Results of a 90-day safety assurance study with rats fed grain from corn rootworm-protected corn

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Abstract

The results of a 90-day rat feeding study with YieldGard® (YieldGard Rootworm Corn is a registered trademark of Monsanto Technology, LLC.) Rootworm corn (MON 863) grain that is protected against feeding damage caused by corn rootworm larvae are presented. Corn rootworm-protection was accomplished through the introduction of a cry3Bb1 coding sequence into the corn genome for in planta production of a modified Cry3Bb1 protein from Bacillus thuringiensis. Grain from MON 863 and its near isogenic control were separately formulated into rodent diets at levels of 11% and 33% (w/w) by Purina Mills, Inc. Additionally, six groups of rats were fed diets containing grain from different conventional (non-biotechnology-derived) reference varieties. The responses of rats fed diets containing MON 863 were compared to those of rats fed grain from conventional corn varieties. All diets were nutritionally balanced and conformed to Purina Mills, Inc. specifications for Certified LabDiet 5002. There were a total of 400 rats in the study divided into 10 groups of 20 rats/sex/group. Overall health, body weight gain, food consumption, clinical pathology parameters (hematology, blood chemistry, urinalysis), organ weights, gross and microscopic appearance of tissues were comparable between groups fed diets containing MON 863 and conventional corn varieties. This study complements extensive agronomic, compositional and farm animal feeding studies with MON 863 grain, confirming that it is as safe and nutritious as existing conventional corn varieties.

Introduction

Global regulatory authorities require that food derived from crops produced through biotechnology be as safe as food produced from conventionally bred crops. There must be “reasonable certainty that no harm will result from intended uses under the anticipated conditions of consumption” (OECD, 1993).

The World Health Organization (WHO, 1995), the United Nations Food and Agricultural Organization (WHO, 1991, FAO, 1996), the Organization for Economic Cooperation and Development (OECD, 1993, OECD, 1997), the Codex Alimentarius Commission (Codex, 2003) and the European Food Safety Agency (EFSA, 2004a) established a safety assessment process to assure that foods produced from these new products are as safe as food produced from conventionally bred crops. This assessment process considers two main categories of potential risk; those related to the properties and function of the introduced protein(s), and those resulting from insertion of the introduced gene(s) into the plant genome that might theoretically cause unintended (pleiotropic) effects. The risk assessment for a new biotechnology-derived crop is a comparative safety assessment using conventional food with a history of safe consumption as the reference point for all comparisons. The outcome of this assessment is to determine whether the genetically modified crop is comparable to the existing conventionally bred crop. Newly introduced proteins are subject to a separate safety assessment.

Corn rootworm-protected corn event MON 863 (hereafter referred to as MON 863) marketed under the brand name YieldGard®1 Rootworm corn was produced by insertion of DNA sequences that encode a modified Bacillus thuringiensis (subspecies kumamotoensis) Cry3Bb1 protein that is selectively toxic to Coleopteran species such as corn rootworm larvae (Diabrotica sp.). The genetic insert in MON 863 also contains the coding sequence for the selectable marker, neomycin phosphotransferase type II (NPTII) that is also produced in the plant. Corn varieties containing MON 863 are afforded a level of root protection from corn rootworm (CRW) larval feeding that is comparable or superior to that offered by currently available conventional chemical insecticides, thereby enabling the reduction in the use of these chemical insecticides.

The deduced amino acid sequence of the B.t. Cry protein (653 amino acids) produced in MON 863 is >98.9% identical to that of the Cry3Bb1 protein contained in the foliar-applied commercial B.t. microbial product, Raven® Oil Flowable Bioinsecticide (Ecogen, Inc.). The United States Environmental Protection Agency (EPA) recently established an exemption from the requirement of a tolerance specifically for Cry3Bb1 protein in corn commodities (EPA, 2004). The conclusion of reasonable certainty of no harm and the resultant tolerance exemptions for this protein in food or feed is based on the lack of adverse effects in mammals in numerous toxicological studies. EPA has granted tolerance exemptions for many B.t. Cry proteins based on the results of extensive toxicity testing which show no adverse effects and a history of safe use in agriculture for over 45 years for B.t. microbial sprays (McClintock et al., 1995, EPA, 1998, Betz et al., 2000, Siegel, 2001, WHO, 1999, Federici, 2002).

