Rod A. Herman

Recommendations for the design of laboratory studies on non-target arthropods for risk assessment of genetically engineered plants

This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.

Unintended compositional changes in genetically modified (GM) crops: 20 years of research.

The compositional equivalency between genetically modified (GM) crops and nontransgenic comparators has been a fundamental component of human health safety assessment for 20 years. During this time, a large amount of information has been amassed on the compositional changes that accompany both the transgenesis process and traditional breeding methods; additionally, the genetic mechanisms behind these changes have been elucidated. After two decades, scientists are encouraged to objectively assess this body of literature and determine if sufficient scientific uncertainty still exists to continue the general requirement for these studies to support the safety assessment of transgenic crops. It is concluded that suspect unintended compositional effects that could be caused by genetic modification have not materialized on the basis of this substantial literature. Hence, compositional equivalence studies uniquely required for GM crops may no longer be justified on the basis of scientific uncertainty.

Application of food and feed safety assessment principles to evaluate transgenic approaches to gene modulation in crops

New crop varieties containing traits such as enhanced nutritional profiles, increased yield, and tolerance to drought are being developed. In some cases, these new traits are dependent on small RNAs or regulatory proteins such as transcription factors (TF) that modify the expression of endogenous plant genes. To date, the food and feed safety of genetically modified (GM) crops has been assessed by the application of a set of internationally accepted procedures for evaluating the safety of GM crops. The goal of this paper is to review the main aspects of the current safety assessment paradigm and to recommend scientifically sound principles for conducting a safety assessment for GM crops that are developed by technologies that modify endogenous plant gene expression. Key considerations for such a safety assessment include the following: (1) RNA and TF are generally recognized as safe (GRAS); (2) Genes encoding RNAi and regulatory proteins such as TFs are an important component of the plantgenome; (3) Crops engineered using RNAi modifications are not expected to produce heterologous proteins; (4) The modulation of TFs may result in quantitative differences in endogenous plant components,which can be assessed through agronomic performance and compositional analysis on a caseby-case basis.

Binary Insecticidal Crystal Protein from Bacillus thuringiensis, Strain PS149B1: Effects of Individual Protein Components and Mixtures in Laboratory Bioassays

Abstract A family of novel binary insecticidal crystal proteins, with activity against western corn rootworm, Diabrotica virgifera virgifera LeConte, was identified from Bacillus thuringiensis Berliner. A binary insecticidal crystal protein (bICP) from B. thuringiensis strain PS149B1 is composed of a 14-kDa protein (Cry34Ab1) and a 44-kDa protein (Cry35Ab1). These proteins have been co-expressed in transgenic maize plants, Zea mays L., and effectively control western corn rootworm larvae under field conditions. Laboratory experiments were conducted to better understand the contribution of each component protein to the in vivo activity of the bICP. The 14-kDa protein is active alone against southern corn rootworm, Diabrotica undecimpunctata howardi Barber, and was synergized by the 44-kDa protein. In mixtures, the concentration of the 14-kDa protein had a greater impact on efficacy than the 44-kDa component. Although both proteins are clearly required for maximal insecticidal activity, laboratory results did not support the formation of a stable, fixed-ratio complex of the two component proteins.

Compositional assessment of transgenic crops: an idea whose time has passed

Compositional studies comparing transgenic crops with non-transgenic crops are almost universally required by governmental regulatory bodies to support the safety assessment of new transgenic crops. Here we discuss the assumptions that led to this requirement and lay out the theoretical and empirical evidence suggesting that such studies are no more necessary for evaluating the safety of transgenic crops than they are for traditionally bred crops.

Acute and repeated dose (28 day) mouse oral toxicology studies with Cry34Ab1 and Cry35Ab1 Bt proteins used in coleopteran resistant DAS-59122-7 corn.

Expression of the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 in genetically modified maize (event DAS-59122-7) protects the crop from damage due to feeding by Diabrotica larvae including the western corn rootworm (Diabrotica virgifera virgifera). As part of the safety assessment of this maize, mammalian toxicology studies were conducted with heterologously produced Cry34Ab1 and Cry35Ab1 proteins. No evidence of acute toxicity was observed in mice following oral exposure to either the Cry34Ab1 or Cry35Ab1 proteins individually (2700 and 1850 mg/kg, respectively) or concomitantly (482 and 1520 mg/kg, respectively; 1:1 molar ratio). Similarly, no adverse effects were observed in mice in a repeated dose (28 day) dietary toxicity study that incorporated these proteins into diets at concentrations corresponding up to 1000-fold greater than the highest estimate of human exposure based on the concentrations of these proteins expressed in 59122 maize grain. These studies demonstrate that the Cry34Ab1 and Cry35Ab1 proteins do not represent a risk to human health and support previous studies indicating that 59122 maize grain is as safe and wholesome as non-GM maize grain.

Stability of a set of allergens and non-allergens in simulated gastric fluid

Stability in simulated gastric fluid has been suggested as a parameter for consideration in the allergenicity assessment of transgenic proteins. However, the relationship between the stability of proteins in simulated gastric fluid and allergenicity has been inconsistent among studies conducted with reference allergens and non-allergens. Differences in laboratory methods and data interpretation have been implicated as possible causes for conflicting study results. We attempted to mitigate some of the methodological inconsistencies among laboratory methods by applying a kinetic interpretation to results of digestion experiments conducted with a set of known allergens and putative non-allergens. We found that pepsinolysis in simulated gastric fluid generally followed an exponential (pseudo-first-order) pattern of decay, at least during the terminal (slower) phase of digestion, allowing the calculation of digestion half-lives. While digestibility estimates were reproducible and robust, results for the proteins evaluated in this study did not support a significant association between stability in simulated gastric fluid and allergenicity.

Should digestion assays be used to estimate persistence of potential allergens in tests for safety of novel food proteins

Food allergies affect an estimated 3 to 4% of adults and up to 8% of children in developed western countries. Results from in vitro simulated gastric digestion studies with purified proteins are routinely used to assess the allergenic potential of novel food proteins. The digestion of purified proteins in simulated gastric fluid typically progresses in an exponential fashion allowing persistence to be quantified using pseudo-first-order rate constants or half lives. However, the persistence of purified proteins in simulated gastric fluid is a poor predictor of the allergenic status of food proteins, potentially due to food matrix effects that can be significant in vivo. The evaluation of the persistence of novel proteins in whole, prepared food exposed to simulated gastric fluid may provide a more correlative result, but such assays should be thoroughly validated to demonstrate a predictive capacity before they are accepted to predict the allergenic potential of novel food proteins.

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.

Digestion assays in allergenicity assessment of transgenic proteins

The food-allergy risk assessment for transgenic proteins expressed in crops is currently based on a weight-of-evidence approach that holistically considers multiple lines of evidence. This approach recognizes that no single test or property is known to distinguish allergens from nonallergens. The stability of a protein to digestion, as predicted by an in vitro simulated gastric fluid assay, currently is used as one element in the risk assessment process. A review of the literature on the use of the simulated gastric fluid assay to predict the allergenic status of proteins suggests that more extensive kinetic studies with well-characterized reference proteins are required before the predictive value of this assay can be adequately judged.