey bee (Apis mellifera L.) adults and larvae in dietary feeding tests.

The honey bee (Apis mellifera L.) is the most important managed pollinator species worldwide and plays a critical role in the pollination of a diverse range of economically important crops. This species is important to agriculture and historically has been used as a surrogate species for pollinators to evaluate the potential adverse effects for conventional, biological, and microbial pesticides, as well as for genetically engineered plants that produce pesticidal products. As part of the ecological risk assessment of MON 87411 maize, which expresses a double-stranded RNA targeting the Snf7 ortholog (DvSnf7) in western corn rootworm (Diabrotica virgifera virgifera), dietary feeding studies with honey bee larvae and adults were conducted. Based on the mode of action of the DvSnf7 RNA in western corn rootworm, the present studies were designed to be of sufficient duration to evaluate the potential for adverse effects on larval survival and development through emergence and adult survival to a significant portion of the adult stage. Testing was conducted at concentrations of DvSnf7 RNA that greatly exceeded environmentally relevant exposure levels based on expression levels in maize pollen. No adverse effects were observed in either larval or adult honey bees at these high exposure levels, providing a large margin of safety between environmental exposure levels and no-observed-adverse-effect levels.

A novel method of demonstrating the molecular and functional equivalence between in vitro and plant-produced double-stranded RNA.

A biotechnology-derived corn variety, MON 87411, containing a suppression cassette that expresses an inverted repeat sequence that matches the sequence of western corn rootworm (WCR; Diabrotica virgifera virgifera) has been developed. The expression of the cassette results in the formation of a double-stranded RNA (dsRNA) transcript containing a 240 bp fragment of the WCR Snf7 gene (DvSnf7) that confers resistance to corn rootworm by suppressing levels of DvSnf7 mRNA in WCR after root feeding. Internationally accepted guidelines for the assessment of genetically modified crop products have been developed to ensure that these plants are as safe for food, feed, and environmental release as their non-modified counterparts (Codex, 2009). As part of these assessments MON 87411 must undergo an extensive environmental assessment that requires large quantities of DvSnf7 dsRNA that was produced by in vitro transcription (IVT). To determine if the IVT dsRNA is a suitable surrogate for the MON 87411-produced DvSnf7 dsRNA in regulatory studies, the nucleotide sequence, secondary structure, and functional activity of each were characterized and demonstrated to be comparable. This comprehensive characterization indicates that the IVT DvSnf7 dsRNA is equivalent to the MON 87411-produced DvSnf7 dsRNA and it is a suitable surrogate for regulatory studies.

The food and environmental safety of Bt crops.

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.