The bacteria Escherichia coli was the source of the nptII gene isolated from prokaryotic transposon Tn5. The enzyme (264 amino acids) encoded by this gene (i.e. NPTII) is a widely used dominant selectable marker that was used in the development of MON 863. E. coli is ubiquitous in nature and found in the digestive tracts of vertebrate species, including humans (Jefferson et al., 1986). E. coli strains are commonly used as protein production systems in many commercial applications (Bogosian and Kane, 1991). Safety of the donor organism, E. coli, has previously been assessed by FDA as part of the consultation process for other transformed crops that contain the same nptII gene (FDA, 1998). In a recent publication from the European Network on Safety Assessment of Genetically Modified (GM) Food Crops (ENTRANSFOOD), the NPTII protein was assigned to group 1, antibiotic markers that pose the least risk for spread of antibiotic resistance genes in the environment (van den Eede et al., 2004). The authors acknowledged a 13 year history of safe use of the NPTII marker in agricultural food crops.

The second aspect of this safety assessment includes testing for potential pleiotropic effects resulting from insertion of the cry3Bb1 and nptII coding sequence into the MON 863 genome. Testing involves a comparative safety assessment of MON 863 with conventionally bred corn varieties (Dybing et al., 2002). The comparative safety assessment includes three main components: (1) field assessments of key agronomic parameters, (2) compositional assessment of corn grain and forage, and (3) nutritional/safety assessment of MON 863 grain and forage fed to livestock.

Agronomic assessments for corn include examination of various parameters such as yield, plant height, silk date, dropped ears, stalk rating, root strength, plant vigor, and susceptibility to pathogens/pests, etc. (Astwood and Fuchs, 2000). These parameters have been measured by corn breeders following many years of conventional breeding of corn, and are used in selecting varieties that have optimal agronomic characteristics to ensure performance in the market place. Agronomic equivalency trials with multiple MON 863 varieties conducted at multiple locations demonstrate that MON 863 is agronomically equivalent to its near isogenic conventional counterparts except for the intentionally added corn rootworm-protected phenotype (Ward, 2001).

Compositional assessments included a comparison of 51 nutritional biochemical components of MON 863 to conventional corn grown during 1999 in the United States, and Argentina. The field trials and compositional analyses were carried out in compliance with EPA Good Laboratory Practice (GLP) standards. Grain was collected from replicated field trials and analyzed for proximates (protein, fat, ash, etc.), fiber, amino acids, fatty acids, vitamin E, mineral content, phytic acid, trypsin inhibitor, and selected secondary metabolites. Forage was also collected and analyzed for proximates and fiber. All comparisons showed that MON 863 is compositionally equivalent to its near-isogenic conventional counterpart (George et al., 2004).

Agronomic and compositional equivalence studies confirm the absence of “unintended effects” in MON 863 when compared to conventional corn. Animal feeding studies have been undertaken to provide confirmation of MON 863 safety and nutritional equivalence. These include feeding studies in ruminants, swine and poultry that demonstrate MON 863 supports the performance of farm animals comparable to that of animals fed conventional corn varieties. A 90-day feeding study was carried out in rats that provided confirmatory evidence of the safety of MON 863 for human consumption. The results of the 90-day “safety-assurance” study will be presented and discussed in the context of the aforementioned studies to provide confirmatory evidence of the safety of MON 863 corn grain for human consumption.

Section snippets

Materials and methods

The study design was adapted from OECD Guideline No. 408 (1981) and was conducted in general compliance with OECD Good Laboratory Practice (GLP) guidelines at Covance Laboratories, Vienna, Virginia, US.

Results

Compositional, contaminant, and nutritional analysis of the experimental diets showed that they met the specifications for Certified Rodent LabDiet 5002 established by PMI. The levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphate insecticides were below detection limits. For chlordane, the Covance Laboratories’ limit of detection was higher (250 ppb) than the maximum specified concentration of 50 ppb, but this was not considered to have an impact on the study. PCR

Discussion

During the course of this study, animals fed MON 863 grain in the diet had similar body weights, body weight gains and food consumption when compared to animals fed diets containing grain from control or six different conventional reference varieties. In addition, there were no differences in hematology, serum chemistry, and urinalysis parameters for animals fed MON 863 that were considered test article related. Similarly, there were no test article related changes in organ weights or gross and

Conclusion

In conclusion, the findings from rats fed diets containing MON 863 grain were similar to those fed diets containing grain from control and conventional reference varieties. The results of this 90-day rat study have been subsequently reviewed in their entirety by multiple global regulatory agencies. The summary prepared by the GMO Panel of the European Food Safety Authority best captures the prevailing scientific conclusion regarding the findings from this study. EFSA concluded that the results

Acknowledgments

Formulas used for the preparation of rodent diets were developed by Dr. Dorrance Haught, Purina Mills, Inc., St Louis, MO. Appreciation is expressed to Drs. Roy Fuchs, Shiela Schuette, and Joel Kronenberg for their review and comments on this manuscript and to Mark Naylor for assistance on the tables and figures.

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